TEAM

Physical and Molecular Principles Governing Cytoskeletal Organization

Group leader : A. Michelot

Our team has two main objectives.
Our first objective is to understand how eukaryotic cells control the assembly of an organized cytoskeleton (mainly actin microfilaments). Our second objective is to understand how actin networks are recycled and renewed in response to cellular needs.
Our approaches are multidisciplinary, using tools from physics and chemistry to answer fundamental biological questions.

FOR NON-SPECIALISTS

The proper functioning of a cell within its environment is linked to its ability to exert forces and resist mechanical constraints. A main actor is its cytoskeleton, which is composed of a set of biological polymers organized in networks. By their polymerization, or by the action of molecular motors, these filamentous networks ensure that cellular processes such as migration, adhesion, division or endocytosis take place correctly.

The properties of each of these networks are constantly remodeled by the action of regulatory proteins, which influence the assembly, disassembly or spatial organization of these polymers. Any change in the activity of these regulatory proteins has a significant impact on the organization of the cytoskeleton, and consequently on the behavior of the cell. For example, the transition of a tumor to malignancy is associated with numerous genetic and epigenetic modifications, often resulting in defects in cytoskeleton assembly. This alters the motile and biomechanical properties of cancer cells, allowing them to spread during metastasis.

The objective of our team is to identify the general principles by which cells organize their cytoskeleton, and how it is reorganized under various perturbations. This implies a deep knowledge of the molecular partners involved, as well as an ability to understand how all these molecules work together in a complex cellular environment.

 

FOR SPECIALISTS

Our work is mainly focused on actin, which polymerizes as a double helix to form semi-rigid filaments. The geometrical organization of these filaments allows to build networks with various mechanical properties.

Actin cytoskeleton organization

Our research on the organization of the actin cytoskeleton is centered around two main questions.

The first question is how cells control the level of assembly of multiple actin filament structures from a common and limiting pool of monomeric actin. The cell must ensure an optimal distribution of this resource in space and time according to its needs.

Our research is organized around two existing mechanisms. First, by activating specific signaling pathways, the cell controls the activation or inhibition of factors involved in the generation of new actin filaments within specific structures. Then, by modulating the activity of additional factors specifically involved in the assembly of particular structures, the cell has a second powerful mechanism to precisely control actin fluxes.

The second question is to understand how cells precisely address, spatially and temporally, multiple families of actin regulatory proteins to particular networks. Surprisingly, we have shown that the recruitment of these proteins is not due to a specific signaling network, or to a local cellular context such as pH or salt concentration. On the contrary, the segregation mechanism of ABPs is due to a finely controlled biochemical regulation.

We explore various mechanisms to explain how actin filaments acquire a particular identity. The first mechanism is that the binding of actin regulatory proteins is generally directly sensitive to the geometrical organization of actin filaments. The second mechanism is related to the fact that a particular identity can be assigned to actin filaments, for example by the addition of particular modifications. Finally, the last mechanism is related to the fact that the simultaneous binding of multiple regulatory proteins can be promoted or limited by collaborative or competitive effects.

Mecanismes-contribuant-segregation-cellulaire-proteines-liaison-actine

Mechanisms contributing to the cellular segregation of actin-binding proteins

Some recent publications related to this project:

  • ANTKOWIAK A, GUILLOTIN A, BOEIRO SANDERS M, COLOMBO J, VINCENTELLI R, MICHELOT A. Sizes of actin networks sharing a common environment are determined by the relative rates of assembly. PLoS Biol. 2019 Jun 10 ;17(6) :e3000317
  • BOIERO SANDERS M, ANTKOWIAK A, MICHELOT A. Diversity from Similarity: Cellular Strategies for Assigning Particular Identities to Actin Filaments and Networks. Open Biol. 2020 Sep;10(9):200157
  • BOIERO-SANDERS M, TORET CP, ANTKOWIAK A, GUILLOTIN A, ROBINSON RC, MICHELOT A.  Specialization of actin isoforms derived from the loss of key interactions with regulatory factors. Available in Bioarxiv: https://doi.org/10.1101/2021.02.09.430555
Actin dynamics

Most actin networks in the cell are renewed on time scales of the order of minutes. In other words, actin alternates between its monomeric and polymeric states, and this rapid dynamic is being maintained by the energy released from the hydrolysis of actin-bound ATPs. Multiple actin regulators are involved in this cycle, the underlying molecular mechanisms of which are poorly understood. This lack of knowledge leads to our inability to reconstruct dynamic actin networks in small cell volumes from purified proteins. Overcoming this limitation will be essential for future reconstitutions of more complex actin-based processes.

Our understanding of actin dynamics has made little progress in the absence of efficient markers to measure the recycling rate of actin monomers. Because actin monomer recycling correlates with the exchange of one ADP molecule for one ATP, we sought to identify nucleotide analogues linked to bright fluorescent probes in order to accurately measure actin-bound nucleotide exchange dynamics. We determined that a family of molecules, N6-(6-Amino)hexyl-ATPs, have chemical properties compatible with actin binding. We have shown that these molecules have exchange dynamics comparable to that of ATP and maintain functional interactions with a number of important actin-binding proteins. We also determined that several colors of fluorophores can be used, that actin polymerization is possible with these fluorescent nucleotides, and that they are hydrolyzed by actin to provide energy for these reactions.

(A)Modélisation structurale d’ATP-ATTO-488 lié à un monomère d’actine (B)Filaments d’actine marqués par ATP-ATTO-488 et observés en microscopie à onde évanescente.

(A) Structural modeling of ATP-ATTO-488 bound to an actin monomer
(B) Actin filaments labeled with ATP-ATTO-488 and observed by evanescent wave microscopy

Some recent publications related to this project:

  • COLOMBO J*, ANTKOWIAK A*, KOGAN K, KOTILA T, ELLIOTT J, GUILLOTIN A, LAPPALAINEN P, MICHELOT A. A Functional Family of Fluorescent Nucleotide Analogues to Investigate Actin Dynamics and Energetics. Nat Commun. 2021 Jan 22;12(1):548 (* equal contribution)
  • GRESSIN L, GUILLOTIN A, GUERIN C, BLANCHOIN L, MICHELOT A*. Architecture Dependence of Actin Filament Network Disassembly. Curr Biol. 2015 Jun 1;25(11):1437-47
Our tools in the lab

To address detailed mechanistic questions, traditional biochemical, cell biological and genetic experiments need to be complemented by engineering-inspired reconstitution approaches. The use of simplified systems and the ability to modify individual components without the active contribution and complexity of the cell has proven beneficial in understanding emerging properties of a variety of biological systems.

Outil-laboratoire

We usually use two complementary ways to reconstitute our biological processes of interest. With “bottom-up” reconstitutions, we purify the essential components, and assemble them in a test tube. Components are added one-by-one into the system, and it allows for testing the role of a molecule in the presence of a limited number of partners. However, for cellular processes involving the participation of large numbers of families of molecules, “bottom-up” approaches can be limited, because they require the identification of all essential components and their purification in an active form. In that situation, we use protein extracts, because they contain a mixture of all the proteins that are essential for the process. We are experts in genetic depletions in protein extracts, in order to experiment ”top-down” approaches, where components are removed one-by-one from the extracts, in order to test for their functions in a near-physiological environment. Both “bottom-up” and “top-down” approaches are used in various experimental setups to bridge the gap between simple biochemistry and the complexity of a cell.

Photo d’équipe pré-covid De gauche à droite: Christopher Toret, Adrien Antkowiak, Agathe de Neufville, Jessica Colombo, Audrey Guillotin, Micaela Boiero-Sanders, Thomas Le Goff, Alphée Michelot and Reda Belbahri

Pre-covid lab picture.
From left to right: Christopher Toret, Adrien Antkowiak, Agathe de Neufville, Jessica Colombo, Audrey Guillotin, Micaela Boiero-Sanders, Thomas Le Goff, Alphée Michelot and Reda Belbahri


Selected publications

PUBLICATION

A Functional Family of Fluorescent Nucleotide Analogues to Investigate Actin Dynamics and Energetics

Jessica Colombo, Adrien Antkowiak, Konstantin Kogan, Tommi Kotila, Jenna Elliott, Audrey Guillotin, Pekka Lappalainen, Alphée Michelot
Nat Commun . 2021 Jan 22;12(1):548. doi: 10.1038/s41467-020-20827-4. PMID: 33483497

PUBLICATION

Diversity from Similarity: Cellular Strategies for Assigning Particular Identities to Actin Filaments and Networks

Micaela Boiero Sanders, Adrien Antkowiak, Alphée Michelot
Open Biol . 2020 Sep;10(9):200157. doi: 10.1098/rsob.200157. Epub 2020 Sep 2. PMID: 32873155

PUBLICATION

Mechanical stiffness of reconstituted actin patches correlates tightly with endocytosis efficiency

Planade J, Belbahri R, Boiero Sanders M, Guillotin A, du Roure O, Michelot A, Heuvingh J.
PLoS Biol. 2019 Oct 25;17(10):e3000500. doi: 10.1371/journal.pbio.3000500. eCollection 2019 Oct. PMID: 31652255

PUBLICATION

Sizes of actin networks sharing a common environment are determined by the relative rates of assembly

Adrien Antkowiak, Audrey Guillotin , Micaela Boiero Sanders, Jessica Colombo, Renaud Vincentelli, Alphée Michelot
PLoS Biol . 2019 Jun 10;17(6):e3000317. doi: 10.1371/journal.pbio.3000317. eCollection 2019 Jun. PMID: 31181075

PUBLICATION

Tropomyosin Isoforms Specify Functionally Distinct Actin Filament Populations In Vitro

Gergana Gateva, Elena Kremneva, Theresia Reindl, Tommi Kotila, Konstantin Kogan, Laurène Gressin, Peter W Gunning, Dietmar J Manstein, Alphée Michelot, Pekka Lappalainen
Curr Biol . 2017 Mar 6;27(5):705-713. doi: 10.1016/j.cub.2017.01.018. Epub 2017 Feb 16. PMID: 28216317

PUBLICATION

Architecture dependence of actin filament network disassembly

Laurène Gressin, Audrey Guillotin, Christophe Guérin, Laurent Blanchoin, Alphée Michelot
Curr Biol . 2015 Jun 1;25(11):1437-47. doi: 10.1016/j.cub.2015.04.011. Epub 2015 Apr 23. PMID: 25913406

PUBLICATION

Actin filament elongation in Arp2/3-derived networks is controlled by three distinct mechanisms

Michelot A, Grassart A, Okreglak V, Costanzo M, Boone C, Drubin DG.
Dev Cell. 2013 Jan 28;24(2):182-95. PMID: 23333351

PUBLICATION

Actin cytoskeleton: a team effort during actin assembly

Blanchoin L, Michelot A.
Curr Biol. 2012 Aug 21;22(16):R643-5. PMID: 22917514

PUBLICATION

Building distinct actin filament networks in a common cytoplasm

Michelot A, Drubin DG.
Curr Biol. 2011 Jul 26;21(14):R560-9. PMID: 21783039

PUBLICATION

Reconstitution and protein composition analysis of endocytic actin patches

Michelot A, Costanzo M, Sarkeshik A, Boone C, Yates JR 3rd, Drubin DG.
Curr Biol. 2010 Nov 9;20(21):1890-9. PMID: 21035341

PUBLICATION

Actin-filament stochastic dynamics mediated by ADF/cofilin.

Michelot A, Berro J, Guérin C, Boujemaa-Paterski R, Staiger CJ, Martiel JL, Blanchoin L.
Curr Biol. 2007 May 15;17(10):825-33. PMID: 17493813

PUBLICATION

A novel mechanism for the formation of actin-filament bundles by a nonprocessive formin.

Michelot A, Derivery E, Paterski-Boujemaa R, Guérin C, Huang S, Parcy F, Staiger CJ, Blanchoin L.
Curr Biol. 2006 Oct 10;16(19):1924-30. PMID: 17027489

PUBLICATION

The formin homology 1 domain modulates the actin nucleation and bundling activity of Arabidopsis FORMIN1.

Michelot A, Guérin C, Huang S, Ingouff M, Richard S, Rodiuc N, Staiger CJ, Blanchoin L.
Plant Cell. 2005 Aug;17(8):2296-313 PMID: 15994911

PUBLICATION

Linking single-cell decisions to collective behaviours in social bacteria

Céline Dinet, Alphée Michelot, Julien Herrou, Tâm Mignot
Philos Trans R Soc Lond B Biol Sci . 2021 Mar 15;376(1820):20190755. PMID: 33487114

PUBLICATION

Amoeboid Swimming Is Propelled by Molecular Paddling in Lymphocytes

Laurene Aoun, Alexander Farutin, Nicolas Garcia-Seyda , Paulin Nègre , Mohd Suhail Rizvi, Sham Tlili, Solene Song, Xuan Luo, Martine Biarnes-Pelicot , Rémi Galland, Jean-Baptiste Sibarita, Alphée Michelot, Claire Hivroz, Salima Rafai, Marie-Pierre Valignat , Chaouqi Misbah , Olivier Theodoly
Biophys J . 2020 Sep 15;119(6):1157-1177. doi: 10.1016/j.bpj.2020.07.033. Epub 2020 Aug 12. PMID: 32882187

PUBLICATION

Force Production by a Bundle of Growing Actin Filaments Is Limited by Its Mechanical Properties

Jean-Louis Martiel, Alphée Michelot, Rajaa Boujemaa-Paterski, Laurent Blanchoin, Julien Berro
Biophys J . 2020 Jan 7;118(1):182-192. doi: 10.1016/j.bpj.2019.10.039. Epub 2019 Nov 6. PMID: 31791547

PUBLICATION

Site-specific cation release drives actin filament severing by vertebrate cofilin

Kang H, Bradley MJ, Cao W, Zhou K, Grintsevich EE, Michelot A, Sindelar CV, Hochstrasser M, De La Cruz EM.
Proc Natl Acad Sci U S A. 2014 Dec 16;111(50):17821-6. PMID: 25468977

PUBLICATION

Cofilin-2 controls actin filament length in muscle sarcomeres

Kremneva E, Makkonen MH, Skwarek-Maruszewska A, Gateva G, Michelot A, Dominguez R, Lappalainen P.
Dev Cell. 2014 Oct 27;31(2):215-26. PMID: 25373779

PUBLICATION

Membrane-sculpting BAR domains generate stable lipid microdomains.

Zhao H, Michelot A, Koskela EV, Tkach V, Stamou D, Drubin DG, Lappalainen P.
Cell Rep. 2013 Sep 26;4(6):1213-23. PMID: 24055060

PUBLICATION

Lsb1 is a negative regulator of las17 dependent actin polymerization involved in endocytosis.

Spiess M, de Craene JO, Michelot A, Rinaldi B, Huber A, Drubin DG, Winsor B, Friant S.
PLoS One. 2013;8(4):e61147 PMID: 23577202

PUBLICATION

Mechanism and cellular function of Bud6 as an actin nucleation-promoting factor.

Graziano BR, DuPage AG, Michelot A, Breitsprecher D, Moseley JB, Sagot I, Blanchoin L, Goode BL.
Mol Biol Cell. 2011 Nov;22(21):4016-28. PMID: 21880892

PUBLICATION

Determinants of endocytic membrane geometry, stability, and scission

Kishimoto T, Sun Y, Buser C, Liu J, Michelot A, Drubin DG.
Proc Natl Acad Sci U S A. 2011 Nov 1;108(44):E979-88. PMID: 22006337

PUBLICATION

The formin DAD domain plays dual roles in autoinhibition and actin nucleation.

Gould CJ1, Maiti S, Michelot A, Graziano BR, Blanchoin L, Goode BL.
Curr Biol. 2011 Mar 8;21(5):384-90. PMID: 21333540

PUBLICATION

A "primer"-based mechanism underlies branched actin filament network formation and motility.

Achard V, Martiel JL, Michelot A, Guérin C, Reymann AC, Blanchoin L, Boujemaa-Paterski R.
Curr Biol. 2010 Mar 9;20(5):423-8. PMID: 20188562

PUBLICATION

Stochastic severing of actin filaments by actin depolymerizing factor/cofilin controls the emergence of a steady dynamical regime.

Roland J, Berro J, Michelot A, Blanchoin L, Martiel JL.
Biophys J. 2008 Mar 15;94(6):2082-94. PMID: 18065447

PUBLICATION

Attachment conditions control actin filament buckling and the production of forces.

Berro J, Michelot A, Blanchoin L, Kovar DR, Martiel JL.
Biophys J. 2007 Apr 1;92(7):2546-58. PMID: 17208983

PUBLICATION

Villin Severing Activity Enhances Actin-Based Motility in vivo

Revenu C, Courtois M, Michelot A, Sykes C, Louvard D, Robine S.
Mol Biol Cell. 2007 Mar;18(3):827-38. PMID: 17182858

Interactions

NATIONAL
  • Olivia du Roure et Julien Heuvingh, ESPCI Paris
  • Clément Campillo, Université d’Evry
  • Tam Mignot, LCB, Marseille
  • Loïc Le Goff, Institut Fresnel, Marseille
  • Renaud Vincentelli, AFMB, Marseille
INTERNATIONAL
  • Pekka Lappalainen, University of Helsinki
  • Robert Robinson, Okayama University
  • Marka Kaksonen, University of Geneva

Funding

ERC

ANR

CENTURI

Members more

annafrancesca Rigato Juna Como Céline Dinet Audrey Guillotin
Alphée Michelot
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Alphée Michelot

Researcher

Alphée Michelot studied physics and chemistry at the Ecole Normale Supérieure de Lyon, France. After a PhD in biophysics and biochemistry at the University of Grenoble, France, he worked as a post-doc in the department of Molecular and Cell Biology at the University of California, Berkeley, USA. He joined the CNRS as a research scientist at the Institut de Recherches en Technologies et Sciences pour le Vivant, Grenoble, France, in 2012. Group leader at the Institute for Developmental Biology of Marseille since 2015,his research is focused on the molecular and physical principles of actin assembly.

annafrancesca Rigato
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annafrancesca Rigato

Researcher

Juna Como
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Juna Como

PhD student

Céline Dinet
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Céline Dinet

Postdoctoral fellow

Audrey Guillotin
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Audrey Guillotin

Technical staff