Mechanisms underlying cell migration

It moves, it’s alive! In the micro-cosmos of our body tissues movement is likewise vital to life- and can also contribute to death! Organ development, wound repair and immune defense all rely on the movement of single cells or cell groups. And in metastasis, renegade cells that escape from primary tumours find their way, by migration, to propagate in multiple sites elsewhere. Discovering how cells move is therefore important for understanding normal and pathological processes, with perspectives of bringing unwanted events under control. We already know that cell movement relies on the turnover of the protein filament systems comprising the so-called “cytoskeleton”. Our research is directed towards understanding the structural basis of cytoskeleton turnover and the underlying molecular mechanisms. To this end we are combining molecular biology techniques with live cell imaging technologies and electron microscopy, including electron tomography for 3D imaging of cytoskeleton networks.


Moving with actin

There is no life without motion at any level of metazoan organization from - individual cells to animal forms. During development, cells migrate from the germ layers and establish a blueprint of the body. In the adult organism, migrating cells play a key role in immune defense and tissue repair. Tumor dissemination and atherosclerosis also involve cell migration. In addition, several bacterial and viral pathogens recruit the motile machinery of cells to propagate their infection. Our studies focus on unraveling the structural basis of these processes and using this information to develop mathematical models of motility.

We know that cell motility is initiated by the extension of thin sheets of cytoplasm, so-called lamellipodia. However, the mechanism responsible for generating a 100- to 200-nm-thick sheet is not known. Cryo-electron tomography of B16 and NIH 3T3 cells, observed in vitreous ice, was used to provide single filament coordinates as well as determine membrane morphology at the leading edge. A stochastic mathematical model in which polymerization, branching, and capping depend on the local membrane curvature, determined by BAR proteins, was shown to mimic the flat shape of the membrane during cellular protrusion without any prescribed geometric restrictions. A feedback loop of local activation of filament polymerization and the dependence of branching on membrane curvature, together with a deformation of the membrane by growing filaments, adequately explained the 3D organization of actin filaments and the flat morphology of lamellipodia.

One class of pathogens, including the Gram-positive bacterium Listeria and the Vaccinia virus, hijack the motile apparatus of cells they infect in order to spread infection. They achieve this by nucleating comet tails of actin to propel them from one cell to the next. The smallest member of this class of pathogens is an insect baculovirus. Baculovirus measures just 40 nm in diameter (contrasted with the Vaccinia virus, which has a diameter of 300 nm) and generates actin comets small enough to permit resolution of all actin filaments constituting the comet tail by electron tomography.

We developed a mathematical model that simulates the structural parameters of the comet tails determined by electron tomography, as well as the tracking characteristics of baculovirus in cytoplasm. In current conflicting models of pathogen propulsion, there is disagreement concerning the extent to which filaments may be bound to the pathogen surface as they polymerize and push. Our simulations support a model of propulsion in which filaments are continuously tethered to the pathogen surface. Analysis of cryo-electron tomograms of actin comets propelling Vaccinia virus reveals the same basic structural features of comet tail organization as determined for baculovirus, but with correspondingly more filaments involved in pushing. The mathematical modelling was performed in collaboration with Christian Schmeiser and Christoph Winkler at RICAM, Austrian Academy of Sciences and the Faculty of Mathematics, University of Vienna.

In separate studies electron tomography was used to obtain new insights into the role of the Arp2/3 complex and co-regulators in the formation and turnover of lamellipodia (Flynn et al., 2012; Koestler et al, 2012), as well as the relative roles of lamellipodia and filopodia in protrusion (Steffen et al.,2012). In further collaboration, we contributed to the characterization of Arpin, a protein inhibitor of the Arp2/3 complex implicated in cell steering (Dang et al.,2012), and the structural organization of lamellipodia in dendritic cells (with Michael Sixt at IST, Austria).

Selected Publications

Mueller, J., Pfanzelter, J., Winkler, C., Narita, A., Le Clainche, C., Nemethova, M., Carlier, MF., Maeda, Y., Welch, MD., Ohkawa, T., Schmeiser, C., Resch, GP., Small, JV. (2014). Electron tomography and simulation of baculovirus actin comet tails support a tethered filament model of pathogen propulsion. PLoS Biol. 12(1):e1001765

Vinzenz, M., Nemethova, M., Schur, F., Mueller, J., Narita, A., Urban, E., Winkler, C., Schmeiser, C., Koestler, SA., Rottner, K., Resch, GP., Maeda, Y., Small, JV. (2012). Actin branching in the initiation and maintenance of lamellipodia. J Cell Sci. 125(Pt 11):2775-85

Breitsprecher, D., Kiesewetter, AK., Linkner, J., Vinzenz, M., Stradal, TE., Small, JV., Curth, U., Dickinson, RB., Faix, J. (2011). Molecular mechanism of Ena/VASP-mediated actin-filament elongation. EMBO J. 30(3):456-67
Breitsprecher, D., Koestler, SA., Chizhov, I., Nemethova, M., Mueller, J., Goode, BL., Small, JV., Rottner, K., Faix, J. (2011). Cofilin cooperates with fascin to disassemble filopodial actin filaments. J Cell Sci. 124(Pt 19):3305-18
Oelkers, JM., Vinzenz, M., Nemethova, M., Jacob, S., Lai, FP., Block, J., Szczodrak, M., Kerkhoff, E., Backert, S., Schlüter, K., Stradal, TE., Small, JV., Koestler, SA., Rottner, K. (2011). Microtubules as platforms for assaying actin polymerization in vivo. PLoS One. 6(5):e19931
Small, J.V., Winkler, C., Vinzenz, M. and Schmeiser, S (2011). Reply: Visualizing branched actin filaments in lamellipodia by electron tomography Nature Cell Biology. doi:10.1038/ncb2322

Small, JV. (2010). Dicing with dogma: de-branching the lamellipodium. Trends Cell Biol. 20(11):628-33
Small, J.V. and Rottner, K.R (2010). Elementary Cellular Processes Driven by Actin Assembly: Lamellipodia and Filopodia In “Actin-based Motility” Ed by M.F. Carlier, Springer Science. :pp 3-33
Urban, E., Jacob, S., Nemethova, M., Resch, GP., Small, JV. (2010). Electron tomography reveals unbranched networks of actin filaments in lamellipodia. Nat Cell Biol. 12(5):429-35