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FORCASTER Report Summary

Project ID: 647073
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - FORCASTER (Force, Motion and Positioning of Microtubule Asters)

Reporting period: 2015-07-01 to 2016-12-31

Summary of the context and overall objectives of the project

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 performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Work Package 1.
In this first 18 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”. 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 are developing 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. In our initial phase, we have almost completed aim1 of the WP2, through the combined efforts of Hirokazu Tanimoto and Jeremy Sallé, 2 post-docs hired on the project. Indeed we van now reproducibly inject beads and attach them to the aster center, and have begun to use this assay to compute the forces associated with aster motion, and aster centration (part of Aim2). Those data are currently being assembled, to publish one first important manuscript on these approaches.

To perform measurement throughout development, we are currently hiring a PhD student that will exploit those established methods, to rigorously map forces throughout development.

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 Wuhr 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.

In more recent unpublished work, we are analyzing by 3D imaging the precise patterns of developing embryos using imaging and cell manipulation, this part is performed by Jeremy Sallé. We also continue the development of our models, through the work of Dmitry Ershov a post-doc hired on the project. This is done in collaboration with several groups (C.P. Heisenberg at IST Austria, and Fiona Francis at IFM in Paris).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

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.
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