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Force, Motion and Positioning of Microtubule Asters

Periodic Reporting for period 3 - FORCASTER (Force, Motion and Positioning of Microtubule Asters)

Reporting period: 2018-07-01 to 2019-12-31

This project focuses on the physics and biology of cell division, especially in the context of the development of early embryos. After fertilization, all animal’s eggs undergo a rapid set of reductive division which exhibit precise geometrical patterns, fundamental for the organization of embryos. These divisions are specified from the forces generated by microtubule asters , which are star-shaped structures made of microtubules, that move and position inside cells.
In this project, we aim to (i) study directly in vivo how aster moves and position themselves as a function of cell geometry and other cues inside cells; (ii) to directly measure the forces produced by asters in vivo and (iii) to establish quantitative methods such as modeling to be able to predict the position of microtubule asters in 3D and the consequent multi-cellular patterns of division in different embryos and tissues.

These studies will generate fundamental information on the processes of cell division and embryo development. As such this information may have value for diagnosis and drug development in many human diseases, which are related to development and cell division, such as cancer.

The detailed work packages of this project are:

1- In Work Package 1 (WP1), we will perform quantitative 3D imaging of microtubule aster trajectory and speeds during sperm aster centration, after fertilization, during which asters migrate rapidly to the egg center. We will monitor motion in eggs with altered geometries and sizes and map micro-scale force generation events. Combining 3D modelling with these quantitative data will serve to elucidate multi-scale principles underlying aster motion.
2- WP2 will focus on developing in vivo magnetic tweezers to quantitate forces exerted by asters in vivo. This development combined with cell manipulation, and used at various stages of development, will serve to address how forces depend on aster size and shape, or vary in symmetric vs. asymmetric positioning situations. These force measurement will be instrumental to assess aster friction in the cytoplasm and relate it to motion studies from WP1.
3- In WP3, we will couple 3D imaging of division cleavage geometry, cell manipulation and modeling to provide a systematic understanding of mechanical laws governing aster-pairs orientation which determine division axis, cleavage patterns and tissue morphogenesis.
Work Package 1.
In this first 36 months of the project, we have completed a significant of aim1 and aim3, by publishing a first paper in J. Cell Biology: Tanimoto et al. 2016. We have also recently published a supportive method paper “Tanimoto and Minc, Methods Cell Biology 2017”, and a review article in J. Cell Science “Haupt and Minc, 2018”. In those papers we study the centration of sperm asters in sea urchin embryos. These MT asters grow and move to the exact center of the cell. This motion is ubiquitous to all animal’s embryos, and essential for embryogenesis and karyogamy. We find that asters move in a self-organized manner, by continuously probing the geometry of eggs to faithfully target the cell center. We also propose a model for how the speed of MT asters may be determined from the growth rates of MTs in the aster. This part of the work was performed by Hirokazu Tanimoto a post-doc hired on the project, and in collaboration with Akatsuki Kimura (NIG, Japan).

We are now completing aim2, by performing detailed tracking of vesicle movement associated with the centration of sperm asters. We could establish precise maps of vesicle centration, for different endomembranes compartments (Endosomes, Lysososmes, ER etc..) . This analysis is being finished, and has been performed by Aurélien Guillou , an engineer hired on the project, and will be completed by Héliciane Palenzuela a PhD student hired on the project. A manuscript on those aspects should be assembled in the coming months. In addition, we have initiated in the context of the thesis of Héliciane Palenzuela a novel framework using in vitro approaches to test some of our hypothesis.


Work Package 2.
For this part of the project, we have pioneered the use of in vivo magnetic tweezers, in which magnetic beads are attached to the center of MT asters, and serve to apply calibrated forces to moving asters inside cells. A first key publication on this part, was recently published in Nature Physics “Tanimoto, Sallé, Dodin and Minc, 2018”. In this paper, we use in vivo tweezers to compute forces associated with aster motion and positioning. A second manuscript is now being assembled, on the use of those magnetic beads and tweezers to control the position and orientation of cell division in embryos. This work is mainly led by Jeremy Sallé a post-doc working on the project, with the help of Jing Xie, Dmitry Ershov and Serge Dmitrieff, who have also been hired on the project. A final direction in the context of Jing Xie PhD thesis (initially funded by the ERC grant, and now through an individual fellowship from the Chinese council), is to use tweezers to systematically map forces throughout embryogenesis as cells become smaller and smaller and exhibit rich patterns of oriented divisions. Those 2 aspects shall allow to complete aim 2.

Work Package 3.
The first aim of the WP, is also partially completed, through the recent publication of a modelling paper in which we can model and predict the overall division patterns of different embryos: “Pierre et al. Developmental Cell, 2016”. This work was achieved by Anaëlle Pierre, a PhD student with an independent fellowship, and with the help of Jeremy Sallé, and in collaboration with Martin Wühr at Princeton University. Using those models, we made important predictions on the simple mechanical rules controlling the position and orientation of subsequent divisions during embryonic development. A second manuscript from Anaëlle Pierre’s thesis is currently being finished and consists of analyzing by 3D imaging the precise patterns of developing embryos using imaging and cell manipulation. This manuscript should be published in the coming months. Finally, Dmitry Ershov a post-doc hired on the project, recently improved the modelling framework, to create a simple open-access platform for predicting cell divisions in 3D, this work is now in press, published as a book chapter in “Vertebrate Embryogenesis: E
In general, our studies at the forefront of cell biophysics are introducing a number of novel methods, and outlining important new concept in the mechanics and dynamics of cells and embryos. In particular, our work so far has allowed to bring important novel understanding on the mechanisms of cell divisions and MT aster organization, motion and forces. The modelling approaches we have introduced in WP3, is the very first attempt to model division position in 3 dimensions, and should have important value for diagnosis as well as fundamental research. In WP2 the development of in vivo magnetic tweezers is very novel, and may serve as a template for screening cytoskeletal drugs that may influence the forces exerted by MTs inside cells.
Centering Microtubule asters after fertilization. Microtubules are in green and DNA in Red