Brian J. Galletta

Roles for Actin Assembly in Endocytosis

Endocytosis includes a number of processes by which cells internalize segments of their plasma membrane, enclosing a wide variety of material from outside the cell. Endocytosis can contribute to uptake of nutrients, regulation of signaling molecules, control of osmotic pressure, and function of synapses. The actin cytoskeleton plays an essential role in several of these processes. Actin assembly can create protrusions that encompass extracellular materials. Actin can also support the processes of invagination of a membrane segment into the cytoplasm, elongation of the invagination, scission of the new vesicle from the plasma membrane, and movement of the vesicle away from the membrane. We briefly discuss various types of endocytosis, including phagocytosis, macropinocytosis, and clathrin-independent endocytosis. We focus mainly on new findings on the relative importance of actin in clathrin-mediated endocytosis (CME) in yeast versus mammalian cells.

Distinct roles for Arp2/3 regulators in actin assembly and endocytosis

The Arp2/3 complex is essential for actin assembly and motility in many cell processes, and a large number of proteins have been found to bind and regulate it in vitro. A critical challenge is to understand the actions of these proteins in cells, especially in settings where multiple regulators are present. In a systematic study of the sequential multicomponent actin assembly processes that accompany endocytosis in yeast, we examined and compared the roles of WASp, two type-I myosins, and two other Arp2/3 activators, along with that of coronin, which is a proposed inhibitor of Arp2/3. Quantitative analysis of high-speed fluorescence imaging revealed individual functions for the regulators, manifested in part by novel phenotypes. We conclude that Arp2/3 regulators have distinct and overlapping roles in the processes of actin assembly that drive endocytosis in yeast. The formation of the endocytic actin patch, the creation of the endocytic vesicle, and the movement of the vesicle into the cytoplasm display distinct dependencies on different Arp2/3 regulators. Knowledge of these roles provides insight into the in vivo relevance of the dendritic nucleation model for actin assembly.

Actin and endocytosis: mechanisms and phylogeny.

The regulated assembly of actin filament networks is a crucial part of endocytosis, with crucial temporal and spatial relationships between proteins of the endocytic and actin assembly machinery. Of particular importance has been a wealth of studies in budding and fission yeast. Cell biology approaches, combined with molecular genetics, have begun to uncover the complexity of the regulation of actin dynamics during the endocytic process. In a wide range of organisms, clathrin-mediated endocytosis appears to be linked to Arp2/3-mediated actin assembly. The conservation of the components, across a wide range eukaryotic species, suggests that the partnership between endocytosis and actin may be evolutionarily ancient.

Actin dynamics and endocytosis in yeast and mammals

Tight regulation of the actin cytoskeleton is critical for many cell functions, including various forms of cellular uptake. Clathrin-mediated endocytosis (CME) is one of the main methods of uptake in many cell types. An intact and properly regulated actin cytoskeleton is required for CME in Saccharomyces cerevisiae. Yeast CME requires the proper regulation of actin polymerization, filament cross-linking, and filament disassembly. Recent studies also point to a role for F-BAR and BAR-domain containing proteins in linking the processes of generating and sensing plasma membrane curvature with those regulating the actin cytoskeleton. Many of these same proteins are conserved in mammalian CME. However, until recently the requirement for actin in mammalian CME was less clear. Several recent studies in mammalian cells provide new support for an actin requirement in the invagination and late stages of CME. This review focuses on the regulation of the actin cytoskeleton during CME in yeast and the emerging evidence for a role for actin during mammalian CME.

Overlapping and distinct functions for cofilin, coronin and Aip1 in actin dynamics in vivo

Actin-filament disassembly is crucial for actin-based motility, to control filament network architecture and to regenerate subunits for assembly. Here, we examined the roles of three actin cytoskeletal proteins, coronin, cofilin and Aip1, which have been suggested to combine in various ways to control actin dynamics by promoting or regulating disassembly. We studied their functions during the endocytosis process in budding yeast, where actin-filament dynamics at the cortical actin ‘patch’ contribute to the formation and movement of endocytic vesicles. We found that all three proteins were recruited during the late phase of the life of the actin patch. They all arrived at the same time, when actin and other actin-associated proteins were leaving the patch. Cofilin point mutations influenced the localization of coronin and Aip1, but the complete loss of coronin had no effect on localization of cofilin or Aip1. Using quantitative patch motion analysis and comparing mutant alleles, the phenotypes for mutations of the three genes showed some commonalities, but also some striking differences. Cofilin was clearly the most important; it displayed the most severe mutant phenotypes affecting actin-patch assembly and movement. Together, the results suggest that all three proteins work together to promote actin disassembly, but not in a simple way, and not with equal importance.

Two Polo-like kinase 4 binding domains in Asterless perform distinct roles in regulating kinase stability

The Asterless N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas its C terminus stabilizes Plk4 during mitosis.

Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function

Cnn and PLP directly interact at two defined sites to coordinate the cell cycle–dependent rearrangement and scaffolding activity of the centrosome to permit normal centrosome organization, cell division, and embryonic viability.

Drosophila pericentrin requires interaction with calmodulin for its function at centrosomes and neuronal basal bodies but not at sperm basal bodies.

Pericentrin (PLP) is a centrosomal protein required for organizing pericentriolar material. It requires interaction with calmodulin (CaM) for proper centrosome targeting both in vitro and in vivo. In addition, the PLP-CaM interaction is critical for mechanosensory neuron function but not for functional sperm.

Asterless is required for centriole length control and sperm development

Loss of the centriole protein Asterless (Asl) prevents centriole duplication, which has limited the study of its function at centrioles. Here, Galletta et al. show that Asl controls centriole length and ensures proper basal body functions during spermatogenesis.

Actin-Regulator Feedback Interactions during Endocytosis.

Endocytosis mediated by clathrin, a cellular process by which cells internalize membrane receptors and their extracellular ligands, is an important component of cell signaling regulation. Actin polymerization is involved in endocytosis in varying degrees depending on the cellular context. In yeast, clathrin-mediated endocytosis requires a pulse of polymerized actin and its regulators, which recruit and activate the Arp2/3 complex. In this article, we seek to identify the main protein-protein interactions that 1) cause actin and its regulators to appear in pulses, and 2) determine the effects of key mutations and drug treatments on actin and regulator assembly. We perform a joint modeling/experimental study of actin and regulator dynamics during endocytosis in the budding yeast Saccharomyces cerevisiae. We treat both a stochastic model that grows an explicit three-dimensional actin network, and a simpler two-variable Fitzhugh-Nagumo type model. The models include a negative-feedback interaction of F-actin onto the Arp2/3 regulators. Both models explain the pulse time courses and the effects of interventions on actin polymerization: the surprising increase in the peak F-actin count caused by reduced regulator branching activity, the increase in F-actin resulting from slowing of actin disassembly, and the increased Arp2/3 regulator lifetime resulting from latrunculin treatment. In addition, they predict that decreases in the regulator branching activity lead to increases in accumulation of regulators, and we confirmed this prediction with experiments on yeast harboring mutations in the Arp2/3 regulators, using quantitative fluorescence microscopy. Our experimental measurements suggest that the regulators act quasi-independently, in the sense that accumulation of a particular regulator is most strongly affected by mutations of that regulator, as opposed to the others.