Actin polymerization plays important roles in many biological processes, such as establishing cell polarization, accommodating protruding and retracting activities of motile cells, maintaining the physical integrity of the cell, and sensing environmental forces. All these processes are facilitated by the dynamical and mechanical properties of actin filaments, and their ability to exert or resist against forces generated in a cellular environment.
Actin filaments can assemble into a variety of architectures, including branched and crosslinked networks, parallel bundles, and anti-parallel contractile fibers. These structures provide architectural specificities for different regions of the cell, and can also organize into more complex actin-based machineries. We aim to understand how the native molecular architecture of actin-based cellular processes gives rise to force generation and rigidity-sensing. We use cryo-electron tomography in combination with cryo-focused ion beam milling, and develop methodologies allowing for quantitative analysis of actin network architecture.
Figure 2. Cryo-electron tomography workflow (A-D) and actin architecture (E-G) in a thin protrusion assembled by Dictyostelium cells in response to a hole.[less]
Figure 2. Cryo-electron tomography workflow (A-D) and actin architecture (E-G) in a thin protrusion assembled by Dictyostelium cells in response to a hole.
Recent and on-going studies in our group include:
Actin cytoskeleton reorganization during bacterial infection
In collaboration with the Cossart lab (Pasteur Institute), we visualized the three-dimensional architecture of Listeria monocytogenes comet tails during intracellular motility and cell-to-cell spread (Jasnin et al., PNAS 2013). Quantitative analysis of their actin network architecture revealed the existence of bundles of hexagonally packed filaments with spacings of 12–13 nm. Similar configurations were observed in stress fibers and filopodia, suggesting that nanoscopic bundles are a generic feature of actin filament assemblies involved in motility. In collaboration with Alvaro Crevenna (LMU Munich), we also provided a quantitative description of filament branch orientation in Listeria comet tails in cell extracts (Jasnin and Crevenna, Biophys J 2016).
Actin ring formation in response to a perforated surface
In collaboration with the Gerisch group (MPIB), we found that Dictyostelium cells migrating on a perforated film explore its holes by forming actin rings around their border and extending protrusions through the free space (Jasnin et al, Structure 2016). Actin rings were identified by correlative cryo-fluorescence and cryo-electron microscopy, and thin wedges of relevant regions were obtained by cryo-focused ion-beam milling. Retracting stages were distinguished from protruding ones by the accumulation of myosin-II. Early actin rings consists of filaments pointing upright from the membrane, entangled with a meshwork of filaments close to the membrane. Branches identified at later stages suggest that formin-based nucleation of filaments is followed by Arp2/3 complex-mediated network stabilization, which prevents buckling of the force-generating filaments. Additionally, we also performed cryo-electron tomography on D. discoideum cells undergoing membrane pearling upon destruction of the actin cortex (Heinrich et al, Biophys J 2014).
The architecture of traveling actin waves
Actin waves are dynamic supramolecular structures involved in cell migration, cytokinesis, adhesion, and neurogenesis. Although actin waves are recognized as a widespread phenomenon, the architecture critical for their propagation is unknown. In collaboration with the Gerisch group (MPIB), we used in situ cryo-electron tomography of Dictyostelium cells to unveil the wave architecture in a native state. We resolve branch junctions associated with the Arp2/3 complex within the wave network, and quantitatively analyze the organization of branching (Jasnin et al., submitted).
Architecture/force relationship in macrophage podosomes
In collaboration with the Maridonneau-Parini group (IPBS), we explore the three-dimensional architecture of podosomes in human primary monocyte-derived macrophages. Our objective is to reveal the molecular organization of actin filaments and actin-related proteins within podosomes, and to formulate its relationship to force generation. From the mechanical and architectural insights we will build a physical model of force generation by actin polymerization in podosomes, which will be provide a unique basis for understanding nN-scale force generation by cellular actin networks.
Jasnin M., Beck F., Ecke M., Fukuda Y., Martinez-Sanchez A., Baumeister W., Gerisch G.: The architecture of traveling actin waves revealed by cryo-electron tomography. Submitted.
Jasnin M., Ecke M., Baumeister W., Gerisch G.: Actin organization in cells responding to a perforated surface, revealed by live imaging and cryo-electron tomography. Structure, 24:1031-43, 2016
Jasnin M., Crevenna A.H.: Quantitative analysis of filament branch orientation in Listeria actin comet tails. Biophys J., 110:817-26, 2016
Heinrich D., Ecke M., Jasnin M., Engel U., Gerisch G.: Reversible membrane pearling in live cells upon destruction of the actin cortex. Biophys J., 106:1079-91, 2014
Jasnin M., Asano S., Gouin E., Hegerl R., Plitzko J.M., Villa E., Cossart P., Baumeister W.: Three-dimentional architecture of actin filaments in Listeria monocytogenes comet tails. Proc Natl Acad Sci U S A, 10:20521-6, 2013