The human actin-related protein hArp5: Nucleo-cytoplasmic shuttling and involvement in DNA repair

Certain actin-related proteins (Arps) of budding yeast are localized in the nucleus, and have essential roles as stoichiometric components of histone acetyltransferase (HAT) and chromatin remodeling complexes. On the other hand, identification of vertebrate nuclear Arps and their functional analyses are just beginning. We show that human Arp5 (hArp5) proteins are localized in the nucleus, and that arp5Δ yeast cells are partially complemented by hArp5. Thus, hArp5 is a novel member of the nuclear Arps of vertebrates, which possess evolutionarily conserved functions from yeast to humans. We show here that hArp5 shuttles between the nucleus and the cytoplasm. Furthermore, after the induction of DNA double strand breaks (DSB), cell growth and the accumulation of phosphorylated histone H2AX (γ-H2AX) are impaired by hArp5 depletion. Association of hArp5 with the hIno80 chromatin remodeling enzyme and decrease of chromatin-bound hIno80 by hArp5-depletion indicate that hArp5 may have a role in the recruitment of the hINO80 complex to chromatin. Overexpression of hArp5 and hIno80 enhanced γ-H2AX accumulation. These observations suggest that hArp5 is involved in the process of DSB repair through the regulation of the chromatin remodelling machinery.     

Novel actin-related proteins in vertebrates: similarities of structure and expression pattern to Arp6 localized on Drosophila heterochromatin

Actin-related proteins (Arps), which share a basal structure with actin isoforms but possess different functions, have been identified in a wide variety of organisms. The Arps are classified into subfamilies based on the relatedness of their sequences and functions. Recently, several Arp subfamilies have been shown to be localized in the nucleus and included in protein complexes involved in the organization of chromatin structure, for example, in chromatin remodeling and histone acetyltransferase complexes. A member of the Arp6 subfamily in Drosophila, dArp6, is localized on centric heterochromatin together with heterochromatin protein 1 (HP1). We have identified the first examples of the Arp6 subfamily in vertebrates, novel human and chicken Arps, hArp6 and gArp6, respectively. They are closely related to each other (98% similar) and show apparent similarity to dArp6 (70%). In addition, the hArp6 gene possesses evolutionarily conserved exon/intron structures compared with genes for members of the Arp6 subfamily in invertebrates. Like Drosophila dArp6, gArp6 is expressed abundantly in the early developmental stages, when heterochromatin condensation and nuclear maturation occur. The finding of a conserved Arp6 subfamily in vertebrates will contribute to the understanding of molecular mechanisms of heterochromatin organization.     

Involvement of actin-related proteins in ATP-dependent chromatin remodeling

Actin-related proteins (Arps) and conventional actin are enigmatic components of many chromatin-remodeling enzyme complexes. The yeast INO80 ATP-dependent chromatin-remodeling complex contains stoichiometric amounts of Arp4, Arp5, Arp8, and actin. Here we have revealed functions of Arp5 and Arp8 by analysis of mutants. arp5 Delta and arp8 Delta mutants display an ino80 Delta phenotype. Purification of INO80 complexes from arp5 Delta and arp8 Delta cells shows that protein complexes remain intact but are compromised for INO80 ATPase activity, DNA binding, and nucleosome mobilization. The INO80 (arp8 Delta) complex is strikingly deficient, not only for the Arp8 subunit, but also for Arp4 and actin, suggesting an ordered assembly of Arps. Binding of Arp8 to the INO80 complex requires an N-terminal region of Ino80 adjacent to the conserved ATPase domain. GST-Arp8 binds preferentially to histones H3 and H4 in vitro, suggesting a histone chaperone function. These findings show direct involvement of Arps in the chromatin-remodeling process.     

Structural biochemistry of nuclear actin-related proteins 4 and 8 reveals their interaction with actin

Nuclear actin and actin-related proteins (Arps) are integral components of various chromatin-remodelling complexes. Actin in such nuclear assemblies does not form filaments but associates in defined complexes, for instance with Arp4 and Arp8 in the INO80 remodeller. To understand the relationship between nuclear actin and its associated Arps and to test the possibility that Arp4 and Arp8 help maintain actin in defined states, we structurally analysed Arp4 and Arp8 from Saccharomyces cerevisiae and tested their biochemical effects on actin assembly and disassembly. The solution structures of isolated Arp4 and Arp8 indicate them to be monomeric and the crystal structure of ATP-Arp4 reveals several differences to actin that explain why Arp4 does not form filaments itself. Remarkably, Arp4, assisted by Arp8, influences actin polymerization in vitro and is able to depolymerize actin filaments. Arp4 likely forms a complex with monomeric actin via the barbed end. Our data thus help explaining how nuclear actin is held in a discrete complex within the INO80 chromatin remodeller.     

Structure of Actin-related protein 8 and its contribution to nucleosome binding

Nuclear actin-related proteins (Arps) are subunits of several chromatin remodelers, but their molecular functions within these complexes are unclear. We report the crystal structure of the INO80 complex subunit Arp8 in its ATP-bound form. Human Arp8 has several insertions in the conserved actin fold that explain its inability to polymerize. Most remarkably, one insertion wraps over the active site cleft and appears to rigidify the domain architecture, while active site features shared with actin suggest an allosterically controlled ATPase activity. Quantitative binding studies with nucleosomes and histone complexes reveal that Arp8 and the Arp8–Arp4–actin-HSA sub-complex of INO80 strongly prefer nucleosomes and H3–H4 tetramers over H2A–H2B dimers, suggesting that Arp8 functions as a nucleosome recognition module. In contrast, Arp4 prefers free (H3–H4)₂ over nucleosomes and may serve remodelers through binding to (dis)assembly intermediates in the remodeling reaction.     

The Arp2/3 complex: a multifunctional actin organizer

The actin-related proteins (Arps) constitute a recently characterized family of proteins, many of which function as members of multiprotein complexes. The discovery that two family members, Arp2 and Arp3, act as multifunctional organizers of actin filaments in all eukaryotes has generated much excitement. Over the past two years, newly discovered properties of the Arp2/3 complex have suggested a central role in the control of actin polymerization. First, it promotes actin assembly on the surface of the motile intracellular pathogen Listeria monocytogenes. Second, it can nucleate and cross-link actin filaments in vitro. Third, it localizes with dynamic actin-rich spots of mammalian cells suggesting a role in protrusion; it is found in cortical actin patches in the budding and fission yeasts where it may control patch movement and cortical actin function. Clearly, the complex has a central role in actin cytoskeletal function and will be the subject of much research in the coming years.     

Sequences, structural models, and cellular localization of the actin- related proteins Arp2 and Arp3 from Acanthamoeba

We cloned and sequenced the two actin-related proteins (Arps) present in the profilin-binding complex of Acanthamoeba (Machesky, L. M., S. J. Atkinson, C. Ampe, J. Vandekerckhove, and T. D. Pollard. 1994, J. Cell Biol. 127:107-115). The sequence of Arp2 is more similar to other Arp2s than to actin, while the sequence of Arp3 is more similar to other Arp3s than to actin. Phylogenetic analysis of all known Arps demonstrates that most group into three major families, which are likely to be shared across all eukaryotic phyla. Together with conventional actins, the Arps form a larger family distinct from structurally related ATPases such as Hsp70's and sugar kinases. Atomic models of the Arps based on their sequences and the structure of actin provide some clues about function. Both Arps have atoms appropriately placed to bind ATP and divalent cation. Arp2, but not Arp3, has a conserved profilin-binding site. Neither Arp has the residues required to copolymerize with actin, but an Arp heterodimer present in the profilin-binding complex might serve as a pointed end nucleus for actin polymerization. Both Acanthamoeba Arps are soluble in cell homogenates, and both are concentrated in the cortex of Acanthamoeba. The cellular concentrations are 1.9 microM Arp2 and 5.1 microM Arp3, substoichiometric to actin (200 microM) but comparable to many actin- binding proteins.     

Characterization of a human SWI2/SNF2 like protein hINO80: demonstration of catalytic and DNA binding activity

The proteins belonging to SWI2/SNF2 family of DNA dependent ATPases are important members of the chromatin remodeling complexes that are implicated in epigenetic control of gene expression. We have identified a human gene with a putative DNA binding domain, which belongs to the INO80 subfamily of SWI2/SNF2 proteins. Here we report the cloning, expression, and functional activity of the domains from hINO80 gene both in terms of the DNA dependent ATPase as well as DNA binding activity. A differential expression of the various domains within this gene is detected in human tissues while a ubiquitous expression is detected in mice. The intranuclear localization is demonstrated using antibodies directed against the DBINO domain of hINO80.     

Molecular recognition of the Tes LIM2-3 domains by the actin-related protein Arp7A

Actin-related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized "orphan" Arps, which appear to be mostly testis-specific. Here we show that Arp7A, which has 43% sequence identity with β-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65-residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1-65(Arp7A)·LIM2-3(Tes)·EVH1(Mena) complex reveals that residues 28-49 of Arp7A contact the LIM2-3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2-3 junction are critical for the Arp7A-Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.     

Actin-related protein Arp4 functions in kinetochore assembly

The actin-related proteins (Arps) comprise a conserved protein family. Arp4p is found in large multisubunits of the INO80 and SWR1 chromatin remodeling complexes and in the NuA4 histone acetyltransferase complex. Here we show that arp4 (arp4S23A/D159A) temperature-sensitive cells are defective in G2/M phase function. arp4 mutants are sensitive to the microtubule depolymerizing agent benomyl and arrest at G2/M phase at restrictive temperature. Arp4p is associated with centromeric and telomeric regions throughout cell cycle. Ino80p, Esa1p and Swr1p, components of the INO80, NuA4 and SWR1 complexes, respectively, also associate with centromeres. The association of many kinetochore components including Cse4p, a component of the centromere nucleosome, Mtw1p and Ctf3p is partially impaired in arp4 cells, suggesting that the G2/M arrest of arp4 mutant cells is due to a defect in formation of the chromosomal segregation apparatus.     

Actin-related proteins (Arps): conformational switches for chromatin-remodeling machines?

The actin superfamily of ATPases includes cytoskeletal actins, the stress 70 proteins (e.g. hsc70), sugar kinases, glycerol kinase, and several prokaryotic cell cycle proteins. Although these proteins share limited sequence identity, they all appear to maintain a similar tertiary structure, the "actin fold", which may serve to couple ATP hydrolysis to protein conformational changes. Recently, an actin-related protein (Arp) subfamily has been identified based on sequence homology to conventional actin. Although some Arps are clearly involved in cytoskeletal functions, both actin and/or Arps have been found as stoichiometric subunits of several nuclear chromatin-remodeling enzymes. Here we present two related models in which actin and/or Arps function as conformational switches that control either the activity or the assembly of chromatin-remodeling machines.     

Vertebrate Arp6, a novel nuclear actin-related protein, interacts with heterochromatin protein 1

Actin-related proteins (Arps) were recently shown to contribute to the organization and regulation of chromatin structures. The nuclear functions of Arps have been investigated principally in budding yeast in which six of the ten Arp subfamilies are localized in the nucleus. In vertebrates, only two isoforms of Arp4 have so far been identified as showing localization to the nucleus. Here we show the predominant nuclear localization of another Arp subfamily, Arp6, in vertebrate cells. Vertebrate Arp6 directly interacted with heterochromatin protein 1 (HP1) orthologs and the two proteins colocalized in pericentric heterochromatin. Yeast Arp6 is involved in telomere silencing, while Drosophila Arp6 is localized in the pericentric heterochromatin. Our data strongly suggest that Arp6 has an evolutionarily conserved role in heterochromatin formation and also provide new insights into the molecular organization of heterochromatin.     

Identification of a novel actin-related protein in Tetrahymena cilia

Actin is an ancient cytoskeletal protein that plays many essential roles in cell motility. In eukaryotes, its gene belongs to a highly conserved gene family, while the protein is also a member of an actin superfamily comprising various kinds of actin-related proteins (Arps). A ciliate, Tetrahymena, has a unique conventional actin. Data from the TIGR Tetrahymena genome project and our own research suggest the existence of 12 actin-like sequences: four conventional actins, two of Arp4, one each of Arp1, Arp2, Arp3, Arp5, and Arp6, and a novel actin-related protein, tArp. We cloned the entire cDNA sequences of Tetrahymena Arp2 (tArp2), Tetrahymena Arp3 (tArp3), and tArp for the work described herein. In phylogenetic analyses, tArp was not included in any Arp subfamily. Unlike other known Arps, tArp localizes in cilia, and its expression was upregulated after deciliation. To see the precise localization of tArp, cilia were fractionated and analyzed using specific antibodies. tArp was observed preferentially in the "outer-doublet" fraction, while actin was found in the "crude-dynein" fraction. In immunoelectron microscopy, most of the gold particles were found either on the outer-doublet or central-pair microtubules. These results suggest that tArp is a ciliary component and that it has a unique function in the formation and maintenance of cilia.     

Brain-specific expression of the nuclear actin-related protein ArpNalpha and its involvement in mammalian SWI/SNF chromatin remodeling complex

Actin-related proteins share significant homology with conventional actins and are classified into subfamilies based on the similarity of their sequences and functions. The Arp4 subfamily of Arps is localized in the nucleus, and a mammalian isoform, ArpNbeta (also known as BAF53), is a component of the chromatin remodeling and histone acetyltransferase complexes. Another isoform identified in humans, ArpNalpha has scarcely been characterized yet. We identified mouse ArpNalpha, and showed that ArpNalpha is more similar between humans and mice than ArpNbeta. No difference was observed between ArpNalpha and beta in subcellular localization and interaction with BRM, which is an ATPase subunit of mammalian SWI/SNF chromatin remodeling complex. However, ArpNalpha was expressed exclusively in the brain and its expression was induced during neural differentiation of P19 mouse embryonic carcinoma cells. ArpNalpha is the first brain-specific component of a chromatin remodeling complex to be identified, suggesting that ArpNalpha has conserved and important roles in the differentiation of neural cells through regulation of chromatin structure.     

Direct involvement of yeast type I myosins in Cdc42-dependent actin polymerization

The generation of cortical actin filaments is necessary for processes such as cell motility and cell polarization. Several recent studies have demonstrated that Wiskott-Aldrich syndrome protein (WASP) family proteins and the actin-related protein (Arp) 2/3 complex are key factors in the nucleation of actin filaments in diverse eukaryotic organisms. To identify other factors involved in this process, we have isolated proteins that bind to Bee1p/Las17p, the yeast WASP-like protein, by affinity chromatography and mass spectroscopic analysis. The yeast type I myosins, Myo3p and Myo5p, have both been identified as Bee1p-interacting proteins. Like Bee1p, these myosins are essential for cortical actin assembly as assayed by in vitro reconstitution of actin nucleation sites in permeabilized yeast cells. Analysis using this assay further demonstrated that the motor activity of these myosins is required for the polymerization step, and that actin polymerization depends on phosphorylation of myosin motor domain by p21-activated kinases (PAKs), downstream effectors of the small guanosine triphosphatase, Cdc42p. The type I myosins also interact with the Arp2/3 complex through a sequence at the end of the tail domain homologous to the Arp2/3-activating region of WASP-like proteins. Combined deletions of the Arp2/3-interacting domains of Bee1p and the type I myosins abolish actin nucleation sites at the cortex, suggesting that these proteins function redundantly in the activation of the Arp2/3 complex.