Pekka Lappalainen

Filopodia: molecular architecture and cellular functions

Filopodia are thin, actin-rich plasma-membrane protrusions that function as antennae for cells to probe their environment. Consequently, filopodia have an important role in cell migration, neurite outgrowth and wound healing and serve as precursors for dendritic spines in neurons. The initiation and elongation of filopodia depend on the precisely regulated polymerization, convergence and crosslinking of actin filaments. The increased understanding of the functions of various actin-associated proteins during the initiation and elongation of filopodia has provided new information on the mechanisms of filopodia formation in distinct cell types.

Stress fibers are generated by two distinct actin assembly mechanisms in motile cells

Stress fibers play a central role in adhesion, motility, and morphogenesis of eukaryotic cells, but the mechanism of how these and other contractile actomyosin structures are generated is not known. By analyzing stress fiber assembly pathways using live cell microscopy, we revealed that these structures are generated by two distinct mechanisms. Dorsal stress fibers, which are connected to the substrate via a focal adhesion at one end, are assembled through formin (mDia1/DRF1)–driven actin polymerization at focal adhesions. In contrast, transverse arcs, which are not directly anchored to substrate, are generated by endwise annealing of myosin bundles and Arp2/3-nucleated actin bundles at the lamella. Remarkably, dorsal stress fibers and transverse arcs can be converted to ventral stress fibers anchored to focal adhesions at both ends. Fluorescence recovery after photobleaching analysis revealed that actin filament cross-linking in stress fibers is highly dynamic, suggesting that the rapid association–dissociation kinetics of cross-linkers may be essential for the formation and contractility of stress fibers. Based on these data, we propose a general model for assembly and maintenance of contractile actin structures in cells.

Cofilin promotes rapid actin filament turnover in vivo

The ability of actin filaments to function in cell morphogenesis and motility is coupled to their capacity for rapid assembly and disassembly. Because disassembly in vitro is much slower than in vivo, cellular factors that stimulate disassembly have long been assumed to exist. Although numerous proteins can affect actin dynamics in vitro, demonstration of in vivo relevance of these effects has not been achieved. We have used genetics and an actin-inhibitor in yeast to demonstrate that rapid cycles of actin assembly and disassembly depend on the small actin-binding protein cofilin, and that cofilin stimulates filament disassembly. These results may explain why cofilin is ubiquitous in eukaryotes and is essential for viability in every organism in which its function has been tested genetically. Magnitudes of disassembly defects in cofilin mutants in vivo were found to be correlated closely with the magnitudes of disassembly defects observed in vitro, supporting our conclusions. Furthermore, these cofilin mutants provided an opportunity to distinguish in living cells those actin functions that depend specifically on filament turnover (endocytosis) from those that do not (cortical actin patch motility).

Regulation of the Actin Cytoskeleton-Plasma Membrane Interplay by Phosphoinositides

The plasma membrane and the underlying cortical actin cytoskeleton undergo continuous dynamic interplay that is responsible for many essential aspects of cell physiology. Polymerization of actin filaments against cellular membranes provides the force for a number of cellular processes such as migration, morphogenesis, and endocytosis. Plasma membrane phosphoinositides (especially phosphatidylinositol bis- and trisphosphates) play a central role in regulating the organization and dynamics of the actin cytoskeleton by acting as platforms for protein recruitment, by triggering signaling cascades, and by directly regulating the activities of actin-binding proteins. Furthermore, a number of actin-associated proteins, such as BAR domain proteins, are capable of directly deforming phosphoinositide-rich membranes to induce plasma membrane protrusions or invaginations. Recent studies have also provided evidence that the actin cytoskeleton-plasma membrane interactions are misregulated in a number of pathological conditions such as cancer and during pathogen invasion. Here, we summarize the wealth of knowledge on how the cortical actin cytoskeleton is regulated by phosphoinositides during various cell biological processes. We also discuss the mechanisms by which interplay between actin dynamics and certain membrane deforming proteins regulate the morphology of the plasma membrane.

Actin stress fibers--assembly, dynamics and biological roles.

Actin filaments assemble into diverse protrusive and contractile structures to provide force for a number of vital cellular processes. Stress fibers are contractile actomyosin bundles found in many cultured non-muscle cells, where they have a central role in cell adhesion and morphogenesis. Focal-adhesion-anchored stress fibers also have an important role in mechanotransduction. In animal tissues, stress fibers are especially abundant in endothelial cells, myofibroblasts and epithelial cells. Importantly, recent live-cell imaging studies have provided new information regarding the mechanisms of stress fiber assembly and how their contractility is regulated in cells. In addition, these studies might elucidate the general mechanisms by which contractile actomyosin arrays, including muscle cell myofibrils and cytokinetic contractile ring, can be generated in cells. In this Commentary, we discuss recent findings concerning the physiological roles of stress fibers and the mechanism by which these structures are generated in cells.

Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis

Dendritic spines are small protrusions along dendrites where the postsynaptic components of most excitatory synapses reside in the mature brain. Morphological changes in these actin-rich structures are associated with learning and memory formation. Despite the pivotal role of the actin cytoskeleton in spine morphogenesis, little is known about the mechanisms regulating actin filament polymerization and depolymerization in dendritic spines. We show that the filopodia-like precursors of dendritic spines elongate through actin polymerization at both the filopodia tip and root. The small GTPase Rif and its effector mDia2 formin play a central role in regulating actin dynamics during filopodia elongation. Actin filament nucleation through the Arp2/3 complex subsequently promotes spine head expansion, and ADF/cofilin-induced actin filament disassembly is required to maintain proper spine length and morphology. Finally, we show that perturbation of these key steps in actin dynamics results in altered synaptic transmission.

Essential functions and actin‐binding surfaces of yeast cofilin revealed by systematic mutagenesis

Cofilin stimulates actin filament turnover in vivo. The phenotypes of twenty yeast cofilin mutants generated by systematic mutagenesis were determined. Ten grew as well as the wild type and showed no cytoskeleton defects, seven were recessive‐lethal and three were conditional‐lethal and caused severe actin organization defects. Biochemical characterization of interactions between nine mutant yeast cofilins and yeast actin provided evidence that F‐actin binding and depolymerization are essential cofilin functions. Locating the mutated residues on the yeast cofilin molecular structure allowed several important conclusions to be drawn. First, residues required for actin monomer binding are proximal to each other. Secondly, additional residues are required for interactions with actin filaments; these residues might bind an adjacent subunit in the actin filament. Thirdly, despite striking structural similarity, cofilin interacts with actin in a different manner from gelsolin segment‐1. Fourthly, a previously unrecognized cofilin function or interaction is suggested by identification of spatially proximal residues important for cofilin function in vivo, but not for actin interactions in vitro. Finally, mutation of the cofilin N‐terminus suggests that its sequence is conserved because of its critical role in actin interactions, not because it is sometimes a target for protein kinases.

Molecular Mechanisms of Membrane Deformation by I-BAR Domain Proteins

BACKGROUND Generation of membrane curvature is critical for the formation of plasma membrane protrusions and invaginations and for shaping intracellular organelles. Among the central regulators of membrane dynamics are the BAR superfamily domains, which deform membranes into tubular structures. In contrast to the relatively well characterized BAR and F-BAR domains that promote the formation of plasma membrane invaginations, I-BAR domains induce plasma membrane protrusions through a poorly understood mechanism. RESULTS We show that I-BAR domains induce strong PI(4,5)P(2) clustering upon membrane binding, bend the membrane through electrostatic interactions, and remain dynamically associated with the inner leaflet of membrane tubules. Thus, I-BAR domains induce the formation of dynamic membrane protrusions to the opposite direction than do BAR and F-BAR domains. Strikingly, comparison of different I-BAR domains revealed that they deform PI(4,5)P(2)-rich membranes through distinct mechanisms. IRSp53 and IRTKS I-BARs bind membranes mainly through electrostatic interactions, whereas MIM and ABBA I-BARs additionally insert an amphipathic helix into the membrane bilayer, resulting in larger tubule diameter in vitro and more efficient filopodia formation in vivo. Furthermore, FRAP analysis revealed that whereas the mammalian I-BAR domains display dynamic association with filopodia, the C. elegans I-BAR domain forms relatively stable structures inside the plasma membrane protrusions. CONCLUSIONS These data define I-BAR domain as a functional member of the BAR domain superfamily and unravel the mechanisms by which I-BAR domains deform membranes to induce filopodia in cells. Furthermore, our work reveals unexpected divergence in the mechanisms by which evolutionarily distinct groups of I-BAR domains interact with PI(4,5)P(2)-rich membranes.

WH2 domain: a small, versatile adapter for actin monomers.

The actin cytoskeleton plays a central role in many cell biological processes. The structure and dynamics of the actin cytoskeleton are regulated by numerous actin‐binding proteins that usually contain one of the few known actin‐binding motifs. WH2 domain ( ASP omology domain‐ ) is a ∼35 residue actin monomer‐binding motif, that is found in many different regulators of the actin cytoskeleton, including the β‐thymosins, ciboulot, WASP ( iskott ldrich yndrome rotein), verprolin/WIP ( ASP‐ nteracting rotein), Srv2/CAP (adenylyl yclase‐ ssociated rotein) and several uncharacterized proteins. The most highly conserved residues in the WH2 domain are important in β‐thymosins interactions with actin monomers, suggesting that all WH2 domains may interact with actin monomers through similar interfaces. Our sequence database searches did not reveal any WH2 domain‐containing proteins in plants. However, we found three classes of these proteins: WASP, Srv2/CAP and verprolin/WIP in yeast and animals. This suggests that the WH2 domain is an ancient actin monomer‐binding motif that existed before the divergence of fungal and animal lineages.

IRSp53: crossing the road of membrane and actin dynamics in the formation of membrane protrusions

A tight spatiotemporal coordination of the machineries controlling membrane bending and trafficking, and actin dynamics is crucial for the generation of cellular protrusions. Proteins that are simultaneously capable of regulating actin dynamics and sensing or inducing membrane curvature are predicted to have a prominent role. A prototypical example of this type of proteins is the insulin receptor tyrosine kinase substrate of 53kDa, the founding member of a recently discovered family of proteins, including missing-in-metastasis and ABBA (actin-bundling protein with BAIAP2 homology). Structural, biochemical and cell biological experiments support the unique role of this family as transducers of signalling, linking the protruding membrane to the underlying actin cytoskeleton.