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ABSTRACT: Microtubule (MT) dynamic instability is fundamental to many cell functions, but its mechanism remains poorly understood, in part because it is difficult to gain information about the dimer-scale events at the MT tip. To address this issue, we used a dimer-scale computational model of MT assembly that is consistent with tubulin structure and biochemistry, displays dynamic instability, and covers experimentally relevant spans of time. It allows us to correlate macroscopic behaviors (dynamic instability parameters) with microscopic structures (tip conformations) and examine protofilament structure as the tip spontaneously progresses through both catastrophe and rescue. The model's behavior suggests that several commonly held assumptions about MT dynamics should be reconsidered. Moreover, it predicts that short, interprotofilament "cracks" (laterally unbonded regions between protofilaments) exist even at the tips of growing MTs and that rapid fluctuations in the depths of these cracks influence both catastrophe and rescue. We conclude that experimentally observed microtubule behavior can best be explained by a "stochastic cap" model in which tubulin subunits hydrolyze GTP according to a first-order reaction after they are incorporated into the lattice; catastrophe and rescue result from stochastic fluctuations in the size, shape, and extent of lateral bonding of the cap.
Molecular biology of the cell 12/2011; 23(4):642-56. · 5.98 Impact Factor
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ABSTRACT: A link between dimer-scale processes and microtubule (MT) dynamics at macroscale is studied by comparing simulations obtained using computational dimer-scale model with its mean-field approximation. The novelty of the mean-field model (MFM) is in its explicit representation of inter-protofilament cracks, as well as in the direct incorporation of the dimer-level kinetics. Due to inclusion of both longitudinal and lateral dimer interactions, the MFM is two dimensional, in contrast to previous theoretical models of MTs. It is the first analytical model that predicts and quantifies crucial features of MT dynamics such as (i) existence of a minimal soluble tubulin concentration needed for the polymerization (with concentration represented as a function of model parameters), (ii) existence of steady-state growth and shortening phases (given with their respective velocities), and (iii) existence of an unstable pause state near zero velocity. In addition, the size of the GTP cap of a growing MT is estimated. Theoretical predictions are shown to be in good agreement with the numerical simulations.
Physical Review E 04/2011; 83(4 Pt 1):041905. · 2.26 Impact Factor
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ABSTRACT: Ring-C modified alkaloids were synthesized from colchicine using iminonitroso Diels-Alder reactions in a highly regio- and stereoselective fashion. Several analogs exhibited cytotoxic activity similar to that of colchicine itself against PC-3 and MCF-7 cancer cell lines, by serving as prodrugs of colchicine through retro Diels-Alder reactions under the assayed conditions. In vitro microtubule polymerization assays indicated that these modifications affected their interaction with tubulin.
Bioorganic & medicinal chemistry letters 06/2010; 20(12):3831-3. · 2.65 Impact Factor
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ABSTRACT: Experimental cell biology, biochemistry, and structural biology have provided a wealth of information about microtubule function and mechanism, but we are reaching a limit as to what can be understood from experiment alone. Standard biochemical approaches are not sufficient to make quantitative predictions about microtubule behavior, and they are limited in their ability to test existing conceptual models of microtubule mechanism. Because microtubules are so complex, achieving a deep understanding of microtubule behavior and mechanism will require the input of mathematical and computational modeling. However, this type of analysis can be daunting to the uninitiated. The purpose of this chapter is to provide a straightforward introduction to the various types of modeling and how they can be used to study microtubule function, dynamics, and mechanism.
Methods in cell biology 01/2010; 95:175-88. · 2.05 Impact Factor
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ABSTRACT: Plus end tracking proteins (+TIPs) are a unique group of microtubule binding proteins that dynamically track microtubule (MT)
plus ends. EB1 is a highly conserved +TIP with a fundamental role in MT dynamics, but it remains poorly understood in part
because reported EB1 activities have differed considerably. One reason for this inconsistency could be the variable presence
of affinity tags used for EB1 purification. To address this question and establish the activity of native EB1, we have measured
the MT binding and tubulin polymerization activities of untagged EB1 and EB1 fragments and compared them with those of His-tagged
EB1 proteins. We found that N-terminal His tags directly influence the interaction between EB1 and MTs, significantly increasing
both affinity and activity, and that small amounts of His-tagged proteins act synergistically with larger amounts of untagged
proteins. Moreover, the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple
binding models. These observations demonstrate that EB1 binding is not limited to the MT seam, and they suggest that EB1 binds
cooperatively to MTs. Finally, we found that removal of tubulin C-terminal tails significantly reduces EB1 binding, indicating
that EB1-tubulin interactions are mediated in part by the same tubulin acidic tails utilized by other MAPs. These binding
relationships are important for helping to elucidate the complex of proteins at the MT tip.
Journal of Biological Chemistry 11/2009; 284(47):32651-32661. · 4.77 Impact Factor
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ABSTRACT: Plus end tracking proteins (+TIPs) are a unique group of microtubule binding proteins that dynamically track microtubule (MT) plus ends. EB1 is a highly conserved +TIP with a fundamental role in MT dynamics, but it remains poorly understood in part because reported EB1 activities have differed considerably. One reason for this inconsistency could be the variable presence of affinity tags used for EB1 purification. To address this question and establish the activity of native EB1, we have measured the MT binding and tubulin polymerization activities of untagged EB1 and EB1 fragments and compared them with those of His-tagged EB1 proteins. We found that N-terminal His tags directly influence the interaction between EB1 and MTs, significantly increasing both affinity and activity, and that small amounts of His-tagged proteins act synergistically with larger amounts of untagged proteins. Moreover, the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple binding models. These observations demonstrate that EB1 binding is not limited to the MT seam, and they suggest that EB1 binds cooperatively to MTs. Finally, we found that removal of tubulin C-terminal tails significantly reduces EB1 binding, indicating that EB1-tubulin interactions are mediated in part by the same tubulin acidic tails utilized by other MAPs. These binding relationships are important for helping to elucidate the complex of proteins at the MT tip.
Journal of Biological Chemistry 09/2009; 284(47):32651-61. · 4.77 Impact Factor
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ABSTRACT: EB1 is a microtubule plus-end tracking protein that plays a central role in the regulation of microtubule (MT) dynamics. GFP-tagged EB1 constructs are commonly used to study EB1 itself and also as markers of dynamic MT plus ends. To properly interpret these studies, it is important to understand the impact of tags on the behavior of EB1 and other proteins in vivo. To address this problem and improve understanding of EB1 function, we surveyed the localization of expressed EB1 fragments and investigated whether GFP tags alter these localizations. We found that neither N-terminal nor C-terminal tags are benign: tagged EB1 and EB1 fragments generally behave differently from their untagged counterparts. N-terminal tags significantly compromise the ability of expressed EB1 proteins to bind MTs and/or track MT plus ends, although they leave some MT-binding ability intact. C-terminally tagged EB1 constructs have localizations similar to the untagged constructs, initially suggesting that they are benign. However, most constructs tagged at either end cause CLIP-170 to disappear from MT plus ends. This effect is opposite to that of untagged full-length EB1, which recruits CLIP-170 to MTs. These observations demonstrate that although EB1-GFP can be a powerful tool for studying microtubule dynamics, it should be used carefully because it may alter the system that it is being used to study. In addition, some untagged fragments had unexpected localizations. In particular, an EB1 construct lacking the coiled-coil tracks MT plus ends, though weakly, providing evidence against the idea that EB1 +TIP behavior requires dimerization.
Cytoskeleton 09/2009; 67(1):1-12.
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ABSTRACT: Proline rich RNA-binding protein (Prrp), which associates with mRNAs that employ the late pathway for localization in Xenopus oocytes, was used as bait in a yeast two-hybrid screen of an expression library. Several independent clones were recovered that correspond to a paralog of 40LoVe, a factor required for proper localization of Vg1 mRNA to the vegetal cortex. 40LoVe is present in at least three alternatively spliced isoforms; however, only one, corresponding to the variant identified in the two-hybrid screen, can be crosslinked to Vg1 mRNA. In vitro binding assays revealed that 40LoVe has high affinity for RNA, but exhibits little binding specificity on its own. Nonetheless, it was only found associated with localized mRNAs in oocytes. 40LoVe also interacts directly with VgRBP71 and VgRBP60/hnRNP I; it is the latter factor that likely determines the binding specificity of 40LoVe. Initially, 40LoVe binds to Vg1 mRNA in the nucleus and remains with the RNA in the cytoplasm. Immunohistochemical staining of oocytes shows that the protein is distributed between the nucleus and cytoplasm, consistent with nucleocytoplasmic shuttling activity. 40LoVe is excluded from the mitochondrial cloud, which is used by RNAs that localize through the early (METRO) pathway in stage I oocytes; nonetheless, it is associated with at least some early pathway RNAs during later stages of oogenesis. A phylogenetic analysis of 2xRBD hnRNP proteins combined with other experimental evidence suggests that 40LoVe is a distant homolog of Drosophila Squid.
Mechanisms of development 05/2009; 126(7):523-38. · 2.83 Impact Factor
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ABSTRACT: Cytoplasmic linker protein 170 (CLIP-170) is the prototype microtubule (MT) plus-end tracking protein (+TIP) and is involved in regulating MT dynamics. A comprehensive understanding of the process by which CLIP-170 tracks MT plus ends would provide insight into its function. However, the precise molecular mechanism of CLIP-170 +TIP behavior is unknown, and many potential models have been presented. Here, by separating the two CLIP-170 CAP-Gly domains and their adjacent serine-rich regions into fragments of varied size, we have characterized the minimal plus-end tracking unit of CLIP-170 in vivo. Each CLIP-170 fragment was also characterized for its tubulin polymerization activity in vitro. We found that the two CAP-Gly domains have different activities, whereas CAP-Gly-1 appears incompetent to mediate either +TIP behavior or MT nucleation, a CLIP-170 fragment consisting of the second CAP-Gly domain and its adjacent serine-rich region can both track MT plus ends in vivo and induce tubulin polymerization in vitro. These observations complement recent work on CLIP-170 fragments, demonstrate that CAP-Gly motifs do not require dimerization for +TIP and polymerization-promoting activities, and provide insight into CLIP-170 function and mechanism.
Journal of Biological Chemistry 01/2009; 284(11):6735-42. · 4.77 Impact Factor
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ABSTRACT: Although the dynamic self-assembly behavior of microtubule ends has been well characterized at the spatial resolution of light microscopy (~200 nm), the single-molecule events that lead to these dynamics are less clear. Recently, a number of in vitro studies used novel approaches combining laser tweezers, microfabricated chambers, and high-resolution tracking of microtubule-bound beads to characterize mechanochemical aspects of MT dynamics at nanometer scale resolution. In addition, computational modeling is providing a framework for integrating these experimental results into physically plausible models of molecular scale microtubule dynamics. These nanoscale studies are providing new fundamental insights about microtubule assembly, and will be important for advancing our understanding of how microtubule dynamic instability is regulated in vivo via microtubule-associated proteins, therapeutic agents, and mechanical forces.
Current Opinion in Cell Biology 03/2008; 20(1):64-70. · 12.90 Impact Factor
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ABSTRACT: Microtubule dynamic instability plays a fundamental role in cell biology, enabling microtubules to find and interact with randomly distributed cargo and spatially localized signals. In vitro, microtubules transition between growth and shrinkage symmetrically, consistent with the theoretical understanding of the mechanism of dynamic instability. In vivo, however, microtubules commonly exhibit asymmetric dynamic instability, growing persistently in the cell interior and experiencing catastrophe near the cell edge. What is the origin of this behavior difference? One answer is that the cell edge causes the asymmetry by inducing catastrophe in persistently growing microtubules. However, the origin of the persistent growth itself is unclear. Using a simplified coarse-grained stochastic simulation of a system of dynamic microtubules, we provide evidence that persistent growth is a predictable property of a system of nucleated, dynamic, microtubules containing sufficient tubulin in a confined space--MAP activity is not required. Persistent growth occurs because cell-edge-induced catastrophe increases the concentration of free tubulin at steady-state. Our simulations indicate that other aspects of MT dynamics thought to require temporal or spatial changes in MAP activity are also predictable, perhaps unavoidable, outcomes of the "systems nature" of the cellular microtubule cytoskeleton. These include the mitotic increase in microtubule dynamics and the observation that defects in nucleation cause changes in the behavior of microtubule plus ends. These predictions are directly relevant to understanding of the microtubule cytoskeleton, but they are also attractive from an evolutionary standpoint because they provide evidence that apparently complex cellular behaviors can originate from simple interactions without a requirement for intricate regulatory machinery.
Journal of Cell Science 12/2006; 119(Pt 22):4781-8. · 6.11 Impact Factor
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ABSTRACT: A theoretical model of dynamic instability of a system of linear one-dimensional microtubules (MTs) in a bounded domain is introduced for studying the role of a cell edge in vivo and analyzing the effect of competition for a limited amount of tubulin. The model differs from earlier models in that the evolution of MTs is based on the rates of single-mesoscopic-unit (e.g., a heterodimer per protofilament) transformations, in contrast to postulating effective rates and frequencies of larger-scale macroscopic changes, extracted, e.g., from the length history plots of MTs. Spontaneous GTP hydrolysis with finite rate after polymerization is assumed, and theoretical estimates of an effective catastrophe frequency as well as other parameters characterizing MT length distributions and cap size are derived. We implement a simple cap model which does not include vectorial hydrolysis. We demonstrate that our theoretical predictions, such as steady-state concentration of free tubulin and parameters of MT length distributions, are in agreement with the numerical simulations. The present model establishes a quantitative link between mesoscopic parameters governing the dynamics of MTs and macroscopic characteristics of MTs in a closed system. Last, we provide an explanation for nonexponential MT length distributions observed in experiments. In particular, we show that the appearance of such nonexponential distributions in the experiments can occur because a true steady state has not been reached and/or due to the presence of a cell edge.
Physical Review E 11/2006; 74(4 Pt 1):041920. · 2.26 Impact Factor
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ABSTRACT: CLASPs are spatially regulated microtubule plus end tracking proteins involved in forming polarized microtubule arrays. Work in this issue of Developmental Cell identifies the protein LL5beta as a key CLASP binding platform that mediates communication between the cell cortex and the microtubule cytoskeleton.
Developmental Cell 08/2006; 11(1):4-5. · 14.03 Impact Factor
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Proceedings of the National Academy of Sciences 04/2006; 103(10):3498-9. · 9.68 Impact Factor
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ABSTRACT: Recent work has underscored the importance of membrane trafficking events during cytokinesis. For example, targeted membrane secretion occurs at the cleavage furrow in animal cells, and proteins that regulate endocytosis also influence the process of cytokinesis. Nonetheless, the prevailing dogma is that endosomal membrane trafficking ceases during mitosis and resumes after cell division is complete. In this study, we have characterized endocytic membrane trafficking events that occur during mammalian cell cytokinesis. We have found that, although endocytosis ceases during the early stages of mitosis, it resumes during late mitosis in a temporally and spatially regulated pattern as cells progress from anaphase to cytokinesis. Using fixed and live cell imaging, we have found that, during cleavage furrow ingression, vesicles are internalized from the polar region and subsequently trafficked to the midbody area during later stages of cytokinesis. In addition, we have demonstrated that cytokinesis is inhibited when clathrin-mediated endocytosis is blocked using a series of dominant negative mutants. In contrast to previous thought, we conclude that endocytosis resumes during the later stages of mitosis, before cytokinesis is completed. Furthermore, based on our findings, we propose that the proper regulation of endosomal membrane traffic is necessary for the successful completion of cytokinesis.
Journal of Biological Chemistry 01/2006; 280(50):41628-35. · 4.77 Impact Factor
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ABSTRACT: CLIP-170 belongs to a group of proteins (+TIPs) with the enigmatic ability to dynamically track growing microtubule plus-ends. CLIP-170 regulates microtubule dynamics in vivo and has been implicated in cargo-microtubule interactions in vivo and in vitro. Though plus-end tracking likely has intimate connections to +TIP function, little is known about the mechanism(s) by which this dynamic localization is achieved. Using a combination of biochemistry and live cell imaging, we provide evidence that CLIP-170 tracks microtubule plus-ends by a preassociation, copolymerization, and regulated release mechanism. As part of this analysis, we find that CLIP-170 has a stronger affinity for tubulin dimer than for polymer, and that CLIP-170 can distinguish between GTP- and GDP-like polymer. This work extends the previous analysis of CLIP-170 behavior in vivo and complements the existing fluorescence microscope characterization of CLIP-170 interactions with microtubules in vitro. In particular, these data explain observations that CLIP-170 localizes to newly polymerized microtubules in vitro but cannot track microtubule plus-ends in vitro. These observations have implications for the functions of CLIP-170 in regulating microtubule dynamics.
Molecular Biology of the Cell 12/2005; 16(11):5373-84. · 4.94 Impact Factor
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ABSTRACT: Toxoplasma gondii and its apicomplexan relatives (such as Plasmodium falciparum, which causes malaria) are obligate intracellular parasites that rely on sequential protein release from specialized secretory organelles for invasion and multiplication within host cells. Because of the importance of these unusual membrane trafficking pathways for drug development and comparative cell biology, characterizing them is essential. In particular, it is unclear what role retrieval mechanisms play in parasite membrane trafficking or where they operate. Previously, we showed that T. gondii's beta-COP (TgBetaCOP; a subunit of coatomer protein complex I, COPI) and retrieval reporters localize exclusively to the zone between the parasite endoplasmic reticulum (ER) and Golgi apparatus. This suggested the existence of an HDEL receptor in T. gondii. We have now identified, cloned, and sequenced this receptor, TgERD2. TgERD2 localizes in a Golgi or ER pattern suggestive of the HDEL retrieval reporter (K. M. Hager, B. Striepen, L. G. Tilney, and D. S. Roos, J. Cell Sci. 112:2631-2638, 1999). A functional assay reveals that TgERD2 is able to complement the Saccharomyces cerevisiae ERD2 null mutant. Retrieval studies reveal that stable expression of a fluorescent exogenous retrieval ligand results in a dispersal of betaCOP signal throughout the cytoplasm and, surprisingly, results in betaCOP staining of the vacuolar space of the parasite. In contrast, stable expression of TgERD2GFP does not appear to disturb betaCOP staining. In addition to TgERD2, Toxoplasma contains two more divergent ERD2 relatives. Phylogenetic analysis reveals that these proteins belong to a previously unrecognized ERD2 subfamily common to plants and alveolate organisms and as such could represent mediators of parasite-specific retrieval functions. No evidence of class 2 ERD2 proteins was found in metazoan organisms or fungi.
Eukaryotic Cell 03/2005; 4(2):432-42. · 3.60 Impact Factor
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Carolyn J Lawrence,
R Kelly Dawe,
Karen R Christie,
Don W Cleveland,
Scott C Dawson,
Sharyn A Endow,
Lawrence S B Goldstein, Holly V Goodson,
Nobutaka Hirokawa,
Jonathon Howard, [......],
Harukata Miki,
Timothy J Mitchison,
Yasushi Okada,
Anireddy S N Reddy,
William M Saxton,
Manfred Schliwa,
Jonathan M Scholey,
Ronald D Vale,
Claire E Walczak,
Linda Wordeman
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ABSTRACT: In recent years the kinesin superfamily has become so large that several different naming schemes have emerged, leading to confusion and miscommunication. Here, we set forth a standardized kinesin nomenclature based on 14 family designations. The scheme unifies all previous phylogenies and nomenclature proposals, while allowing individual sequence names to remain the same, and for expansion to occur as new sequences are discovered.
The Journal of Cell Biology 11/2004; 167(1):19-22. · 10.26 Impact Factor
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ABSTRACT: Actin is an ancient and abundant protein with well-established roles in fundamental processes ranging from cell migration to membrane transport. Most eukaryotic cells also contain at least eight actin-related proteins (ARPs) that are, themselves, conserved between organisms as divergent as yeast and mammals. Although many ARPs are cytoskeletal, recent biochemical and genetic work has demonstrated that some ARPs function largely or entirely in the nucleus. Evidence for the participation of both actin and ARPs in chromatin remodeling is becoming conclusive, and support for the still controversial involvement of actin in processes ranging from transcription to nuclear assembly is growing. The existence of conserved nuclear ARPs, together with accumulating biochemical, genetic and cell biology data, points to ancient and fundamental roles of actin in the nucleus, but the nature of these roles is just beginning to be revealed.
Trends in Cell Biology 09/2004; 14(8):435-42. · 12.35 Impact Factor
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ABSTRACT: Histone deacetylases (HDACs) modify core histones and participate in large regulatory complexes that both suppress and enhance transcription. Recent studies indicate that some HDACs can act on non-histone proteins as well. Interest in these enzymes is growing because HDAC inhibitors appear to be promising therapeutic agents against cancer and a variety of other diseases. Thus far, 11 members of the HDAC family have been identified in humans, but few have been characterized in detail. To better define the biological function of these proteins, make maximal use of studies performed in other systems, and assist in drug development efforts, we have performed a phylogenetic analysis of all HDAC-related proteins in all fully sequenced free-living organisms. Previous analyses have divided non-sirtuin HDACs into two groups, classes 1 and 2. We find that HDACs can be divided into three equally distinct groups: class 1, class 2, and a third class consisting of proteins related to the recently identified human HDAC11 gene. We term this novel group "class 4" to distinguish it from the unrelated "class 3" sirtuin deacetylases. Analysis of gene duplication events indicates that the common ancestor of metazoan organisms contained two class 1, two class 2, and a single class 4 HDAC. Examination of HDAC characteristics in light of these evolutionary relationships leads to functional predictions, among them that self-association is common among HDAC proteins. All three HDAC classes (including class 4) exist in eubacteria. Phylogenetic analysis of bacterial HDAC relatives suggests that all three HDAC classes precede the evolution of histone proteins and raises the possibility that the primary activity of some "histone deacetylase" enzymes is directed against non-histone substrates.
Journal of Molecular Biology 05/2004; 338(1):17-31. · 4.00 Impact Factor
