The shape of many eukaryotic cells depends on the actin cytoskeleton, and changes in actin assembly dynamics underlie many changes in cell shape. Ena/VASP-family actin polymerases, for example, modulate cell shape by locally accelerating actin filament assembly and slowing filament capping. When concentrated into discrete foci at the leading edge, VASP promotes filopodia assembly, and forms part of a poorly understood molecular complex that remains associated with growing filopodia tips. Here we identify precursors of this filopodia tip complex in migrating B16F1 cells: small leading-edge clusters of the adaptor protein lamellipodin (Lpd) which subsequently recruit VASP and initiate filopodia formation. Dimerization, membrane association, and VASP binding are all required for lamellipodin to incorporate into filopodia tip complexes, and over-expression of monomeric, membrane-targeted lamellipodin mutants disrupts tip complex assembly. Once formed, VASP and lamellipodin-containing tip complexes grow by fusing with each other, but their growth is limited by a size-dependent, dynamic instability. Our results demonstrate that assembly and disassembly dynamics of filopodia tip complexes are determined, in part, by a network of multivalent interactions between Ena/VASP proteins, EVH1 ligands, and actin filaments.

The proper organization and coordination of cells is essential to the integrity and function of tissues, organs, and multicellular organisms. While diverse organs and organisms have been used to study these foundational principles, the shear structural and compositional complexity of many in vivo experimental models raises numerous challenges to dissecting the cellular mechanisms underlying their formation and function. Conversely, in vitro models, despite limiting structural complexity, frequently fail to recapitulate the complete physiology of the organs from which they are derived. To ameliorate these tradeoffs, we exploited the anatomical simplicity and extraordinary regenerative capacity of the basal metazoan Hydra vulgaris to probe the mechanisms of tissue patterning and coordinated cellular physiology. In Chapter 2, we apply live microscopy and physical cellular manipulation to investigate a decades-old observation in which a disordered aggregate of Hydra cells sorts into inner and outer epithelial compartments, a prerequisite to regenerating a functional body plan. We show that differences in the rates of epithelialization between two primary cell types give rise to the bilayered structure of the intact animal. In Chapter 3, we investigate the function of these assembled tissues. Specifically, through the identification and dissection of a fluid engulfment phenomenon known as macropinocytosis, we reveal a role for tissue mechanics in regulating fluid uptake and membrane recycling in homeostatic Hydra epithelia. Together, these studies illustrate how the emergent properties of cell collectives, such as cell polarity and tissue mechanics, can contribute to the structure and function of living animals.

The ParM protein, encoded by the parMRC locus from plasmid R1, is the best-characterized actin homolog present in prokaryotes. In conjunction with a nucleoprotein complex formed by a DNA binding protein ParR and a DNA centromere parC, ParM constructs a simple mitotic spindle that pushes plasmid DNA to opposite poles of a bacterial cell for DNA segregation. Like actin, ParM assembles into ATP-dependent double-stranded, helical filaments via a nucleation condensation reaction. However, unlike actin, it displays three kinetic properties that are essential to its function: rapid spontaneous nucleation, bilateral elongation and ATPase dependent dynamic instability: the stochastic switching between filament elongation and rapid catastrophic shrinkage. In this dissertation, we investigated three questions related to ParM's evolution and function. First, many divergent R1 ParM homologs are encoded by other plasmids, and function in plasmid DNA segregation. We asked whether the filament architecture and assembly dynamics are conserved in a divergent member of the ParM family, the ParM protein from plasmid pB171. Using microscopic and biophysical techniques, we compared and contrasted the architecture and assembly of these related proteins. Despite being only 41% identical, we find that R1 and pB171 ParM polymerize into nearly identical filaments that display similar assembly dynamics, suggesting that structure and function are conserved in the ParM family. Second, we asked whether ParM prefers ATP or GTP in vitro. Using biochemical assays and TIRF microscopy, we find that ParM displays similar polymerization kinetics and stability in the presence of either nucleotide, but binds to ATP ten times tighter than GTP. Third, the structural basis for ParM filament dynamic instability remains poorly understood. Using cryo-electron microscopy to evaluate ParM filaments, we find that the nucleotide binding pocket of ParM protomers exist in an open or closed state, and that ATP hydrolysis shifts the distribution of protomers states; these findings suggest a mechanism for dynamic instability.

The par operon of the R1 plasmid constructs a DNA segregating spindle from three components, parC, ParR, and ParM. parC is a centromeric stretch of DNA which is bound by the protein ParR. ParM is an actin homologue that polymerizes into two stranded filaments that can interact with the ParR/parC complex. These three components are both necessary and sufficient to segregate parC containing plasmids, implying that the par operon is a biochemically self-contained DNA segregation apparatus. To elucidate how this system functions to segregate plasmid DNA, we wished to determine the intrinsic kinetic properties of ParM assembly, and how this assembly is regulated by the interaction with its cargo, the ParR/parC complex.

We find that ParM displays three properties that distinguish its assembly from that of eukaryotic actins. 1) ParM displays hydrolysis dependent dynamic instability, the stochastic switching between states of growth and rapid depolymerization. 2) ParM filaments display a very rapid rate of nucleation, occurring at a rate 200-fold faster than actin. 3) ParM filaments exhibit no kinetic polarity, with equal rates of growth at each filament end.

By combining ParM with bead immobilized ParR/parC we demonstrate that these three components can create bipolar spindles in vitro similar to those observed in vivo. ParR/parC coated microspheres are connected by bundles of ParM filaments that elongate over time, pushing the microspheres apart over long distances. Speckle microscopy and photobleaching experiments demonstrate that this motility is driven by insertional polymerization at the bead surface. We find that the ParR/parC complex prevents ParM filament ends from catastrophe by stabilizing filaments down to the ATP-ParM critical concentration. Futhermore, we show that the dynamic instability of ParM is critical to the spatial regulation of the R1 spindle, as filament ends that are not bound at each end by ParR/parC are destined to undergo catastrophe. The dynamic instability of ParM also provides an energetic differential to power spindle elongation, as the turnover of background, unbound filaments provide the monomer excess required for the growth of stabilized, spindle associated filaments.

Tropomyosin, which binds along the length of actin filaments, has long been considered the master regulator over the binding of other proteins to actin. In particular, non-muscle tropomyosin is considered the key to the transition between the lamellipod and the lamellum in motile cells by inhibiting Arp2/3 complex nucleation and cofilin disassembly. This in vitro study of a D. melangaster tropomyosin isoform, TM1A, which localizes to the lamellum of S2 cells, shows that Arp2/3 and cofilin both affect the binding of TM1A. Labeled TM1A binds preferentially near the pointed end of actin filaments, and Arp2/3 blocks TM1A from binding to branched networks. Surprisingly, cofilin promotes the binding of TM1A to a branched actin network. Our data provides an exciting look at how actin, tropomyosin, cofilin and Arp2/3 complex together can self-organize to create two structurally and dynamically different actin networks at the leading edge. We also explore the actin-binding characteristics of another non-muscle tropomyosin isoform, TM1J, and show that its binding is dependent on TM1A. Capping protein also contributes to the exclusion of tropomyosin from a branched network. Attempts at measuring the mechanical properties of a branched network ultimately failed, but are presented for potential future inspiration. Finally, the results of two collaborations are presented.

Polymerization of actin into filaments powers diverse cellular functions, and a key way that cells build distinct types of actin networks is by using different actin nucleation factors. We identified a new vertebrate actin nucleation factor, junction-mediating and regulatory protein (JMY, pronounced "jammy"). JMY combines de novo nucleation by a Spire-like mechanism with activation of the Arp2/3 complex to rapidly generate branched actin networks. JMY localizes to the leading edge of motile cells, and contributes to cell motility. Since the Arp2/3 complex requires pre-existing filaments for its branching nucleation activity, we hypothesize that JMY contributes to cell motility by nucleating filaments, which then serve as substrates for JMY-activated Arp2/3 nucleation.