Dissecting Microtubule Dynamics in Mitosis by Reconstituting Spindle Submodules – MITOTUBES

To study the mechanisms underlying the mitotic spindle dynamics at the molecular level, our strategy is based on reconstituting in vitro the minimal microtubule networks in the presence of key proteins in biochemically controled conditions and under precise TIRF (evanescent wave) microscopy. Controlling the geometry of microtubule assembly is achieved by a method of micro-engineering that enables the nucleation of microtubules from areas whose shapes, size and distribution with respect to each others are tightly controled.

Reconstitution of the central area of the mitotic spindle submodule:
We have developed conditions for observation of microtubules on micro-structured materials (micro-printing) which impose a geometry for microtubule assembly (analysis in constrained 2D system) within limits that are similar to those observed in cells. With this method, we have forced the assembly of microtubules from two opposing discs to mimic the two poles of the spindle (microtubules radially believe their end '+'). Using this method, we have assembled a subset of the mitotic spindle: the central center or «midzone«. Two «pseudo-asters« are positioned face to face in order to mimic the two poles of the mitotic spindle. The addition of the microtubule-associated protein MAP65-1/Ase1 in this system induces the formation of bundles of antiparallel microtubules and interdigitated between the two «asters«, generating a structure that corresponds to the minimal spindle midzone in anaphase. This module will be used to study the role of key proteins that act synergistically or in opposition to MAP65 to organize a dynamic midzone, such as the ‘+’ end tracking protein CLASP. In parallel, the regulation of the activity of MAP65 by Aurora kinase is underway.

The printing technique of MTs (micro-patterning) can reconstruct networks of MTs in defined conditions where nucleation, polarity and biochemistry of MTs are fully controlled. This system can be easily modulated to characterize the essential elements in building a network of MTs that mimic that of the cell environment. The research project we develop proposes to study the role of key proteins (purified and / or present in Xenopus egg extracts) in the construction / operation of the mitotic spindle using sub-modules of microtubule nucleation centers. This system will provide the missing link between the in vitro reconstitution assays using purified proteins in solution and the inherent complexity of the organization of MTs in cellulo. It should allow us to take a step further in understanding the mechanisms by which the cells segregate their genome. The success of this project depends entirely on the presence of staff mastering these new technologies.

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Investigation of the fine dynamics of microtubules (MTs) within the mitotic spindle has been precluded in living cells due to the density and complexity of MT networks. We therefore thought to reconstitute a minimal module of mitotic microtubule networks in a biochemically-controlled medium (biomimetic system). Indeed, the mitotic spindle can be conceptualized as integrated sub-arrays among which figure the radial MTs at the poles (astral MTs), the overlapping MTs at the midzone (MT midzone) and the MTs connected to the kinetochores (kinetochore fibres), or to the chromatin. To impose geometries of assembly for MTs that will mimic these different units in a simplified fashion, we are currently developing novel micro-engineering techniques, i.e. a micro-patterning method that allows to precisely control the position of MT nucleation sites at scales corresponding to cellular dimensions (Figure 1). Furthermore, we are performing state-of-the-art measurements of MT dynamics using dual view TIRF microscopy.
We will first try to reconstitute a minimal midzone structure using geometrically constrained anti-parallel MTs in the presence of one or two proteins that promote MT bundling (tasks 1 & 2). We will also determine how phosphorylation of these proteins by candidate mitotic kinases can affect their bundling activity. Meanwhile, and in order to get a clearer picture of spindle organization, we will develop a novel assay to explore the relative contribution of the overlapping nucleation centres that are known to act in parallel pathways in vivo. To this end, we will individualize the three main nucleation centres: centrosome, kinetochore and chromatin onto the patterned surface using Xenous egg extracts (in extracto, tasks 3 & 4). The extract will provide the complexity of the cytoplasm and reconstitute its natural viscosity. We will then analyze the relative contribution of several mitotic kinases in the regulation of MT dynamics from individual submodules using depletion/complementation studies. In addition, we will explore how the geometries of the patterns affect spindle organization and dynamics. Whenever required, the dynamic behaviour of MTs that are connected within bundles will be analyzed using a computational model developed by Team 2. For in extracto studies we will use both speckle microscopy and fluorescent labelling of the MT plus-end.
The geometrically constrained in vitro assay that is being developed by Team 2 will bring MT dynamics studies one-step forward. The next step developed by Team 1 will increase the intricacy of the system by using MT nucleation centres submodules in Xenopus egg extracts. Altogether, these studies that will impose geometries similar to the ones observed in living cells will bridge the gap between in vitro reconstitution assays using soluble proteins and the inextricable complexity of cellular MT arrays. Thus, we are proposing to set-up the optimal context to dissect the parallel pathways required to assemble a robust mitotic spindle.