Actin-Binding Protein Function and Its Relation to Disease Pathology

5 Overview: Actin-Binding Protein Function and Its Relation to Disease Pathology Mira Krendel and Enrique M. De La Cruz Introduction The actin cytoskeleton generates force and movement responsible for many critical and fundamental cellular processes (see Chap. 1). Force generation and motility are produced by two distinct mechanisms (1) the self-assembly of actin monomers into filaments, which can exert forces against boundaries and particles such as cell membranes, vesicles, organelles, or pathogenic bacteria, and generate movement of these boundaries and (2) through the activity of contractile motor proteins of the myosin family, which generate force and motility along actin filaments. Both mechanisms utilize chemical energy in the form of ATP although hydrolysis of ATP by actin does not contribute to the force generated by actomyosin. Each monomer incorporated into a filament and each myosin mechanical “step” consumes one ATP molecule, generating ADP and Pi as the hydrolysis products. The actin cytoskeleton also helps form stable structures, often linked to membranes and the extracellular matrix (ECM) through membrane- associated proteins, which give the cell mechanical strength and polarity. Although these structures are dynamic and constantly undergoing remodel- ing through the addition and exchange of filament subunits, the principal functions of these structures are attributed to the large-scale, mechanical properties of actin filaments and filament networks. The assembly and three-dimensional organization of actin filaments in cells is controlled through interactions with a large number of distinct, regulatory actin- binding proteins (ABPs) that are classified as monomer- or filament-binding based on which assembly state of actin they bind more strongly. Well-characterized disease phenotypes in humans and higher vertebrates that arise from dis- turbed ABP function generally interfere with one or more of the following cellular functions of the actin cytoskeleton (1) polymerization and assembly dynamics; (2) formation of filament assemblies and networks; (3) interaction with membranes and the ECM; and/or (4) actomyosin-based contractility. In this overview, we turn our attention to these processes and general functions of the actin cytoskeleton, and how they contribute to disease 65 66 M. Krendel and E. M. De La Cruz development and pathology. We focus on specific, well-characterized examples, understanding that proteins with related functions in different organisms will likely contribute to analogous phenotypes, depending on the specific function of the actin cytoskeleton in those cells. Modulation of Actin Assembly and Polymerization Dynamics Because actin filaments elongate only from their ends, any process that changes the number of accessible filament ends will influence actin assembly driven processes. The Arp2/3 complex and members of the formin protein family nucleate actin filaments. Isolated Arp2/3 complex is a weak nucleator, but is activated by regulatory, WASp/SCAR family proteins that bind the complex and promote formation of the active, nucleating conformation. Rho family GTPase proteins activate WASp/SCAR family proteins in response to extracellular cues and signals. Formin activity is also regulated through Rho GTPases. Capping proteins bind terminal subunits at filament ends and reduce the rates of filament polymerization and depolymerization. The affinity of capping proteins for filament ends depends on calcium, phospholipid binding (see chapter by dos Remedios), and various other signals. Cofilin (see chapter by Maloney et al.) and members of the villin/gelsolin family (see chapter by Burtnick and Robinson) sever filaments and increase the number of filament ends. The actin monomer concentration determines whether these ends grow or shrink. Cofilin is regulated through phosphorylation by LIM kinase, which in turn, is regulated by Rho GTPases through PAK kinase activity. Actin filament binding and severing activity of villin/gelsolin family proteins is regulated by calcium and phospholipids. Thus, both actin polymerization and depolymerization are regulated via complex signaling pathways (Fig. 1), which create numerous possibilities for finely tuned modulation of cytoskeletal organization and cell motility. Inactivation or upregulation of any of the components of these pathways can result in disruption of actin organization and cell motility. Such modulation of actin assembly is associated with a number of diverse disease phenotypes and pathologies. Pathogenic Bacteria and Viruses Many cell pathogens (see chapter by Bearer) do not disrupt ABP function, but rather utilize normal functions of proteins involved in regulating actin polymerization to promote infectivity. Listeria, Shigella, Rickettsia, Burkholderia, and Mycobacterium bacterial pathogens enter human cells and use growing actin filament “tails” to propel themselves through the 5. Overview: Actin-Binding Protein Function 67 FIG. 1. Regulation of actin dynamics and organization by actin-binding proteins. Assembly of actin monomers into a filament is dependent on actin monomer concentration (the fast-growing, or barbed, end of the filament assembles at a lower critical concentration than the slow-growing, or pointed, end) and on ATP exchange and hydrolysis (ATP-bound monomers tend to associate with the filament and ADP-bound monomers readily dissociate from the filament). These properties of actin result in the process known as treadmilling, in which actin monomers are added to the barbed end of the filament and removed from the pointing end, allowing the barbed end of the filament to exert pushing force against the plasma membrane or other structures. Actin assembly and disassembly are regulated by actin-binding proteins that modulate effective concentration of actin monomers (monomer sequestering proteins) or exchange of monomer- bound nucleotides, as well as by proteins that prevent monomer addition to the barbed end (end capping proteins) and promote filament depolymerization (filament severing and depolymerizing proteins). Assembled filaments are organized into rigid structures by filament bundling and crosslinking proteins. Reproduced with permission from Macmillan Publishers Ltd (Revenu et al. 2004) cytoplasm (Fig. 2) and to induce protrusions at the plasma membrane that allow invasion of adjacent cells (Stevens, Galyov and Stevens 2006). Enteropathogenic E. coli and enterohemorrhagic E. coli do not enter host cells, but instead induce formation of large membrane protrusions filled with actin filaments, called “pedestals.” Pedestals move along the cell surface and may promote spreading of bacteria from one enterocyte to another. A similar strategy for movement along the cell surface using submembrane actin assemblies is employed by Vaccinia virus to move from one cell to another, whereas for intracellular movement this virus utilizes microtubule- dependent transport (Smith, Murphy and Law 2003). Some bacterial pathogens, such as Listeria and Rickettsia, express on their surface proteins that recruit Arp2/3 complex and serve as nucleation-promoting 68 M. Krendel and E. M. De La Cruz FIG. 2. Bacterial pathogens hijack cellular actin and actin-binding proteins. Upon cell entry, pathogenic bacteria Listeria monocytogenes (a) and Rickettsia conorii (b) induce formation of actin tails, shown here in the transmission electron micrographs. Actin tails induced by these intracellular pathogens represent either branched and crosslinked meshworks of short actin filaments (Listeria) or parallel bundles of long actin filaments (Rickettsia). Actin filaments are more densely packed near the surface of the bacterium than in the rest of the tail (boxes). (c) Attachment of enteropathogenic Escherichia coli to the surface of enterocytes promotes formation of actin-rich pedestals that can be observed by scanning electron microscopy. Formation of actin tails and pedestals relies on the functions of intracellular actin-binding proteins that regulate actin assembly and cross-linking. Reproduced with permission from Macmillan Publishers Ltd (Stevens, Galyov and Stevens 2004), Company of Biologists (Gouin et al. 1999) (a, b), and Wiley Interscience (Rottner et al. 2004) (c) factors. Others, including Shigella, Mycobacterium marinum, Vaccinia, and E. coli, recruit endogenous, host cellular nucleation-promoting factors, prima- rily N-WASp. Both RickA protein of Rickettsia and ActA protein of Listeria contain protein domains similar to those in the WASp family proteins: proline- rich motifs, WH2 domains that bind actin monomers, and a CA domain that interacts with the Arp2/3 complex. Actin tails of Shigella, Listeria, and Mycobacterium consist of short, orthog- onally crosslinked actin filament networks, whereas actin tails of Rickettsia contain long parallel bundles of actin filaments, similar to the actin bundles of microvilli or filopodia. It is unclear what factors are responsible for this differ- ence in actin tail organization. One of the explanations that has been proposed for the unusual morphology of Rickettsia actin tails is that VASP protein, which is known to promote formation of long actin filaments and decrease the density of actin branches, is located along the entire length of Rickettsia actin tail, and not just on the surface of the bacteria, as is the case for Listeria. Defects in Cell Motility Induced by Changes in Expression or Activity of ABPs Mutations in the gene encoding WASp that abrogate expression cause Wiskott–Aldrich

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