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ABSTRACT: BACKGROUND: It is widely believed that integral outer membrane (OM) proteins in bacteria are able to diffuse laterally in the OM. However, stable, immobile proteins have been identified in the OM of Escherichia coli. In explaining the observations, a hypothesized interaction of the immobilized OM proteins with the underlying peptidoglycan (PG) cell wall played a prominent role. RESULTS: OmpA is an abundant outer membrane protein in E. coli containing a PG-binding domain. We use FRAP to investigate whether OmpA is able to diffuse laterally over long-range (> ~100 nm) distances in the OM. First, we show that OmpA, containing a PG binding domain, does not exhibit long-range lateral diffusion in the OM. Then, to test whether PG interaction was required for this immobilization, we genetically removed the PG binding domain and repeated the FRAP experiment. To our surprise, this did not increase the mobility of the protein in the OM. CONCLUSIONS: OmpA exhibits an absence of long-range (> ~100 nm) diffusion in the OM that is not caused by its PG binding domain. Therefore, other mechanisms are needed to explain this observation, such as the presence of physical barriers in the OM, or strong interactions with other elements in the cell envelope.
BMC Microbiology 03/2013; 13(1):66. · 3.04 Impact Factor
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ABSTRACT: Microtubules organize into a set of distinct patterns with the help of associated molecules that control nucleation, polymerization, crosslinking, and transport. These patterns, alone or in combination with each other, define the functional architecture of the microtubule cytoskeleton in living cells. In vitro experiments of increasing complexity help understand, in combination with theoretical models, the basic mechanisms by which elementary microtubule patterns arise, how they are maintained, and how they position themselves with respect to the confining geometry of living cells.
Current opinion in cell biology 12/2012; · 14.15 Impact Factor
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ABSTRACT: During important cellular processes such as centrosome and spindle positioning, dynein at the cortex interacts with dynamic microtubules in an apparent "end-on" fashion. It is well-established that dynein can generate forces by moving laterally along the microtubule lattice, but much less is known about dynein's interaction with dynamic microtubule ends. In this paper, we review recent in vitro experiments that show that dynein, attached to an artificial cortex, is able to capture microtubule ends, regulate microtubule dynamics and mediate the generation of pulling forces on shrinking microtubules. We further review existing ideas on the involvement of dynein-mediated cortical pulling forces in the positioning of microtubule organizing centers such as centrosomes. Recent in vitro experiments have demonstrated that cortical pulling forces in combination with pushing forces can lead to reliable centering of microtubule asters in quasi two-dimensional microfabricated chambers. In these experiments, pushing leads to slipping of microtubule ends along the chamber boundaries, resulting in an anisotropic distribution of cortical microtubule contacts that favors centering, once pulling force generators become engaged. This effect is predicted to be strongly geometry-dependent, and we therefore finally discuss ongoing efforts to repeat these experiments in three-dimensional, spherical and deformable geometries.
Cell cycle (Georgetown, Tex.) 10/2012; 11(20):3750-7. · 5.36 Impact Factor
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ABSTRACT: Dynein at the cortex contributes to microtubule-based positioning processes such as spindle positioning during embryonic cell division and centrosome positioning during fibroblast migration. To investigate how cortical dynein interacts with microtubule ends to generate force and how this functional association impacts positioning, we have reconstituted the 'cortical' interaction between dynein and dynamic microtubule ends in an in vitro system using microfabricated barriers. We show that barrier-attached dynein captures microtubule ends, inhibits growth, and triggers microtubule catastrophes, thereby controlling microtubule length. The subsequent interaction with shrinking microtubule ends generates pulling forces up to several pN. By combining experiments in microchambers with a theoretical description of aster mechanics, we show that dynein-mediated pulling forces lead to the reliable centering of microtubule asters in simple confining geometries. Our results demonstrate the intrinsic ability of cortical microtubule-dynein interactions to regulate microtubule dynamics and drive positioning processes in living cells.
Cell 02/2012; 148(3):502-14. · 32.40 Impact Factor
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ABSTRACT: The ability of growing microtubules to undergo catastrophes--abrupt switches from growth to shortening--is one of the key aspects of microtubule dynamics important for shaping cellular microtubule arrays. Gardner et al. show that catastrophes occur at a microtubule age-dependent rate and that depolymerizing kinesins can affect this process in fundamentally different ways.
Cell 11/2011; 147(5):966-8. · 32.40 Impact Factor
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ABSTRACT: Biopolymers are essential for cellular organization. They bridge the cell interior, forming a framework that is used as a reference for different cellular organelles. This framework, called the cytoskeleton, is not static but constantly reorganizes. The dynamics of the cytoskeleton allows the cell to rearrange its interior for various processes, such as cell division. This dynamic reorganization relies at least partly on forces that arise from the assembly and disassembly of cytoskeletal biopolymers. In many cases, these forces are generated when biopolymers interact with the cell boundary. This chapter focuses on force generation by and regulation of microtubules (MTs) that interact with growth-opposing barriers. We describe three in vitro assays that can be used to mimic MT interactions with the cell boundary. The essential components in each of our minimal systems are (functionalized) microfabricated barriers against which we grow MTs under different conditions. We describe in detail the different methods and assays necessary to realize these in vitro experiments.
Methods in molecular biology (Clifton, N.J.) 01/2011; 777:147-65.
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ABSTRACT: We present a detailed study on the formation of neighboring β-strands during the folding of a monomeric integral membrane protein of the β-barrel type. β-Strand and β-barrel formations were investigated for the eight-stranded transmembrane domain of outer membrane protein A (OmpA) with single-tryptophan (W), single-cysteine (C) OmpA mutants. Based on the OmpA structure, W and C were introduced in two neighboring β-strands oriented toward the hydrocarbon core of the membrane. Replaced residue pairs were closer to either the periplasmic turns (named cis-side) or the outer loops (named trans-side) of the strand. W(n)C(m) OmpA mutants containing W at position n and C at position m along the polypeptide chain were labeled at the C by a nitroxyl spin label, which is a short-range fluorescence quencher. To monitor the association of neighboring β-strands, we determined the proximity between fluorescent W and labeled C in OmpA folding experiments by intramolecular fluorescence quenching. Formation of native β-strand contacts in folding experiments required the lipid membrane. Residues in the trans-side of strands β(1), β(2), and β(3), represented by mutants W(15)C(35) (β(1)β(2), trans) and W(57)C(35) (β(3)β(2), trans), reached close proximity prior to residues in the N(β(1))- and C(β(8))-terminal strands as examined for mutants W(15)C(162) (β(1)β(8), trans) and W(7)C(170) (β(1)β(8), cis). Tryptophan and cysteine converged slightly faster in W(15)C(162) (β(1)β(8), trans) than in W(7)C(170) (β(1)β(8), cis). The last folding step was observed for residues at the cis-ends of strands β(1) and β(2) for the mutant W(7)C(43) (β(1)β(2), cis). The data also demonstrate that the neighboring β-strands associate upon insertion into the hydrophobic core of the lipid bilayer.
Journal of Molecular Biology 01/2011; 407(2):316-32. · 4.00 Impact Factor
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ABSTRACT: Collections of motors dynamically organize to extract membrane tubes. These tubes grow but often pause or change direction as they traverse an underlying microtubule (MT) network. In vitro, membrane tubes also stall: they stop growing in length despite a large group of motors available at the tip to pull them forward. In these stationary membrane tubes in vitro, we find that clusters of processive kinesin motors form and reach the tip of the tube at regular time intervals. The average times between cluster arrivals depends on the time over which motors depart from the tip, suggesting that motors are recycled toward the tip. Numerical simulations of the motor dynamics in the membrane tube and on the MTs show that the presence of cooperative binding between motors quantitatively accounts for the clustering observed experimentally. Cooperative binding along the length of the MT and a nucleation point at a distance behind the tip define the recycling period. Based on comparison of the numerical results and experimental data, we estimate a cooperative binding probability and concentration regime where the recycling phenomenon occurs.
Biophysical Journal 09/2010; 99(6):1835-41. · 3.65 Impact Factor
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ABSTRACT: Microtubules (MTs) are dynamic protein polymers that change their length by switching between growing and shrinking states in a process termed dynamic instability. It has been suggested that the dynamic properties of MTs are central to the organization of the eukaryotic intracellular space, and that they are involved in the control of cell morphology, but the actual mechanisms are not well understood. Here, we present a theoretical analysis in which we explore the possibility that a system of dynamic MTs and MT end-tracking molecular motors is providing specific positional information inside cells. We compute the MT length distribution for the case of MT-length-dependent switching between growing and shrinking states, and analyze the accumulation of molecular motors at the tips of growing MTs. Using these results, we show that a transport system consisting of dynamic MTs and associated motor proteins can deliver cargo proteins preferentially to specific positions within the cell. Comparing our results with experimental data in the model organism fission yeast, we propose that the suggested mechanisms could play important roles in setting length scales during cellular morphogenesis.
Biophysical Journal 08/2010; 99(3):726-35. · 3.65 Impact Factor
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ABSTRACT: Key cellular processes such as cell division, membrane compartmentalization, and intracellular transport rely on motor proteins. Motors have been studied in detail on the single motor level such that information on their step size, stall force, average run length, and processivity are well known. However, in vivo, motors often work together, so that the question of their collective coordination has raised great interest. Here, we specifically attach motors to giant vesicles and examine collective motor dynamics during membrane tube formation. Image correlation spectroscopy reveals directed motion as processive motors walk at typical speeds (< or = 500 nm/s) along an underlying microtubule and accumulate at the tip of the growing membrane tube. In contrast, nonprocessive motors exhibit purely diffusive behavior, decorating the entire length of a microtubule lattice with diffusion constants at least 1000 times smaller than a freely-diffusing lipid-motor complex in a lipid bilayer (1 microm(2)/s); fluorescence recovery after photobleaching experiments confirm the presence of the slower-moving motor population at the microtubule-membrane tube interface. We suggest that nonprocessive motors dynamically bind and unbind to maintain a continuous interaction with the microtubule. This dynamic and continuous interaction is likely necessary for nonprocessive motors to mediate bidirectional membrane tube dynamics reported previously.
Biophysical Journal 01/2010; 98(1):93-100. · 3.65 Impact Factor
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ABSTRACT: Biopolymers are essential for cellular organization. They bridge the cell interior, forming a framework that is used as a reference for different cellular organelles. Interestingly, this framework called the cytoskeleton is not static but constantly reorganizes. The dynamics of the cytoskeleton allows the cell to rearrange its interior for various processes such as cell division. This dynamic reorganization relies at least partly on forces that arise from assembly and disassembly of the cytoskeletal polymers. In many cases, these forces are generated when cytoskeletal polymers interact with the cell boundary. This chapter focuses on force generation by and regulation of microtubules (MTs) that interact with opposing barriers. In this chapter we describe four in vitro assays to study how MT interactions with the cell boundary play a role in cellular organization. In our minimal systems, (functionalized) microfabricated barriers mimic cell boundaries. We carefully design experiments where we grow MTs against these microfabricated structures to study a specific cellular process. Furthermore in this chapter different methods and assays necessary to realize these in vitro experiments are described. Section II describes the materials used, and Section III elaborates on the microfabrication. In Section III.C we explain how we specifically label our microfabricated structures, and in Section III.D we present how these functionalized microfabricated structures are incorporated into assays, with a discussion of the details of the assays themselves. Finally in Section IV we give examples of data obtained with these assays, and in Section V we discuss the assays in a general context.
Methods in cell biology 01/2010; 95:617-39. · 2.05 Impact Factor
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ChemBioChem 12/2009; 11(2):175-9. · 3.94 Impact Factor
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Yulia Komarova,
Christian O De Groot,
Ilya Grigoriev,
Susana Montenegro Gouveia,
E Laura Munteanu,
Joseph M Schober,
Srinivas Honnappa,
Rubén M Buey,
Casper C Hoogenraad, Marileen Dogterom,
Gary G Borisy,
Michel O Steinmetz,
Anna Akhmanova
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ABSTRACT: End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.
The Journal of Cell Biology 04/2009; 184(5):691-706. · 10.26 Impact Factor
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ABSTRACT: Microtubules (MTs) are central to the organisation of the eukaryotic intracellular space and are involved in the control of cell morphology. For these purposes, MT polymerisation dynamics are tightly regulated. Using automated image analysis software, we investigate the spatial dependence of MT dynamics in interphase fission yeast cells with unprecedented statistical accuracy. We find that MT catastrophe frequencies (switches from polymerisation to depolymerisation) strongly depend on intracellular position. We provide evidence that compressive forces generated by MTs growing against the cell pole locally reduce MT growth velocities and enhance catastrophe frequencies. Furthermore, we find evidence for an MT length-dependent increase in the catastrophe frequency that is mediated by kinesin-8 proteins (Klp5/6). Given the intrinsic susceptibility of MT dynamics to compressive forces and the widespread importance of kinesin-8 proteins, we propose that similar spatial regulation of MT dynamics plays a role in other cell types as well. In addition, our systematic and quantitative data should provide valuable input for (mathematical) models of MT organisation in living cells.
Molecular Systems Biology 02/2009; 5:250. · 8.63 Impact Factor
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ABSTRACT: Peptide libraries or antigenic determinants can be displayed on the surface of bacteria through insertion in a suitable outer membrane scaffold protein. Here, we inserted the well-known antibody epitopes 3xFLAG and 2xmyc in exterior loops of the transmembrane (TM) domain of OmpA. Although these highly charged epitopes were successfully displayed on the cell surface, their levels were 10-fold reduced due to degradation. We verified that the degradation was not caused by the absence of the C-terminal domain of OmpA. In contrast, a peptide that was only moderately charged (SA-1) appeared to be stably incorporated in the outer membrane at normal protein levels. Together, these results suggest that the display efficiency is sensitive to the charge of the inserted epitopes. In addition, the high-level expression of OmpA variants with surface-displayed epitopes adversely affected growth in a strain dependent, transient manner. In a MC4100 derived strain growth was affected, whereas in MC1061 derived strains growth was unaffected. Finally, results obtained using a gel-shift assay to monitor beta-barrel folding in vivo show that the insertion of small epitopes can change the heat modifiability of the OmpA TM domain from 'aberrant' to normal, and predict that some beta-barrels will not display any significant heat-modifiability at all.
PLoS ONE 02/2009; 4(8):e6739. · 4.09 Impact Factor
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ABSTRACT: Individual dynamic microtubules can generate pushing or pulling forces when their growing or shrinking ends are in contact with cellular objects such as the cortex or chromosomes. These microtubules can operate in parallel bundles, for example when interacting with mitotic chromosomes. Here, we investigate the force-generating capabilities of a bundle of growing microtubules and study the effect that force has on the cooperative dynamics of such a bundle. We used an optical tweezers setup to study microtubule bundles growing against a microfabricated rigid barrier in vitro. We show that multiple microtubules can generate a pushing force that increases linearly with the number of microtubules present. In addition, the bundle can cooperatively switch to a shrinking state, due to a force-induced coupling of the dynamic instability of single microtubules. In the presence of GMPCPP, bundle catastrophes no longer occur, and high bundle forces are reached more effectively. We reproduce the observed behavior with a simple simulation of microtubule bundle dynamics that takes into account previously measured force effects on single microtubules. Using this simulation, we also show that a constant compressive force on a growing bundle leads to oscillations in bundle length that are of potential relevance for chromosome oscillations observed in living cells.
Proceedings of the National Academy of Sciences 08/2008; 105(26):8920-5. · 9.68 Impact Factor
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ABSTRACT: In cells, membrane tubes are extracted by molecular motors. Although individual motors cannot provide enough force to pull a tube, clusters of such motors can. Here, we investigate, using a minimal in vitro model system, how the tube pulling process depends on fundamental properties of the motor species involved. Previously, it has been shown that processive motors can pull tubes by dynamic association at the tube tip. We demonstrate that, remarkably, nonprocessive motors can also cooperatively extract tubes. Moreover, the tubes pulled by nonprocessive motors exhibit rich dynamics as compared to those pulled by their processive counterparts. We report distinct phases of persistent growth, retraction, and an intermediate regime characterized by highly dynamic switching between the two. We interpret the different phases in the context of a single-species model. The model assumes only a simple motor clustering mechanism along the length of the entire tube and the presence of a length-dependent tube tension. The resulting dynamic distribution of motor clusters acts as both a velocity and distance regulator for the tube. We show the switching phase to be an attractor of the dynamics of this model, suggesting that the switching observed experimentally is a robust characteristic of nonprocessive motors. A similar system could regulate in vivo biological membrane networks.
Proceedings of the National Academy of Sciences 07/2008; 105(23):7993-7. · 9.68 Impact Factor
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ABSTRACT: We demonstrate the simultaneous trapping of multiple high-refractive index (n > 2) particles in a dynamic array of counterpropagating optical tweezers in which the destabilizing scattering forces are canceled. These particles cannot be trapped in single-beam optical tweezers. The combined use of two opposing high-numerical aperture objectives and micrometer-sized high-index titania particles yields an at least threefold increase in both axial and radial trap stiffness compared to silica particles under the same conditions. The stiffness in the radial direction is obtained from measured power spectra; calculations are given for both the radial and the axial force components, taking spherical aberrations into account. A pair of acousto-optic deflectors allows for fast, computer-controlled manipulation of the individual trapping positions in a plane, while the method used to create the patterns ensures the possibility of arbitrarily chosen configurations. The manipulation of high-index particles finds its application in, e.g., creating defects in colloidal photonic crystals and in exerting high forces with low laser power in, for example, biophysical experiments.
Applied Optics 06/2008; 47(17):3196-202. · 1.41 Impact Factor
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ABSTRACT: Microtubules (MTs) are cytoskeletal polymers whose spatial organization is dynamically regulated, depending on their biological function during different cell cycle stages. Growing MT ends are, for example, specifically targeted towards the cortex of motile or growing cells during interphase or towards chromosomal attachment sites during mitosis. An important parameter that cells use to control the average length of MTs, and thus the distance over which these targeting processes may operate, is the so-called catastrophe frequency f(cat): the rate at which MTs switch from a growing to a shrinking state. To understand how spatial targeting and the local control of f(cat) are related, quantitative in vivo measurements are needed that allow for the measurement of f(cat) in a spatially resolved way. Since catastrophes are intrinsically stochastic events, it is essential to acquire enough statistics to obtain the underlying rate constant f(cat). Here, we present automated image processing methodology, developed using GFP-tubulin expressing fission yeast cells, that makes it possible to measure f(cat) both spatially resolved and with high statistical accuracy. Although certain aspects of the analysis are specific to the system under investigation the basic concepts of the methodology are applicable to any kind of movies of fluorescently labeled MTs.
Methods in cell biology 02/2008; 89:521-38. · 2.05 Impact Factor
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ABSTRACT: The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.
Nature 01/2008; 450(7172):1100-5. · 36.28 Impact Factor
