François Nédélec

Self-organization of microtubules and motors

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’.

Mechanisms for focusing mitotic spindle poles by minus end–directed motor proteins

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.

Crosslinkers and Motors Organize Dynamic Microtubules to Form Stable Bipolar Arrays in Fission Yeast

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.

Computer simulations reveal motor properties generating stable antiparallel microtubule interactions

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.

Mechanism of phototaxis in marine zooplankton

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.

Cortical Microtubule Contacts Position the Spindle in C. elegans Embryos

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.

Katanin Contributes to Interspecies Spindle Length Scaling in Xenopus

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.

Self-organisation and forces in the microtubule cytoskeleton

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.

Modelling microtubule patterns

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.

A computational model predicts Xenopus meiotic spindle organization

Spatially dispersed nucleation and minus end–directed transport of microtubule end disassembly activity can lead to bipolar spindle assembly.