There is no life without movement, at all levels of metazoan organization, from individual cells to the animal form. During development, individual cells migrate from the germ layers to lay down the body plan and in the adult organism migrating cells play key roles in immune defense and tissue repair. Pathological processes, including tumor dissemination and atherosclerosis, likewise involve cell migration. Our studies focus on unraveling the structural basis of cell movement.
Cells move by recycling the protein polymers that make up the so-called cytoskeleton, primarily the actin filaments and the microtubules. The turnover of actin filaments, together with associated proteins provides the forces required for movement, whereas microtubules serve a role in polarity determination and guidance.
We currently focus on elucidating the structural reorganizations of actin filaments required for cells to move. Actin filaments are used in two ways to create movement. As in muscle cells, actin filaments in non-muscle cells can slide over myosin filaments to produce contraction and this property is exploited during the later stages of cell migration, when the cell body catches up with the advancing front. To initiate movement in the first place a cell must protrude in the direction it wants to go, which it does by extending thin sheets of cytoplasm called lamellipodia. This protrusion is achieved by exploiting the other ability of actin filaments to push, by polymerization. Our current studies focus on determining the structural basis of this protrusive activity. Towards this aim we have developed procedures to correlate the movement of living cells in the light microscope with the structure of the same cells in the electron microscope. An important advance in this approach has been the introduction of electron tomography that allows visualization of cellular architecture at nanometer resolution (Figure 1).
A current debate in the field centers on the question of how actin networks in lamellipodia are established and maintained. To study lamellipodia initiation, we have exploited an experimental model in which a microneedle is used to produce a hole in the cytoplasm that is then repaired by lamellipodia. By capturing the earliest stages of repair we have shown by electron tomography that lamellipodia initiation involves the branching of actin filaments from the sides of filaments aligned parallel to the periphery of the membrane bordering the hole (Figure 2). We have also provided the first 3D maps of actin filament organization in established lamellipodia, which show that the actin networks are constructed from actin filaments of variable length, linked into subsets by branch junctions (Figure 3). The heptameric actin related protein complex, the “Arp2/3 complex” promotes actin branching in vitro and the structure of the in vitro branch junction has been determined by electron microscopy.
From image analysis in collaboration with Akihiro Narita in Nagoya we have recently obtained the first model of branch junctions in lamellipodia in vivo, at 3.5nm resolution. Our data reveal a close structural homology of in vivo branches with those formed in vitro from actin and the Arp2/3 complex. In ongoing studies we are probing the dependence of lamellipodia protrusion on Arp2/3 complex activity and the roles of other actin regulators in the reorganization and turnover of actin networks necessary for movement and are developing mathematical models of protrusion.