Structural relationships of actin-binding proteins.

The sequences of a large number of actin-binding proteins have been compared. These findings, together with the results of protein-chemical analysis, peptide synthesis and site-directed and deletion mutagenesis, have led to the assignment of actin-binding sites. Within these segments, small actin-binding motifs have been delineated. Most actin-binding proteins interact with actin subdomain-1 but our analyses reveal neither primary nor secondary structure homology among these proteins, suggesting that actin binding does not follow simple structural principles.     

Roles of actin binding proteins in mammalian oocyte maturation and beyond

Actin nucleation factors, which promote the formation of new actin filaments, have emerged in the last decade as key regulatory factors controlling asymmetric division in mammalian oocytes. Actin nucleators such as formin-2, spire, and the ARP2/3 complex have been found to be important regulators of actin remodeling during oocyte maturation. Another class of actin-binding proteins including cofilin, tropomyosin, myosin motors, capping proteins, tropomodulin, and Ezrin-Radixin-Moesin proteins are thought to control actin cytoskeleton dynamics at various steps of oocyte maturation. In addition, actin dynamics controlling asymmetric-symmetric transitions after fertilization is a new area of investigation. Taken together, defining the mechanisms by which actin-binding proteins regulate actin cytoskeletons is crucial for understanding the basic biology of mammalian gamete formation and pre-implantation development.     

Identification of Obscure yet Conserved Actin-Associated Proteins in Giardia lamblia

Consistent with its proposed status as an early branching eukaryote, Giardia has the most divergent actin of any eukaryote and lacks core actin regulators. Although conserved actin-binding proteins are missing from Giardia, its actin is utilized similarly to that of other eukaryotes and functions in core cellular processes such as cellular organization, endocytosis, and cytokinesis. We set out to identify actin-binding proteins in Giardia using affinity purification coupled with mass spectroscopy (multidimensional protein identification technology [MudPIT]) and have identified >80 putative actin-binding proteins. Several of these have homology to conserved proteins known to complex with actin for functions in the nucleus and flagella. We validated localization and interaction for seven of these proteins, including 14-3-3, a known cytoskeletal regulator with a controversial relationship to actin. Our results indicate that although Giardia lacks canonical actin-binding proteins, there is a conserved set of actin-interacting proteins that are evolutionarily indispensable and perhaps represent some of the earliest functions of the actin cytoskeleton.     

A novel role for 14-kDa phosphohistidine phosphatase in lamellipodia formation

Cell migration involves dynamic regulation of the actin cytoskeleton, which exhibits rapid actin polymerization at the leading edge of migrating cells. This process relies on regulated recruitment of actin nucleators and actin-binding proteins to the leading edge to polymerize new actin filaments. Many of these proteins have been identified, including the actin-related protein (Arp) 2/3 complex, which has emerged as the core player in the initiation of actin polymerization. However, the functional coordination of these proteins is unclear. Previously, we have demonstrated that the 14-kDa phosphohistidine phosphatase (PHP14) is involved in cell migration regulation and affects actin cytoskeleton reorganization. Here, we show that PHP14 may regulate actin remodeling directly and play an important role in dynamic regulation of the actin cytoskeleton. We observed a colocalization of PHP14 with Arp3 and F-actin at the leading edge of migrating cells. Moreover, PHP14 was recruited to the actin remodeling sites in parallel with Arp3 during lamellipodia formation. Furthermore, PHP14 knockdown impaired Arp3 localization at the leading edge of lamellipodia, as well as lamellipodia formation. Most importantly, we found that PHP14 was a novel F-actin-binding protein, displaying an Arp2/3-dependent localization to the leading edge. Collectively, our results indicated a crucial role for PHP14 in the dynamic regulation of the actin cytoskeleton and cell migration.     

Biochemical and cell biological analysis of actin in the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans has long been a useful model organism for muscle research. Its body wall muscle is obliquely striated muscle and exhibits structural similarities with vertebrate striated muscle. Actin is the core component of the muscle thin filaments, which are highly ordered in sarcomeric structures in striated muscle. Genetic studies have identified genes that regulate proper organization and function of actin filaments in C. elegans muscle, and sequence of the worm genome has revealed a number of conserved candidate genes that may regulate actin. To precisely understand the functions of actin-binding proteins, such genetic and genomic studies need to be complemented by biochemical characterization of these actin-binding proteins in vitro. This article describes methods for purification and biochemical characterization of actin from C. elegans. Although rabbit muscle actin is commonly used to characterize actin-binding proteins from many eukaryotic organisms, we detect several quantitative differences between C. elegans actin and rabbit muscle actin, highlighting that use of actin from an appropriate source is important in some cases. Additionally, we describe probes for cell biological analysis of actin in C. elegans.     

Coronin proteins as multifunctional regulators of the cytoskeleton and membrane trafficking

Coronins constitute an evolutionarily conserved family of WD-repeat actin-binding proteins, which can be clearly classified into two distinct groups based on their structural features. All coronins possess a conserved basic N-terminal motif and three to ten WD repeats clustered in one or two core domains. Dictyostelium and mammalian coronins are important regulators of the actin cytoskeleton, while the fly Dpod1 and the yeast coronin proteins crosslink both actin and microtubules. Apart from that, several coronins have been shown to be involved in vesicular transport. C. elegans POD-1 and Drosophila coro regulate the actin cytoskeleton, but also govern vesicular trafficking as indicated by mutant phenotypes. In both organisms, defects in cytoskeleton and trafficking lead to severe developmental defects ranging from abnormal cell division to aberrant formation of morphogen gradients. Finally, mammalian coronin 7 appears not to execute any cytoskeleton-related functions, but rather participates in regulating Golgi trafficking. Here, we review recent data providing more insight into molecular mechanisms underlying the regulation of F-actin structures, cytoskeletal rearrangements and intracellular membrane transport by coronin proteins and the way that they might link cytoskeleton with trafficking in development and disease.     

Intrasteric inhibition mediates the interaction of the I/LWEQ module proteins Talin1, Talin2, Hip1, and Hip12 with actin

The I/LWEQ module superfamily is a class of actin-binding proteins that contains a conserved C-terminal actin-binding element known as the I/LWEQ module. I/LWEQ module proteins include the metazoan talins, the cellular slime mold talin homologues TalA and TalB, fungal Sla2p, and the metazoan Sla2 homologues Hip1 and Hip12 (Hip1R). These proteins possess a similar modular organization that includes an I/LWEQ module at their C-termini and either a FERM domain or an ENTH domain at their N-termini. As a result of this modular organization, I/LWEQ module proteins may serve as linkers between cellular compartments, such as the plasma membrane and the endocytic machinery, and the actin cytoskeleton. Previous studies have shown that I/LWEQ module proteins bind to F-actin. In this report, we have determined the affinity of the I/LWEQ module proteins Talin1, Talin2, huntingtin interacting protein-1 (Hip1), and the Hip1-related protein (Hip1R/Hip12) for F-actin and identified a conserved structural element that interferes with the actin binding capacity of these proteins. Our data support the hypothesis that the actin-binding determinants in native talin and other I/LWEQ module proteins are cryptic and indicate that the actin binding capacities of Talin1, Talin2, Hip1, and Hip12 are regulated by intrasteric occlusion of primary actin-binding determinants within the I/LWEQ module. We have also found that the I/LWEQ module contains a dimerization motif and stabilizes actin filaments against depolymerization. This activity may contribute to the function of talin in cell adhesion and the roles of Hip1, Hip12 (Hip1R), and Sla2p in endocytosis.     

Evidence for a Conformational Change in Actin Induced by Fimbrin (N375) Binding

Fimbrin belongs to a superfamily of actin cross-linking proteins that share a conserved 27-kD actin-binding domain. This domain contains a tandem duplication of a sequence that is homologous to calponin. Calponin homology (CH) domains not only cross-link actin filaments into bundles and networks, but they also bind intermediate filaments and some signal transduction proteins to the actin cytoskeleton. This fundamental role of CH domains as a widely used actin-binding domain underlines the necessity to understand their structural interaction with actin. Using electron cryomicroscopy, we have determined the three-dimensional structure of F-actin and F-actin decorated with the NH₂-terminal CH domains of fimbrin (N375). In a difference map between actin filaments and N375-decorated actin, one end of N375 is bound to a concave surface formed between actin subdomains 1 and 2 on two neighboring actin monomers. In addition, a fit of the atomic model for the actin filament to the maps reveals the actin residues that line, the binding surface. The binding of N375 changes actin, which we interpret as a movement of subdomain 1 away from the bound N375. This change in actin structure may affect its affinity for other actin-binding proteins and may be part of the regulation of the cytoskeleton itself. Difference maps between actin and actin decorated with other proteins provides a way to look for novel structural changes in actin.     

Spire and Cordon-bleu: multifunctional regulators of actin dynamics

WASP-homology 2 (WH2) domains, which were first identified in the WASP/Scar (suppressor of cAMP receptor)/WAVE (WASP-family verprolin homologous protein) family of proteins, are multifunctional regulators of actin assembly. Two recently discovered actin-binding proteins, Spire and Cordon-bleu (Cobl), which have roles in axis patterning in developmental processes, use repeats of WH2 domains to generate a large repertoire of novel regulatory activities, including G-actin sequestration, actin-filament nucleation, filament severing and barbed-end dynamics regulation. We describe how these multiple functions selectively operate in a cellular context to control the dynamics of the actin cytoskeleton. In vivo, Spire and Cobl can synergize with other actin regulators. As an example, we outline potential methods to gain insight into the functional basis for reported genetic interactions among Spire, profilin and formin.     

Actin deficiency induces cofilin phosphorylation: proteome analysis of HeLa cells after beta-actin gene silencing

Actin-binding proteins regulate the dynamic structure and function of actin filaments in the cell. Much is known about how manipulation of the actin-binding proteins affects the structure and function of actin filaments; however, little is known about how manipulation of actin in the cell affects actin-binding proteins. We addressed this question by utilizing two technologies: RNA interference and 2-dimensional gel electrophoresis. We knocked down beta-actin expression in HeLa cells using short interfering RNA and applied 2-DGE to examine alterations in the HeLa cell proteome. We revealed a 2-5 fold increases of four protein spots on 2-D gels and identified these proteins by mass spectrometry. Three of the four proteins were actin-binding proteins, including cofilin, which promotes both disassembly and assembly of actin filaments but becomes inactivated when phosphorylated. Further examination revealed that the cofilin total protein level barely increased, but the phosphorylated cofilin level increased dramatically in HeLa cells after beta-actin siRNA treatment. These results suggest that in response to siRNA-induced beta-actin deficiency HeLa cells inactivate cofilin by phosphorylation rather than down-regulate its protein expression level. This study also demonstrates that the combination of RNA interference and 2-dimensional gel electrophoresis technologies provides a valuable method to study protein interactions in a specific cellular pathway.     

Dissecting regulatory networks of filopodia formation in a Drosophila growth cone model

F-actin networks are important structural determinants of cell shape and morphogenesis. They are regulated through a number of actin-binding proteins. The function of many of these proteins is well understood, but very little is known about how they cooperate and integrate their activities in cellular contexts. Here, we have focussed on the cellular roles of actin regulators in controlling filopodial dynamics. Filopodia are needle-shaped, actin-driven cell protrusions with characteristic features that are well conserved amongst vertebrates and invertebrates. However, existing models of filopodia formation are still incomplete and controversial, pieced together from a wide range of different organisms and cell types. Therefore, we used embryonic Drosophila primary neurons as one consistent cellular model to study filopodia regulation. Our data for loss-of-function of capping proteins, enabled, different Arp2/3 complex components, the formin DAAM and profilin reveal characteristic changes in filopodia number and length, providing a promising starting point to study their functional relationships in the cellular context. Furthermore, the results are consistent with effects reported for the respective vertebrate homologues, demonstrating the conserved nature of our Drosophila model system. Using combinatorial genetics, we demonstrate that different classes of nucleators cooperate in filopodia formation. In the absence of Arp2/3 or DAAM filopodia numbers are reduced, in their combined absence filopodia are eliminated, and in genetic assays they display strong functional interactions with regard to filopodia formation. The two nucleators also genetically interact with enabled, but not with profilin. In contrast, enabled shows strong genetic interaction with profilin, although loss of profilin alone does not affect filopodia numbers. Our genetic data support a model in which Arp2/3 and DAAM cooperate in a common mechanism of filopodia formation that essentially depends on enabled, and is regulated through profilin activity at different steps.     

Impacts of dystrophin and utrophin domains on actin structural dynamics: implications for therapeutic design

We have used time-resolved phosphorescence anisotropy (TPA) of actin to evaluate domains of dystrophin and utrophin, with implications for gene therapy in muscular dystrophy. Dystrophin and its homolog utrophin bind to cytoskeletal actin to form mechanical linkages that prevent muscular damage. Because these proteins are too large for most gene therapy vectors, much effort is currently devoted to smaller constructs. We previously used TPA to show that both dystrophin and utrophin have a paradoxical effect on actin rotational dynamics-restricting amplitude while increasing rate, thus increasing resilience, with utrophin more effective than dystrophin. Here, we have evaluated individual domains of these proteins. We found that a "mini-dystrophin," lacking one of the two actin-binding domains, is less effective than dystrophin in regulating actin dynamics, correlating with its moderate effectiveness in rescuing the dystrophic phenotype in mice. In contrast, we found that a "micro-utrophin," with more extensive internal deletions, is as effective as full-length dystrophin in the regulation of actin dynamics. Each of utrophin's actin-binding domains promotes resilience in actin, while dystrophin constructs require the presence of both actin-binding domains and the C-terminal domain for full function. This work supports the use of a utrophin template for gene or protein therapy designs. Resilience of the actin-protein complex, measured by TPA, correlates remarkably well with previous reports of functional rescue by dystrophin and utrophin constructs in mdx mice. We propose the use of TPA as an in vitro method to aid in the design and testing of emerging gene therapy constructs.     

Separases: biochemistry and function

Tight regulation of cell cycle is of critical importance for eukaryotic biology and is achieved through a combined action of a large number of highly specialized proteins. Separases are evolutionarily conserved caspase-like proteases playing a crucial role in cell cycle regulation, as they execute sister chromatid separation at metaphase to anaphase transition. In contrast to extensively studied yeast and metazoan separases, very little is known about the role of separases in plant biology. Here we describe the molecular mechanisms of separase-mediated chromatid segregation in yeast and metazoan models, discuss new emerging but less-understood functions of separases and highlight major gaps in our knowledge about plant separases.     

Holding back the microfilament--structural insights into actin and the actin-monomer-binding proteins of apicomplexan parasites

Parasites from the phylum Apicomplexa are responsible for several major diseases of man, including malaria and toxoplasmosis. These highly motile protozoa use a conserved actomyosin-based mode of movement to power tissue traversal and host cell invasion. The mode termed as 'gliding motility' relies on the dynamic turnover of actin, whose polymerisation state is controlled by a markedly limited number of identifiable regulators when compared with other eukaryotic cells. Recent studies of apicomplexan actin regulator structure-in particular those of the core triad of monomer-binding proteins, actin-depolymerising factor/cofilin, cyclase-associated protein/Srv2, and profilin-have provided new insights into possible mechanisms of actin regulation in parasite cells, highlighting divergent structural features and functions to regulators from other cellular systems. Furthermore, the unusual nature of apicomplexan actin itself is increasingly coming into the spotlight. Here, we review recent advances in understanding of the structure and function of actin and its regulators in apicomplexan parasites. In particular we explore the paradox between there being an abundance of unpolymerised actin, its having a seemingly increased potential to form filaments relative to vertebrate actin, and the apparent lack of visible, stable filaments in parasite cells.     

Involvement of the Rab27 binding protein Slac2c/MyRIP in insulin exocytosis

Rab27a is a GTPase associated with insulin-containing secretory granules of pancreatic beta-cells. Selective reduction of Rab27a expression by RNA interference did not alter granule distribution and basal secretion but impaired exocytosis triggered by insulin secretagogues. Screening for potential effectors of the GTPase revealed that the Rab27a-binding protein Slac2c/MyRIP is associated with secretory granules of beta-cells. Attenuation of Slac2c/MyRIP expression by RNA interference did not modify basal secretion but severely impaired hormone release in response to secretagogues. Although beta-cells express Myosin-Va, a potential partner of Slac2c/MyRIP, no functional link between the two proteins could be demonstrated. In fact, overexpression of the Myosin-Va binding domain of Slac2c/MyRIP did not affect granule localization and hormone exocytosis. In contrast, overexpression of the actin-binding domain of Slac2c/MyRIP led to a potent inhibition of exocytosis without detectable alteration in granule distribution. This effect was prevented by point mutations that abolish actin binding. Taken together our data suggest that Rab27a and Slac2c/MyRIP are part of a complex mediating the interaction of secretory granules with cortical actin cytoskeleton and participate to the regulation of the final steps of insulin exocytosis.     

Regulation of the Cortical Actin Cytoskeleton in Budding Yeast by Twinfilin,a Ubiquitous Actin Monomer-sequestering Protein

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)–twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.     

Functional genomic analysis reveals the utility of the I/LWEQ module as a predictor of protein:actin interaction

The I/LWEQ module is a conserved sequence that we have identified as an actin-binding motif in the metazoan focal adhesion protein talin and the yeast protein Sla2p. Both of these proteins are associated with the actin cytoskeleton in cells. To better establish the value of the I/LWEQ module for prediction of actin-binding function, we have applied a functional genomics approach. Analysis of the 23 available I/LWEQ module sequences supports the division of I/LWEQ protein superfamily into four groups: (1) metazoan talin, (2) Dictyostelium discoideum talin homologs TalA/B, (3) metazoan Hip1p, and (4) yeast Sla2p. We show here that I/LWEQ modules from each major group bind to F-actin in vitro and that GFP-fusion proteins of the I/LWEQ modules of talin and Sla2p bind to F-actin in vivo. Therefore, the presence of an I/LWEQ module is strongly predictive of protein-actin interactions. The structural and functional conservation of the I/LWEQ module across the phylogenetic distance between cellular slime molds and mammals implies that the role of the I/LWEQ module is to connect diverse proteins involved in distinct cellular processes, including cell adhesion, cytoskeletal organization, and cell differentiation, to the actin cytoskeleton.