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Structural Basis for the Activation of Microtubule Assembly by the EB1 and p150Glued Complex
Abstract
Plus-end tracking proteins, such as EB1 and the dynein/dynactin complex, regulate microtubule dynamics. These proteins are thought to stabilize microtubules by forming a plus-end complex at microtubule growing ends with ill-defined mechanisms. Here we report the crystal structure of two plus-end complex components, the carboxy-terminal dimerization domain of EB1 and the microtubule binding (CAP-Gly) domain of the dynactin subunit p150Glued. Each molecule of the EB1 dimer contains two helices forming a conserved four-helix bundle, while also providing p150Glued binding sites in its flexible tail region. Combining crystallography, NMR, and mutational analyses, our studies reveal the critical interacting elements of both EB1 and p150Glued, whose mutation alters microtubule polymerization activity. Moreover, removal of the key flexible tail from EB1 activates microtubule assembly by EB1 alone, suggesting that the flexible tail negatively regulates EB1 activity. We, therefore, propose that EB1 possesses an auto-inhibited conformation, which is relieved by p150Glued as an allosteric activator.
... Recently, a new signaling motif, LxxPTPh (x for any amino acid and h represents the hydrophobic residue) was also observed in various +TIPs [23]. The +TIPS bind to EB1c domain and subsequently target the respective proteins toward the MTs [24][25][26]. EB1 protein also showed a significant increase in their MT localization with the addition of these peptide signals [25,27,28]. Hence, characterizing the EB1 interaction with the signalling motifs of the +TIPS is vital for understanding the mechanism of recruitment of the protein to the plus end of the microtubule. ...
... The +TIPS bind to EB1c domain and subsequently target the respective proteins toward the MTs [24][25][26]. EB1 protein also showed a significant increase in their MT localization with the addition of these peptide signals [25,27,28]. Hence, characterizing the EB1 interaction with the signalling motifs of the +TIPS is vital for understanding the mechanism of recruitment of the protein to the plus end of the microtubule. ...
... The crystal structure of the complex formed by EB1c and GKNDG signalling motif of P150N protein showed that the GKNDG signals specifically recognized the extreme C-terminal EEY/F amino acid sequence of EB1 [25]. A proposed model for the activation of EB1 by GKNDG signalling motif described the apo EB1 in an autoinhibitory state with the extreme Cterminal end of EB1 masking the microtubule-binding site of EB1n. ...
SxIP is a microtubule tip localizing signal found in many +TIP proteins that bind to the hydrophobic cavity of C-terminal domain of End binding protein 1 (EB1), and then positively regulate the microtubule plus-end tracking of EBs. However, the exact mechanism of microtubule activation of EBs in the presence of SxIP signaling motif is not known. Here, we studied the effect of SxIP peptide on the native conformation of EB1 in solution. Using various NMR experiments, we found that SxIP peptide promoted the dissociation of natively formed EB1 dimer. We also discovered that I224A mutation of EB1 resulted in an unfolded C-terminal domain, which upon binding with SxIP motif, folded to its native structure. MD simulation also confirmed the relative structural stability of EB1 monomer in the SxIP bound state. Residual dipolar couplings and heteronuclear NOE analysis suggested that the binding of SxIP peptide at the C-terminal domain of EB1, decreased the dynamics and conformational flexibility of N-terminal domain involved in EB1-microtubule interaction. The SxIP induced disruption of the dimeric interactions in EB1, coupled with the reduction in conformational flexibility of N-terminal domain of EB1 might facilitate the microtubule association of EB1.
... In vitro studies with purified components established that p150 and EB1 directly interact with each other (Fig.6d) and a co crystal structure between the interacting fragments could be achieved. P150 makes contacts to flexible acidic tail of EBs as well as to the hydrophobic cavity of EBs, in close proximity to were SXIP motifs bind to (Hayashi et al., 2005, Honnappa et al., 2006, Honnappa et al., 2009a. Even though EB1 and p150 ...
... Purified full length p150 derived from baculovirus infected insect cells was not soluble on its own (not shown, also reported in ) but fragments lacking the dynactin integration domain have been shown to be soluble and comprise the ability to bind MTs, the dynein complex and EB1 in vitro (Miura et al., 2010, Culver-Hanlon et al., 2006, Hayashi et al., 2005. I therefore tested if fragments lacking the dynactin integration domain could be used as surrogates as they should comprise all necessary features with respect to plus end tracking and dynein interaction. ...
... A direct interaction between EB1 and p150 is well described in vitro and has been further supported by co-immunoprecipitations from cell extracts (Hayashi et al., 2005, Sun et al., 2008, Honnappa et al., 2006. In cells however, EB1 is necessary but not sufficient for p150 end tracking. ...
Microtubules are non-covalent polymers that form an essential part of the cytoskeleton in eukaryotic cells. Alternating phases of growth and shortening are essential for space exploration, force generation and facilitate rearrangements of the microtubule cytoskeleton in response to various stimuli. Microtubule associated proteins regulate filament dynamics and can transport cargoes. The mechanism of how microtubules grow, what triggers the transition from a growing to a shrinking microtubule, and the interplay between the various microtubule-associated proteins is only poorly understood. In vitro reconstitution approaches from purified components in combination with microfluidics techniques and simultaneous multi-colour total internal reflection florescence microscopy were employed to shed new light on the mechanism of microtubule dynamics and the interplay of proteins that bind specifically to growing microtubule ends. Tubulin undergoes conformational changes during incorporation into the polymer. Using a conformation-sensitive designed ankyrin repeat protein probe, it has been found here that these conformational changes occur at much later steps during incorporation into the polymer than previously appreciated. Growing microtubules switch to a rapid shortening phase unless their ends contain a stabilizing structure whose nature is not fully understood. The decay of this stabilizing structure was directly measured by rapid tubulin dilutions and predictions from several theoretical models have been tested. The density of a particular tubulin conformation recognized by microtubule End Binding proteins (EB1/Mal3) could be linked to filament stability. Microtubule end tracking proteins form a dynamic protein interaction network. Here, the molecular mechanism of several main players of these proteins that lead to growing microtubule end accumulation of the motor protein dynein has been elucidated by in vitro reconstitutions. The bottom up approach applied in this thesis yielded new information about fundamental properties of microtubule dynamics and gained new insight into the interplay of an important class of microtubule associated proteins.
... The CH domain binds the microtubule lattice between four tubulin heterdimers where it specifically engages tubulin in a transition state / post GTP-hydrolysis state just distal to the microtubule's polymerizing GTP cap (7). Both the EB1 dimerization domain and the EEY motif serve to bind and recruit factors to the microtubule plus-end (12)(13)(14)(15). ...
... Over 50 proteins have been identified that contain SxIP-motifs and show EB1dependent microtubule plus-end tracking activity (3). The EB1 C-terminal EEY motif is capable of binding CAP-Gly domains such as those found in p150 Glued of the dynactin complex as well as CLIP-115 and CLIP-170 ( Fig. 1C) (14,15,18). The p150 Glued CAP-Gly domain dually engages the EB1 EEY motif as well as the EBHD where its binding site overlaps with the binding site for SxIP and LxxPTPh motifs, suggesting mutually exclusive binding per site on the EB1 dimer (13)(14)(15)(16)(17). Whether CLIP-170 CAP-Gly domains engage the EBHD in a similar manner remains to be determined. ...
... The EB1 C-terminal EEY motif is capable of binding CAP-Gly domains such as those found in p150 Glued of the dynactin complex as well as CLIP-115 and CLIP-170 ( Fig. 1C) (14,15,18). The p150 Glued CAP-Gly domain dually engages the EB1 EEY motif as well as the EBHD where its binding site overlaps with the binding site for SxIP and LxxPTPh motifs, suggesting mutually exclusive binding per site on the EB1 dimer (13)(14)(15)(16)(17). Whether CLIP-170 CAP-Gly domains engage the EBHD in a similar manner remains to be determined. ...
Cytoplasmic linker protein (CLIP)4-170 is a microtubule plus-end factor that links vesicles to microtubules and recruits the dynein/dynactin complex to microtubule plus ends. CLIP-170 plus-end localization is end binding 1 (EB1)-dependent. CLIP-170 contains two N-terminal cytoskeleton-associated protein glycine-rich (CAP-Gly) domains flanked by serine-rich regions. The CAP-Gly domains are known EB1-binding domains, and the serine-rich regions have also been implicated in CLIP-170's microtubule plus-end localization mechanism. However, the determinants in these serine-rich regions have not been identified. Here we elucidated multiple EB1-binding modules in the CLIP-170 N-terminal region. Using isothermal titration calorimetry and size-exclusion chromatography, we mapped and biophysically characterized these EB1-binding modules, including the two CAP-Gly domains, a bridging SxIP motif, and a unique array of divergent SxIP-like motifs located N-terminally to the first CAP-Gly domain. We found that unlike the EB1-binding mode of the CAP-Gly domain in the dynactin-associated protein p150Glued, which dually engages the EB1 C-terminal EEY motif, as well as the EB homology domain (EBHD), and sterically occludes SxIP motif binding, the CLIP-170 CAP-Gly domains engage only the EEY motif, enabling the flanking SxIP and SxIP-like motifs to bind the EBHD. These multivalent EB1-binding modules provided avidity to the CLIP-170-EB1 interaction, likely clarifying why CLIP-170 preferentially binds EB1 rather than the α-tubulin C-terminal EEY motif. Our finding that CLIP-170 has multiple non-CAP-Gly EB1-binding modules may explain why autoinhibition of CLIP-170 GAP-Gly domains does not fully abrogate its microtubule plus end-localization. This work expands our understanding of EB1-binding motifs and their multivalent networks.
... The p150 Glued N-terminus may also play a role in the initiation of dynein transport, particularly at the microtubule plus-end, where a pool of dynactin has long been observed (Lloyd et al., 2012;Moore et al., 2009;Moughamian and Holzbaur, 2012;Vaughan et al., 2002;Xiang et al., 2000;Zhang et al., 2003). Finally, at the microtubule plus-end, the p150 Glued N-terminus may directly modify dynamics through interactions with the microtubule or through other plus end-tracking proteins (Berrueta et al., 1999;Goodson et al., 2003;Hayashi et al., 2005;Lansbergen et al., 2004;Ligon et al., 2003;Ligon et al., 2006). ...
... Because p150 Glued binds directly to two other canonical +TIPs, EB1 and CLIP-170, it may be uniquely positioned to coordinate microtubule dynamics (Goodson et al., 2003;Hayashi et al., 2005;Honnappa et al., 2006;Ligon et al., 2003). Previous biochemical studies concluded that p150 Glued alone does not markedly affect microtubule dynamics, but these investigations were limited to consideration of a monomeric CAP-Gly domain lacking the flanking basic domain (Hayashi et al., 2005;Manna et al., 2008). ...
... Because p150 Glued binds directly to two other canonical +TIPs, EB1 and CLIP-170, it may be uniquely positioned to coordinate microtubule dynamics (Goodson et al., 2003;Hayashi et al., 2005;Honnappa et al., 2006;Ligon et al., 2003). Previous biochemical studies concluded that p150 Glued alone does not markedly affect microtubule dynamics, but these investigations were limited to consideration of a monomeric CAP-Gly domain lacking the flanking basic domain (Hayashi et al., 2005;Manna et al., 2008). ...
Microtubules are cytoskeletal polymers that serve as long-distance tracks for intracellular transport. However, microtubules are not static tracks; they undergo dynamic instability, a non-equilibrium behavior which allows them to continually remodel. Microtubules must remodel to adapt to the changing needs of the cell, yet they must also be stable enough to allow long-distance transport by kinesins and by cytoplasmic dynein and its partner complex dynactin. Microtubule dynamics are strongly influenced by microtubule-associated proteins, which bind to microtubules and bias them toward growth or shrinkage. A pool of dynein and dynactin localizes to the microtubule plus end and to the cortex, where we hypothesized that they might not be solely poised to initiate retrograde runs, but might also act to modify microtubule dynamics. To test this, we reconstituted microtubule dynamic instability in vitro and visualized it using TIRF microscopy. We show that dynein, when immobilized in an orientation recapitulating its membrane recruitment, can tether growing microtubules, reducing their lateral diffusion and delaying catastrophe. This effect does not represent mere microtubule binding; dynein tethers microtubules more effectively than kinesin or the plus end-tracking protein EB1, and we show that this ability of dynein is dependent on its ATPase activity. Modeling suggests that dynein may delay catastrophe by actively applying tension to straighten, and thus stabilize the microtubule. We also have examined the effects of the p150Glued subunit of dynactin on microtubule dynamics. We find that to modify dynamics, p150Glued must be dimerized, and it must bind tubulin, an interaction that requires its CAP-Gly and basic domains in tandem. p150Glued is alternatively spliced in vivo, with the isoform including both these domains expressed primarily in neurons. Accordingly, depletion of p150Glued in a non-polarized cell line does not alter microtubule dynamics, while p150Glued RNAi in neurons leads to a dramatic increase in microtubule catastrophe. Strikingly, a Parkinson syndrome-associated mutation blocks this microtubule-stabilizing activity both in vitro and in neurons. Together, these data provide novel mechanistic insight into how cytoplasmic dynein and dynactin, the principle minus end-directed motor complex in the cell, also act at the plus end to coordinate microtubule dynamics.
... in Schizosaccharomyces pombe (Busch and Brunner, 2004). Similarly, in vitro studies have described that EB1 can promote polymerization of pure tubulin (Ligon et al., 2003;Sandblad et al., 2006;Bieling et al., 2007;Manna et al., 2008), whereas other studies suggested that EB1 requires a binding partner to accomplish this, such as APC or p150 Glued (Nakamura et al., 2001;Hayashi et al., 2005). ...
... This EB1 C-terminal tail is rich in Glu residues and seems to be strikingly similar to the tail of α-tubulin. The EB1 tail is also involved in the interaction with other +TIP proteins, such as CLIPs (Komarova et al., 2005) and p150 Glued (Hayashi et al., 2005;Manna et al., 2008). It has been suggested that the flexible tail negatively regulates EB1 activity, and that this auto-inhibition is relieved by interaction with other +TIPs, such as p150 Glued (Hayashi et al., 2005). ...
... The EB1 tail is also involved in the interaction with other +TIP proteins, such as CLIPs (Komarova et al., 2005) and p150 Glued (Hayashi et al., 2005;Manna et al., 2008). It has been suggested that the flexible tail negatively regulates EB1 activity, and that this auto-inhibition is relieved by interaction with other +TIPs, such as p150 Glued (Hayashi et al., 2005). ...
In recent years it has become apparent that major cell biological processes are not dependent on individual proteins, but are carried out by assemblies of protein molecules. Different proteins work together in a highly coordinated way, resembling “molecular machines”. Several of these molecular machines can be found during division, when protein complexes precisely orchestrate profound changes in the structure and physiology of the cell. The plant microtubule (MT) cytoskeleton plays an essential role in cell division and undergoes fast rearrangements from cortical microtubules to a preprophase band, spindle and phragmoplast. This process requires the cooperation of several MT-associated proteins (MAPs). Although the knowledge on plant MAPs is gradually increasing, not much is known yet about the interactions between these MAPs, nor about the processes that regulate their activity, such as phosphorylation. In animal cells it is already described that the mitotic Aurora kinase is an important player in the cytoskeletal organization during division. Aurora kinase is therefore often compared to a conductor of a symphonic orchestra, interacting with several microtubule-binding partners, and coordinating the transitions through the different phases of the mitotic symphony. In plants however, not much is known yet about the function of the Aurora kinases or their interacting proteins. Consequently, in this research we aimed to identify MAP protein complexes that function together in the successful execution of mitosis and cytokinesis, specifically focusing on AURORA1 complexes. Yeast two-hybrid library screens and Tandem Affinity Purification experiments were performed to identify interaction partners (Chapter 2). To narrow down the resulting set of candidate interacting proteins, their GFP-localization was followed in dividing BY-2 cells. This strategy resulted in identification of candidate proteins that associate with the cytoskeleton or cell plate during cell division. In Chapter 3, the interaction between AURORA1 (AUR1) and its novel interacting partner, ARCTICA1 (ARC1) is analyzed in more detail. We provide evidence that ARC1 is an in vitro substrate of AUR1. Besides localizing to kinetochores and the cell plate during cell division, ARC1 associated with the plasma membrane in a polar manner. This membrane association was further characterized in Chapter 4. Finally, a similar strategy was followed to study the binding partners of the EB1 (End Binding 1) protein, that is known to form an interaction hub at the microtubule plus end in human cells (Chapter 5). Our interaction assays identified that the EB1 family of microtubule plus-end binding proteins dimerize in plants, and we further investigated the function of EB1 dimerization in the EB1 plus-end complex assembly.
... As anticipated, the overall structural topology of the protein was retained upon binding to MTs, with the core of the protein comprised of four β-sheets and a short C-terminal α-helix. At the same time, as shown in Fig. 3D, loop regions in CAP-Gly underwent pronounced conformational changes upon the formation of a complex with MTs, compared with the free CAP-Gly and CAP-Gly/EB1 complex (15,23,26). These changes resulted in sidechains adopting different orientations in free and MTand EB1-bound states (Fig. 3E). ...
... The β3-β4 loop and the GKNDG motif within this loop constitute the primary interface with microtubules. This finding corroborates prior hypotheses, made on the basis of structural analysis of CAP-Gly complexes with EB1 and CLIP170, that the GKNDG motif likely constitutes a binding interface, with MT being a specific recognition sequence for EEY/F motifs ubiquitously present in CLIP170 and EB proteins, as well as in α-tubulin (26,39). Our current results also clearly indicate that the bean-shaped CAP-Gly molecule binds to the microtubules with its flat surface comprised by its loops, the C-terminal β4-strand, and the short α-helix. ...
... Our results reveal that the intermolecular interfaces of CAP-Gly with microtubules and EB1 partly overlap (β3-β4 loop and S92-Q93 at helix turn). The binding interface of CAP-Gly with EB1 is known from previous studies (15,26). The dREDOR-based spectra of the U-13 C, 15 N-CAP-Gly/EB1 complex shown in Fig. 4C corroborate the previous findings and validate the dREDOR approach as applied to the CAP-Gly/MT complex. ...
Significance
Microtubules and their associated proteins are central to most cellular functions. They have been extensively studied at multiple levels of resolution; however, significant knowledge gaps remain. Structures of microtubule-associated proteins bound to microtubules are not known at atomic resolution. We used magic angle spinning NMR to solve a structure of dynactin’s cytoskeleton-associated protein glycine-rich (CAP-Gly) domain bound to microtubules and to determine the intermolecular interface, the first example, to our knowledge, of the atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. The results reveal remarkable structural plasticity of CAP-Gly, which enables CAP-Gly’s binding to microtubules and other binding partners. This approach offers atomic-resolution information of microtubule-binding proteins on microtubules and opens up the possibility to study critical parameters such as protonation states, strain, and dynamics on multiple time scales.
... The acidic tails of EB1 are also essential in the association with another group of +TIPs -the CAP-Gly (cytoskeleton associated protein with a conserved glycine) domain containing proteins, which include the CLIPs and p150 Glued , the large subunit of the dynein accessory complex dynactin. p150 Glued contains a single NH 2 -terminal CAP-Gly domain per monomeric subunit, which is essential for the binding to MTs as well as EB1 (16,(29)(30)(31). The MT plus-end accumulation of p150 Glued may partly result from the direct MT binding; however, it probably mostly occurs via hitchhiking on CLIP-170 and EB1 (29,(31)(32)(33). ...
... p150 Glued contains a single NH 2 -terminal CAP-Gly domain per monomeric subunit, which is essential for the binding to MTs as well as EB1 (16,(29)(30)(31). The MT plus-end accumulation of p150 Glued may partly result from the direct MT binding; however, it probably mostly occurs via hitchhiking on CLIP-170 and EB1 (29,(31)(32)(33). ...
... A recent structural study has shown that p150 Glued makes contact predominantly with the tail of EB1 (31). This result is in complete agreement with other studies, including our own, on the critical binding region of EB1 for both CLIPs and p150 Glued . ...
... Another feature of Skeletonema EB proteins is the presence of two conservative runs of negatively charged aspartate (D) and glutamate (E) residues, DDDDDE and DDDDDDE, on the C terminus (Supplements_EB.fast). The presence of these amino acids in the C-terminal area is common for all EB proteins and was noted to be similar to a flexible acidic tail of tubulin (Hayashi et al. 2005), but negatively charged boxes of this size were not previously documented. ...
... It is known that charged amino acids in the C-tail of EB proteins are essential for their interactions with microtubule ends (Hayashi et al. 2005), even though their removal does not affect the general protein conformation (Buey et al. 2011). To determine whether the structure of the diatom C-tail could affect the functioning of the EB protein in diatoms, we compared the distributions of charge along the sequence between diatom EB proteins, human EB1, and A. thaliana EBA (Fig. 5). ...
The end-binding proteins are a family of microtubule-associated proteins; this family belongs to plus-end-tracking proteins (+TIPs) that regulate microtubule growth and stabilisation. Although the genes encoding EB proteins are found in all eukaryotic genomes, most studies of them have centred on one or another taxonomic group, without a broad comparative analysis. Here, we present a first phylogenetic analysis and a comparative analysis of domain structures of diatom EB proteins in comparison with other phyla of Chromista, red and green algae, as well as model organisms A. thaliana and H. sapiens. Phylogenetically, diatom EB proteins are separated into six clades, generally corresponding to the phylogeny of their respective organisms. The domain structure of this family is highly variable, but the CH and EBH domains responsible for binding tubulin and other MAPs are mostly conserved. Homologous modelling of the F. cylindrus EB protein shows that conserved motifs of the CH domain are positioned on the protein surface, which is necessary for their functioning. We hypothesise that high variance of the diatom C-terminal domain is caused by previously unknown interactions with a CAP-GLY motif of dynactin subunit p150. Our findings contribute to wider possibilities for further investigations of the cytoskeleton in diatoms.
... The dynactin subunit p150Glued plays a key role in the release of the MT plus end protein EB1 auto-inhibition and subsequent engagement of the dynein/dynactin complex at MT plus ends (Hayashi et al, 2005;Akhmanova & Steinmetz, 2015;McKenney et al, 2016;McKenney, 2018). However, how those molecular dynamics, so far investigated in in vitro studies with recombinant proteins or in crystals (Duellberg et al, 2014), are regulated in intact living cells is unknown. ...
... To verify the possible involvement of STIM1 SxIP motif in the interaction with p150Glued, we employed a mutant version of STIM1 unable to bind EB1 (Grigoriev et al, 2008;Honnappa et al, 2009) and dubbed GFP-STIM1 NN ( Fig 1C) as the Ile and Pro residues of the SxIP motif were replaced by two Asn (N). Since STIM1 also owns three CC domains (Novello et al, 2018), which are known ubiquitous protein-protein interaction domains (Woolfson et al, 2012) and key in releasing the auto-inhibition of MT plus end proteins (Hayashi et al, 2005), we asked whether STIM1 CC motifs may also mediate its association with p150Glued. Hence, we generated GFP-STIM1 mutants lacking each of the CC domain (DCC1, DCC2, and DCC3; Fig EV1C) or all of them (DCC1-3; Fig 1E) in both wild-type (WT) or NN-STIM1 backbones (DCC1-3/WT and DCC1-3/NN), cotransfected them with mCherry-p150Glued WT in HEK239T cells, and verified their interactions. ...
Although subcellular positioning of endosomes significantly impacts on their functions, the molecular mechanisms governing the different steady-state distribution of early endosomes (EEs) and late endosomes (LEs)/lysosomes (LYs) in peripheral and perinuclear eukaryotic cell areas, respectively, are still unsolved. We unveil that such differences arise because, while LE retrograde transport depends on the dynein microtubule (MT) motor only, the one of EEs requires the cooperative antagonism of dynein and kinesin-14 KIFC1, a MT minus end-directed motor involved in cancer progression. Mechanistically, the Ser-x-Ile-Pro (SxIP) motif-mediated interaction of the endoplasmic reticulum transmembrane protein stromal interaction molecule 1 (STIM1) with the MT plus end-binding protein 1 (EB1) promotes its association with the p150Glued subunit of the dynein activator complex dynactin and the distinct location of EEs and LEs/LYs. The peripheral distribution of EEs requires their p150Glued-mediated simultaneous engagement with dynein and SxIP motif-containing KIFC1, via HOOK1 and HOOK3 adaptors, respectively. In sum, we provide evidence that distinct minus end-directed MT motor systems drive the differential transport and subcellular distribution of EEs and LEs in mammalian cells. © 2020 The Authors. Published under the terms of the CC BY 4.0 license
... MT nucleation and anchoring activities at the centrosome are regulated by multiple protein complexes, but the processes involved have not been fully elucidated. One of the complexes that is important for MT nucleation and anchoring at the centrosome is the dynactin complex (Delgehyr et al., 2005;Hayashi et al., 2005;Strickland et al., 2005). Interaction between p150, a component of the dynactin complex, and end-binding 1 (EB1), an MT plus-end tracking protein, has been shown to be necessary for the assembly of a radial MT array during the formation of the centrosome (Askham et al., 2002;Louie et al., 2004;Vaughan, 2005). ...
... A previous report indicated that the formation of radial MT arrays from the centrosome requires interaction between EB1 and p150 (Askham et al., 2002;Hayashi et al., 2005;Strickland et al., 2005). We therefore examined whether the interaction between EB1 and p150 is affected by the EB1-A7 mutation. ...
ADP-ribosylation factor (Arf)-like 4D (Arl4D), one of the Arf-like small GTPases, functions in the regulation of cell morphology, cell migration, and actin cytoskeleton remodeling. EB1 is a microtubule plus-end tracking protein that preferentially localizes at the tips of the plus ends of growing microtubules and at the centrosome. EB1 depletion results in many centrosome-related defects. Here, we report that Arl4D promotes the recruitment of EB1 to the centrosome and regulates microtubule nucleation. We first showed that Arl4D interacts with EB1 in a GTP-dependent manner. This interaction is dependent on the C-terminal EBH region of EB1 and partially dependent on an SxLP motif of Arl4D. We found that Arl4D colocalized with γ-tubulin in centrosomes and the depletion of Arl4D resulted in a centrosomal microtubule nucleation defect. We further demonstrated that abolishing Arl4D-EB1 interaction decreased microtubule nucleation rate and diminished the centrosomal recruitment of EB1 without affecting MT growth rate. In addition, Arl4D binding to EB1 increased the association between the p150 subunit of dynactin and EB1, which is important for microtubule stabilization. Together, our results indicate that Arl4D modulates microtubule nucleation through regulation of the EB1-p150 association at the centrosome.
[Media: see text]
... Here, we performed a systematic investigation of the accuracy and precision attainable in protein structures determined from MAS NMR-derived carbon-carbon distances. To this end, we carried out model calculations for four proteins depicted in Fig. 1, (i) the CAP-Gly domain of dynactin, an 89-residue protein whose structures, free and bound to several target proteins, have been determined by X-ray crystallography (Bjelic et al. 2012;Honnappa et al. 2006;Weisbrich et al. 2007;Hayashi et al. 2005Hayashi et al. , 2007, by solution NMR (Hayashi et al. 2005(Hayashi et al. , 2007, and by MAS NMR (Yan et al. , 2015; (ii) the 133 residue agglutinin from Oscillatoria agardhii (OAA), whose structure and carbohydrate interactions were studied by solution NMR and crystallography (Koharudin and Gronenborn 2011), and for which both solution and MAS NMR resonance assignments are available (Carneiro et al. 2015;Fritz et al. 2017); (iii) the carbohydrate binding domain of galectin-3C (Galectin CBD), which comprises 138 amino acids and for which extensive structural information is available including solution and MAS NMR chemical shifts (Kraus et al. 2018) and 48 X-ray crystal structures of various resolutions in the apo and ligand-bound states (Sorme et al. 2005;Atmanene et al. 2017;Bum-Erdene et al. 2013;Collins et al. 2012Collins et al. , 2014Hsieh et al. 2016;Rajput et al. 2016;Saraboji et al. 2012); and (iv) a tubular assembly of the 231 amino acid HIV-1 capsid protein (CA), which has been studied extensively in our laboratories by solution and MAS NMR Han et al. 2010Han et al. , 2013Byeon et al. 2012;Gupta et al. 2016;Liu et al. 2016;Zhang et al. 2016;Quinn et al. 2018 Our results establish the criteria for determining accurate protein structures on the basis of the distance restraints that are typically recorded in MAS NMR experiments for 13 C labeled proteins, following a common protocol for the preparation of isotopically labeled samples using either 1,6-13 C-glucose or 2-13 C-glucose as the carbon source. For compact, single domain proteins, at least 3-5 C-C restraints per residue are required to derive the global fold of the molecule, based on distances up to 7 Å, and 15 random restraints per residue are required for attaining maximum accuracy. ...
... Here, we performed a systematic investigation of the accuracy and precision attainable in protein structures determined from MAS NMR-derived carbon-carbon distances. To this end, we carried out model calculations for four proteins depicted in Fig. 1, (i) the CAP-Gly domain of dynactin, an 89-residue protein whose structures, free and bound to several target proteins, have been determined by X-ray crystallography (Bjelic et al. 2012;Honnappa et al. 2006;Weisbrich et al. 2007;Hayashi et al. 2005Hayashi et al. , 2007, by solution NMR (Hayashi et al. 2005(Hayashi et al. , 2007, and by MAS NMR (Yan et al. , 2015; (ii) the 133 residue agglutinin from Oscillatoria agardhii (OAA), whose structure and carbohydrate interactions were studied by solution NMR and crystallography (Koharudin and Gronenborn 2011), and for which both solution and MAS NMR resonance assignments are available (Carneiro et al. 2015;Fritz et al. 2017); (iii) the carbohydrate binding domain of galectin-3C (Galectin CBD), which comprises 138 amino acids and for which extensive structural information is available including solution and MAS NMR chemical shifts (Kraus et al. 2018) and 48 X-ray crystal structures of various resolutions in the apo and ligand-bound states (Sorme et al. 2005;Atmanene et al. 2017;Bum-Erdene et al. 2013;Collins et al. 2012Collins et al. , 2014Hsieh et al. 2016;Rajput et al. 2016;Saraboji et al. 2012); and (iv) a tubular assembly of the 231 amino acid HIV-1 capsid protein (CA), which has been studied extensively in our laboratories by solution and MAS NMR Han et al. 2010Han et al. , 2013Byeon et al. 2012;Gupta et al. 2016;Liu et al. 2016;Zhang et al. 2016;Quinn et al. 2018 Our results establish the criteria for determining accurate protein structures on the basis of the distance restraints that are typically recorded in MAS NMR experiments for 13 C labeled proteins, following a common protocol for the preparation of isotopically labeled samples using either 1,6-13 C-glucose or 2-13 C-glucose as the carbon source. For compact, single domain proteins, at least 3-5 C-C restraints per residue are required to derive the global fold of the molecule, based on distances up to 7 Å, and 15 random restraints per residue are required for attaining maximum accuracy. ...
We present a systematic investigation into the attainable accuracy and precision of protein structures determined by heteronuclear magic angle spinning solid-state NMR for a set of four proteins of varied size and secondary structure content. Structures were calculated using synthetically generated random sets of C-C distances up to 7 Å at different degrees of completeness. For single-domain proteins, 9–15 restraints per residue are sufficient to derive an accurate model structure, while maximum accuracy and precision are reached with over 15 restraints per residue. For multi-domain proteins and protein assemblies, additional information on domain orientations, quaternary structure and/or protein shape is needed. As demonstrated for the HIV-1 capsid protein assembly, this can be accomplished by integrating MAS NMR with cryoEM data. In all cases, inclusion of TALOS-derived backbone torsion angles improves the accuracy for small number of restraints, while no further increases are noted for restraint completeness above 40%. In contrast, inclusion of TALOS-derived torsion angle restraints consistently increases the precision of the structural ensemble at all degrees of distance restraint completeness.
... However, the existence of autoinhibition among EB proteins is not reliably established and remains a matter of controversy. This hypothesis was first proposed by Hayashi et al. [39] based on data on tubulin polymer ization in solution in the presence of a high concentration of EB1. The mutant protein EB1 lacking 20 a.a. on the C end was shown to be more favorable for tubulin polymer ization than the full length protein EB1. ...
... The mutant protein EB1 lacking 20 a.a. on the C end was shown to be more favorable for tubulin polymer ization than the full length protein EB1. This observation became the basis for the hypothesis of EB1 autoinhibition through a direct intramolecular interaction between the calponin homology domains and flexible C terminal tails [39]. But later it became clear that removal of the nega tively charged C terminal tail increased the affinity of EB1 for the whole microtubule body, lowering the "pref erence" for the microtubule growing end [32]. ...
Microtubules are polymers of tubulin protein, one of the key components of cytoskeleton. They are polar filaments whose plus-ends usually oriented toward the cell periphery are more dynamic than their minus-ends, which face the center of the cell. In cells, microtubules are organized into a network that is being constantly rebuilt and renovated due to stochastic switching of its individual filaments from growth to shrinkage and back. Because of these dynamics and their mechanical properties, microtubules take part in various essential processes, from intracellular transport to search and capture of chromosomes during mitosis. Microtubule dynamics are regulated by many proteins that are located on the plus-ends of these filaments. One of the most important and abundant groups of plus-end-interacting proteins are EB-family proteins, which autonomously recognize structures of the microtubule growing plus-ends, modulate their dynamics, and recruit multiple partner proteins with diverse functions onto the microtubule plus-ends. In this review, we summarize the published data about the properties and functions of EB-proteins, focusing on analysis of their mechanism of interaction with the microtubule growing ends.
... Recent studies reveal that CLIP-170 and endbinding protein 1 (EB1), two important microtubule plusend tracking proteins (+TIPs), can form dimer to exert their roles in the regulation of microtubule assembly [19,20]. Interestingly, structural characterization of the CYLD USP domain reveals a small zinc-binding B-box module [21]. ...
... In cells, CYLD acts in concert with the +TIPs protein EB1 to regulate microtubule dynamics [22]. Recent study reveals that EB1 can form dimers by an intermolecular interaction, which leads to the autoinhibition of EB1 activity [19,23]. The dimerization-caused autoinhibition of EB1 prompts us to investigate whether CYLD has similar behavior. ...
The tumor suppressor protein cylindromatosis (CYLD), as a microtubule-associated deubiquitinase, plays a pivotal role in a wide range of cellular activities, including innate immunity, cell division, and ciliogenesis. Structural characterization reveals a small zinc-binding B-box inserted within the ubiquitin specific protease (USP) domain of CYLD; however, the exact role for this module remains yet to be elucidated. Here we identify a critical role for the B-box in facilitating the intermolecular interaction and subcellular localization of CYLD. By co-immunoprecipitation assays we uncover that CYLD has the ability to form an intermolecular complex. Native gel electrophoresis analysis and pull down assays show that the USP domain of CYLD is essential for its intermolecular interaction. Further investigation reveals that deletion of the B-box from the USP domain disrupts the intermolecular interaction of CYLD. Importantly, although loss of the B-box has no obvious effect on the deubiquitinase activity of CYLD, it abolishes the USP domain-mediated retention of CYLD in the cytoplasm. Collectively, these data demonstrate an important role for the B-box module of CYLD in mediating its assembly and subcellular distribution, which might be related to the functions of CYLD in various biological processes.
... Residues V47, T50 and K56 are in close contacts with the EB1 C-terminal α helix while H40-T43 and I61 are in proximity to the β3-β4 loop, which contains the GKNDG binding motif. The C-terminal α-helix (S92-Q93) exhibits obvious NMR chemical shifts changes upon complex formation in both the microcrystalline and solution states and was shown to have hydrogen bonding interaction with tripeptide segment EEY at the C-terminal tail of EB1 (Hayashi et al. 2005;Honnappa et al. 2006;Yan et al. 2013a). The rest of the residues detected by the dREDOR-based experiments are located in the β3-β4 loop. ...
... The rest of the residues detected by the dREDOR-based experiments are located in the β3-β4 loop. These results are generally consistent with the previous X-ray diffraction studies (Hayashi et al. 2005;Honnappa et al. 2006). We note that some of these residues detected by dREDOR (i.e. ...
REDOR-based experiments with simultaneous ¹H–¹³C and ¹H−¹⁵N dipolar dephasing are explored for investigating intermolecular protein–protein interfaces in complexes formed by a U–¹³C,¹⁵N-labeled protein and its natural abundance binding partner. The application of a double-REDOR filter (dREDOR) results in a complete dephasing of proton magnetization in the U–¹³C,¹⁵N-enriched molecule while the proton magnetization of the unlabeled binding partner is not dephased. This retained proton magnetization is then transferred across the intermolecular interface by ¹H–¹³C or ¹H–¹⁵N cross polarization, permitting to establish the residues of the U–¹³C,¹⁵N-labeled protein, which constitute the binding interface. To assign the interface residues, this dREDOR-CPMAS element is incorporated as a building block into ¹³C–¹³C correlation experiments. We established the validity of this approach on U–¹³C,¹⁵N-histidine and on a structurally characterized complex of dynactin’s U–¹³C,¹⁵N-CAP-Gly domain with end-binding protein 1 (EB1). The approach introduced here is broadly applicable to the analysis of intermolecular interfaces when one of the binding partners in a complex cannot be isotopically labeled.
... Its plus end-tracking ability depends on both EB1 and CLIP-170 [12]. In vitro assays have demonstrated that p150 glued stabilizes microtubules by associating with EB1 [13]. Similarly, binding of EB1 to p150 glued is also required for astral microtubule elongation and cleavage furrow initiation during anaphase onset, underscoring the importance of the EB1-p150 glued interaction in connecting astral microtubules to the cell cortex [14]. ...
... Through structural analysis, we found that S40D formed a new hydrogen bond with Y71, but that S40 and S40A did not. This new connection between S40D and Y71 might stabilize the CH domain and contribute to microtubule plus end binding, since S40 and Y71 are located in two adjacent helices that form a nearly parallel bundle [13]. In contrast to S40, T154 and T206 are located in the flexible linker region and the linker region close to the EBH domain, respectively, neither of which is known to directly participate in microtubule binding or recruitment of CLIP-170 and p150 glued to microtubule plus ends. ...
Phosphorylation of end-binding protein 1 (EB1), a key member of microtubule plus end-tracking proteins (+TIPs), by apoptosis signal-regulating kinase 1 (ASK1) has been demonstrated to promote the stability of astral microtubules during mitosis by stimulating the binding of EB1 to microtubule plus ends. However, the roles of other members of the +TIPs family in ASK1/EB1-mediated regulation of astral microtubules are unknown. Herein, we show that ASK1-mediated phosphorylation of EB1 enhances the localization of cytoplasmic linker protein 170 (CLIP-170) and p150glued to the plus ends of astral microtubules. Depletion of ASK1 or expression of phospho-deficient or phospho-mimetic EB1 mutants results in changes in the levels of plus-end localized CLIP-170 or p150glued. Mechanistic studies reveal that EB1 phosphorylation promotes its interactions with CLIP-170 and p150glued, thereby recruiting these +TIPs to microtubules. Structural analysis suggests that serine-40 is the primary phosphorylation site on EB1 that exerts these effects. Together, these findings provide novel insight into the molecular mechanisms that regulate the interactions of EB1 with other +TIPs.
... The p150Glued protein can interact with EB1, a microtubuleassociated protein involved in microtubule nucleation (33,34). This interaction, between p150Glued and EB1, is necessary for microtubule binding to centrosomes (33) and for microtubule nucleation (35)(36)(37). The C-terminus of EB1 binds to the Nterminus of p150Glued, and this event decreases microtubule shortening and increases rescue frequency and the growth rate of microtubules, thereby favoring microtubule elongation (33,36). ...
... Aurora A depletion results in the disconnection of centrosomes from mitotic spindle poles in Drosophila (16), and inhibition of Aurora A seems to be involved in central spindle microtubule nucleation (29). Both of these effects resemble those of EB1 inactivation (33,(35)(36)(37). Consequently, phosphorylation of p150Glued serine 19 by Aurora A could be involved in central spindle assembly through an EB1 function. ...
Until recently, the knowledge of Aurora A kinase functions during mitosis was limited to pre-metaphase events, particularly centrosome maturation, G2/M transition, and mitotic spindle assembly. However, an involvement of Aurora A in post-metaphase events was also suspected, but not clearly demonstrated due to the technical difficulty to perform the appropriate experiments. Recent developments of both an analog-specific version of Aurora A and small molecule inhibitors have led to the first demonstration that Aurora A is required for the early steps of cytokinesis. As in pre-metaphase, Aurora A plays diverse functions during anaphase, essentially participating in astral microtubules dynamics and central spindle assembly and functioning. The present review describes the experimental systems used to decipher new functions of Aurora A during late mitosis and situate these functions into the context of cytokinesis mechanisms.
... L'interaction de Bim1p et Bik1p a été décrite en double hybride (Wolyniak et al., 2006). De plus les fondements structuraux de l'interaction de EB1 avec le domaine CAP-Gly sont détaillés (Hayashi et al., 2005;Honnappa et al., 2006). Cette interaction se fait par deux contacts : un premier entre la queue C-terminale de EB1 qui est similaire à celui de la tubuline α (les tous derniers acides aminés sont EEY) et un deuxième entre la partie C-terminale de EB1 ...
... Leurs homologues, EB1 et CLIP-170, interagissent aussi. Cette interaction fait intervenir le domaine CAP-Gly de CLIP-170 et la queue C-terminale EB1(Hayashi et al., 2005;Honnappa et al., 2006;Komarova et al., 2005). La déplétion de EB1 et EB3 par interférence ARN dans des cellules CHO-K1 entraîne une diminution de la quantité de CLIP-170 aux bouts plus des microtubules. ...
Microtubules (MT) are dynamic structures involved in different essential processes of cell architecture.
Particularly, MT plus ends (+ends) can accumulate specific proteins controlling MT dynamics and MT interaction with the cell cortex. These interactions are implicated in the correct positionning of the mitotic spindle. Description of the mechanisms of protein association with MT +ends is important to understand MT functions. C-terminal tyrosine of α-tubulin is crucial for the interaction of CAP-Gly domain containing proteins.
Indeed, CLIP-170 and its S. cerevisiae homolog Bik1p bind less efficiently to MT deleted for the C-terminal tyrosine (Glu MT).
In this work, we studied the consequences of low Bik1p association with Glu MT +ends.
Current models propose that Bik1p is targeted towards MT +ends by the kinesin Kip2p. Dynein (Dyn1p) is,
then, recruited by Bik1p at MT +ends and offloaded at the cortex. Here, we show that, in yeast cells expressing only
detyrosinated tubulin (tub1-Glu strain), Kip2p and Dyn1p are correctly associated with MT +ends, despite the
decreased Bik1p binding. We propose that, in wild type cells, the Kip2p/Bik1p complex transports Dyn1p along MT towards MT +ends. Then, Kip2p, Bik1p and Dyn1p track MT +ends independently.
Moreover, we show that constituvely active forms of the small G protein Rho1p favour Bik1p binding to MT +ends. These data will be important to understand the role of Rho GTPases in the regulation of MT, for instance during migration of mammalian cells.
Finally, we systematically searched for mutations that are lethal in combination with the tub1-Glu mutation
(synthetic lethality). This screen identified components important in the formation of cell membrane and cell wall.
These genes could be involved, at the cortex, in the establishment of microtubule-cortex interactions, and could show the importance of the α-tubulin C-terminal tyrosine in this function.
... This is unexpected, since S4 is located at the very N-terminus, and it is difficult to imagine how this would affect overall mal3p structure. However, it has recently been suggested that mal3p undergoes a conformational change after release of a head to tail auto-inhibitory mechanism (Hayashi et al., 2005). It is possible that this mechanism is affected in the mal3p S4A mutant. ...
... Another possibility is that S4 has a structural role. Hayashi and co-workers had proposed that the EB1 C-terminal tail is part of an auto-inhibitory process that involves EB1 head to tail association (Hayashi et al., 2005). This is possible due to the existence of the large mal3p flexible linker region that would enable such a conformation. ...
Mikrotubuli sind hochdynamische Polymere, die wesentliche Funktionen in einer Reihe von grundlegenden zellulären Prozessen haben. Zum Beispiel sind das Generieren und Erhaltenvon Zellpolarität, die Chromosomensegregation während der Mitose oder die Positionierung und der Transport von Organellen ohne Mikrotubuli nicht möglich. Um all diese Aufgaben zu erfüllen, müssen Mikrotubuli Organisation und Dynamik genau geregelt werden. Angehörige der EB (end binding) Proteinfamilie sind vor kurzem als zentrale Regulatoren der wachsenden Mikrotubuli Plus-Enden bekannt geworden. Trotzdem sind die molekularen Mechanismen, welche die vielfältigen Aktivitäten dieser Proteine in den verschiedenen biologischenZusammenhängen und Prozessen kontrollieren noch weitgehend unverstanden. In dieser Studie zeigen wir, dass mal3p, das EB1 (end binding protein 1) Homologe der Spalthefe, an einem Cluster von fünf Serinen phosphoryliert wird. Das Cluster liegt in einer flexiblen Proteinregion, welche die mikrotubulibindende Calponinhomologie-domäne mit der konservierten EB1-Domäne verbindet. Von den Serinen sind S144 und S145 „dominant“, da die Absenz von Phosphorylierung an diesen Serinen, die Phosphorylierung der Serine S147, S148 und S151 verhindert. Darüber hinaus zeigen wir, daß sich die Phosphorylierung von mal3p während des Zellzyklus in einem bestimmten Muster ändert. Dabei ist mal3p am Übergang von der Interphase zur Mitose dephosphoryliert. Zu diesem Zeitpunkt bilden sich anstelle der zytoplasmatischen Interphasenmikrotubuli, intranukleäre Mikrotubuli welche die mitotische Spindel aufbauen. Mal3p wird anschließend, am Übergang von der Metaphase zu Anaphase, wieder phosphoryliert. Das geschieht wenn die Spindelmikrotubuli stabilisiert werden müssen damit die Spindel sich verlängern kann. Diese mal3p Phosphorylierung scheint sowohl an der Regulation der Anaphase als auch an der richtigen Mikrotubuli-Organisation im Mittelbereich der Spindel beteiligt zu sein. Parallel zum Phosphorylierungsstand, ist die Menge an mal3p Protein zu Beginn der Mitose am höchsten und nimmt dann während der Mitose sukzessive ab wobei sie zum Zeitpunktder Zytokinese ein Minimum erreicht. Zusammen mit der Feststellung, daß die mal3p Proteinmenge in mutanten Zellen, in denen mal3p nicht phosphoryliert werden kann, generell erhöht ist, legt dies nahe, daß die Phosphorylierung den mal3p Proteingehalt der Zelle reguliert. Überraschenderweise haben Mikrotubuli in Gegenwart von höheren Mengen von unphosphoryliertem mal3p eine erhöhte Katastrophenrate. Dies widerspricht der bisherigen Ansicht, wonach mal3p Protein für Mikrotubuli wachstumsfördernd ist. Unsere Ergebnisse geben somit neue Einblicke in die pleiotropen Funktionen der EB1 Proteine und in die regulatorischen Mechanismen, die diese Funktionen kontrollieren.
... Much information has been gathered about the roles of APC and EB1 with regard to their interactions with MTs in interphase cells [5,6]. Recent studies also imply that the binding of APC to EB1 relieves a self-inhibitory EB1 configuration, allowing the localisation of EB1 to MT tips where it promotes plus end growth by inhibiting MT catastrophe [26]. However, the roles of APC and EB1 and the potential functional significance of their interaction in mitotic cells are not fully understood. ...
... DNA was counterstained using DAPI (blue GST-EB1 bound to glutathione-agarose beads was preincubated with 1A11 or non-specific IgG (4 U) before being used in pull-downs from cell extracts. Bound proteins in each pull down were revealed by APC appears to require an EB1 region more N-terminal to that used by p150Glued [17,26,44]. A screen against these three proteins therefore covers the major binding modes currently identified for EB1 ligands. ...
... The EB1 protein plays a significant role in the targeted transport of Cx43 [91]. EB1 has a dual binding site with one end directly binding to the microtubule orthotope and the other end binding to the p150(Glued), a subunit of dynactin, to form the dynein/dynactin complex [111][112][113], which then combines with β-catenin [114] to tether microtubules to AJs [115,116], in turn facilitating hemichannel transport to the cell membrane ( Figure 1). N-cadherin mediates homologous interactions between adjacent cells, while β-catenin acts as a cytoplasmic enforcer of N-cadherin/Ncadherin interactions. ...
The connexin gene family is the most prevalent gene that contributes to hearing loss. Connexins 26 and 30, encoded by GJB2 and GJB6, respectively, are the most abundantly expressed connexins in the inner ear. Connexin 43, which is encoded by GJA1, appears to be widely expressed in various organs, including the heart, skin, the brain, and the inner ear. The mutations that arise in GJB2, GJB6, and GJA1 can all result in comprehensive or non-comprehensive genetic deafness in newborns. As it is predicted that connexins include at least 20 isoforms in humans, the biosynthesis, structural composition, and degradation of connexins must be precisely regulated so that the gap junctions can properly operate. Certain mutations result in connexins possessing a faulty subcellular localization, failing to transport to the cell membrane and preventing gap junction formation, ultimately leading to connexin dysfunction and hearing loss. In this review, we provide a discussion of the transport models for connexin 43, connexins 30 and 26, mutations affecting trafficking pathways of these connexins, the existing controversies in the trafficking pathways of connexins, and the molecules involved in connexin trafficking and their functions. This review can contribute to a new way of understanding the etiological principles of connexin mutations and finding therapeutic strategies for hereditary deafness.
... The EB1 protein plays a significant role in the targeted transport of Cx43 [69]. EB1 has a dual binding site with one end directly binding to the microtubule orthotope and the other end binding to the p150(Glued), a subunit of dynactin, to form the dynein/dynactin complex [89][90][91], which then combines with β-catenin [92] to tether microtubules to AJs [93,94], in turn facilitating hemichannel transport to the cell membrane ( Figure 1). N-cadherin mediates homologous interactions between adjacent cells, while β-catenin acts as a cytoplasmic enforcer of N-cadherin/Ncadherin interactions. ...
The connexin gene family is the most prevalent gene that contributes to hearing loss. Connexins 26 and 30, encoded by GJB2 and GJB6 respectively, are the most abundant expressed connexins in the inner ear. Connexin 43 which is encoded by GJA1 appears to be widely expressed in various organs, including the heart, skin, brain, and inner ear. The mutations that arise in GJB2, GJB6 and GJA1 can all result in comprehensive or non-comprehensive genetic deafness in newborns. As it is predicted that connexins include at least 20 isoforms in humans, the biosynthesis, structural composition, and degradation of connexins must be precisely regulated so that the gap junctions can operate properly. Certain mutations result in connexins possessing a faulty subcellular localization, failing to transport to the cell membrane and preventing gap junction formation, ultimately leading to connexin dysfunction and hearing loss. In this review, we provide a discussion of the transport models for connexin 43, connexins 30 and 26, mutations affecting trafficking pathways of connexin 26, the existing controversies in trafficking pathways of connexins, and the molecules involved in connexin trafficking and their functions. This review can contribute to a new way of understanding the etiological principles of connexin mutations and finding therapeutic strategies for hereditary deafness.
... However, affinity of Mal3 for microtubules is independent of Tea2 and Tip1. Tip1 has been shown to interact with the EB homology domain of Mal3 through its CAP-Gly domain , as also shown for Tip1 homolog CLIP170 and other plus-end tracking proteins (+TIPs) interacting with EB proteins (Bieling et al., 2008;Hayashi et al., 2005;Honnappa et al., 2009;Stangier et al., 2018). Tea2 interacts with Mal3 through its N-terminal extension and with Tip1 through its coiled-coil region (Browning and Hackney, 2005;Brunner and Nurse, 2000;. ...
Growing microtubule ends provide platforms for the accumulation of plus-end tracking proteins that organize into comets of mixed protein composition. Using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions appear to facilitate the non-stoichiometric accumulation of cargo Tip1. We dissected the contribution of two disordered protein regions in Mal3 and found that both are required for the ability to form droplets and Tip1 accumulation, while autonomous Mal3 comet formation only requires one of them. Using theoretical modeling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends.
... The microtubule dynamics rely on the assembly or disintegration of tubulins on plusends of microtubules [462]. These dynamics are regulated by the presence or absence of factors and proteins such as the GTP-bound tubulin dimers [463], tau protein [464,465], or plus-end tracking proteins [466]. The two plus-end tracking proteins are known to directly interact with tau, the microtubule-associated protein RP/EB family member 1 (MAPRE1) [467] and 3 (MAPRE3) [467]. ...
Tau protein plays a critical role in the assembly, stabilization, and modulation of microtubules, which are important for the normal function of neurons and the brain. In diseased conditions, several pathological modifications of tau protein manifest. These changes lead to tau protein aggregation and the formation of paired helical filaments (PHF) and neurofibrillary tangles (NFT), which are common hallmarks of Alzheimer's disease and other tauopathies. The accumulation of PHFs and NFTs results in impairment of physiological functions, apoptosis, and neuronal loss, which is reflected as cognitive impairment, and in the late stages of the disease, leads to death. The causes of this pathological transformation of tau protein haven't been fully understood yet. In both physiological and pathological conditions, tau interacts with several proteins which maintain their proper function or can participate in their pathological modifications. Interaction partners of tau protein and associated molecular pathways can either initiate and drive the tau pathology or can act neuroprotective, by reducing pathological tau proteins or inflammation. In this review, we focus on the tau as a multifunctional protein and its known interacting partners active in regulations of different processes and the roles of these proteins in Alzheimer's disease and tauopathies.
... On the contrary, the EB-1 localization on the plus-ends of MTs may occur similarly on both tyrosinated and detyrosinated polymers (Badin-Larcon et al., 2004;Erck et al., 2005). It was reported that the link of EB-1 with MTs is capable of influencing the functioning of CLIP-170, which is by itself unable to bind to growing MT ends (Arnal et al., 2004;Bieling et al., 2008;Hayashi et al., 2005;Komarova et al., 2005). ...
Glioblastoma multiforme is the most lethal brain tumor. In the study of mechanisms underlying its development attention has been paid to the microtubular network of its cells, mainly on βIII tubulin, considered as a marker of malignancy. In the present work, we chose to investigate the tubulin code in glioblastoma cells, analyzing the degree of interaction between tubulin post-translational modifications and different proteins associated with them. The pattern of diverse associated proteins such as EB-1, CLIP-170 and kinesin-1 and their degree of co-distribution with the most abundant post-translational tubulin modifications (tyrosination, acetylation and polyglutamylation) were evaluated. Through immunofluorescence we have shown that EB-1, CLIP-170 and kinesin-1 were well detectable in glioblastoma cells. The double fluorescence and colocalization index between the post-translational modifications of tubulin and associated proteins showed that tyrosinated α-tubulin has significantly high affinity with EB-1, CLIP-170 and kinesin-1, while for acetylated and polyglutamylated tubulin, the degree of interaction with the three associated proteins evaluated was less apparent. Data presented in this paper underline the importance of a thorough analysis of the microtubular mechanics in glioblastoma cells. This may suggest new experimental therapeutic approaches able to act more selectively on the microtubular network of cells in this type of cancer.
... Likewise, MAPRE1 (microtubule associated protein RP/ EB family member 1, also known as EB1), found overexpressed in PANC-1 KO-miR-93 protein extracts and confirmed as a direct target of miR-93, is a microtubule (MT) plus end-tracking protein (+TIPS) that promotes cytoplasmic MT nucleation and elongation 39 , is involved in spindle function by stabilizing microtubules and anchoring them at centrosomes 40 and suppresses catastrophes 41 . The remodeling of MT, filamentous structures required for cellular processes such as intracellular transport, cell division, and locomotion, depends on MT dynamic instability, spontaneous switching between episodes of growth and shortening 42 . ...
Biomarkers and effective therapeutic agents to improve the dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) are urgently required. We aimed to analyze the prognostic value and mechanistic action of miR-93 in PDAC. Correlation of miR-93 tumor levels from 83 PDAC patients and overall survival (OS) was analyzed by Kaplan–Meier. MiR-93 depletion in PANC-1 and MIA PaCa-2 cells was achieved by CRISPR/Cas9 and miR-93 overexpression in HPDE cells by retroviral transduction. Cell proliferation, migration and invasion, cell cycle analysis, and in vivo tumor xenografts in nude mice were assessed. Proteomic analysis by mass spectrometry and western-blot was also performed. Finally, miR-93 direct binding to candidate mRNA targets was evaluated by luciferase reporter assays. High miR-93 tumor levels are significantly correlated with a worst prognosis in PDAC patients. MiR-93 abolition altered pancreatic cancer cells phenotype inducing a significant increase in cell size and a significant decrease in cell invasion and proliferation accompanied by a G2/M arrest. In vivo, lack of miR-93 significantly impaired xenograft tumor growth. Conversely, miR-93 overexpression induced a pro-tumorigenic behavior by significantly increasing cell proliferation, migration, and invasion. Proteomic analysis unveiled a large group of deregulated proteins, mainly related to G2/M phase, microtubule dynamics, and cytoskeletal remodeling. CRMP2, MAPRE1, and YES1 were confirmed as direct targets of miR-93. MiR-93 exerts oncogenic functions by targeting multiple genes involved in microtubule dynamics at different levels, thus affecting the normal cell division rate. MiR-93 or its direct targets (CRMP2, MAPRE1, or YES1) are new potential therapeutic targets for PDAC.
... Although the information about the dynamics of the microtubules polymerization is limited, the expression levels from the genes that code for EB1, EB2 and CLASP1 (+TIPs proteins), in human platelets, are high (http://www.plateletomics.com/plateletomics/). In others cells, it has been described that both C-terminal and N-terminal EB1, can affect the dynamics of microtubules polymerization negatively, inhibiting the polymerizing activity [22] or by suppressing the shortening of the microtubule, respectively [23]. Additionally, CLASP1 can bind directly with EB1 and can regulate microtubule plus-end dynamics at the mammalian inner face of the cell membrane [24], acting as a microtubule-stabilizing factor. ...
Platelets are anuclear cells with a short lifespan that play an essential role in many pathophysiological processes, including haemostasis, inflammation, infection, vascular integrity, and metastasis. Billions of platelets are produced daily from megakaryocytes (platelet precursors). Despite this high production, the number of circulating platelets is stable and, under resting conditions, they maintain their typical discoid shape thanks to cytoskeleton proteins. The activation of platelets is associated with dynamic and rapid changes in the cytoskeleton. Two cytoskeletal polymer systems exist in megakaryocytes and platelets: actin filaments and microtubules, based on actin, and α- and β-tubulin heterodimers, respectively. Herein, we will focus on platelet-specific tubulins and their alterations and role of the microtubules skeleton in platelet formation (thrombopoiesis). During this process, microtubules mediate elongation of the megakaryocyte extensions (proplatelet) and granule trafficking from megakaryocytes to nascent platelets. In platelets, microtubules form a subcortical ring, the so-called marginal band, which confers the typical platelet discoid shape and is also responsible for changes in platelet morphology upon activation. Molecular alterations in the gene encoding β1 tubulin and microtubules post-translational modifications may result in quantitative or qualitative changes in tubulin, leading to altered cytoskeleton reorganization that may induce changes in the platelet number (thrombocytopenia), morphology or function. Consequently, β1-tubulin modifications may participate in pathological and physiological processes, such as development.
... Similar to the effect of GTP-binding in the wild type EB1 protein, the mutation can relieve auto-inhibitory interaction between EB1 CH domain and EB1 C-terminus. As the GTP-binding site residues in the CH domain are also known to be involved in EB1-microtubule binding 7,8,26 , the increased exposure of those residues could allow more EB1-binding to the microtubules. Together, our findings suggest that GTP-binding activity of EB1 could be physiologically relevant. ...
Microtubule plus end-binding protein, EB1 is a key regulator of microtubule dynamics. Auto-inhibitory interaction in EB1 has previously been shown to inhibit its ability to bind to microtubules and regulate microtubule dynamics. However, the factors that promote its microtubule regulatory activity by over-coming the auto-inhibition are less known. Here, we show that GTP plays a critical role in promoting the microtubule-targeting activity of EB1 by suppressing its auto-inhibition. Our biophysical data demonstrate that GTP binds to EB1 at a distinct site in its conserved N-terminal domain. Detailed analyses reveal that GTP-binding suppresses the intra-molecular inhibitory interaction between the globular N-terminus and the C-terminal coiled-coil domain. We further show that mutation of the GTP-binding site residues in N-terminus weakens the affinity for GTP, but also for the C-terminus, indicating overlapping binding sites. Confocal imaging and biochemical analysis reveal that EB1 localization on the microtubules is significantly increased upon mutations of the GTP-binding site residues. The results demonstrate a unique role of GTP in facilitating EB1 interaction with the microtubules by relieving its intra-molecular inhibition. They also implicate that GTP-binding may regulate the functions of EB1 on the cellular microtubules.
... EBs are involved in establishing a protein network at the '+tips' through recruitment of other proteins. Specifically, EB proteins interact with the CAP-Gly domain proteins (such as cytoplasmic linker and p150-Glued proteins) and proteins containing the short EB-binding motif S/TxIP [315][316][317][318][319][320]. ...
The endothelium, a monolayer of endothelial cells lining vessel walls, maintains tissue-fluid homeostasis by restricting the passage of the plasma proteins and blood cells into the interstitium. The ion Ca²⁺, a ubiquitous secondary messenger, initiates signal transduction events in endothelial cells that is critical to control of vascular tone and endothelial permeability. The ion Ca²⁺ is stored inside the intracellular organelles and released into the cytosol in response to environmental cues. The inositol 1,4,5-trisphosphate (IP3) messenger facilitates Ca²⁺ release through IP3 receptors which are Ca²⁺-selective intracellular channels located within the membrane of the endoplasmic reticulum. Binding of IP3 to the IP3Rs initiates assembly of IP3R clusters, a key event responsible for amplification of Ca²⁺ signals in endothelial cells. This review discusses emerging concepts related to architecture and dynamics of IP3R clusters, and their specific role in propagation of Ca²⁺ signals in endothelial cells.
... The p150 CAP-Gly domain recognizes the C-terminal EEY/F motif present in α-tubulin and EB/CLIP-170 [19][20][21][22]. The C-terminal tyrosine of α-tubulin can be removed and religated in a tyrosination-detyrosination cycle and is proposed to regulate the interactions with molecular motors and other MT binding proteins [23,24]. ...
Author summary
Animal cells rely on molecular motor proteins to distribute intracellular components and organize their cytoplasmic content. The motor cytoplasmic dynein 1 (dynein) uses microtubule filaments as tracks to transport cargo from the cell periphery to the cell center, where the microtubule minus ends are embedded at the centrosome. Conversely, when dynein is anchored at the cell cortex or on organelles in the cytoplasm, the motor can pull on microtubules to position centrosomes within the cell. The intracellular location of centrosomes determines cell geometry and cell fate, and studying the underlying mechanisms will help us understand polarized cell behaviors such as cell migration or neurite outgrowth, and how cleavage plane orientation is established during cell division. Here, we show in C. elegans embryos that dynactin, an essential dynein regulator, uses its microtubule binding activity to help dynein pull on microtubules for centrosome positioning during the first mitotic division. Our results with engineered dynactin and tubulin mutants suggest that microtubule binding by dynactin increases the efficiency with which dynein can initiate the transport of small organelles towards centrosomes. More organelles moving along microtubules through the viscous cytoplasm means that correspondingly larger pulling forces act on centrosomes. Thus, our work provides evidence for a novel functional link between dynactin's role in initiating transport of dynein cargo and the generation of cytoplasmic pulling forces critical for the positioning of centrosomes.
... CAP-Gly interacts with its binding partners on the flat side of the protein, where most of the surface-exposed hydrophobic residues are located. dREDOR experiments of the CAP-Gly/EB1 complex were consistent with the known binding interface for this complex, which has been determined previously (Hayashi et al. 2005;Honnappa et al. 2006;Yan et al. 2013a), validating the approach for characterizing the CAP-Gly/MT interface. ...
In recent years, exciting developments in instrument technology and experimental methodology have advanced the field of magic-angle spinning (MAS) nuclear magnetic resonance (NMR) to new heights. Contemporary MAS NMR yields atomic-level insights into structure and dynamics of an astounding range of biological systems, many of which cannot be studied by other methods. With the advent of fast MAS, proton detection, and novel pulse sequences, large supramolecular assemblies, such as cytoskeletal proteins and intact viruses, are now accessible for detailed analysis. In this review, we will discuss the current MAS NMR methodologies that enable characterization of complex biomolecular systems and will present examples of applications to several classes of assemblies comprising bacterial and mammalian cytoskeleton as well as human immunodeficiency virus 1 and bacteriophage viruses. The body of work reviewed herein is representative of the recent advancements in the field, with respect to the complexity of the systems studied, the quality of the data, and the significance to the biology.
... Some of these recruited proteins have pronounced effects on the dynamic properties of microtubules (Komarova et al., 2002;Montenegro Gouveia et al., 2010), suggesting that misregulation of cellular microtubule dynamics following EB loss of function (Busch and Brunner, 2004;Komarova et al., 2009;Tirnauer et al., 2002) arises primarily from defects in recruitment of these proteins. High-resolution structural studies have provided detailed information about the interaction of the EB homology domain with proteins containing either CAP-Gly domains (Hayashi et al., 2005;Honnappa et al., 2006) or SxIP motifs ), but the structural basis of selective EB binding to growing microtubule ends is unknown. ...
... However, nearly all the EB1 248 fragment pelleted in the presence (but not the absence) of 2 μM F-actin under these conditions ( Fig. 1c). These observations demonstrate that EB1 can bind to actin and that EB1:F-actin binding, like EB1:MT binding, is negatively regulated by the EB1 tail (i.e., removal of the tail increases the affinity of EB1 for both actin and MTs) [10,18]. In addition, the monomeric MT binding "head" domain of EB1 (which contains the CH domain) also pelleted with F-actin ( Fig. 1c). ...
Many cellular processes including cell division and cell migration require coordination between the actin and microtubule (MT) cytoskeletons. This coordination is as-yet poorly understood, but proteins such as formins and IQGAP1 are known to be involved. We show that the MT binding protein EB1, a key regulator of microtubule dynamics, can bind directly to F-actin. We determined that the EB1:F-actin interaction is salt sensitive and weak under physiological salt concentrations but might be relevant in contexts where the local concentration of actin is high. Using bioinformatics and mutagenesis we found that the EB1:F-actin binding site partially overlaps the well characterized EB1:MT binding interface. Congruently, competition experiments indicate that EB1 can bind to F-actin or MTs but not both simultaneously. These observations suggest that EB1:F-actin interactions may negatively regulate EB1:MT interactions, and we speculate that this interaction may assist cells in differentially regulating microtubule stability in the actin-rich cortex as opposed to the cell interior.
... His-tagged human tau (1N4R tau-412 isoform) and GST-tagged human EB1 plasmids were kindly provided by Drs N. Sergeant (Lille, France) and I. Hayashi (Yokohama, Japan) (Hayashi et al. 2005) respectively. Proteins were purified either on Glutathione Sepharose (Thermo scientific, Walthman, MA, USA) and/or Talon metal affinity resin (Clontech, Mountain View, CA, USA), according to the manufacturer 0 s instructions. ...
The axonal microtubule-associated protein tau is a well-known regulator of microtubule stability in neurons. However, the putative interplay between tau and End-binding proteins 1 and 3 (EB1/3), the core microtubule plus-end tracking proteins, has not been elucidated yet. Here, we show that a cross-talk between tau and EB1/3 exists in developing neuronal cells. Tau and EBs partially colocalize at extending neurites of N1E-115 neuroblastoma cells and axons of primary hippocampal neurons, as shown by confocal immunofluorescence analyses. Tau down-regulation leads to a reduction of EB1/3 comet length, as observed in shRNA-stably depleted neuroblastoma cells and TAUÀ/À neurons. EB1/3 localization depends on the expression levels and localization of tau protein. Over-expression of tau at high levels induces EBs relocalization to microtubule bundles at extending neurites of N1E-115 cells. In differentiating primary neurons, tau is required for the proper accumulation of EBs at stretches of microtubule bundles at the medial and distal regions of the axon. Tau interacts with EB proteins, as shown by immunoprecipitation in different non-neuronal and neuronal cells and in whole brain lysates. A tau/ EB1 direct interaction was corroborated by in vitro pull-down assays. Fluorescence recovery after photobleaching assays performed in neuroblastoma cells confirmed that tau modulates EB3 cellular mobility. In summary, we provide evidence of a new function of tau as a direct regulator of EB proteins in developing neuronal cells. This cross-talk between a classical microtubule-associated protein and a core microtubule plus-end tracking protein may contribute to the fine-tuned regulation of microtubule dynamics and stability during neuronal differentiation.
... The more dynamic end is the growing plus end. The main protein components involved in growth of microtu bules are the MAP (microtubule associated protein) and dynactin, the dinein protein complex (Hayashi et al., 2005;Akhmanova and Steinmetz, 2008). The complex of proteins involved in ensuring growth of microtubule plus ends was named "+ TIPs" (plus end tracking proteins) (Schuyler and Pellman, 2001;Howard and Hyman, 2003). ...
The syncytial development is a feature of early embryogenesis and spermatogenesis in Drosophila melanogaster. All elements of syncytium are interconnected by single cytoskeletal network that enables equal conditions and provides synchronic development. The cytoskeleton is essential for the formation and functioning of the mitotic spindle, cytoskeletal elements are the main structural component of cilia and flagella. Intra- and intercellular transport, morphogenesis processes depend from cytoskeleton on both within a single cell, and at the level of the whole organism. The sbr (small bristles) gene of D. melanogaster belongs to the NXF (nuclear export factor) evolutionarily conservative proteins family. Gene Dm nxf1 (sbr), as well as its orthologs in other organisms, controls the export of poly(A)-containing RNA from the nucleus to the cytoplasm, and the corresponding proteins are usually localized in the nucleus or in the nuclear envelope. For SBR protein we have shown the localization not only in the nucleus, but in the cytoplasm marking of characteristic cytoplasmic structures. A breach of the cytoskeleton in the sbr (Dm nxf1) mutant in D. melanogaster shown by us and cytoplasmic localization of the protein SBR allow us to link the specific functions of this protein with the dynamics of the cytoskeleton.
... The EB1 is capable of binding to the end of microtubules to regulate the dynamics of the microtubule cytoskeleton, specifically promoting cytoplasmic microtubule nucleation and elongation. Their role in spindle function is thought to occur through stabilization of microtubules, anchoring them at centrosomes (Askham et al. 2002;Hayashi et al. 2005;Honnappa et al. 2009;van der Vaart et al. 2011). The reduced levels of EB1 perhaps cause dysregulation of the actin cytoskeletal network. ...
Radiation-induced bystander signaling has been found to occur in live rainbow trout fish (Oncorhynchus mykiss). This article reports identification of key proteomic changes in a bystander reporter cell line (HaCaT) grown in low-dose irradiated tissue-conditioned media (ITCM) from rainbow trout fish. In vitro explant cultures were generated from the skin of fish previously exposed to low doses (0.1 and 0.5 Gy) of X-ray radiation in vivo. The ITCM was harvested from all donor explant cultures and placed on recipient HaCaT cells to observe any change in protein expression caused by the bystander signals. Proteomic methods using 2-dimensional (2D) gel electrophoresis and mass spectroscopy were employed to screen for novel proteins expressed. The proteomic changes measured in HaCaT cells receiving the ITCM revealed that exposure to 0.5 Gy induced an upregulation of annexin A2 and cingulin and a downregulation of Rho-GDI2, F-actin-capping protein subunit beta, microtubule-associated protein RP/EB family member, and 14-3-3 proteins. The 0.1 Gy dose also induced a downregulation of Rho-GDI2, hMMS19, F-actin-capping protein subunit beta, and microtubule-associated protein RP/EB family member proteins. The proteins reported may influence apoptotic signaling, as the results were suggestive of an induction of cell communication, repair mechanisms, and dysregulation of growth signals.
... This motif partially overlaps with the end binding homology (EBH) domain, which is involved in the interaction with EB binding partners ). An acidic tail at the C-terminus contains a highly conserved EEY sequence motif, similar to those found in α-tubulin and CLIP170 (Komarova Y et al. 2005;Honnappa S et al. 2006), that is implicated in the interaction with other proteins (Honnappa S et al. 2005;Hayashi I et al. 2005). EB1 and EB3 are able to interact with a large number of proteins (Bu W & Su LK, 2003). ...
Le cancer du sein touche une femme sur huit dans le monde et représente un problème majeur de santé publique. Alors que la majorité des tumeurs du sein sont aujourd'hui la cible de traitement efficaces, il reste une sous-population de tumeurs (dites triple-négatives) à fort potentiel métastatique qui ne sont pas accessibles aux thérapies ciblées et demeurent de mauvais pronostic. L'élucidation des processus impliqués dans la progression tumorale et la formation de métastases reste un challenge majeur dans la recherche de nouvelles thérapies contre le cancer du sein de mauvais pronostic. ATIP3 (AT2-interacting protein 2), produit du gène candidat suppresseur des tumeurs MTUS1 (Microtubule-Associated Tumor Suppressor), a été identifiée par le laboratoire comme étant un biomarqueur des tumeurs du sein les plus agressives. En effet, les résultats précédents de notre équipe ont montré que l'expression d'ATIP3 est diminuée dans 85% des tumeurs de haut grade, 83% des tumeurs triples négatives et dans 62% des tumeurs métastatiques. Il a également été montré qu'ATIP3 inhibe la prolifération cellulaire in vitro, ainsi que la croissance tumorale in vivo. Au niveau moléculaire, ATIP3 a été identifiée comme étant une nouvelle protéine associée aux microtubules (MAP) localisée au centrosome, le long de microtubules dans les cellules en interphase, au fuseau mitotique pendant la division cellulaire et au pont intercellulaire lors de la cytokinèse. La localisation cellulaire d'ATIP3, étroitement associée aux microtubules, prend toute son importance du fait du rôle de ce cytosquelette dans la division et migration cellulaire, deux étapes essentielles du processus tumoral.Mon projet de thèse a pour objectif principal d'évaluer le rôle potentiel d'ATIP3 dans la migration cellulaire et la formation de métastases tumorales. Dans un premier temps, les niveaux d'expression d'ATIP3 ont été analysés dans des séries de puces à ADN issues de trois cohortes indépendantes de patientes atteintes d'un cancer du sein invasif, et les données ont été comparées avec les caractéristiques cliniques des patientes. Ces analyses transcriptomiques ont permis de montrer que l'expression d'ATIP3 est un marqueur pronostic de la survie des patientes et de façon intéressante, qu'ATIP3 est un nouvel indicateur de la progression métastatique. L'effet d'ATIP3 sur la progression des métastases a alors été évalué dans un modèle de bioluminescence in vivo, ce qui a permis de montrer que cette protéine est une molécule anti-métastatique qui réduit la progression, le nombre et la taille des foyers métastatiques. La colonisation métastatique inclut la migration des cellules cancéreuses à travers la matrice extracellulaire (invasion) et l'endothélium vasculaire (extravasation), puis leur prolifération au site secondaire. L'évaluation détaillée de ces étapes a montré qu'ATIP3 diminue tous ces processus. En ce qui concerne la migration, une réduction de vitesse, de la direction de migration et possiblement de la polarité cellulaire, pourraient expliquer les effets inhibiteurs d'ATIP3 sur la migration des cellules. Comme de nombreuses études ont démontré que la migration et la polarité cellulaires dépendent du cytosquelette de microtubules, je me suis intéressée aux effets d'ATIP3 sur la dynamique microtubulaire. Des expériences de vidéomicroscopie ont permis de montrer que l'extinction d'ATIP3 augmente la dynamique des microtubules en incrémentant les épisodes et la vitesse de croissance et en diminuant le temps passé en pause et la fréquence des catastrophes. Ces résultats, couplés à des expériences de dépolymérisation et de re-croissance ont permis de conclure qu'ATIP3 est une MAP qui stabilise les microtubules et diminue leur dynamique pour contrôler la polarité et la migration cellulaires. L'ensemble de ces travaux font l'objet d'une publication parue en 2013 (Molina et al., Cancer Res 73, 2905). (...)
... His-tagged human tau (1N4R tau-412 isoform) and GST-tagged human EB1 plasmids were kindly provided by Drs N. Sergeant (Lille, France) and I. Hayashi (Yokohama, Japan) (Hayashi et al. 2005) respectively. Proteins were purified either on Glutathione Sepharose (Thermo scientific, Walthman, MA, USA) and/or Talon metal affinity resin (Clontech, Mountain View, CA, USA), according to the manufacturer 0 s instructions. ...
The axonal microtubule-associated protein (MAP) tau is a well-known regulator of microtubule stability in neurons. However, the putative interplay between tau and End-binding proteins 1 and 3 (EB1/3), the core microtubule plus-end tracking proteins (+TIPs), has not been elucidated yet. Here we show that a crosstalk between tau and EB1/3 exists in developing neuronal cells. Tau and EBs partially colocalize at extending neurites of N1E-115 neuroblastoma cells and axons of primary hippocampal neurons, as shown by confocal immunofluorescence analyses. Tau downregulation leads to a reduction of EB1/3 comet length, as observed in shRNA-stably depleted neuroblastoma cells and TAU-/- neurons. EB1/3 localization depends on the expression levels and localization of tau protein. Overexpression of tau at high levels induces EBs relocalization to microtubule bundles at extending neurites of N1E-115 cells. In differentiating primary neurons, tau is required for the proper accumulation of EBs at stretches of microtubule bundles at the medial and distal regions of the axon. Tau interacts with EB proteins, as shown by immunoprecipitation in different non-neuronal and neuronal cells and in whole brain lysates. A tau/EB1 direct interaction was corroborated by in vitro pull-down assays. FRAP assays performed in neuroblastoma cells confirmed that tau modulates EB3 cellular mobility. In summary, we provide evidence of a new function of tau as a direct regulator of EB proteins in developing neuronal cells. This crosstalk between a classical MAP and a core +TIP may contribute to the fine-tuned regulation of microtubule dynamics and stability during neuronal differentiation. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
... The three-dimensional structures of CAP-Gly free (13,14) and in complex with the various binding partners (8,(15)(16)(17) reveal unusually high loop content and significant conformational flexibility in the immediate vicinity of these loop regions (14,18). To understand these observations, we examine here the role of conformational dynamics occurring on multiple time scales for the ability of CAP-Gly to interact with its binding partners and with microtubules. ...
CAP-Gly domain of dynactin, a microtubule-associated activator of dynein motor, participates in multiple cellular processes and its point mutations are associated with neurodegenerative diseases. Recently, we have demonstrated that conformational plasticity is an intrinsic property of CAP-Gly. To understand its origin, we addressed internal dynamics of CAP-Gly assembled on polymeric microtubules, bound to end-binding protein EB1, and free, by magic-angle-spinning NMR and molecular-dynamics simulations. The analysis of residue-specific dynamics of CAP-Gly on timescales spanning nano- through milliseconds reveals its unusually high mobility, both free and assembled on polymeric microtubules. On the contrary, CAP-Gly bound to EB1 is significantly more rigid. MD simulations indicate that these motions are strongly temperature dependent, and loop regions are surprisingly mobile. These findings establish the connection between conformational plasticity and internal dynamics in CAP-Gly, which is essential for CAP-Gly's biological functions and ability to bind to polymeric microtubules and multiple binding partners. In this work, we establish an approach, for the first time, to probe atomic-resolution dynamic profiles of a microtubule-associated protein assembled on polymeric microtubules. More broadly, the methodology established here can be applied for atomic-resolution analysis of dynamics in other microtubule-associated protein assemblies, including but not limited to dynactin, dynein, and kinesin motors assembled on microtubules.
Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
... Interactions with helix H3 and the compacting region of -tubulin also enable EB1 to accelerate the transitions of tubulin from the GTP state to the GDP state; in other words, EB1 acts as a "maturation factor" for the microtubule end (Maurer et al., 2014). EB1 recruits a large network of plus-end-tracking proteins through interactions with the EB1 C terminus (Hayashi et al., 2005;Honnappa et al., 2006) and EB1 homology domain (Honnappa et al., 2009). This diverse and complex protein network is essential for the regulation of microtubule dynamics, the capture of microtubule ends by the cell cortex (Kodama et al., 2003) and endoplasmic reticulum (Grigoriev et al., 2008), and the positioning of the mitotic spindle (Liakopoulos et al., 2003). ...
Microtubules are dynamic polymers of αβ-tubulin that form diverse cellular structures, such as the mitotic spindle for cell division, the backbone of neurons, and axonemes. To control the architecture of microtubule networks, microtubule-associated proteins (MAPs) and motor proteins regulate microtubule growth, shrinkage, and the transitions between these states. Recent evidence shows that many MAPs exert their effects by selectively binding to distinct conformations of polymerized or unpolymerized αβ-tubulin. The ability of αβ-tubulin to adopt distinct conformations contributes to the intrinsic polymerization dynamics of microtubules. αβ-Tubulin conformation is a fundamental property that MAPs monitor and control to build proper microtubule networks.
In the axon terminal, microtubule stability is decreased relative to the axon shaft. The dynamic microtubule plus ends found in the axon terminal have many functions, including serving as a docking site for the Cytoplasmic dynein motor. Here, we report an unexplored function of dynein in microtubule regulation in axon terminals: regulation of microtubule stability. Using a forward genetic screen, we identified a mutant with abnormal axon terminal structure due to a loss of function mutation in NudC. We show that, in the axon terminal, NudC is a chaperone for the protein Lis1. Decreased Lis1 in nudc axon terminals causes dynein/dynactin accumulation and increased microtubule stability. Microtubule dynamics can be restored by pharmacologically inhibiting dynein, implicating excess dynein motor function in microtubule stabilization. Together, our data support a model in which local NudC-Lis1 modulation of the dynein motor is critical for regulation of microtubule stability in the axon terminal.
End binding protein 1 (EB1) is a key regulator of microtubule dynamics that orchestrates hierarchical interaction networks at microtubule plus ends to control proper cell division. EB1 activity is known to be regulated by serine/threonine phosphorylation; however, how tyrosine phosphorylation affects EB1 activity remains poorly understood. In this study, we mapped the tyrosine phosphorylation pattern of EB1 in synchronized cells and identified two tyrosine phosphorylation sites (Y217 and Y247) in mitotic cells. Using phospho-deficient (Y/F) and phospho-mimic (Y/D) mutants, we revealed that Y247, but not Y217, is critical for astral microtubule stability. The Y247D mutant contributed to increased spindle angle, indicative of defects in spindle orientation. Time-lapse microscopy revealed that the Y247D mutant significantly delayed mitotic progression by increasing the duration times of prometaphase and metaphase. Structural analysis suggests that Y247 mutants lead to instability of the hydrophobic cavity in the EB homology (EBH) domain, thereby affecting its interactions with p150glued, a protein essential for Gαi/LGN/NuMA complex capture. These findings uncover a crucial role for EB1 phosphorylation in the regulation of mitotic spindle orientation and cell division.
The centrosome executes diverse functions including the nucleation and organization of microtubules. A subset of centrosomal proteins is found to be involved in regulating the nucleation, stability, and dynamics of microtubules. Literature is flooded with reports of centrosomal proteins regulating microtubule nucleation. However, the centrosomal proteins that regulate microtubule stability are underexplored. Here, we review the centrosomal proteins, which either enhance or reduce the stability of microtubules and thereby regulate microtubule dynamics. We also discuss unexplored aspects of the centrosomal proteins that influence microtubule stability.
Dynactin (. dynein activator) is a large multiprotein complex that is required for function of the cytoplasmic dynein-1 motor. In this chapter, we discuss new structure-based insights into the mechanism of dynein activation.
Microtubules are polar cytoskeleton filaments that extend via growth at their plus ends. Microtubule plus-end-tracking proteins (+TIPs) accumulate at these growing plus ends to control microtubule dynamics and attachment. The +TIP end-binding protein 1 (EB1) and its homologs possess an autonomous plus-end-tracking mechanism and interact with other known +TIPs, which then recruit those +TIPs to the growing plus ends. A major +TIP class contains the SxIP (Ser-x-Ile-Pro, with x denoting any amino acid residue) motif, known to interact with EB1 and its homologs for plus-end tracking, but the role of SxIP in regulating EB1 activities is unclear. We show here that an interaction of EB1 with the SxIP-containing +TIP CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) regulates several EB1 activities, including microtubule plus-end tracking, dynamics at microtubule plus ends, microtubule and α/β-tubulin binding, and microtubule polymerization. The SxIP motif fused with a dimerization domain from CDK5RAP2 significantly enhanced EB1 plus-end-tracking and microtubule-polymerizing and bundling activities, but the SxIP motif alone failed to do so. An SxIP-binding-deficient EB1 mutant displayed significantly lower microtubule plus-end tracking than the wild-type protein in transfected cells. These results suggest that EB1 cooperates with CDK5RAP2 and perhaps other SxIP-containing +TIPs in tracking growing microtubule tips. We also generated plus-end-tracking chimeras of CDK5RAP2 and the adenomatous polyposis coli protein (APC) and found that overexpression of the dimerization domains interfered with microtubule plus-end tracking of their respective SxIP-containing chimeras. Our results suggest that disruption of SxIP dimerization enables detailed investigations of microtubule plus-end-associated functions of individual SxIP-containing +TIPs.
Dynactin is a multi-subunit complex that was first identified as a biochemical activity required for dynein-driven vesicle movement along microtubules in vitro. It has since been found to be required for most types of dynein-based motility in vivo. Dynein and dynactin also play essential roles in mitotic processes, including transport of checkpoint proteins from kinetochores to spindle poles, spindle pole organization, and chromosome movement. The known activities of dynactin have been observed in microtubule binding and dynein binding. Dynactin and dynein are components of the same genetic pathway, and mutations in subunits of either molecule appear to have a similar set of cellular consequences. This chapter explains dynactic function in dynein-based motility. These functions of dynactin include the role of dynactin as a cargo adaptor, cargo interactions of the arp1 filament, cargo interactions with the shoulder-sidearm complex, and the role of dynactin as a processivity enhancer. Following this, the study provides an understanding of the modes of dynein activation. Under this, it also describes how the chemical and mechanical cycles of cytoplasmic dynein are coupled to allow the motor to walk along the microtubule lattice. Further studies surrounding dynactin and the roles it plays in cytoplasmic dynein function are being undertaken.
Allostery is a ubiquitous mechanism to control biological function and arises from the coupling of inhibitory and binding equilibria. The extent of coupling reflects the inactive vs. active state selectivity of the allosteric effector. Hence, dissecting allosteric determinants requires quantification of state-specific association constants. However, observed association constants are typically population-averages, reporting on overall affinities but not on allosteric coupling. Here we propose a general method to measure state-specific association constants in allosteric sensors based on three key elements, i.e. state-selective molecular stapling through disulphide bridges, competition binding saturation transfer experiments and chemical shift correlation analyses to gauge state populations. The proposed approach was applied to the prototypical cAMP-dependent protein kinase (PKA-RIα), for which the structures of the inactive and active states are available, as needed to design the state-selective disulphide bridges. Surprisingly, the PKA-RIα state-specific association constants are comparable to those of a structurally homologous domain with ~10(3)-fold lower cAMP-affinity, suggesting that the affinity difference arises primarily from changes in the position of the dynamic apo inhibitory equilibrium.
Das COP9-Signalosom (CSN) ist ein evolutionär konservierter Proteinkomplex. Er besteht aus acht Untereinheiten und wird als Paralog des Lid-Subkomplexes des 26S Proteasoms angesehen. Das CSN verfügt über diverse enzymatische Aktivitäten, die es zu einem regulatorischen Faktor des Ubiquitin-Proteasom-Systems (UPS) machen. Das UPS ist für den Abbau von einem Großteil der zellulären Proteine notwendig. Für die Proteolyse bestimmter Proteine werden diese mit einer Polyubiquitinkette markiert. Dies geschieht über eine Enzymkaskade von E1, E2s und E3-Ligasen, wobei die E3s die Substratspezifität bestimmen. Die Interaktion von E3s mit dem CSN ist für deren Assemblierung und Aktivität von entscheidender Bedeutung. Des Weiteren bindet das CSN eine Vielzahl von proteasomalen Substraten und scheint deren Abbau direkt zu kontrollieren. In dieser Arbeit konnte eine Interaktion des CSN mit dem Mikrotubuli-bindenden Protein EB1 nachgewiesen werden. EB1 wirkt präferentiell an den (+)-Enden von Mikrotubuli und fördert die Polymerisierung und Stabilität von Mikrotubulifilamenten. EB1 bindet über die Untereinheit CSN5 an das CSN. Die Interaktion von EB1 mit dem CSN findet im Centrosom statt und führt zur Phosphorylierung und Stabilisierung von EB1. Eine verminderte Bindung von EB1 an das CSN oder eine reduzierte Phosphorylierung von EB1 führt zu einem beschleunigten Abbau. Die Funktion der Interaktion zwischen EB1 und dem CSN wurde in CSN-siRNA-Zelllinien untersucht. Dazu wurden die Untereinheiten CSN1, 3 und 5 in HeLa-Zellen permanent herunterreguliert. Die siRNAs gegen CSN1 und 3 (siCSN1, siCSN3) führen zur Reduktion des gesamten CSN Komplexes, der Knockdown von CSN5 (siCSN5) nur zur Verminderung von CSN5. In allen drei Zelllinien ist der Abbau von EB1 beschleunigt, was auf eine verminderte Bindung an, bzw. Phosphorylierung durch das CSN zurückzuführen ist. Dies hat Konsequenzen für die Stabilität von Mikrotubulifilamenten in siCSN1- und siCSN3-Zellen. Diese zeigen eine erhöhte Sensibilität gegenüber Nocodazol, welches die Polymerisierung von Mikrotubuli inhibiert. Des Weiteren konnte ein durch Nocodazol ausgelöster Zellzyklusarrest durch die Überexpression von EB1 oder CSN1 in HeLa-Zellen überwunden werden.
CLIP-associated proteins (CLASPs) are mammalian microtubule plus-end tracking proteins (+TIPs) that promote microtubule rescue in vivo. Their plus-end localization is dependent on other +TIPs, EB1 and CLIP-170, but in the leading edge of the cell, CLASPs display lattice-binding activity. Microtubule association of CLASPs is suggested to be regulated by multiple TOG domains and by the serine-arginine rich (SR) region, which contains binding sites for EB1. Here we report the crystal structures of the two TOG domains of CLASP2. Both domains consist of six HEAT repeats, which are similar to the canonical paddle-like tubulin-binding TOG domains, but have arched conformations. The degrees and directions of curvature are different between the two TOG domains, implying that they have distinct roles in microtubule binding. Using biochemical, molecular modeling and cell biological analyses, we have investigated the interactions between the TOG domains and αβ-tubulin, and found that each domain associates differently with αβ-tubulin. Our findings suggest that, by varying the degrees of domain curvature, the TOG domains may distinguish the structural conformation of the tubulin dimer, discriminate between different states of microtubule dynamic instability, and thereby function differentially as stabilizers of microtubules.
Copyright © 2015. Published by Elsevier Ltd.
The evolutionarily conserved protein EB1 originally was identified by its physical association with the carboxyl-terminal
portion of the adenomatous polyposis coli (APC) tumor suppressor protein, an APC domain commonly mutated in familial and sporadic
forms of colorectal neoplasia. The subcellular localization of EB1 in epithelial cells was studied by using immunofluorescence
and biochemical techniques. EB1 colocalized both to cytoplasmic microtubules in interphase cells and to spindle microtubules
during mitosis, with pronounced centrosome staining. The cytoskeletal array detected by anti-EB1 antibody was abolished by
incubation of the cells with nocodazole, an agent that disrupts microtubules; upon drug removal, EB1 localized to the microtubule-organizing
center. Immunofluorescence analysis of SW480, a colon cancer cell line that expresses only carboxyl-terminal-deleted APC unable
to interact with EB1, demonstrated that EB1 remained localized to the microtubule cytoskeleton, suggesting that this pattern
of subcellular distribution is not mediated by its interaction with APC. In vitro cosedimentation with taxol-stabilized microtubules demonstrated that a significant fraction of EB1 associated with microtubules.
Recent studies of the yeast EB1 homologues Mal3 and Bim1p have demonstrated that both proteins localize to microtubules and
are important in vivo for microtubule function. Our results demonstrate that EB1 is a novel component of the microtubule cytoskeleton in mammalian
cells. Associating with the mitotic apparatus, EB1 may play a physiologic role connecting APC to cellular division, coordinating
the control of normal growth and differentiation processes in the colonic epithelium.
Publisher Summary X-ray data can be collected with zero-, one-, and two-dimensional detectors, zero-dimensional (single counter) being the simplest and two-dimensional the most efficient in terms of measuring diffracted X-rays in all directions. To analyze the single-crystal diffraction data collected with these detectors, several computer programs have been developed. Two-dimensional detectors and related software are now predominantly used to measure and integrate diffraction from single crystals of biological macromolecules. Macromolecular crystallography is an iterative process. To monitor the progress, the HKL package provides two tools: (1) statistics, both weighted (χ 2 ) and unweighted (R-merge), where the Bayesian reasoning and multicomponent error model helps obtain proper error estimates and (2) visualization of the process, which helps an operator to confirm that the process of data reduction, including the resulting statistics, is correct and allows the evaluation of the problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and reduction has been reached. At that point, the effort should be directed to solving the structure. The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.
The NMRPipe system is a UNIX software environment of processing, graphics, and analysis tools designed to meet current routine and research-oriented multidimensional processing requirements, and to anticipate and accommodate future demands and developments. The system is based on UNIX pipes, which allow programs running simultaneously to exchange streams of data under user control. In an NMRPipe processing scheme, a stream of spectral data flows through a pipeline of processing programs, each of which performs one component of the overall scheme, such as Fourier transformation or linear prediction. Complete multidimensional processing schemes are constructed as simple UNIX shell scripts. The processing modules themselves maintain and exploit accurate records of data sizes, detection modes, and calibration information in all dimensions, so that schemes can be constructed without the need to explicitly define or anticipate data sizes or storage details of real and imaginary channels during processing. The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks.
Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the beta-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.
Although cytoplasmic dynein is known to attach to microtubules and translocate toward their minus ends, dynein's ability to serve in vitro as a minus end-directed transporter of membranous organelles depends on additional soluble factors. We show here that a approximately 20S polypeptide complex (referred to as Activator I; Schroer, T. A., and M.P. Sheetz. 1991a. J. Cell Biol. 115:1309-1318.) stimulates dynein-mediated vesicle transport. A major component of the activator complex is a doublet of 150-kD polypeptides for which we propose the name dynactin (for dynein activator). The 20S dynactin complex is required for in vitro vesicle motility since depletion of it with a mAb to dynactin eliminates vesicle movement. Cloning of a brain specific isoform of dynactin from chicken reveals a 1,053 amino acid polypeptide composed of two coiled-coil alpha-helical domains interrupted by a spacer. Both this structural motif and the underlying primary sequence are highly conserved in vertebrates with 85% sequence identity within a central 1,000-residue domain of the chicken and rat proteins. As abundant as dynein, dynactin is ubiquitously expressed and appears to be encoded by a single gene that yields at least three alternative isoforms. The probable homologue in Drosophila is the gene Glued, whose protein product shares 50% sequence identity with vertebrate dynactin and whose function is essential for viability of most (and perhaps all) cells in the organism.
Mutations of the APC gene play a critical role in both sporadic and familial forms of colorectal cancer. The vast majority of these mutations result in the loss of the carboxyl terminus of the protein. To further elucidate the function of APC, we searched for cellular proteins that associate with its carboxyl terminus. One million human cDNA clones were screened with the use of the interaction trap two-hybrid system, and 67 clones were found to have a phenotype suggestive of an APC-interacting protein. Nucleotide sequence analysis revealed that 48 of these clones were derived from a single novel named EBI. The association of APC and EB1 proteins was confirmed with in vitro binding assays. mAbs against EB1 were then produced and used to demonstrate the association of APC and EB1 in vivo. The EB1 gene was predicted to encode a 268-amino acid protein without significant homology to proteins with known function. However, searches of nucleotide databases did identify evidence for at least two related human genes and a yeast homologue. This conservation suggests an essential function for EB1 that might provide clues to the mechanism through which APC suppresses colonic neoplasia.
p150Glued was first identified as a polypeptide that copurifies with cytoplasmic dynein, the minus-end-directed microtubule-based motor protein, and has more recently been shown to be present as a member of the oligomeric dynactin complex, which includes the actin-related protein centractin (Arp-1). Dynactin is thought to mediate dynein-driven vesicle motility, as well as nuclear transport, in lower eukaryotes. The mechanism by which dynactin may function in these cellular processes is unknown. To examine the role of the dynactin complex in vivo, we overexpressed the rat cDNA encoding p150Glued in Rat-2 fibroblasts. Overexpression of full-length, as well as C-terminal deletion, constructs resulted in the decoration of microtubules with the p150Glued polypeptides. This cellular evidence for microtubule association was corroborated by in vitro microtubule-binding assays. Amino acids 39-150 of p150Glued were determined to be sufficient for microtubule association. We also tested for a direct interaction between p150Glued and centractin. In vitro translated centractin was specifically retained by a p150Glued affinity column, and this interaction was blocked by a synthetic peptide which corresponds to a highly conserved motif from the C terminus of p150Glued. These results demonstrate that p150Glued, a protein implicated in cytoplasmic dynein-based microtubule motility, is capable of direct binding to both microtubules and centractin.
Defects in the APC gene occur frequently in patients with familial adenomatous polyposis coli and are associated with the progression of sporadic tumors of the colon and stomach. We examined the subcellular location of adenomatous polyposis coli (APC) protein resulting from transient expression of full length and partial APC complementary DNAs in epithelial cells. Immunofluorescent detection revealed an association of APC with cytoplasmic microtubules. Expression of partial complementary DNA constructs indicated that the carboxy-terminal region of the APC protein, typically deleted in cancers, is essential for this association. The same APC polypeptides that associated with microtubules in vivo also dramatically promoted their assembly in vitro. These results suggest that wild-type APC protein binds to and affects the assembly of microtubules, whereas the mutants identified in tumors have lost this activity.
The alphabeta tubulin heterodimer is the structural subunit of microtubules, which are cytoskeletal elements that are essential for intracellular transport and cell division in all eukaryotes. Each tubulin monomer binds a guanine nucleotide, which is nonexchangeable when it is bound in the alpha subunit, or N site, and exchangeable when bound in the beta subunit, or E site. The alpha- and beta-tubulins share 40% amino-acid sequence identity, both exist in several isotype forms, and both undergo a variety of posttranslational modifications. Limited sequence homology has been found with the proteins FtsZ and Misato, which are involved in cell division in bacteria and Drosophila, respectively. Here we present an atomic model of the alphabeta tubulin dimer fitted to a 3.7-A density map obtained by electron crystallography of zinc-induced tubulin sheets. The structures of alpha- and beta-tubulin are basically identical: each monomer is formed by a core of two beta-sheets surrounded by alpha-helices. The monomer structure is very compact, but can be divided into three functional domains: the amino-terminal domain containing the nucleotide-binding region, an intermediate domain containing the Taxol-binding site, and the carboxy-terminal domain, which probably constitutes the binding surface for motor proteins.
We have identified a Saccharomyces cerevisiae protein, Cyk1p, that exhibits sequence similarity to the mammalian IQGAPs. Gene disruption of Cyk1p results in a failure in cytokinesis without affecting other events in the cell cycle. Cyk1p is diffused throughout most of the cell cycle but localizes to a ring structure at the mother-bud junction after the initiation of anaphase. This ring contains filamentous actin and Myo1p, a myosin II homologue. In vivo observation with green fluorescent protein-tagged Myo1p showed that the ring decreases drastically in size during cell division and therefore may be contractile. These results indicate that cytokinesis in budding yeast is likely to involve an actomyosin-based contractile ring. The assembly of this ring occurs in temporally distinct steps: Myo1p localizes to a ring that overlaps the septins at the G1-S transition slightly before bud emergence; Cyk1p and actin then accumulate in this ring after the activation of the Cdc15 pathway late in mitosis. The localization of myosin is abolished by a mutation in Cdc12p, implicating a role for the septin filaments in the assembly of the actomyosin ring. The accumulation of actin in the cytokinetic ring was not observed in cells depleted of Cyk1p, suggesting that Cyk1p plays a role in the recruitment of actin filaments, perhaps through a filament-binding activity similar to that demonstrated for mammalian IQGAPs.
A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.
The characteristics of the adenomatous polyposis coli (APC) associated protein EB1 were examined in mammalian cells. By immunocytochemistry EB1 was shown to be closely associated with the microtubule cytoskeleton throughout the cell cycle. In interphase cells EB1 was associated with microtubules along their full length but was often particularly concentrated at their tips. During early mitosis, EB1 was localized to separating centrosomes and associated microtubules, while at metaphase it was associated with the spindle poles and associated microtubules. During cytokinesis EB1 was strongly associated with the midbody microtubules. Treatment with nocodazole caused a diffuse redistribution of EB1 immunoreactivity, whereas treatment with cytochalasin D had no effect. Interestingly, treatment with taxol abolished the EB1 association with microtubules. In nocodazole washout experiments EB1 rapidly became associated with the centrosome and repolymerizing microtubules. In taxol wash-out experiments EB1 rapidly re-associated with the microtubule cytoskeleton, resembling untreated control cells within 10 min. Immunostaining of SW480 cells, which contain truncated APC incapable of interaction with EB1, showed that the association of EB1 with microtubules throughout the cell cycle was not dependent upon an interaction with APC. These results suggest a role for EB1 in the control of microtubule dynamics in mammalian cells.
Obtaining an electron-density map from X-ray diffraction data can be difficult and time-consuming even after the data have been collected, largely because MIR and MAD structure determinations currently require many subjective evaluations of the qualities of trial heavy-atom partial structures before a correct heavy-atom solution is obtained. A set of criteria for evaluating the quality of heavy-atom partial solutions in macromolecular crystallography have been developed. These have allowed the conversion of the crystal structure-solution process into an optimization problem and have allowed its automation. The SOLVE software has been used to solve MAD data sets with as many as 52 selenium sites in the asymmetric unit. The automated structure-solution process developed is a major step towards the fully automated structure-determination, model-building and refinement procedure which is needed for genomic scale structure determinations.
In protein crystallography, much time and effort are often required to trace an initial model from an interpretable electron density map and to refine it until it best agrees with the crystallographic data. Here, we present a method to build and refine a protein model automatically and without user intervention, starting from diffraction data extending to resolution higher than 2.3 A and reasonable estimates of crystallographic phases. The method is based on an iterative procedure that describes the electron density map as a set of unconnected atoms and then searches for protein-like patterns. Automatic pattern recognition (model building) combined with refinement, allows a structural model to be obtained reliably within a few CPU hours. We demonstrate the power of the method with examples of a few recently solved structures.
Microtubule dynamics vary during the cell cycle, and microtubules appear to be more dynamic in vivo than in vitro. Proteins that promote dynamic instability are therefore central to microtubule behavior in living cells. Here, we report that a yeast protein of the highly conserved EB1 family, Bim1p, promotes cytoplasmic microtubule dynamics specifically during G1. During G1, microtubules in cells lacking BIM1 showed reduced dynamicity due to a slower shrinkage rate, fewer rescues and catastrophes, and more time spent in an attenuated/paused state. Human EB1 was identified as an interacting partner for the adenomatous polyposis coli (APC) tumor suppressor protein. Like human EB1, Bim1p localizes to dots at the distal ends of cytoplasmic microtubules. This localization, together with data from electron microscopy and a synthetic interaction with the gene encoding the kinesin Kar3p, suggests that Bim1p acts at the microtubule plus end. Our in vivo data provide evidence of a cell cycle-specific microtubule-binding protein that promotes microtubule dynamicity.
Adenomatous polyposis coli (APC) tumor suppressor protein has been shown to be localized near the distal ends of microtubules (MTs) at the edges of migrating cells. We expressed green fluorescent protein (GFP)-fusion proteins with full-length and deletion mutants of Xenopus APC in Xenopus epithelial cells, and observed their dynamic behavior in live cells. During cell spreading and wound healing, GFP-tagged full-length APC was concentrated as granules at the tip regions of cellular extensions. At higher magnification, APC appeared to move along MTs and concentrate as granules at the growing plus ends. When MTs began to shorten, the APC granules dropped off from the MT ends. Immunoelectron microscopy revealed that fuzzy structures surrounding MTs were the ultrastructural counterparts for these GFP signals. The COOH-terminal region of APC was targeted to the growing MT ends without forming granular aggregates, and abruptly disappeared when MTs began to shorten. The APC lacking the COOH-terminal region formed granular aggregates that moved along MTs toward their plus ends in an ATP-dependent manner. These findings indicated that APC is a unique MT-associated protein that moves along selected MTs and concentrates at their growing plus ends through their multiple functional domains.
Mutations in the adenomatous polyposis coli (APC) gene are associated with most colorectal cancers. The APC protein has been implicated in many aspects of tumour development. This article will discuss recent data suggesting that APC may have multiple functions in the cell. First, APC is a component of the Wnt signalling pathway; second, APC may have a role in cell migration; finally, APC may regulate proliferation and apoptosis.
Cytoplasmic linker proteins (CLIPs) bind to microtubules and are proposed to link this cytoskeletal network to other intracellular structures. We are interested in CLIP-115, since this protein is enriched in neuronal dendrites and may operate in the control of brain-specific organelle translocations. Each CLIP monomer is characterized by two microtubule-binding (MTB) motifs, surrounded by basic, serine-rich regions. This head domain is connected to the C-terminal tail through a long coiled-coil structure. The MTB domains are conserved as a single domain in other proteins involved in microtubule based transport and dynamics, such as p150(Glued). Here we provide evidence that efficient binding of CLIP-115 to microtubules is sensitive to phosphorylation and is not mediated by the conserved MTB domains alone, but requires the presence of the basic, serine rich regions in addition to the MTB motifs. In transfected COS-1 cells, CLIP-115 initially accumulates at the distal ends of microtubules and coincides with CLIP-170, indicating that both proteins mark growing microtubule ends. However, when expressed at higher levels, CLIP-115 and -170 affect the microtubule network differently. This might be partly due to the divergent C-termini of the two proteins. We demonstrate that, similar to CLIP-170, CLIP-115 forms homodimers, which, at least in vitro, are linked by disulfide bridges. Cysteine(391) of CLIP-115, however, is specific in that it controls the microtubule bundling capacity of certain mutant CLIP-115 molecules. Therefore, both similar and specific mechanisms appear to regulate the conformation of CLIPs as well as their binding to microtubules.
End-binding protein (EB) 1 binds to the C-terminus of adenomatous polyposis coli (APC) protein and to the plus ends of microtubules (MT) and has been implicated in the regulation of APC accumulation in cortical clusters at the tip of extending membranes. We investigated which APC domains are involved in cluster localization and whether binding to EB1 or MTs is essential for APC cluster localization. Armadillo repeats of APC that lack EB1- and MT-binding domains are necessary and sufficient for APC localization in cortical clusters; an APC fragment lacking the armadillo repeats, but containing MT- and EB1-binding domains, does not localize to the cortical clusters but instead co-aligns with MTs throughout the cell. Significantly, analysis of endogenous proteins reveals that EB1 does not accumulate in the APC clusters. However, overexpressed EB1 does accumulate in APC clusters; the APC-binding domain in EB1 is located in the C-terminal region of EB1 between amino acids 134 and 268. Overexpressed APC- or MT-binding domains of EB1 localize to APC cortical clusters and MT, respectively, without affecting APC cluster formation itself. These results show that localization of APC in cortical clusters is different from that of EB1 at MT plus ends and appears to be independent of EB1.
A subset of microtubule-associated proteins, including cytoplasmic linker protein (CLIP)-170, dynactin, EB1, adenomatous polyposis coli, cytoplasmic dynein, CLASPs, and LIS-1, has been shown recently to target to the plus ends of microtubules. The mechanisms and functions of this binding specificity are not understood, although a role in encouraging microtubule elongation has been proposed. To extend previous work on the role of dynactin in organelle transport, we analyzed p150(Glued) by live-cell imaging. Time-lapse analysis of p150(Glued) revealed targeting to the plus ends of growing microtubules, requiring the NH2-terminal cytoskeleton-associated protein-glycine rich domain, but not EB1 or CLIP-170. Effectors of protein kinase A modulated microtubule binding and suggested p150(Glued) phosphorylation as a factor in plus-end binding specificity. Using a phosphosensitive monoclonal antibody, we mapped the site of p150(Glued) phosphorylation to Ser-19. In vivo and in vitro analysis of phosphorylation site mutants revealed that p150(Glued) phosphorylation mediates dynamic binding to microtubules. To address the function of dynamic binding, we imaged GFP-p150(Glued) during the dynein-dependent transport of Golgi membranes. Live-cell analysis revealed a transient interaction between Golgi membranes and GFP-p150(Glued)-labeled microtubules just prior to transport, implicating microtubules and dynactin in a search-capture mechanism for minus-end-directed organelles.
Cytoskeleton-associated proteins (CAPs) are involved in the organization of microtubules and transportation of vesicles and organelles along the cytoskeletal network. A conserved motif, CAP-Gly, has been identified in a number of CAPs, including CLIP-170 and dynactins. The crystal structure of the CAP-Gly domain of Caenorhabditis elegans F53F4.3 protein, solved by single wavelength sulfur-anomalous phasing, revealed a novel protein fold containing three beta-sheets. The most conserved sequence, GKNDG, is located in two consecutive sharp turns on the surface, forming the entrance to a groove. Residues in the groove are highly conserved as measured from the information content of the aligned sequences. The C-terminal tail of another molecule in the crystal is bound in this groove.
The end-binding protein 1 (EB1) family is a highly conserved group of proteins that localizes to the plus-ends of microtubules.
EB1 has been shown to play an important role in regulating microtubule dynamics and chromosome segregation, but its regulation
mechanism is poorly understood. We have determined the 1.45-Å resolution crystal structure of the amino-terminal domain of
EB1, which is essential for microtubule binding, and show that it forms a calponin homology (CH) domain fold that is found
in many proteins involved in the actin cytoskeleton. The functional CH domain for actin binding is a tandem pair, whereas
EB1 is the first example of a single CH domain that can associate with the microtubule filament. Although our biochemical
study shows that microtubule binding of EB1 is electrostatic in part, our mutational analysis suggests that the hydrophobic
network, which is partially exposed in our crystal structure, is also important for the association. We propose that, like
other actin-binding CH domains, EB1 employs the hydrophobic interaction to bind to microtubules.
Although cytoplasmic dynein is known to attach to microtubules and translocate toward their minus ends, dynein's ability to serve in vitro as a minus end-directed transporter of membranous organelles depends on additional soluble factors. We show here that a approximately 20S polypeptide complex (referred to as Activator I; Schroer, T. A., and M.P. Sheetz. 1991a. J. Cell Biol. 115:1309-1318.) stimulates dynein-mediated vesicle transport. A major component of the activator complex is a doublet of 150-kD polypeptides for which we propose the name dynactin (for dynein activator). The 20S dynactin complex is required for in vitro vesicle motility since depletion of it with a mAb to dynactin eliminates vesicle movement. Cloning of a brain specific isoform of dynactin from chicken reveals a 1,053 amino acid polypeptide composed of two coiled-coil alpha-helical domains interrupted by a spacer. Both this structural motif and the underlying primary sequence are highly conserved in vertebrates with 85% sequence identity within a central 1,000-residue domain of the chicken and rat proteins. As abundant as dynein, dynactin is ubiquitously expressed and appears to be encoded by a single gene that yields at least three alternative isoforms. The probable homologue in Drosophila is the gene Glued, whose protein product shares 50% sequence identity with vertebrate dynactin and whose function is essential for viability of most (and perhaps all) cells in the organism.
The CCP4 (Collaborative Computational Project, number 4) program suite is a collection of programs and associated data and subroutine libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims and so there may be more than one program to cover each function. The programs are written mainly in standard Fortran77. They are from a wide variety of sources but are connected by standard data file formats. The package has been ported to all the major platforms under both Unix and VMS. The suite is distributed by anonymous ftp from Daresbury Laboratory and is widely used throughout the world.
EB1 targets to polymerizing microtubule ends, where it is favorably positioned to regulate microtubule polymerization and confer molecular recognition of the microtubule end. In this study, we focus on two aspects of the EB1–microtubule interaction: regulation of microtubule dynamics by EB1 and the mechanism of EB1 association with microtubules. Immunodepletion of EB1 from cytostatic factor-arrested M-phaseXenopus egg extracts dramatically reduced microtubule length; this was complemented by readdition of EB1. By time-lapse microscopy, EB1 increased the frequency of microtubule rescues and decreased catastrophes, resulting in increased polymerization and decreased depolymerization and pausing. Imaging of EB1 fluorescence revealed a novel structure: filamentous extensions on microtubule plus ends that appeared during microtubule pauses; loss of these extensions correlated with the abrupt onset of polymerization. Fluorescent EB1 localized to comets at the polymerizing plus ends of microtubules in cytostatic factor extracts and uniformly along the lengths of microtubules in interphase extracts. The temporal decay of EB1 fluorescence from polymerizing microtubule plus ends predicted a dissociation half-life of seconds. Fluorescence recovery after photobleaching also revealed dissociation and rebinding of EB1 to the microtubule wall with a similar half-life. EB1 targeting to microtubules is thus described by a combination of higher affinity binding to polymerizing ends and lower affinity binding along the wall, with continuous dissociation. The latter is likely to be attenuated in interphase. The highly conserved effect of EB1 on microtubule dynamics suggests it belongs to a core set of regulatory factors conserved in higher organisms, and the complex pattern of EB1 targeting to microtubules could be exploited by the cell for coordinating microtubule behaviors.
A new program package, XEASY, was written for interactive computer support of the analysis of NMR spectra for three-dimensional structure determination of biological macromolecules. XEASY was developed for work with 2D, 3D and 4D NMR data sets. It includes all the functions performed by the precursor program EASY, which was designed for the analysis of 2D NMR spectra, i.e., peak picking and support of sequence-specific resonance assignments, cross-peak assignments, cross-peak integration and rate constant determination for dynamic processes. Since the program utilizes the X-window system and the Motif widget set, it is portable on a wide range of UNIX workstations. The design objective was to provide maximal computer support for the analysis of spectra, while providing the user with complete control over the final resonance assignments. Technically important features of XEASY are the use and flexible visual display of strips, i.e., two-dimensional spectral regions that contain the relevant parts of 3D or 4D NMR spectra, automated sorting routines to narrow down the selection of strips that need to be interactively considered in a particular assignment step, a protocol of resonance assignments that can be used for reliable bookkeeping, independent of the assignment strategy used, and capabilities for proper treatment of spectral folding and efficient transfer of resonance assignments between spectra of different types and different dimensionality, including projected, reduced-dimensionality triple-resonance experiments.
TAXOL (Fig. 1) was isolated from the plant Taxus brevifolia (western
yew) by Wani et al., who reported that the molecule has antitumour
activity in several experimental systems1. In our laboratory
we have found that taxol, a low molecular weight neutral compound,
completely inhibits division of exponentially growing HeLa cells at low
concentrations of drug (0.25 µM) that have no significant effects
on DNA, RNA or protein synthesis during a 4-h incubation with the cells.
HeLa cells incubated with taxol for 20 h are blocked in late
G2 and/or M (ref. 2). We report here that taxol acts as a
promoter of calf brain microtubule assembly in vitro, in contrast to
plant products such as colchicine and podophyllotoxin, which inhibit
assembly. Taxol decreases the lag time for microtubule assembly and
shifts the equilibrium for assembly in favour of the microtubule,
thereby decreasing the critical concentration of tubulin required for
assembly. Microtubules polymerised in the presence of taxol are
resistant to depolymerisation by cold (4 °C) and CaCl2 (4
mM).
This chapter presents a collection of the various different ways by which tubulins are modified to generate probes for investigating microtubule (MT) dynamics in vitro and in vivo. Labeling with biotin and various fluorochromes is described, as well as the preparation of N-ethylmaleimide tubulin, which has been used to block minus-end growth in vitro. The use of GTP analogs to prepare stable labeled microtubules has proved very useful in a number of different experiments. The tubulin used in the presented methods was prepared from bovine brain by two cycles of temperature-dependent polymerization, followed by phosphocellulose chromatography. The cycling procedure described in the chapter selects active subunits and removes free nucleotide. This produces a tubulin preparation suitable for use in in vitro assays. The standard biotin-labeled tubulin preparation has been used to determine sites of microtubule elongation in vivo and in vitro. It is difficult to quantitate the stoichiometry of biotin labeling on a routine basis, but early work using radioactive N-hydroxysuccinimide (NHS)-biotin gave a labeling stochiometry of one to three biotins/tubulin dimer. The final yield of twice cycled biotin-tubulin is about 10% of the starting protein. Tetramethylrhodamine-labeled tubulin has been used to follow microtubules in living cells and it is also used for marking microtubules in real-time in vitro assays.
We report here that microtubules in vitro coexist in growing and shrinking populations which interconvert rather infrequently. This dynamic instability is a general property of microtubules and may be fundamental in explaining cellular microtubule organization.
The polymerization dynamics of microtubules are central to their biological functions. Polymerization dynamics allow microtubules to adopt spatial arrangements that can change rapidly in response to cellular needs and, in some cases, to perform mechanical work. Microtubules utilize the energy of GTP hydrolysis to fuel a unique polymerization mechanism termed dynamic instability. In this review, we first describe progress toward understanding the mechanism of dynamic instability of pure tubulin and then discuss the function and regulation of microtubule dynamic instability in living cells.
A number of accessory proteins capable of stabilizing or destabilizing microtubule polymers in dividing cells have been identified recently. Many of these accessory proteins are modified and regulated by cell-cycle-dependent phosphorylation. Through this regulation, microtubule dynamics are modified to generate rapid microtubule turnover during mitosis. In general, although some microtubule-stabilizing proteins are inactivated at entry into mitosis, a critical balance between microtubule stabilizers and destabilizers is necessary for assembly of the mitotic spindle.
A chimera with the green fluorescent protein (GFP) has been constructed to visualize the dynamic properties of the endosome-microtubule linker protein CLIP170 (GFP-CLIP170). GFP-CLIP170 binds in stretches along a subset of microtubule ends. These fluorescent stretches appear to move with the growing tips of microtubules at 0.15-0.4 microm/s, comparable to microtubule elongation in vivo. Analysis of speckles along dynamic GFP-CLIP170 stretches suggests that CLIP170 treadmills on growing microtubule ends, rather than being continuously transported toward these ends. Drugs affecting microtubule dynamics rapidly inhibit movement of GFP-CLIP170 dashes. We propose that GFP-CLIP170 highlights growing microtubule ends by specifically recognizing the structure of a segment of newly polymerized tubulin.
Xfit is a model-building and map viewing program in XtalView that is used by the structural biology community including researchers in the fields of crystallography, molecular modeling, and electron microscopy. Among its distinguishing features are built-in fast Fourier transforms that allow users flexibility in map calculations including the creation of OMIT maps and the updating of structure factors to reflect model changes from within the program. Written in C and using the freely available XView toolkit, it is highly portable to almost any X-windows based workstation including Intel-based LINUX systems. Its user interface is designed to aid in facile model-building and contains a semiautomated fitting system that allows the user to interactively and rapidly build chain de novo into an electron density map. The program is highly optimized to allow such features as interactive contour levels and map calculations to be completed within a few seconds. Features in the latest version including phase-combination, solvent-flattening, automated water addition, and small-probe dot contact surfaces, as well as basic design features, are discussed.
Human EB1 is a highly conserved protein that binds to the carboxyl terminus of the human adenomatous polyposis coli (APC) tumor suppressor protein [1], a domain of APC that is commonly deleted in colorectal neoplasia [2]. EB1 belongs to a family of microtubule-associated proteins that includes Schizosaccharomyces pombe Mal3 [3] and Saccharomyces cerevisiae Bim1p [4]. Bim1p appears to regulate the timing of cytokinesis as demonstrated by a genetic interaction with Act5, a component of the yeast dynactin complex [5]. Whereas the predominant function of the dynactin complex in yeast appears to be in positioning the mitotic spindle [6], in animal cells, dynactin has been shown to function in diverse processes, including organelle transport, formation of the mitotic spindle, and perhaps cytokinesis [7] [8] [9] [10]. Here, we demonstrate that human EB1 can be coprecipitated with p150(Glued), a member of the dynactin protein complex. EB1 was also found associated with the intermediate chain of cytoplasmic dynein (CDIC) and with dynamitin (p50), another component of the dynactin complex, but not with dynein heavy chain, in a complex that sedimented at approximately 5S in a sucrose density gradient. The association of EB1 with members of the dynactin complex was independent of APC and was preserved in the absence of an intact microtubule cytoskeleton. The molecular interaction of EB1 with members of the dynactin complex and with CDIC may be important for microtubule-based processes.
Utrophin is a large multidomain protein that belongs to a superfamily of actin-binding proteins, which includes dystrophin, alpha-actinin, beta-spectrin, fimbrin, filamin and plectin. All the members of this family contain a common actin-binding region at their N termini and perform a wide variety of roles associated with the actin cytoskeleton. Utrophin is the autosomal homologue of dystrophin, the protein defective in the X-linked Duchenne and Becker muscular dystrophies, and upregulation of utrophin has been suggested as a potential therapy for muscular dystrophy patients.
The structure of the actin-binding region of utrophin, consisting of two calponin-homology (CH) domains, has been solved at 3.0 A resolution. It is composed of an antiparallel dimer with each of the monomers being present in an extended dumbell shape and the two CH domains being separated by a long central helix. This extended conformation is in sharp contrast to the compact monomer structure of the N-terminal actin-binding region of fimbrin.
The crystal structure of the actin-binding region of utrophin suggests that these actin-binding domains may be more flexible than was previously thought and that this flexibility may allow domain reorganisation and play a role in the actin-binding mechanism. Thus utrophin could possibly bind to actin in an extended conformation so that the sites previously identified as being important for actin binding may be directly involved in this interaction.
Adenomatous polyposis coli protein (APC) is a well-characterized tumor suppressor protein [1] [2] [3]. We previously showed that APC tagged with green fluorescent protein (GFP) in Xenopus A6 epithelial cells moves along a subset of microtubules and accumulates at their growing plus ends in cell extensions [4]. EB1, which was identified as an APC-binding protein by yeast two-hybrid analysis [5], was also reported to be associated with microtubules [6] [7] [8]. To examine the interaction between APC and EB1 within cells, we compared the dynamic behavior of EB1-GFP with that of APC-GFP in A6 transfectants. Time-lapse microscopy of live cells at interphase revealed that EB1-GFP was concentrated at all of the growing microtubule ends throughout the cytoplasm and abruptly disappeared from the ends when microtubules began to shorten. Therefore, EB1 appeared to be co-localized and interact with APC on the growing ends of a subset of microtubules. When APC-GFP was overexpressed, endogenous EB1 was recruited to APC-GFP, which accumulated in large amounts on microtubules. On the other hand, when microtubules were disassembled by nocodazole, EB1 was not co-localized with APC-GFP, which was concentrated along the basal plasma membrane. During mitosis, APC appeared to be dissociated from microtubules, whereas EB1-GFP continued to concentrate at microtubule growing ends. These findings showed that the APC-EB1 interaction is regulated within cells and is allowed near the ends of microtubules only under restricted conditions.
Microtubule plus end dynamics, as well as interactions with the cell cortex and internal organelles, may be mediated by a 'plus end complex' of interacting proteins. Recent results suggest that centrosomes with different microtubule-releasing and anchoring properties underlie the development of various microtubule arrays.
Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial polyposis, a form of inherited colon cancer. In addition to its role in mediating beta-catenin degradation in the Wnt signaling pathway, APC plays a role in regulating microtubules. This was suggested by its localization to the end of dynamic microtubules in actively migrating areas of cells and by the apparent correlation between the dissociation of APC from polymerizing microtubules and their subsequent depolymerization [1, 2]. The microtubule binding domain is deleted in the transforming mutations of APC [3, 4]; however, the direct effect of APC protein on microtubules has never been examined. Here we show that binding of APC to microtubules increases microtubule stability in vivo and in vitro. Deleting the previously identified microtubule binding site from the C-terminal domain of APC does not eliminate its binding to microtubules but decreases the ability of APC to stabilize them significantly. The interaction of APC with microtubules is decreased by phosphorylation of APC by GSK3 beta. These data confirm the hypothesis that APC is involved in stabilizing microtubule ends. They also suggest that binding of APC to microtubules is mediated by at least two distinct sites and is regulated by phosphorylation.
There are several key unanswered questions about the +TIPs. One is the mechanism of release of +TIPs from the trailing edge of polymerizing MTs. An important barrier to progress is the absence of an in vitro system that faithfully recapitulates both the binding and release that characterize plus end tracking. As +TIP partners/regulators may be important to reconstitute +TIP treadmilling in vitro, future progress may come from systems with additional purified components, or from ones based on crude extracts. Another question is the in vivo relationship between different +TIPs. It appears that CLIP-170 and EB1 can reside on the same growing MT end (Akhmanova et al., 2001xAkhmanova, A., Hoogenraad, C.C., Drabek, K., Stepanova, T., Dortland, B., Verkerk, T., Vermeulen, W., Burgering, B.M., De Zeeuw, C.I., Grosveld, F., and Galjart, N. Cell. 2001; 104: 923–935Abstract | Full Text | Full Text PDF | PubMed | Scopus (290)See all References)(Akhmanova et al., 2001). Is there cooperation or competition between these proteins, and could such interactions be a nodal point for regulating the repertoire of plus end behaviors?In addition, it is not known whether +TIPs can provide stable attachments. It will be important to determine if their association with MTs is stabilized (i.e., if they treadmill less) when MT plus ends interact with target sites. An alternative, suggested for CLIP-170 at the kinetochore, is that +TIPs mediate the initial attachment but then dissociate (Dujardin et al., 1998xDujardin, D., Wacker, U.I., Moreau, A., Schroer, T.A., Rickard, J.E., and De Mey, J.R. J. Cell Biol. 1998; 141: 849–862Crossref | Scopus (116)See all References)(Dujardin et al., 1998). Real-time methods where the turnover of +TIPs at the MT end can be measured may help distinguish between these possibilities. Although budding yeast is not famous for the awesome power of its cytology, the ability to see single MTs interacting with target sites on the membrane provides unique spatial resolution that should facilitate these experiments. Finally, do the +TIPs only function at the MT plus end? Several +TIPs interact with proteins that associate with or regulate the behavior of the MT minus ends (Chen et al. 1998xChen, X.P., Yin, H., and Huffaker, T.C. J. Cell Biol. 1998; 141: 1169–1179Crossref | Scopus (71)See all References, Chen et al. 2000xChen, C.R., Chen, J., and Chang, E.C. Mol. Biol. Cell. 2000; 11: 4067–4077CrossrefSee all References). This raises the possibility that +TIPs might have an additional role in MT nucleation or in anchoring MT minus ends to the centrosome.Like motors and the proteins involved in microtubule nucleation, the CLIP-170 family and EB1 family proteins are highly conserved. We speculate that these proteins evolved because of the need for devices that distinguish the plus end from the body of the MT. Although the plus end has a unique shape, its large size (>25 nm in diameter) makes it an unwieldy object to recognize at the molecular level. +TIPs may solve this problem; if copolymerized with tubulin, +TIPs may “tag” MT plus ends. By interacting with different partners, +TIPs could serve as molecular adaptors, providing links to a large repertoire of signals and target sites. In the cell, this diversity of plus end interactions may be fundamental to the regional control of MT dynamics, MT attachment, and the assembly of complex MT-based structures necessary for cell division and morphogenesis.
Human EB1 was originally cloned as a protein that interacts with the COOH terminus of adenomatous polyposis coli (APC). Interestingly, this interaction is often disrupted in colon cancer, due to mutations in APC. EB1 also interacts with the plus-ends of microtubules and targets APC to microtubule tips. Since APC is detected on the kinetochores of chromosomes, it has been hypothesized that the EB1-APC interaction connects microtubule spindles to the kinetochores and regulates microtubule stability. In yeast, EB1 regulates microtubule dynamics, and its binding domain in APC may be conserved in Kar9, an EB1 binding protein involved in the microtubule-capturing mechanism. These results suggest that the interaction of EB1 and APC is important and may be conserved. However, it is largely unknown whether the EB1-APC interaction affects microtubule dynamics. Here, we show that EB1 potently promotes microtubule polymerization in vitro and in permeabilized cells, but, surprisingly, only in the presence of the COOH-terminal EB1 binding domain of APC (C-APC). Significantly, this C-APC activity is abolished by phosphorylation, which also disrupts its ability to bind to EB1. Furthermore, yeast EB1 protein effectively substitutes for the human protein but also requires C-APC in promoting microtubule polymerization. Finally, C-APC is able to promote microtubule polymerization when stably expressed in APC mutant cells, demonstrating the ability of C-APC to promote microtubule assembly in vivo. Thus, the interaction between EB1 and APC plays an essential role in the regulation of microtubule polymerization, and a similar mechanism may be conserved in yeast.
Many eukaryotic signal transduction proteins have component-based architectures: they are built from combinations of protein interaction domains and catalytic domains. Intact, these proteins display the sophisticated allosteric behavior required for cellular regulation; the protein's output activity is tightly repressed under basal conditions, but can be robustly activated by a specific set of input effector ligands. A combination of structural, biophysical and computational studies is beginning to shed light on the fundamental principles governing this type of modular allostery.
With the refinement of algorithms for the identification of distinct motifs from sequence databases, especially those using secondary structure predictions, new protein modules have been determined in recent years. Calponin homology (CH) domains were identified in a variety of proteins ranging from actin cross-linking to signaling and have been proposed to function either as autonomous actin binding motifs or serve a regulatory function. Despite the overall structural conservation of the unique CH domain fold, the individual modules display a quite striking functional variability. Analysis of the actopaxin/parvin protein family suggests the existence of novel (type 4 and type 5) CH domain families which require special attention, as they appear to be a good example for how CH domains may function as scaffolds for other functional motifs of different properties.
Microtubule polymerization dynamics at kinetochores is coupled to chromosome movements, but its regulation there is poorly understood. The plus end tracking protein EB1 is required both for regulating microtubule dynamics and for maintaining a euploid genome. To address the role of EB1 in aneuploidy, we visualized its targeting in mitotic PtK1 cells. Fluorescent EB1, which localized to polymerizing ends of astral and spindle microtubules, was used to track their polymerization. EB1 also associated with a subset of attached kinetochores in late prometaphase and metaphase, and rarely in anaphase. Localization occurred in a narrow crescent, concave toward the centromere, consistent with targeting to the microtubule plus end-kinetochore interface. EB1 did not localize to kinetochores lacking attached kinetochore microtubules in prophase or early prometaphase, or upon nocodazole treatment. By time lapse, EB1 specifically targeted to kinetochores moving antipoleward, coupled to microtubule plus end polymerization, and not during plus end depolymerization. It localized independently of spindle bipolarity, the spindle checkpoint, and dynein/dynactin function. EB1 is the first protein whose targeting reflects kinetochore directionality, unlike other plus end tracking proteins that show enhanced kinetochore binding in the absence of microtubules. Our results suggest EB1 may modulate kinetochore microtubule polymerization and/or attachment.
An important function of microtubules is to move cellular structures such as chromosomes, mitotic spindles and other organelles around inside cells. This is achieved by attaching the ends of microtubules to cellular structures; as the microtubules grow and shrink, the structures are pushed or pulled around the cell. How do the ends of microtubules couple to cellular structures, and how does this coupling regulate the stability and distribution of the microtubules? It is now clear that there are at least three properties of a microtubule end: it has alternate structures; it has a biochemical transition defined by GTP hydrolysis; and it forms a distinct target for the binding of specific proteins. These different properties can be unified by thinking of the microtubule as a molecular machine, which switches between growing and shrinking modes. Each mode is associated with a specific end structure on which end-binding proteins can assemble to modulate dynamics and couple the dynamic properties of microtubules to the movement of cellular structures.
A crowd of proteins seems to have gathered around the plus-ends of microtubules. A rapidly expanding group of proteins known as plus-end tracking proteins (+TIPs) have been identified that seem to be able to 'surf' the dynamic ends of microtubules. Microtubule plus-ends exist in multiple conformational and chemical states. In principle, altering this plus-end microenvironment is an appealing way for regulators such as the +TIPS to control microtubule dynamics; however, specific mechanisms are poorly defined. Here, we focus on new findings addressing the underlying mechanisms of plus-end tracking and the mechanisms by which +TIPS control microtubule dynamics. We review the evidence that plus-end-binding and the control of microtubule dynamics are mechanistically linked. We also consider the possibility that, by studying +TIPs, we might learn more about the dynamic structural changes at the microtubule ends that are at the heart of dynamic instability.