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• W2003997462 abstract "The regulation of the cytoskeleton is essential for the proper organization and function of eukaryotic cells. For instance, radial arrays of microtubules (MTs), called asters, determine the intracellular localization of organelles [1Kirschner M. Mitchison T. Beyond self-assembly: from microtubules to morphogenesis.Cell. 1986; 45: 329-342Abstract Full Text PDF PubMed Scopus (924) Google Scholar, 2Kirschner M. Gerhart J. Mitchison T. Molecular “vitalism”.Cell. 2000; 100: 79-88Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar]. Asters can be generated through either MT organizing center (MTOC)-dependent regulation or self-organization processes [1Kirschner M. Mitchison T. Beyond self-assembly: from microtubules to morphogenesis.Cell. 1986; 45: 329-342Abstract Full Text PDF PubMed Scopus (924) Google Scholar, 3Nedelec F.J. Surrey T. Maggs A.C. Leibler S. Self-organization of microtubules and motors.Nature. 1997; 389: 305-308Crossref PubMed Scopus (577) Google Scholar, 4Surrey T. Nedelec F. Leibler S. Karsenti E. Physical properties determining self-organization of motors and microtubules.Science. 2001; 292: 1167-1171Crossref PubMed Scopus (409) Google Scholar]. In vivo, this occurs within the cell boundaries. How the properties of these boundaries affect MT organization is unknown. To approach this question, we studied the organization of microtubules inside droplets of eukaryotic cellular extracts with varying sizes and elastic properties. Our results show that the size of the droplet determined the final steady-state MT organization, which changed from symmetric asters to asymmetric semi-asters and, finally, to cortical bundles. A simple physical model recapitulated these results, identifying the main physical parameters of the transitions. The use of vesicles with more elastic boundaries resulted in very different morphologies of microtubule structures, such as asymmetrical semi-asters, “Y-branching” organizations, cortical-like bundles, “rackets,” and bundled organizations. Our results highlight the importance of taking into account the physical characteristics of the cellular confinement to understand the formation of cytoskeleton structures in vivo. The regulation of the cytoskeleton is essential for the proper organization and function of eukaryotic cells. For instance, radial arrays of microtubules (MTs), called asters, determine the intracellular localization of organelles [1Kirschner M. Mitchison T. Beyond self-assembly: from microtubules to morphogenesis.Cell. 1986; 45: 329-342Abstract Full Text PDF PubMed Scopus (924) Google Scholar, 2Kirschner M. Gerhart J. Mitchison T. Molecular “vitalism”.Cell. 2000; 100: 79-88Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar]. Asters can be generated through either MT organizing center (MTOC)-dependent regulation or self-organization processes [1Kirschner M. Mitchison T. Beyond self-assembly: from microtubules to morphogenesis.Cell. 1986; 45: 329-342Abstract Full Text PDF PubMed Scopus (924) Google Scholar, 3Nedelec F.J. Surrey T. Maggs A.C. Leibler S. Self-organization of microtubules and motors.Nature. 1997; 389: 305-308Crossref PubMed Scopus (577) Google Scholar, 4Surrey T. Nedelec F. Leibler S. Karsenti E. Physical properties determining self-organization of motors and microtubules.Science. 2001; 292: 1167-1171Crossref PubMed Scopus (409) Google Scholar]. In vivo, this occurs within the cell boundaries. How the properties of these boundaries affect MT organization is unknown. To approach this question, we studied the organization of microtubules inside droplets of eukaryotic cellular extracts with varying sizes and elastic properties. Our results show that the size of the droplet determined the final steady-state MT organization, which changed from symmetric asters to asymmetric semi-asters and, finally, to cortical bundles. A simple physical model recapitulated these results, identifying the main physical parameters of the transitions. The use of vesicles with more elastic boundaries resulted in very different morphologies of microtubule structures, such as asymmetrical semi-asters, “Y-branching” organizations, cortical-like bundles, “rackets,” and bundled organizations. Our results highlight the importance of taking into account the physical characteristics of the cellular confinement to understand the formation of cytoskeleton structures in vivo. Asters can be produced through cross-linking and moving of microtubules (MTs) by oligomeric motors in vitro [3Nedelec F.J. Surrey T. Maggs A.C. Leibler S. Self-organization of microtubules and motors.Nature. 1997; 389: 305-308Crossref PubMed Scopus (577) Google Scholar, 4Surrey T. Nedelec F. Leibler S. Karsenti E. Physical properties determining self-organization of motors and microtubules.Science. 2001; 292: 1167-1171Crossref PubMed Scopus (409) Google Scholar]. Recent experiments suggest interplay between the cell shape and intracellular cytoskeleton organization [5Carazo-Salas R.E. Nurse P. Self-organization of interphase microtubule arrays in fission yeast.Nat. Cell Biol. 2006; 8: 1102-1107Crossref PubMed Scopus (57) Google Scholar, 6Daga R.R. Lee K.G. Bratman S. Salas-Pino S. Chang F. Self-organization of microtubule bundles in anucleate fission yeast cells.Nat. Cell Biol. 2006; 8: 1108-1113Crossref PubMed Scopus (52) Google Scholar, 7Haase S.B. Lew D.J. Microtubule organization: cell shape is destiny.Curr. Biol. 2007; 17: R249-R251Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar, 8Terenna C.R. Makushok T. Velve-Casquillas G. Baigl D. Chen Y. Bornens M. Paoletti A. Piel M. Tran P.T. Physical mechanisms redirecting cell polarity and cell shape in fission yeast.Curr. Biol. 2008; 18: 1748-1753Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. Yet, the techniques available for investigation of the effects of boundaries are still limited. In vivo studies rely mainly upon recent microfabrication advances that allow the design of microenvironments of well-defined geometrical and surface patterning [8Terenna C.R. Makushok T. Velve-Casquillas G. Baigl D. Chen Y. Bornens M. Paoletti A. Piel M. Tran P.T. Physical mechanisms redirecting cell polarity and cell shape in fission yeast.Curr. Biol. 2008; 18: 1748-1753Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 9Romet-Lemonne G. VanDuijn M. Dogterom M. Three-dimensional control of protein patterning in microfabricated devices.Nano Lett. 2005; 5: 2350-2354Crossref PubMed Scopus (19) Google Scholar, 10Whitesides G.M. Ostuni E. Takayama S. Jiang X. Ingber D.E. Soft lithography in biology and biochemistry.Annu. Rev. Biomed. Eng. 2001; 3: 335-373Crossref PubMed Scopus (2107) Google Scholar, 11Thery M. Racine V. Pepin A. Piel M. Chen Y. Sibarita J.B. Bornens M. The extracellular matrix guides the orientation of the cell division axis.Nat. Cell Biol. 2005; 7: 947-953Crossref PubMed Scopus (609) Google Scholar]. In vitro experiments performed in microfabricated chambers showed the importance of forces produced by the polymerization of MTs on their three-dimensional organization or on self-centering of MTOCs [12Holy T.E. Dogterom M. Yurke B. Leibler S. Assembly and positioning of microtubule asters in microfabricated chambers.Proc. Natl. Acad. Sci. USA. 1997; 94: 6228-6231Crossref PubMed Scopus (155) Google Scholar, 13Dogterom M. Kerssemakers J.W. Romet-Lemonne G. Janson M.E. Force generation by dynamic microtubules.Curr. Opin. Cell Biol. 2005; 17: 67-74Crossref PubMed Scopus (180) Google Scholar, 14Cosentino Lagomarsino M. Tanase C. Vos J.W. Emons A.M. Mulder B.M. Dogterom M. Microtubule organization in three-dimensional confined geometries: evaluating the role of elasticity through a combined in vitro and modeling approach.Biophys. J. 2007; 92: 1046-1057Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar]. Recently, the compartmentalization of biomolecules inside droplets has been successfully used for biochemical assays [15Griffiths A.D. Tawfik D.S. Miniaturising the laboratory in emulsion droplets.Trends Biotechnol. 2006; 24: 395-402Abstract Full Text Full Text PDF PubMed Google Scholar, 16Noireaux V. Libchaber A. A vesicle bioreactor as a step toward an artificial cell assembly.Proc. Natl. Acad. Sci. USA. 2004; 101: 17669-17674Crossref PubMed Scopus (796) Google Scholar] and for studying confined biopolymers [17Elbaum M. Kuchnir Fygenson D. Libchaber A. Buckling microtubules in vesicles.Phys. Rev. Lett. 1996; 76: 4078-4081Crossref PubMed Scopus (150) Google Scholar, 18Claessens M.M. Bathe M. Frey E. Bausch A.R. Actin-binding proteins sensitively mediate F-actin bundle stiffness.Nat. Mater. 2006; 5: 748-753Crossref PubMed Scopus (178) Google Scholar, 19Limozin L. Sackmann E. Polymorphism of cross-linked actin networks in giant vesicles.Phys. Rev. Lett. 2002; 89: 168103Crossref PubMed Scopus (105) Google Scholar]. Here, we extend the compartmentalization techniques to quantitatively examine the effects of size and elasticity of cell boundaries on the self-organization of MTs and motors. Femto-liters of eukaryotic cytoplasmic extracts were enclosed inside emulsion droplets (a droplet of extracts surrounded by oil) or inside vesicles. Although the boundary in both droplets and vesicles lacks the functionalities normally found in the cortex of cell membranes, this bottom-up strategy isolates some general physical principles and allows their quantitative study [20Karsenti E. Self-organization in cell biology: a brief history.Nat. Rev. Mol. Cell Biol. 2008; 9: 255-262Crossref PubMed Scopus (304) Google Scholar, 21Carazo-Salas R. Nurse P. Sorting out interphase microtubules.Mol Syst Biol. 2007; 3: 95Crossref PubMed Scopus (4) Google Scholar]. To investigate the effect of a confined environment on MT self-organization, we developed a method to generate droplets of Xenopus laevis egg extracts dispersed in oil. These metaphase II-arrested oocyte extracts contain all the components necessary for the formation of self-organized MT asters in metaphase [22Desai A. Murray A. Mitchison T.J. Walczak C.E. The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro.Methods Cell Biol. 1999; 61: 385-412Crossref PubMed Scopus (228) Google Scholar, 23Verde F. Berrez J.M. Antony C. Karsenti E. Taxol-induced microtubule asters in mitotic extracts of Xenopus eggs: requirement for phosphorylated factors and cytoplasmic dynein.J. Cell Biol. 1991; 112: 1177-1187Crossref PubMed Scopus (235) Google Scholar]. Fluorescently labeled tubulin was mixed with the egg extracts, allowing for observation of MT formation and organization via fluorescent microscopy. First, we analyzed the ability of MTs to form asters in nonconfined Xenopus cell extracts that were complemented with taxol for the enhancement MT nucleation and stability. We observed that in this system, growing MTs were spontaneously organized into radial arrays, probably by minus-end-directed motors such as dyneins, which are known to be present in extracts [23Verde F. Berrez J.M. Antony C. Karsenti E. Taxol-induced microtubule asters in mitotic extracts of Xenopus eggs: requirement for phosphorylated factors and cytoplasmic dynein.J. Cell Biol. 1991; 112: 1177-1187Crossref PubMed Scopus (235) Google Scholar] (Figures 1A and 1B). Extract-in-oil droplets with a diameter of 0.5 μm to 70 μm were formed within few seconds by the stirring of a mixture of cell extracts (3% v/v) and mineral oil. The extract was supplemented with fluorescently-labeled tubulin, which was initially homogeneous (Figure 1D). After 5 min of incubation, approximately 75% of the droplets contained visible micrometer-sized MT bundles (Figure 1E). In nonconfined extracts, MTs assemble in asters reaching a steady-state diameter of 28 ± 4 μm within 15 min (Figure S1A, available online). To test whether MTs behaved similarly in the confined extracts, we first analyzed droplets larger than 29 μm (average aster diameter) to avoid any perturbation due to the confinement. Our results show that in those droplets, MTs organized mostly in radial arrays similar to those observed in nonconfined extracts (Figures 2A and 2D). The distribution of fluorescence intensity of radial asters in droplets was similar to that seen in bulk measurements (Figure S1B). In addition, time-point measurements suggested that MTs organized in fully developed asters within 15 min for both unconfined and confined extracts (Figure S2B). This suggests that the components of the extracts behaved similarly in droplets and in bulk. Given the wide range of obtained droplet sizes, our system is ideal for examining the effect of variance in the diameter of a spherical confinement on MT self-organization. Indeed, because the surface tension between the oil and the extract is high, the confinement is always rigid and spherical (Figures 1C and 1D). We next analyzed MT organization with regard to the size of the droplets. Remarkably, we noticed three characteristic MT motifs, each associated with a specific range of droplet diameters. In droplets larger than 29 μm, MTs organized in asters that were centered within the droplet (Figure 2A and Figure S4A). For droplets between 11 and 29 μm, MTs organized mostly in asymmetrically focused structures (semi-asters) with their pole near the surface of the droplet (Figure 2B and Figures S4B and S4C). Time-series acquisitions illustrate MT organization and pole movement within a droplet (Figures S2B–S2E) and transition to asymmetric structures (Movies S1 and S2). Once MTs organized in asymmetric structures, their poles were dynamic, showing that they were not physically linked to the oil-extract interface (Movie S3). Finally, for droplet diameters ranging from 4 to 11 μm, microtubules were bent cortically (Figure 2C and Figure S4E). These qualitative observations were confirmed by quantifying, for each diameter category, the percentage of droplets containing each of these three MT structures (Figure 2D). For each diameter category, more than 93% of the droplets contained MTs organized in the characteristic morphology. Coexistence of asters and semi-asters was observed within a narrow range of droplet size (26–30 μm; Figure S3A). This coexistence might be the consequence of the intrinsic variability in aster assembly, as indicated, for instance, in the distribution of aster diameters (Figure S1A). To determine whether the MT arrays are polarized within droplets, we used fluorescent stabilized MT seeds, which allowed us to distinguish the minus end of MT bundles from the plus end, given that assembly is faster at the growing MT plus end. For semi-aster structures, seeds colocalized mainly at the poles, indicating their minus-end nature (Figure 2E). In addition, analysis of semi-aster pole orientation showed that the direction of the polarization is random in space (Figure 2F). To investigate whether a motor activity is required in the MT organizing process, we added to the extract-in-oil droplets either p50/dynamitin, which is a subunit of dynactin known to perturb the dynein-dynactin motor activity [24Wittmann T. Hyman T. Recombinant p50/dynamitin as a tool to examine the role of dynactin in intracellular processes.Methods Cell Biol. 1999; 61: 137-143Crossref PubMed Scopus (56) Google Scholar], or ortho-vanadate, which is an inhibitor of dynein ATPase activity. In these experimental conditions, MTs became organized mostly in randomly oriented bundles or cortically, rather than in asters or semi-asters (Figures S5A–S5H), showing the essential role of dynein-dynactin in assembling MTs into asters in our system. Taken altogether, our results suggest that MT organization by minus-end motors can self-polarize, even in the absence of external cues or structural anisotropy of the environment, for a specific range of confinement size. What are the mechanisms determining MT organization and their transition as a function of the confinement size? We used physical and geometrical arguments to describe how the radius at which MT-based structures switch from one arrangement to another can be linked to the mechanical properties of MTs and of the boundary. Our observations of small droplets showed that MTs do not deform the droplet boundary, in agreement with the high value of the extract-oil surface tension. When growing MTs encounter the rigid droplet boundary, they experience compressive forces exerted by the extract-oil interfacial tension [25Cohen A.E. Mahadevan L. Kinks, rings, and rackets in filamentous structures.Proc. Natl. Acad. Sci. USA. 2003; 100: 12141-12146Crossref PubMed Scopus (150) Google Scholar]. When the MT length is larger than the droplet perimeter (in the micrometer range), the fibers buckle and circle along the spherical confinement to minimize their bending elastic energy (Figure 2C, Supplemental Discussion, and Figure S4E). The transition from an isotropic aster to a semi-aster seems to be the point at which the diameter of the droplet is comparable to the diameter of the aster. Indeed, the transition size of 29 μm matches the size of asters formed in the absence of confinement (Figure 2D and Figure S1A). At this point, the geometric center of a growing aster becomes unstable. In general, predicting the configuration of a confined aster is quite complex. However, in Figure 2G we present a simple model explaining how a radial aster, having a diameter slightly larger than the confinement, becomes asymmetric. This model shows that the aster pole moves away from the geometrical center of the confinement in order to allow most of the MT filaments to adopt a configuration that is the least compressed and bent (Figure 2G). The behavior of a self-organized aster described above is distinct from an aster generated by a MTOC confined in rigid boxes [12Holy T.E. Dogterom M. Yurke B. Leibler S. Assembly and positioning of microtubule asters in microfabricated chambers.Proc. Natl. Acad. Sci. USA. 1997; 94: 6228-6231Crossref PubMed Scopus (155) Google Scholar]. First, whereas in the experiments performed with MTOCs the MTs were confined in square boxes, our experiments were performed with MTs inside spherical droplets without corners. Second, MTs in self-organized asters are constrained in position but still have the capability to pivot around their minus-ends, whereas a MTOC additionally constrains MTs in a particular direction. This can explain why the self-organized semi-asters in our experiments were positioned at the vicinity of the physical boundary, whereas the most favorable position for the geometrical center of an MTOC aster is between the center of the chamber and its edge. Thus, the center of a sphere is an unstable position for a self-organized aster (see Figure 2G). In addition, directed movement of minus-end motors that can transport MTs amplifies any deviation from the geometrical center. Physically, the formation of semi-asters corresponds to a spontaneous symmetry-breaking event, which is expected to occur in a random direction (Figure 2E). This is analogous to the spontaneous cell polarization of the acto-myosin system in budding yeast, in which a built-in positive feedback is thought to amplify small stochastic variations in the concentration of polarity proteins [26Wedlich-Soldner R. Li R. Spontaneous cell polarization: undermining determinism.Nat. Cell Biol. 2003; 5: 267-270Crossref PubMed Scopus (97) Google Scholar, 27Wedlich-Soldner R. Altschuler S. Wu L. Li R. Spontaneous cell polarization through actomyosin-based delivery of the Cdc42 GTPase.Science. 2003; 299: 1231-1235Crossref PubMed Scopus (315) Google Scholar]. To determine whether a simple physical model could explain the observed transitions, we performed numerical simulations taking into account some of the known properties of MTs and motor proteins. MTs were modeled as semi-flexible polar polymers that all share the same fixed length. They were confined in two dimensions in a disc with frictionless boundaries and could be cross-linked by processive minus-end motors. Several parameters, including the bending elasticity of MTs, the concentration and properties of motors, and the size of the droplet, were considered in the simulation (see Table S1). We systematically examined the relationship between the confinement size, MT length, and organization and found that the computed configurations reproduced the patterns observed experimentally (Figures 3A–3C). For each case, we performed several runs of the simulation, all of which differed as a result of the stochastic nature of the model [28Nedelec F. Foethke D. Collective Langevin dynamics of flexible cytoskeletal fibers.New J. Phys. 2007; 9: 24Crossref Scopus (122) Google Scholar]. The transition between a radial array and a semi-aster occurred when the droplet diameter (29 μm) matched the aster length (29 μm). Figure 4D shows that the majority of the structures were radial at 30 μm and asymmetric at 27 μm when MT lengths were fixed to 29 μm, confirming the sharpness of the transition observed experimentally. The variability in the final organization of MTs under confinement arises mostly from the random initial configurations and the stochastic nature of motor activity (Figure 3D and Figures S3 and S3C).Figure 4Self-Organization of Microtubules and Motors Depends on Membrane Bending StiffnessShow full caption(A) Schematic representation of the compartmentalization process of the Xenopus metaphase cell extract inside vesicles.(B–D) Results of microtubule and motor self-organization inside vesicles formed with 5% or 50% cholesterol, corresponding to a membrane stiffness of roughly 1 × 10−19 J or 6 × 10−19 J, respectively.(E) Percentage of morphologies as a function of the membrane stiffness.(F) Fluorescence observations and schematic representations of microtubule-based structures inside vesicles as a function of the bending stiffness. Scale bars represent 10 μm. Below the schematic representation, the percentage of vesicles containing each structure type is given for vesicles containing 5% cholesterol (top) or 50% cholesterol (bottom).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Schematic representation of the compartmentalization process of the Xenopus metaphase cell extract inside vesicles. (B–D) Results of microtubule and motor self-organization inside vesicles formed with 5% or 50% cholesterol, corresponding to a membrane stiffness of roughly 1 × 10−19 J or 6 × 10−19 J, respectively. (E) Percentage of morphologies as a function of the membrane stiffness. (F) Fluorescence observations and schematic representations of microtubule-based structures inside vesicles as a function of the bending stiffness. Scale bars represent 10 μm. Below the schematic representation, the percentage of vesicles containing each structure type is given for vesicles containing 5% cholesterol (top) or 50% cholesterol (bottom). The transition between semi-asters and cortical arrangements occurred when individual MTs were long enough to buckle and circle along the contour of the compartment; this was observed for a confinement diameter of 10 to 13 μm (Figure 3E). Numerical simulations were also used to investigate other parameters inaccessible in our experimental assay. We tested the condition in which MT rotation around the aster pole was constrained, which could mimic a rigid MTOC aster, and showed that in this case radial asters did not undergo the transition toward an asymmetric structure (see Supplemental Discussion and Figure S7D). This suggests that the ability of MTs to pivot around their minus end is a key parameter of the symmetry-breaking process. We also tested the geometry of the confinement. Interestingly, simulations of self-organized asters confined in a square box showed that MTs and motors do not organize into an asymmetric structure, contrary to what is observed with a spherical confinement (Supplemental Discussion and Figure S7F). Thus, the confinement geometry plays a major role in self-organization of MTs and motors. Numerical simulations thus succeed in reproducing both the final steady-state organization of MTs and the radius of transition observed experimentally. Taken altogether, our results confirm that the main parameters controlling transitions in MT and motor self-organization are the MT length and the confinement diameter. Whereas unicellular eukaryotes such as yeasts have rigid cell walls, many higher eukaryotic cells have a deformable cortex. To mimic this, we encapsulated the extracts in a lipid bilayer in a two-step process that allowed a precise control of the cytoplasm-encapsulation procedure (Figure 4A). In addition, with this method, we tuned the membrane bending stiffness from k∼ 1–6 × 10−19 J by changing the cholesterol content from 5% to 50% [29Meleard P. Gerbeaud C. Pott T. Fernandez-Puente L. Bivas I. Mitov M.D. Dufourcq J. Bothorel P. Bending elasticities of model membranes: influences of temperature and sterol content.Biophys. J. 1997; 72: 2616-2629Abstract Full Text PDF PubMed Scopus (253) Google Scholar], thus increasing cohesion between adjacent lipids. As a result of this stiffness, MTs may deform the cell membrane, something that is impossible with extract-in-oil droplets (Supplemental Discussion). Five minutes after the vesicles were generated, a limited number of morphologies of quasi-spherical shape was observed. Some of these structures also exhibited micrometer-sized membrane tubes containing MTs (Figures 4B–4E). The overall membrane shapes were classified as a function of the internal microtubule architectures in asymmetrical semi-asters, “Y-branching” organizations, cortical-like bundles, “rackets,” and linear bundles (Figure 4F). Interestingly, these morphologies depend not only on the confinement size but also on the bending stiffness of the membrane. For a bending stiffness of k∼ 6 × 10−19 J (50% cholesterol), 73% of vesicles were quasi-spherical, having either cortical-like bundles (diameter 4–10 μm; Figure 4C) or semi-asters (12–16 μm; Figure 4D). In contrast, for a bending stiffness of k∼ 1 × 10−19 J (5% cholesterol), 60% of the vesicles underwent important morphological changes associated with the formation of a single (Figure 4B) or multiple (Figure S8) MT-containing protrusions. These morphological transformations were most likely determined by the membrane-rigidity- and force-generating elements, such as MTs [17Elbaum M. Kuchnir Fygenson D. Libchaber A. Buckling microtubules in vesicles.Phys. Rev. Lett. 1996; 76: 4078-4081Crossref PubMed Scopus (150) Google Scholar], which drive filament sliding and overall structural rearrangement. For example, the morphological transition from a spherical vesicle to a vesicle with a single protrusion occurred when a critical force required for membrane buckling was reached; we estimate this as being between 25 and 45 pN (Supplemental Discussion). Previous experiments explained different morphologies of vesicles deformed by microtubule growth by considering the elastic properties of the membrane [30Fygenson D.K. Marko J.F. Libchaber A. Mechanics of Microtubule-Based Membrane Extension.Phys. Rev. Lett. 1997; 79: 4497Crossref Scopus (187) Google Scholar, 31Emsellem V. Cardoso O. Tabeling P. Vesicle deformation by microtubules: A phase diagram.Physical Review E. 1998; 58: 4807-4810Crossref Scopus (35) Google Scholar]. In our experiments, the presence of motors cross-linking several microtubule bundles led to the formation of various intracellular networks (Figures 4D and 4F). Thus, the ability of intracellular organization to deform the membrane increased the number of possible morphological states, compared to those obtained within rigid droplets (Figure 4F). We found that symmetry breaking in MT organization arose for a specific range of confinement size, depending on the rigidity of the boundary. In particular, MT asters can self-polarize within a range of size in the absence of external cues. Softening the rigidity of the boundary led to different morphological states. The morphological transitions observed depend solely on MT length, confinement size, and membrane rigidity. Using a simple model, we showed that the physical characteristics of MTs and of boundaries are sufficient to explain the mechanism of the symmetry breaking observed. In particular, forces generated by MTs and minus-end motors drive filament sliding and overall structural rearrangement." @default.
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• W2003997462 title "Effects of Confinement on the Self-Organization of Microtubules and Motors" @default.
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