Spindle pole body-anchored Kar3 drives the nucleus along microtubules from another nucleus in preparation for nuclear fusion during yeast karyogamy.
Gibeaux, R., Politi, A.Z., Nedelec, F., Antony, C. & Knop, M.
Genes Dev. 2013 Feb 1;27(3):335-49. doi: 10.1101/gad.206318.112.
Nuclear migration during yeast karyogamy, termed nuclear congression, is required to initiate nuclear fusion. Congression involves a specific regulation of the microtubule minus end-directed kinesin-14 motor Kar3 and a rearrangement of the cytoplasmic microtubule attachment sites at the spindle pole bodies (SPBs). However, how these elements interact to produce the forces necessary for nuclear migration is less clear. We used electron tomography, molecular genetics, quantitative imaging, and first principles modeling to investigate how cytoplasmic microtubules are organized during nuclear congression. We found that Kar3, with the help of its light chain, Cik1, is anchored during mating to the SPB component Spc72 that also serves as a nucleator and anchor for microtubules via their minus ends. Moreover, we show that no direct microtubule-microtubule interactions are required for nuclear migration. Instead, SPB-anchored Kar3 exerts the necessary pulling forces laterally on microtubules emanating from the SPB of the mating partner nucleus. Therefore, a twofold symmetrical application of the core principle that drives nuclear migration in higher cells is used in yeast to drive nuclei toward each other before nuclear fusion.
Patterns of molecular motors that guide and sort filaments.
Rupp, B. & Nedelec, F.
Lab Chip. 2012 Nov 21;12(22):4903-10. doi: 10.1039/c2lc40250e.
Molecular motors can be immobilized to transport filaments and loads that are attached to these filaments inside a nano-device. However, if motors are distributed uniformly over a flat surface, the motility is undirected, and the filaments move equally in all directions. For many applications it is important to control the direction in which the filaments move, and two strategies have been explored to achieve this: applying external forces and confining the filaments inside channels. In this article, we discuss a third strategy in which the topography of the sample remains flat, but the motors are distributed non-uniformly over the surface. Systems of filaments and patterned molecular motors were simulated using a stochastic engine that included Brownian motion and filament bending elasticity. Using an evolutionary algorithm, patterns were optimized for their capacity to precisely control the paths of the filaments. We identified patterns of motors that could either direct the filaments in a particular direction, or separate short and long filaments. These functionalities already exceed what has been achieved with confinement. The patterns are composed of one or two types of motors positioned in lines or along arcs and should be easy to manufacture. Finally, these patterns can be easily combined into larger designs, allowing one to precisely control the motion of microscopic objects inside a device.
Katanin contributes to interspecies spindle length scaling in Xenopus.
Loughlin, R., Wilbur, J.D., McNally, F.J., Nedelec, F.J. & Heald, R.
Cell. 2011 Dec 9;147(6):1397-407.
Bipolar spindles must separate chromosomes by the appropriate distance during cell division, but mechanisms determining spindle length are poorly understood. Based on a 2D model of meiotic spindle assembly, we predicted that higher localized microtubule (MT) depolymerization rates could generate the shorter spindles observed in egg extracts of X. tropicalis compared to X. laevis. We found that katanin-dependent MT severing was increased in X. tropicalis, which, unlike X. laevis, lacks an inhibitory phosphorylation site in the katanin p60 catalytic subunit. Katanin inhibition lengthened spindles in both species. In X. tropicalis, k-fiber MT bundles that connect to chromosomes at their kinetochores extended through spindle poles, disrupting them. In both X. tropicalis extracts and the spindle simulation, a balance between k-fiber number and MT depolymerization is required to maintain spindle morphology. Thus, mechanisms have evolved in different species to scale spindle size and coordinate regulation of multiple MT populations in order to generate a robust steady-state structure.
Augmin promotes meiotic spindle formation and bipolarity in Xenopus egg extracts.
Petry, S., Pugieux, C., Nedelec, F.J. & Vale, R.D.
Proc Natl Acad Sci U S A. 2011 Aug 30;108(35):14473-8. Epub 2011 Aug 15.
Female meiotic spindles in many organisms form in the absence of centrosomes, the organelle typically associated with microtubule (MT) nucleation. Previous studies have proposed that these meiotic spindles arise from RanGTP-mediated MT nucleation in the vicinity of chromatin; however, whether this process is sufficient for spindle formation is unknown. Here, we investigated whether a recently proposed spindle-based MT nucleation pathway that involves augmin, an 8-subunit protein complex, also contributes to spindle morphogenesis. We used an assay system in which hundreds of meiotic spindles can be observed forming around chromatin-coated beads after introduction of Xenopus egg extracts. Spindles forming in augmin-depleted extracts showed reduced rates of MT formation and were predominantly multipolar, revealing a function of augmin in stabilizing the bipolar shape of the acentrosomal meiotic spindle. Our studies also have uncovered an apparent augmin-independent MT nucleation process from acentrosomal poles, which becomes increasingly active over time and appears to partially rescue the spindle defects that arise from augmin depletion. Our studies reveal that spatially and temporally distinct MT generation pathways from chromatin, spindle MTs, and acentrosomal poles all contribute to robust bipolar spindle formation in meiotic extracts.
A computational model predicts Xenopus meiotic spindle organization.
Loughlin, R., Heald, R. & Nedelec, F.
J Cell Biol. 2010 Dec 27;191(7):1239-49. Epub 2010 Dec 20.
The metaphase spindle is a dynamic bipolar structure crucial for proper chromosome segregation, but how microtubules (MTs) are organized within the bipolar architecture remains controversial. To explore MT organization along the pole-to-pole axis, we simulated meiotic spindle assembly in two dimensions using dynamic MTs, a MT cross-linking force, and a kinesin-5-like motor. The bipolar structures that form consist of antiparallel fluxing MTs, but spindle pole formation requires the addition of a NuMA-like minus-end cross-linker and directed transport of MT depolymerization activity toward minus ends. Dynamic instability and minus-end depolymerization generate realistic MT lifetimes and a truncated exponential MT length distribution. Keeping the number of MTs in the simulation constant, we explored the influence of two different MT nucleation pathways on spindle organization. When nucleation occurs throughout the spindle, the simulation quantitatively reproduces features of meiotic spindles assembled in Xenopus egg extracts.
Condensins promote chromosome recoiling during early anaphase to complete sister chromatid separation.
Renshaw, M.J., Ward, J.J., Kanemaki, M., Natsume, K., Nedelec, F.J. & Tanaka, T.U.
Dev Cell. 2010 Aug 17;19(2):232-44.
Sister chromatid separation is initiated at anaphase onset by the activation of separase, which removes cohesins from chromosomes. However, it remains elusive how sister chromatid separation is completed along the entire chromosome length. Here we found that, during early anaphase in Saccharomyces cerevisiae, sister chromatids separate gradually from centromeres to telomeres, accompanied by regional chromosome stretching and subsequent recoiling. The stretching results from residual cohesion between sister chromatids, which prevents their immediate separation. This residual cohesion is at least partly dependent on cohesins that have escaped removal by separase at anaphase onset. Meanwhile, recoiling of a stretched chromosome region requires condensins and generates forces to remove residual cohesion. We provide evidence that condensins promote chromosome recoiling directly in vivo, which is distinct from their known function in resolving sister chromatids. Our work identifies residual sister chromatid cohesion during early anaphase and reveals condensins' roles in chromosome recoiling, which eliminates residual cohesion to complete sister chromatid separation.
Computational cell biology at the home of the helix.
Ward, J.J. & Nedelec, F.J.
EMBO Rep. 2010 Jun;11(6):413-5. Epub 2010 May 21.
The Computational Cell Biology Conference, held jointly by the Cold Spring Harbor Laboratory and the Wellcome Trust, was convened in the grand surroundings of Hinxton Hall near Cambridge, UK. The high quality of the research presented at the meeting confirmed that the field of computational cell biology is maturing rapidly, which mirrors the progression of cell biology from being mostly descriptive to a more quantitative discipline.
La chromatine faconne le fuseau mitotique. [This review in French summarises the results of Chromatin shapes the mitotic spindle.]
Pugieux C, Nedelec, F.
Medecine/Sciences 26 (2) 139-142; Feb 2010.
A theory of microtubule catastrophes and their regulation.
Brun, L., Rupp, B., Ward, J.J. & Nedelec, F.
Proc Natl Acad Sci U S A. 2009 Dec 15;106(50):21173-8. Epub 2009 Nov 30.
Dynamic instability, in which abrupt transitions occur between growing and shrinking states, is an intrinsic property of microtubules that is regulated by both mechanics and specialized proteins. We discuss a model of dynamic instability based on the popular idea that growth is maintained by a cap at the tip of the fiber. The loss of this cap is thought to trigger the transition from growth to shrinkage, called a catastrophe. The model includes longitudinal interactions between the terminal tubulins of each protofilament and "gating rescues" between neighboring protofilaments. These interactions allow individual protofilaments to transiently shorten during a phase of overall microtubule growth. The model reproduces the reported dependency of the catastrophe rate on tubulin concentration, the time between tubulin dilution and catastrophe, and the induction of microtubule catastrophes by walking depolymerases. The model also reproduces the comet tail distribution that is characteristic of proteins that bind to the tips of growing microtubules.
Chromatin shapes the mitotic spindle.
Dinarina, A., Pugieux, C., Corral, M.M., Loose, M., Spatz, J., Karsenti, E. & Nedelec, F.
Cell. 2009 Aug 7;138(3):502-13.
In animal and plant cells, mitotic chromatin locally generates microtubules that self-organize into a mitotic spindle, and its dimensions and bipolar symmetry are essential for accurate chromosome segregation. By immobilizing microscopic chromatin-coated beads on slide surfaces using a microprinting technique, we have examined the effect of chromatin on the dimensions and symmetry of spindles in Xenopus laevis cytoplasmic extracts. While circular spots with diameters around 14-18 microm trigger bipolar spindle formation, larger spots generate an incorrect number of poles. We also examined lines of chromatin with various dimensions. Their length determined the number of poles that formed, with a 6 x 18 microm rectangular patch generating normal spindle morphology. Around longer lines, multiple poles formed and the structures were disorganized. While lines thinner than 10 mum generated symmetric structures, thicker lines induced the formation of asymmetric structures where all microtubules are on the same side of the line. Our results show that chromatin defines spindle shape and orientation. For a video summary of this article, see the PaperFlick file available with the online Supplemental Data.
Phospho-regulated interaction between kinesin-6 Klp9p and microtubule bundler Ase1p promotes spindle elongation.
Fu, C., Ward, J.J., Loiodice, I., Velve-Casquillas, G., Nedelec, F.J. & Tran, P.T.
Dev Cell. 2009 Aug;17(2):257-67.
The spindle midzone-composed of antiparallel microtubules, microtubule-associated proteins (MAPs), and motors-is the structure responsible for microtubule organization and sliding during anaphase B. In general, MAPs and motors stabilize the midzone and motors produce sliding. We show that fission yeast kinesin-6 motor klp9p binds to the microtubule antiparallel bundler ase1p at the midzone at anaphase B onset. This interaction depends upon the phosphorylation states of klp9p and ase1p. The cyclin-dependent kinase cdc2p phosphorylates and its antagonist phosphatase clp1p dephosphorylates klp9p and ase1p to control the position and timing of klp9p-ase1p interaction. Failure of klp9p-ase1p binding leads to decreased spindle elongation velocity. The ase1p-mediated recruitment of klp9p to the midzone accelerates pole separation, as suggested by computer simulation. Our findings indicate that a phosphorylation switch controls the spatial-temporal interactions of motors and MAPs for proper anaphase B, and suggest a mechanism whereby a specific motor-MAP conformation enables efficient microtubule sliding.
Effects of confinement on the self-organization of microtubules and motors.
Pinot, M., Chesnel, F., Kubiak, J.Z., Arnal, I., Nedelec, F.J. & Gueroui, Z.
Curr Biol. 2009 Jun 9;19(11):954-60. Epub 2009 May 7.
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. Asters can be generated through either MT organizing center (MTOC)-dependent regulation or self-organization processes. 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.
Force- and length-dependent catastrophe activities explain interphase microtubule organization in fission yeast.
Foethke, D., Makushok, T., Brunner, D. & Nedelec, F.
Mol Syst Biol. 2009;5:241. Epub 2009 Mar 17.
The cytoskeleton is essential for the maintenance of cell morphology in eukaryotes. In fission yeast, for example, polarized growth sites are organized by actin, whereas microtubules (MTs) acting upstream control where growth occurs. Growth is limited to the cell poles when MTs undergo catastrophes there and not elsewhere on the cortex. Here, we report that the modulation of MT dynamics by forces as observed in vitro can quantitatively explain the localization of MT catastrophes in Schizosaccharomyces pombe. However, we found that it is necessary to add length-dependent catastrophe rates to make the model fully consistent with other previously measured traits of MTs. We explain the measured statistical distribution of MT-cortex contact times and re-examine the curling behavior of MTs in unbranched straight tea1Delta cells. Importantly, the model demonstrates that MTs together with associated proteins such as depolymerizing kinesins are, in principle, sufficient to mark the cell poles.
Regulation of microtubule dynamics by reaction cascades around chromosomes.
Athale, C.A., Dinarina, A., Mora-Coral, M., Pugieux, C., Nedelec, F. & Karsenti, E.
Science. 2008 Nov 21;322(5905):1243-1247. Epub 2008 Oct 23.
During spindle assembly, chromosomes generate gradients of microtubule stabilization through a reaction-diffusion process, but how this is achieved is not well understood. We measured the spatial distribution of microtubule aster asymmetry around chromosomes by incubating centrosomes and micropatterned chromatin patches in frog egg extracts. We then screened for microtubule stabilization gradient shapes that would generate such spatial distributions with the use of computer simulations. Only a long-range, sharply decaying microtubule stabilization gradient could generate aster asymmetries fitting the experimental data. We propose a reaction-diffusion model that combines the chromosome generated Ran-guanosine triphosphate-Importin reaction network to a secondary phosphorylation network as a potential mechanism for the generation of such gradients.
Mechanism of phototaxis in marine zooplankton.
Jekely, G., Colombelli, J., Hausen, H., Guy, K., Stelzer, E.H.K., Nedelec, F. & Arendt, D.
Nature. 2008 Nov 20;456(7220):395-9.
The simplest animal eyes are eyespots composed of two cells only: a photoreceptor and a shading pigment cell. They resemble Darwin's 'proto-eyes', considered to be the first eyes to appear in animal evolution. Eyespots cannot form images but enable the animal to sense the direction of light. They are characteristic for the zooplankton larvae of marine invertebrates and are thought to mediate larval swimming towards the light. Phototaxis of invertebrate larvae contributes to the vertical migration of marine plankton, which is thought to represent the biggest biomass transport on Earth. Yet, despite its ecological and evolutionary importance, the mechanism by which eyespots regulate phototaxis is poorly understood. Here we show how simple eyespots in marine zooplankton mediate phototactic swimming, using the marine annelid Platynereis dumerilii as a model. We find that the selective illumination of one eyespot changes the beating of adjacent cilia by direct cholinergic innervation resulting in locally reduced water flow. Computer simulations of larval swimming show that these local effects are sufficient to direct the helical swimming trajectories towards the light. The computer model also shows that axial rotation of the larval body is essential for phototaxis and that helical swimming increases the precision of navigation. These results provide, to our knowledge, the first mechanistic understanding of phototaxis in a marine zooplankton larva and show how simple eyespots regulate it. We propose that the underlying direct coupling of light sensing and ciliary locomotor control was a principal feature of the proto-eye and an important landmark in the evolution of animal eyes.
Spatial regulation improves antiparallel microtubule overlap during mitotic spindle assembly.
Channels, W.E., Nedelec, F., Zheng, Y. & Iglesias, P.A.
Biophys J. 2008 Apr 1;94(7):2598-609. Epub 2007 Dec 20.
The mitotic spindle plays an essential role in chromosome segregation during cell division. Spindle formation and proper function require that microtubules with opposite polarity overlap and interact. Previous computational simulations have demonstrated that these antiparallel interactions could be created by complexes combining plus- and minus-end-directed motors. The resulting spindles, however, exhibit sparse antiparallel microtubule overlap with motor complexes linking only a nominal number of antiparallel microtubules. Here we investigate the role that spatial differences in the regulation of microtubule interactions can have on spindle morphology. We show that the spatial regulation of microtubule catastrophe parameters can lead to significantly better spindle morphology and spindles with greater antiparallel MT overlap. We also demonstrate that antiparallel microtubule overlap can be increased by having new microtubules nucleated along the length of existing astral microtubules, but this increase negatively affects spindle morphology. Finally, we show that limiting the diffusion of motor complexes within the spindle region increases antiparallel microtubule interaction.
Cortical microtubule contacts position the spindle in C. elegans embryos.
Kozlowski, C., Srayko, M. & Nedelec, F.
Cell. 2007 May 4;129(3):499-510.
Interactions between microtubules and the cell cortex play a critical role in positioning organelles in a variety of biological contexts. Here we used Caenorhabditis elegans as a model system to study how cortex-microtubule interactions position the mitotic spindle in response to polarity cues. Imaging EBP-2::GFP and YFP::alpha-tubulin revealed that microtubules shrink soon after cortical contact, from which we propose that cortical adaptors mediate microtubule depolymerization energy into pulling forces. We also observe association of dynamic microtubules to form astral fibers that persist, despite the catastrophe events of individual microtubules. Computer simulations show that these effects, which are crucially determined by microtubule dynamics, can explain anaphase spindle oscillations and posterior displacement in 3D.
Crosslinkers and motors organize dynamic microtubules to form stable bipolar arrays in fission yeast.
Janson, M.E., Loughlin, R., Loiodice, I., Fu, C., Brunner, D., Nedelec, F. & Tran, P.T.
Cell. 2007 Jan 26;128(2):357-68.
Microtubule (MT) nucleation not only occurs from centrosomes, but also in large part from dispersed nucleation sites. The subsequent sorting of short MTs into networks like the mitotic spindle requires molecular motors that laterally slide overlapping MTs and bundling proteins that statically connect MTs. How bundling proteins interfere with MT sliding is unclear. In bipolar MT bundles in fission yeast, we found that the bundler ase1p localized all along the length of antiparallel MTs, whereas the motor klp2p (kinesin-14) accumulated only at MT plus ends. Consequently, sliding forces could only overcome resistant bundling forces for short, newly nucleated MTs, which were transported to their correct position within bundles. Ase1p thus regulated sliding forces based on polarity and overlap length, and computer simulations showed these mechanisms to be sufficient to generate stable bipolar bundles. By combining motor and bundling proteins, cells can thus dynamically organize stable regions of overlap between cytoskeletal filaments.
Modelling microtubule patterns.
Karsenti, E., Nedelec, F. & Surrey, T.
Nat Cell Biol. 2006 Nov;8(11):1204-11.
The cellular cytoskeleton is well studied in terms of its biological and physical properties, making it an attractive subject for systems approaches. Here, we describe the experimental and theoretical strategies used to study the collective behaviour of microtubules and motors. We illustrate how this led to the beginning of an understanding of dynamic cellular patterns that have precise functions.
Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins.
Goshima, G., Nédélec, F. & Vale, R.D.
J Cell Biol 2005 Oct 24;171(2):229-40.
During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end-directed motor proteins. Here, we have characterized the roles of two minus end-directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.
The mitotic spindle and actin tails.
Karsenti, E. & Nedelec, F.
Biol Cell 2004 Apr;96(3):237-40.
To segregate their chromosomes, eukaryotic cells rely on a dynamic structure made of microtubules: the mitotic spindle. This structure can form in cells lacking centrosomes, because their chromosomes also nucleate microtubules. This second assembly pathway is observed even in some cells that naturally have centrosomes, for example when the centrosomes are ablated by laser surgery. Recent results have started to address the complementary question of whether centrosome-nucleated microtubules alone could sustain the formation of a functional mitotic spindle. We wonder in this respect whether lower eukaryotes such as yeasts are different from higher eukaryotes such as vertebrates.
Self-organisation and forces in the microtubule cytoskeleton.
Nedelec, F., Surrey, T. & Karsenti, E.
Curr Opin Cell Biol. 2003 Feb;15(1):118-24.
Modern microscopy techniques allow us to observe specifically tagged proteins in live cells. We can now see directly that many cellular structures, for example mitotic spindles, are in fact dynamic assemblies. Their apparent stability results from out-of-equilibrium stochastic interactions at the molecular level. Recent studies have shown that the spindles can form even after centrosomes are destroyed, and that they can even form around DNA-coated beads devoid of kinetochores. Moreover, conditions have been produced in which microtubule asters interact even in the absence of chromatin. Together, these observations suggest that the spindle can be experimentally deconstructed, and that its defining characteristics can be studied in a simplified context, in the absence of the full division machinery.
Computer simulations reveal motor properties generating stable antiparallel microtubule interactions.
J Cell Biol. 2002 Sep 16;158(6):1005-15. Epub 2002 Sep 16.
An aster of microtubules is a set of flexible polar filaments with dynamic plus ends that irradiate from a common location at which the minus ends of the filaments are found. Processive soluble oligomeric motor complexes can bind simultaneously to two microtubules, and thus exert forces between two asters. Using computer simulations, I have explored systematically the possible steady-state regimes reached by two asters under the action of various kinds of oligomeric motors. As expected, motor complexes can induce the asters to fuse, for example when the complexes consist only of minus end-directed motors, or to fully separate, when the motors are plus end directed. More surprisingly, complexes made of two motors of opposite directionalities can also lead to antiparallel interactions between overlapping microtubules that are stable and sustained, like those seen in mitotic spindle structures. This suggests that such heterocomplexes could have a significant biological role, if they exist in the cell.
Physical properties determining self-organization of motors and microtubules.
Surrey, T., Nédélec, F., Leibler, S. & Karsenti, E.
Science 2001 May 11;292(5519):1167-71.
In eukaryotic cells, microtubules and their associated motor proteins can be organized into various large-scale patterns. Using a simplified experimental system combined with computer simulations, we examined how the concentrations and kinetic parameters of the motors contribute to their collective behavior. We observed self-organization of generic steady-state structures such as asters, vortices, and a network of interconnected poles. We identified parameter combinations that determine the generation of each of these structures. In general, this approach may become useful for correlating the morphogenetic phenomena taking place in a biological system with the biophysical characteristics of its constituents.
Dynamic concentration of motors in microtubule arrays.
Nedelec, F., Surrey, T. & Maggs, A.C.
Phys Rev Lett 2001 Apr 2;86(14):3192-5.
We present experimental and theoretical studies of the dynamics of molecular motors in microtubule arrays and asters. By solving a convection-diffusion equation we find that the density profile of motors in a two-dimensional aster is characterized by continuously varying exponents. Simulations are used to verify the assumptions of the continuum model. We observe the concentration profiles of kinesin moving in quasi-two-dimensional artificial asters by fluorescent microscopy and compare with our theoretical results.
Dynamics of microtubule aster formation by motor complexes.
Nédélec, F. & Surrey, T.
C. R. Acad. Sci 2001; 2 (IV); 841-847.
De l'action collective des moteurs à l'ordre cellulaire.
Nédélec, F. & Surrey, T.
Médecine/Science 2000; 16:739 - 744.
Chromophore-assisted light inactivation and self-organization of microtubules and motors.
Surrey, T., Elowitz, M.B., Wolf, P.E., Yang, F., Nédélec, F., Shokat, K. & Leibler, S.
Proc Natl Acad Sci U S A 1998 Apr 14;95(8):4293-8.
Chromophore-assisted light inactivation (CALI) offers the only method capable of modulating specific protein activities in localized regions and at particular times. Here, we generalize CALI so that it can be applied to a wider range of tasks. Specifically, we show that CALI can work with a genetically inserted epitope tag; we investigate the effectiveness of alternative dyes, especially fluorescein, comparing them with the standard CALI dye, malachite green; and we study the relative efficiencies of pulsed and continuous-wave illumination. We then use fluorescein-labeled hemagglutinin antibody fragments, together with relatively low-power continuous-wave illumination to examine the effectiveness of CALI targeted to kinesin. We show that CALI can destroy kinesin activity in at least two ways: it can either result in the apparent loss of motor activity, or it can cause irreversible attachment of the kinesin enzyme to its microtubule substrate. Finally, we apply this implementation of CALI to an in vitro system of motor proteins and microtubules that is capable of self-organized aster formation. In this system, CALI can effectively perturb local structure formation by blocking or reducing the degree of aster formation in chosen regions of the sample, without influencing structure formation elsewhere.
Self-organization of microtubules and motors.
Nedelec, Fr., Surrey, T., Maggs, A.C. & Leibler, S.
Nature 1997 Sep 18;389(6648):305-8.
Cellular structures are established and maintained through a dynamic interplay between assembly and regulatory processes. Self-organization of molecular components provides a variety of possible spatial structures: the regulatory machinery chooses the most appropriate to express a given cellular function. Here we study the extent and the characteristics of self-organization using microtubules and molecular motors as a model system. These components are known to participate in the formation of many cellular structures, such as the dynamic asters found in mitotic and meiotic spindles. Purified motors and microtubules have previously been observed to form asters in vitro. We have reproduced this result with a simple system consisting solely of multi-headed constructs of the motor protein kinesin and stabilized microtubules. We show that dynamic asters can also be obtained from a homogeneous solution of tubulin and motors. By varying the relative concentrations of the components, we obtain a variety of self-organized structures. Further, by studying this process in a constrained geometry of micro-fabricated glass chambers, we demonstrate that the same final structure can be reached through different assembly 'pathways.