Université PARIS 6 et Université PARIS 7

MASTER 2 - SPÉCIALITÉ"BIOPHYSIQUE MOLÉCULAIRE ET

CELLULAIRE"Proposition de Stage - Année 2008 - 2009

http://www.master.phys.upmc.fr/S_biophysique/master.phys.biophys@upmc.fr

mSujet du stage (sous forme de titre court) :Role of forces exerted on dividing cells: how do they control the orientation of the division axis?LaboratoireNom du Responsable : Bruno Goud

Affiliation administrative (CNRS, INSERM...) et n° l'Unité : UMR 144 InstitutCurie/CNRS

Adresse précise du Laboratoire : Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05(adresse postale)

Laboratoires Constant Burg ; 12 rue Lhomond, 75005Paris (adresse physique)

Équipe d'accueil des DoctorantsNom de l'équipe: Biologie systémique de la polarité et de la division cellulaire

Nom du Responsable : Matthieu Piel

École Doctorale de rattachement : EDGGC, Paris-Sud et EDFDV, Paris-Diderot

Responsable du StageNom : Matthieu Piel

Numéro de téléphone : 01 56 24 63 83

Numéro de télécopie : 01 56 24 63 19

Adresse électronique : Matthieu.piel@curie.fr

Profil de l'étudiant(e) souhaité : Interface physique/biologie

Renseignements complémentairesPerspectives de poursuite de thèse:

X oui Non

Avec une bourse spécifique : oui X Non

si oui précisez : Présentation du dossier aux bourses fléchées et bourses de l’écoledoctorale FDV

Laboratoire d'accueil (Unité CNRS, INSERM, etc..) :

Nombre de chercheurs : 3 6 (dont 1 dans l'équipe d'accueil)

Nombre d'enseignants- chercheurs : 0 (dont 0 dans l'équipe d'accueil)

Nombre de "HDR" : ? (dont 1 dans l'équipe d'accueil)

Nombre d'ITA : 5 5 (dont 2 dans l'équipe d'accueil)

Nombre de "post-docs" : 4 3 (dont 2 dans l'équipe d'accueil)

Nombre de visiteurs étrangers : 2 (dont 0 dans l'équipe d'accueil)Sujet de stage (et principales techniques) :The general goal of our team is to understand the physical principles and the molecularmechanisms driving cell polarity in the context of cell division and cell migration.When eukaroytic cells divide, they assemble a mitotic spindle meant to separate chromosomes. Inmammalian cells, the orientation of this spindle will also determine the division axis of the cell, avery important parameter in developing tissues. We have been studying the extracellular signalsthat guide the orientation of the division axis using a control of cell adhesion geometry thanks tomicro-patterning techniques (refs 1 to 3), and we recently found that forces exerted on cellsduring mitosis are crucial. We would thus like to be able to precisely measure these forces and tobe able to understand how they affect mitotic spindle orientation. This could be achieved by atechnique developed in the field of biomechanics, called traction force microscopy, which hasbeen applied to measure forces cells exert on substrates during interphase, but it has never beenused for dividing cells. Moreover, when cells are platted on elastomeric substrates, it is possible toapply forces on them by stretching the substrate, a technique now set up in our lab. We alsofound that the geometry of cell adhesion guides mitotic spindle orientation by organizing thedistribution of fibers, called retraction fibers (RFs), that connect the mitotic cell body to the culturesubstrate. We would thus like to be able to dynamically modify the distribution of these RFs duringmitosis. An idea is to use photo-cleavable molecules to bind the adhesive molecules on whichRFs are attached. This way, we could simply use UV light from a regular microscope to cleave thebounds that attach the adhesive molecules to the substrate and thus detach the retraction fiberslocally. These experiments will be coupled with high resolution fluorescence microscopy to followthe dynamic reorganisation of the cortical cytoskeleton (actin filaments and myosin motors) uponforce application, or upon re-shaping of the retraction fibers geometry. We will also carefully studyhow this cortical reorganization affects astral microtubules interaction with the cortex, leading tospindle rotation. We wish to infer, from the sub-pixel tracking of spindle pole movements, coupledwith tracking of astral microtubule plus ends interaction with the cell cortex, the forces exerted onthe spindle from various regions of the cell cortex.Through this set of experiments, we wish to provide a physical understanding of how the mitoticspindle is oriented by extra-cellular mechanical cues.

Techniques/méthodes utiliséesOur lab has a good expertise on micro-patterning and other micro-fabrication techniques, as wellas on all kind of live cell microscopy using fluorescently tagged proteins (Institut Curie has a NikonImaging Center and an imaging platform that together provide access to most of up to date typesof microscopy) – which could be extended to other techniques like injection of quantum dotscoupled proteins. We have recently developed techniques like cell stretching and 2-photo laserablation, coupled to micro-patterning. We are in the process of implemented force measurementtechniques on mitotic cells, thanks to a new post-doc in the lab, Olivier Collin, specialist in bio-mechanics (see refs 4) and thanks to a collaboration with Benoit Ladoux, from Paris Diderot (seeref 5).Our team also has two microscopes for exclusive use: a live cell imaging automated microscopefrom Zeiss and an Olympus microscope with a laser ablation and a micro-manipulation set-up.We have access to the clean room in Yong Chen’s lab at ENS, and we are in the process ofassembling a ‘grey room’ for simple soft lithography, with Jean-Louis Viovy and Laurent Malaquinat Institut Curie.

The LabHalf of the lab works on mitotic spindle orientation while the other half works on cell motility, usingmicro-fabricated channels to control the mechanical and geometrical properties of the migrationenvironment. Two first sets of results using this technique to study cell migration, but also yeastcell morphogenesis, have just been accepted for publication in Science and in Current Biology

Références1. Thery M. et al., Nature Cell Biology, 2005 2. Thery M. et al., Nature, 2007 3.Fink J. et al., Lab on a Chip, 2007 4. Collin O. et al., Current Biology, 2008 5. duRoure O et al., PNAS 2005 6. Jiang X et al., PNAS, 2005 7. Nakanishi J.,JACS, 2005