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.
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.
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.
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
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.
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.
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.
January 22nd, 2021
A Functional Family of Fluorescent Nucleotide Analogues to Investigate Actin Dynamics and Energetics
September 10th, 2020
Diversity from Similarity: Cellular Strategies for Assigning Particular Identities to Actin Filaments and Networks
October 25th, 2019
Mechanical stiffness of reconstituted actin patches correlates tightly with endocytosis efficiency
June 10th, 2019
Sizes of actin networks sharing a common environment are determined by the relative rates of assembly
March 6th, 2017
Tropomyosin Isoforms Specify Functionally Distinct Actin Filament Populations In Vitro
June 1st, 2015
Architecture dependence of actin filament network disassembly
January 28th, 2013
Actin filament elongation in Arp2/3-derived networks is controlled by three distinct mechanisms
January 21st, 2012
Actin cytoskeleton: a team effort during actin assembly
July 26th, 2011
Building distinct actin filament networks in a common cytoplasm
November 9th, 2010
Reconstitution and protein composition analysis of endocytic actin patches
May 15th, 2007
Actin-filament stochastic dynamics mediated by ADF/cofilin.
October 10th, 2006
A novel mechanism for the formation of actin-filament bundles by a nonprocessive formin.
August 17th, 2006
The formin homology 1 domain modulates the actin nucleation and bundling activity of Arabidopsis FORMIN1.
January 25th, 2021
Linking single-cell decisions to collective behaviours in social bacteria
September 15th, 2020
Amoeboid Swimming Is Propelled by Molecular Paddling in Lymphocytes
January 7th, 2020
Force Production by a Bundle of Growing Actin Filaments Is Limited by Its Mechanical Properties
December 16th, 2014
Site-specific cation release drives actin filament severing by vertebrate cofilin
October 27th, 2014
Cofilin-2 controls actin filament length in muscle sarcomeres
September 26th, 2013
Membrane-sculpting BAR domains generate stable lipid microdomains.
April 8th, 2013
Lsb1 is a negative regulator of las17 dependent actin polymerization involved in endocytosis.
November 22nd, 2011
Mechanism and cellular function of Bud6 as an actin nucleation-promoting factor.
November 1st, 2011
Determinants of endocytic membrane geometry, stability, and scission
March 8th, 2011
The formin DAD domain plays dual roles in autoinhibition and actin nucleation.
March 9th, 2010
A "primer"-based mechanism underlies branched actin filament network formation and motility.
March 15th, 2008
Stochastic severing of actin filaments by actin depolymerizing factor/cofilin controls the emergence of a steady dynamical regime.
April 1st, 2007
Attachment conditions control actin filament buckling and the production of forces.
March 18th, 2007