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Plant actin-related proteins
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Vol.9 No.4 April 2004
Plant actin-related proteins Muthugapatti K. Kandasamy, Roger B. Deal, Elizabeth C. McKinney and Richard B. Meagher Department of Genetics, University of Georgia, Athens, GA 30602, USA
Actin-related proteins (ARPs) constitute a family of divergent and evolutionarily ancient eukaryotic proteins whose primary sequences display homology to conventional actins. Whereas actins play well-characterized cytoskeletal roles, the ARPs are implicated in various cellular functions in both the cytoplasm and in the nucleus. Cytoplasmic ARPs, for example, are known to participate in the assembly of branched actin filaments and dynein-mediated movement of vesicles in many eukaryotes. Nuclear ARPs, by contrast, are enigmatic components of various chromatin-modifying complexes involved in transcriptional regulation. Here, we review homologs to several known classes of ARPs and two distinct ARP classes in plants, and summarize recent work elucidating the biological functions of ARPs in eukaryotes. Actin-related proteins (ARPs), a novel family of highly conserved proteins that exhibit moderate sequence identity (17 – 60%) to conventional actins, have been found in all eukaryotic kingdoms [1– 3]. The ARPs and actins possess a common tertiary structure centered on the nucleotide-binding pocket known as the actin fold [4]. This fold undergoes major conformational changes in response to the 50 phosphorylation state of the adenine-containing nucleotide, and such changes are thought to be central to the function of the actin superfamily members, including ARPs. However, the various ARPs have divergent surface features caused by insertions, deletions and point mutations, suggesting that they are functionally distinct from actins and from each other [5]. Studies over the past decade have implicated ARPs in several roles in the cytoplasm, notably modulation of actin assembly and the microtubule-based motility of vesicles [2,6]. Recently, ARPs and actins have been discovered in the nucleus as integral components of many chromatin remodeling and histone acetyltransferase (HAT) complexes [7– 12]. These multiprotein complexes are involved in the modulation of chromatin structure, transcription and possibly DNA repair. The ARPs are grouped into several classes or subfamilies that are highly conserved in a wide range of eukaryotes, from yeast to plants and humans [13]. Each class or subfamily is distinguished by its degree of similarity to conventional actin. For example, the ten ARPs of budding yeast are classified into subfamilies 1 – 10, where ARP1 is the most and ARP10 is the least similar to yeast actin [14]. This yeast classification system Corresponding author: Muthugapatti K. Kandasamy ([email protected]).
is used to group the plant and human ARP sequences into various classes (Figure 1, neighbor joining tree). The ARPs exhibit structural and functional diversity, and different subcellular distribution (Table 1). ARP1 – ARP3 and ARP10 in yeast, and their homologs in other organisms ScARP1 ARP1 HsARP1 ScARP2 ARP2 HsARP2 OsARP2 AtARP2 ScARP3 HsARP3 OsARP3 AtARP3
ARP3 AtARP4A AtARP4 OsARP4 HsBAF53A ScARP4
Actin ScACT1 HsACTB AtACT2
ARP4 ARP5 OsARP5 AtARP5 HsARP5 ScARP5 OsARP6 ARP6 AtARP6 HsARP6X ScARP6
OsARP8 AtARP8
OsRAC1
Plant ARP8
ARP8, ARP9, ARP10 OsARP9 AtARP9 HsARP8 ScARP8 ScARP10 ScARP9 HsARP11 ARP7 ScARP7
100 changes
OsARP7 AtARP7
Plant ARP7 TRENDS in Plant Science
Figure 1. Phylogenetic relationships of actin and actin-related proteins. Arabidopsis, rice, human and yeast ARP sequences are compared in a neighbor-joining (NBJ) tree. Clades of ARP sequences are named based primarily on the nomenclature of yeast ARPs or distinct plant ARPs. The tree is rooted to highly conserved conventional actins. Because branch length is sensitive to the degree of sequence divergence in the NBJ tree, the accelerated rate of divergence among ARPs relative to actins is supported by the long horizontal distances among ARPs compared with the short distances among actins. The tree shows that Arabidopsis and rice contain eight ancient and highly divergent classes of ARPs, six classes that have homologs in yeast and human (ARP2– ARP6 and ARP9), and two classes that are specific to plants (ARP7 and ARP8). The accession numbers for the protein sequences used in the tree are as follows. Human: HsACTB, P02570; HsARP1, S29089; HsARP2A, AB64187; HsARP3, P32391; HsBAF53A, 096019; HsARP5, CAD37358; HsARP6X, Q9GZN1; HsARP8, Q9H981; HsARP11, Q9NZ32. Yeast: ScACT1, P02579; ScARP1, P38696; ScARP2, P32381; ScARP3, P47117; ScARP4, P80428; ScARP5, P53946; ScARP6, Q12509; ScARP7, Q12406; ScARP8, Q12386; ScARP9, Q055123; ScARP10, Q04549. Arabidopsis: AtACT2, Q96292 (for ARPs, see Table 2). Rice: OsRAC1, AA038821 (for ARPs, see Table 2). The various yeast, human and plant ARPs and actin are shown in black, purple and green, respectively. The major ARP classes are indicated adjacent to each clade of ARPs; the two novel plant-specific ARP classes are in green.
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Table 1. Summary of properties of Arabidopsis ARPs and those in other kingdoms Arabidopsis ARP – AtARP2 AtARP3 AtARP4 AtARP4A AtARP5 AtARP6 AtARP7 AtARP8 – AtARP9b – –
ARP class ARP1 ARP2 ARP3 ARP4 ARP4 ARP5 ARP6 Novel Novel ARP7 ARP8 ARP9 ARP10
Essential in yeast Yes Yes Yes Yes – Yes No – – Yes No Yes N/A
Localizationa MFY
Cytoplasm CytoplasmMFY CytoplasmMFY NucleusMYA N/A NucleusY NucleusFY NucleusA N/A NucleusY NucleusY NucleusY CytoplasmMY
Complexa DynactinMFY ARP2/3MFYA ARP2/3MFYA SWI/SNFM, INO80Y, NuA4Y, HATMY ? INO80Y Localized to heterochromatinF ? ? SWI/SNFY, RSCY INO80Y SWI/SNFY, RSCY DynactinM
Abbreviations: N/A, data not available; – , not represented in the genome; ?, unknown. a Superscripts (M,F,Y,A) refer to the type of organism (mammal, fly, yeast, Arabidopsis, respectively) in which the observation was made. This table is constructed from information from Refs [3,13]. b AtARP9 groups weakly with the ARP8 class.
are localized to the cytoplasm. These ARPs have functions distinct from those of actin, and each subclass has a similar role across phyla. By contrast, the divergent members of ARP subfamilies 4 –9 are localized to the nucleus in Saccharomyces cerevisiae [9]. Functional analyses of the ARP classes reveal that they, with other proteins, assemble into stable heteromultimeric protein complexes (Table 1). Few, if any, ARPs are known to function outside such complexes. ARP1 and ARP10 are major components of the dynactin complex, which is required for the orientation of mitotic spindle and nuclear migration [2]. ARP2 and ARP3, together with five other novel proteins, make up the ARP2/3 complex, which is implicated in capping, nucleation and branching of actin filaments [6,15,16]. Several of the divergent nuclear ARPs, such as ARP4, ARP5 and ARP7–ARP9, are components of different chromatin remodeling complexes (e.g. SWI/SNF, INO80, RSC, and NuA4) that are required for altering chromatin structure and/or transcriptional regulation [7,8,17,18]. These nuclear multiprotein complexes alter the nucleosome architecture either by covalent modification of the N-terminal histone tails (e.g. the HATs) or by ATP-dependent perturbations of histone-DNA interactions (e.g. SWI/SNF family of protein complexes) [19–21]. Thus, the biological functions of ARPs should be considered in light of their roles as components of larger macromolecular machines. Significant progress has been made in understanding the role of the highly conserved ARPs of classes 1–3 in many eukaryotes [2,22–24]. However, characterization of the function of highly divergent ARPs of animals and yeast has received relatively little attention [11,25–27]. In plants, which contain almost the full complement of the ARP gene family [3], we are just beginning to understand and appreciate the intriguing roles of ARPs in various cellular processes. Structure and evolution of the actin superfamily Actins and ARPs represent two ancient branches of a highly diverse superfamily of ATPases that also includes heat-shock proteins (HSPs) and their cognates (e.g. HSP60, HSC70), sugar kinases (e.g. hexokinase) of both prokaryotes and eukaryotes, and a range of bacterial ATP-binding proteins (e.g. MreB, FtsA, StbA) [1,23]. Although the actin and ARP proteins share no sequence homology with these www.sciencedirect.com
other proteins, all the actin superfamily members contain a common structural motif: the actin fold. This fold has been shown for many of these diverse proteins to bind ATP and/or ADP, and this binding allows major conformational changes in the protein. This reversible structural shift is thought to be crucial to the function of various actin family members (e.g. control of polymerization of F-actin and initiation of protein complex formation for the ARPs and HSPs). Also, the divergent nature of molecular surfaces for each protein is thought to indicate unique functions specific for each family member [1,23]. All the plant actins seem to have originated from a single green-algal ancestral actin gene [28]. Subsequent gene duplications and divergence in vascular plants have resulted in two classes, vegetative and reproductive, and at least five ancient subclasses of actin that pre-date the monocot– dicot separation. In the Crucifer Arabidopsis, further divergence resulted in a total of eight functional actin genes [29]. Both the vegetative and reproductive classes of actins in plants show distinct spatial and temporal patterns of expression. The vegetative actins are strongly expressed in all vegetative organs and cell types, and the reproductive actins are expressed predominantly in the mature pollen, the ovule and, in some cases, the embryo [29]. However, the ARPs of plants have an entirely different phylogeny from the actins and have a much older gene family structure. The ARP protein sequence tree in Figure 1 compares all the known or predicted ARP genes from the completed genomic sequences of Arabidopsis and the available sequences from rice [30,31] with those from human and yeast. There appear to be eight distinct classes of ARPs in these two distant plant genomes. Unlike actin isovariants, which predominate either in vegetative or reproductive tissues, most ARPs in plants are constitutively expressed in all organs and tissues [3,32]. The plant ARP genes are predicted to encode proteins ranging from 146 to 717 amino acids in length, whereas plant actin genes encode proteins containing 377 amino acids [3,32]. ARP gene family in plants Examination of the completed genome sequence of Arabidopsis revealed nine highly divergent ARP genes
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Table 2. Relationships of Arabidopsis and rice ARP homologs Arabidopsisa
Accession number
Riceb
Clone number
Percentage identity
AtARP2 AtARP3 AtARP4 AtARP4A AtARP5 AtARP6 AtARP7 AtARP8 AtARP9
BK000428 BK000427 BK000422 BK000424 AY052346 BK000425 BK000423 BK000426 NM_123716
OsARP2 OsARP3 OsARP4 – OsARP5 OsARP6 OsARP7 OsARP8 OsARP9
OSJNBa0091C18 OJ1568_B05 OJ1613_604 – P0024G09 P0667A10 OSJNBa0091J19 B0811B10 OSJNBa0096F01
87 82 67 – 63 65 73 63 52
Abbreviation: – , not applicable. a TAIR (The Arabidopsis Information Resource) Database (http://www.arabidopsis.org/). b TIGR (The Institute for Genomic Research) Rice Genome Project (http://www.tigr.org/tdb/e2k1/osa1/).
that fall into eight ancient classes (Figure 1). Except for the ARP4 class, which represents two closely related genes (AtARP4 and AtARP4A), all classes of Arabidopsis ARPs (ARP2, ARP3, ARP5 – ARP9) are encoded by single genes. Comparison of Arabidopsis ARP gene sequences with the available rice genome database showed the existence of a closely related homolog for each of the eight Arabidopsis ARP classes. The relationships between these two plants ARP classes are illustrated by various tree-building methods (not shown) and are summarized in Figure 1 and Table 2. Because the monocot rice and dicot Arabidopsis have not shared a common ancestor for 200 million years, these eight ARP sequence clades are probably conserved in all higher plants. Also, six of the eight plant ARP classes (ARP2 – ARP6 and APR9) have orthologs in yeast and humans. However, the plant ARP7 and ARP8 classes represent novel ARPs that are specific to plants. Surprisingly, the yeast ARP1 and ARP10 classes are completely absent from Arabidopsis, rice and possibly other plants [3,13]. Moreover, we do not find homologs for yeast ScARP7 and ScARP9 in plants, yet the plant ARP9 groups with yeast ARP8 in the neighbor joining tree shown in Figure 1. The relationships among the most divergent ARPs (ScARP8 – ScARP10), which are only 17 – 30% identical to conventional actin, are difficult to resolve in the ARP sequence tree owing to low degrees of homology and numerous insertions and deletions. The nine Arabidopsis ARP genes are dispersed on three of the five chromosomes (AtARP2, AtARP5, AtARP6 and AtARP7 on chromosome 3, AtARP3, AtARP4 and AtARP4A on chromosome 1, and AtARP8 and AtARP9 on chromosome 5). Reverse-transcription PCR analysis reveals that seven of the nine Arabidopsis ARP genes (AtARP2, AtARP3, AtARP4, AtARP4A, AtARP6, AtARP7 and AtARP8) are expressed at 10 – 100 times lower levels than actin in most plant organs [3]. In addition, transcripts for AtARP5 and AtARP9 are represented in the expressed sequence tag database of Arabidopsis, suggesting that all nine ARP genes are expressed and are probably functional. In rice, the eight ARP genes are dispersed on five of the 12 chromosomes (OsARP2 and OsARP4 on chromosome 8, OsARP3 on chromosome 2, OsARP5 and OsARP6 on chromosome 1, OsARP7 on chromosome 3, and OsARP8 and OsARP9 on chromosome 4) and nothing is yet known about their cellular expression. www.sciencedirect.com
Properties of ancient ARP classes The existence of the various classes of actin-related proteins in widely divergent eukaryotic organisms and the predominant localization of ARPs to the cytoplasm or nuclear compartment suggests that they have conserved cytoplasmic and nuclear functions across phyla. Because ARPs exist mostly as components of multiprotein complexes, most of our understanding of the roles of various ARPs is based on knowledge of the function of different ARP-containing complexes. However, there is currently no reported evidence that plant ARPs form complexes. Moreover, our understanding of the functional hallmarks of ARPs in plants is still in its infancy and thus we here consider results mainly from yeast and other eukaryotes concerning ARP functions. Cytoplasmic actin-related proteins ARP1 and ARP10 ARP1, also called centractin, was first identified in vertebrate cells as a major component of the dynactin complex [33,34]. Since then, it has been cloned and characterized in a wide range of organisms, from yeast to humans [2,22]. Yeast has a single gene in the ARP1 class, whereas the higher eukaryotes have at least two and perhaps three isoforms of ARP1 (centractin a, b and g) [35]. ARP1 proteins are generally 376 amino acids long in vertebrates and vary from 380 to 385 amino acids in other species, and they are 55 – 60% identical to conventional actins [22]. However, ARP1 orthologs are much closer to each other than to any true actin. A remarkable feature of the ARP1 protein is that it is the only actin-related protein known to polymerize into filaments. On the basis of ultrastructural analysis and stoichiometry measurements, the ARP1 minifilaments within dynactin are predicted to contain only eight to ten monomers each [2,22]. Like ARP1, a second actin-related protein is also found in the dynactin complex – ARP10 (yeast) or ARP11 (vertebrates and fly) – along with nine other protein subunits. The ARP10 class protein is believed to play distinct roles in dynactin. Its predicted structure suggests it might cap the ARP1 filament to disallow further subunit addition or filament annealing [36]. The dynactin complex is suggested to promote dynein-mediated movement of membrane vesicles along microtubules [2] and breakdown of the nuclear envelope during open mitosis [37]. Dynactin structure and
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function in yeast and other organisms are the subject of several reviews [2,38 – 40]. Analysis of the Arabidopsis and rice genomes with yeast or human ARP1 as the query sequence revealed the absence of an ARP1 homolog in both of these highly divergent plants. From our search, it was interesting that ARP10 (the other ARP component of the dynactin complex) was also not obviously present in Arabidopsis [3] or rice. Are members of these subfamilies totally missing or just unrecognizable in flowering plants? Given that Arabidopsis also appears to lack cytoplasmic dynein [41] and that dynactin is an activator of cytoplasmic dynein, it is tempting to speculate that plants lack dynactin and therefore lack the dynactin-associated ARPs (ARP1 and ARP10). The loss of these ARPs is predicted to correlate with the loss of motile sperm in pre-angiosperms [41]. ARP2 and ARP3 ARP2 and ARP3 are present in all eukaryotes and they are probably the best-characterized ARP family members. They are closely related to actin, with 40–50% and 30–40% sequence identity, respectively. However, unlike ARP1, they are incapable of forming actin-like filaments. In animals, protists and yeast, ARP2 and ARP3 are stable components of a seven-subunit ARP2/3 complex that contains a WD repeat protein (ARPC1, also known as p40 or Sop2) and four novel proteins (ARPC2– ARPC5), suggesting that the complex is highly conserved throughout eukaryotic evolution. The ARP2/3 complex was first isolated from Acanthamoeba castellanii based on its affinity for the actin monomer binding protein profilin [15]. In Acanthamoeba, the Arp2/3 complex is abundant, being present at concentrations of ,2 mM in the cytoplasm or 1/100 of the total concentration of actin [6]. This complex was subsequently purified from several organisms including humans [42] and the yeast S. cerevisiae [43]. In yeast cells, the ARP2/3 complex is concentrated in the actin patches, whereas, in migrating animal cells, it is localized to the lamellipodia and actin-rich spots [42,44 – 46]. In Listeria monocytogenes, the Arp2/3 complex localizes along the whole actin tail [47], suggesting that it remains attached to the growing actin filament network after actin nucleation. The Arp2/3 complex has been shown to play a key role in actin filament nucleation and branching, and force production by actin polymerization at the leading edge of motile cells [6,48]. The structure of this complex was ˚ resolution, providing atomic recently determined at 2-A scale insight into its functions [49]. The Arp2/3 complex promotes formation of actin filaments as 708 branches on the sides of older, pre-existing filaments. Molecular models suggest that ARP2 and ARP3 proteins form a stable dimer with a capacity to bind the slow growing ‘pointed’ end of an actin filament and hence to nucleate filaments that grow at the fast, ‘barbed’ end direction [16]. The ARP2/3 complex is inactive until stimulated by activating proteins such as ActA [50], WASP (Wiskott – Aldrich syndrome protein) [51], cortactin [52] or ABP1p [53]. Among these activators, WASP/Scar family proteins are the prominent cellular nucleation factors, but their homologs have not been found in plants. They bring together an actin monomer and the www.sciencedirect.com
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Arp2/3 complex in solution or on the side of an existing actin filament to initiate a new filament that grows in the barbed end direction [46]. Not surprisingly, genetic experiments have indicated a central role for the Arp2/3 complex in actin cytoskeletal function. In yeast, deletion of Arp2/3 complex subunits causes severe growth defects or lethality [44,54,55]. A flurry of new contributions using fruit fly as a model system have recently shown that the Arp2/3 complex is crucial for a diverse range of developmental processes [56,57], including the growth of ring canals during oogenesis, furrow formation during divisions of the syncytial blastoderm, formation of the central nervous system and morphogenesis of the eye and sensory bristles [58]. Helene Gournier et al. [59] reconstituted a recombinant human Arp2/3 complex using a baculovirus expression system to define the biochemical function of its subunits. Analysis of subcomplexes lacking one or more subunits revealed that the filament nucleating and organizing functions of Arp2/3 complex subunits are separable. Genes encoding ARP2 and ARP3 homologs in Arabidopsis have been cloned [3,60]. AtARP2 and AtARP3 are, respectively, , 45% and 37% identical to the Arabidopsis vegetative actin AtACT2, and they group with the yeast and human ARP2 and ARP3 classes (Figure 1). Rice also has single genes encoding ARP2 and ARP3 homologs that are 87% and 82% identical to the Arabidopsis AtARP2 and AtARP3, respectively (Table 2). Recent immunological studies suggest the presence of ARP3 homologs in other plants such as tobacco and maize [61]. In addition to ARP2 and ARP3, the five other subunits of the ARP2/3 complex have also been recently identified in the Arabidopsis sequence database [62,63]. Although the promoter of AtARP2 has been reported to be active only in pollen and a subset of vascular tissues [60], reverse-transcription PCR analysis revealed ubiquitous expression of AtARP2 and AtARP3, as well as the genes encoding other subunits, in most plant tissues [3,62]. However, relative to conventional actin and the other ARPs, AtARP2 and AtARP3 transcripts are present at low levels in all organs of Arabidopsis [3]. Recently the research groups of Elizabeth Lord (University of California, Riverside; CA, USA) [62], Daniel Szymanski (Purdue University, IN, USA) [64] and Martin Hulskamp (University of Koln, Germany) [65], using genetic approaches, have independently demonstrated that, in Arabidopsis, the putative Arp2/3 complex controls cell morphogenesis through its roles in cell expansion and establishing cell polarity. Their studies suggest a function for the complex in the modulation of the spatial distribution of cortical F-actin in specific plant cells. Mutations in the Arabidopsis AtARP2 (WURM) and AtARP3 (DISTORTED) genes cause severe growth defects in the epidermal cells, especially the trichomes [62,64,65]. The potential parallels in the action of ARP2/3 complex in controlling the polymerization and organization of actin in plants and animals have recently been summarized [66]. Thus, the ARP2/3 complex appears to be essential for several actin-mediated cellular and developmental functions in eukaryotes, including plants. A necessary next step is to demonstrate that ARP2 and ARP3, along with
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other protein components, form a functional complex in plants. Nuclear actin-related proteins: ARP4 –ARP9 These divergent actin-related proteins share only 17 –45% sequence homology with actin, yet they are predicted to be structurally similar. Interestingly, all these ARPs are localized to the nucleus in yeast [9,67], and many of their homologs in other organisms such as humans [e.g. HsArpNa and HsArpNb/BAF53 of ARP4 class HsBAF53A (Figure 1) [68]], mouse (e.g. MmArpNa [69]) and flies (e.g. DmArp6 of ARP6 class [70]) are also found in the nucleus. In Drosophila, DmArp6 is localized to heterochromatin together with heterochromatin protein 1 (HP1) [70,71]. Most of the nuclear ARPs are essential components of large multiprotein chromatinmodifying complexes (Table 1). For example, yeast ScARP7 and ScARP9 are important subunits of the related ATP-dependent chromatin remodeling complexes SWI/SNF and RSC. The human SWI/SNF and Drosophila Brm complexes contain one ARP (HsBAF53 in human and DmBAP55 in Drosophila) and one molecule of b-actin. Remarkably, yeast INO80 complex contains three ARPs (ARP4, ARP5 and ARP8) and b-actin [10,26]. ARP4 and b-actin are also present in HAT complexes such as the essential yeast H4 HAT complex NuA4 [11] and human TIP60 [72], which has roles in apoptosis and DNA repair. Although we understand little about the function of the nuclear ARPs within various multiprotein complexes, genetic and biochemical studies point to a structural or direct enzymatic role for ARPs in chromatin-mediated gene regulation. For instance, phenotypic analysis of yeast arp4 mutants revealed epigenetic transcriptional defects consistent with a function for ARPs in transcriptional regulation and chromatin structure [11,25], and purified ARP4 protein has been shown to bind to all four core histones in vitro [73]. Also in yeast, mutations in ARP7 or ARP9 display a swi/snf phenotype, suggesting that they are required for the function of the SWI/SNF complex. However, mutagenesis of residues that were predicted to mediate ATP binding or hydrolysis did not affect ARP7 or ARP9 functions in vivo. Moreover, RSC complex isolated from yeast mutants lacking ARP7 and ARP9 proteins nevertheless displays robust nucleosome remodeling activity. These studies indicate that these ARP proteins provide a structural, rather than an enzymatic, function in the complex [8,27]. However, a screen for suppressors of arp7 and arp9 mutations yielded the DNA bending, architectural transcription factor Nhp6, which interacts physically and functionally with RSC and shows facilitated binding to nucleosomes by RSC [8,27]. This finding suggests that ARP7 and ARP9 connect the RSC complex to interacting proteins or other complexes, allowing functionality in vivo. Moreover, when mutant INO80 complexes lacking ARP5 and APR8 are purified, they are deficient for ARP4 and actin, the other components of the complex. Also, the mutant complexes are compromised for INO80 ATPase activity, DNA binding and nucleosome mobilization. These studies of yeast INO80 complex suggest direct functions for ARPs in chromatin remodeling [26]. www.sciencedirect.com
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In Arabidopsis and rice, four divergent ARP classes (ARP4 –ARP6 and ARP9) are sequence homologs of ARPs that are nuclear in animals and fungi (Figure 1). In addition, plants have two novel ARP classes (ARP7 and ARP8), one of which (ARP7) is also localized to the nucleus during interphase in Arabidopsis. However, in mitotic plant cells lacking a nuclear envelope (e.g. metaphase, anaphase and early telophase stages), AtARP7 is dispersed throughout the cytoplasm [32]. Arabidopsis AtARP4 protein also exhibits similar cell-cycle-dependent subcellular distribution (Figure 2). AtARP4 is likely to be involved in transcriptional regulation via chromatin remodeling, because of its high sequence homology to human BAF53 and yeast ARP4 and its nuclear localization.
(a)
AtARP4 (b)
DNA (c)
AtARP7 (d)
DNA (e)
(f) C T
AtARP4
M
DNA
Figure 2. Nuclear localization of Arabidopsis AtARP4 and AtARP7. Root cells labeled with a monoclonal antibody (mAb) against AtARP4 (a,e) or AtARP7 (c) are shown in green. DAPI (4,6-diamidino-2-phenylindole) staining of DNA is shown in red (b,d,f). Both AtARP4 and AtARP7 are concentrated in the nucleoplasm of interphase cells (a,c). In dividing cells (e), AtARP4 is not associated with the chromosomes but is dispersed throughout the cytoplasm. Abbreviations: C, cytokinesis; M metaphase; T, telophase. AtARP7 also exhibits a similar pattern of subcellular distribution in mitotic cells [32].
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Consistent with this proposed function, knockdown of ARP4 protein by RNA interference or T-DNA insertion results in severe defects in overall plant and flower development (M.K. Kandasamy et al., unpublished). Similarly, knockout mutations in AtARP6 (a homolog of Drosophila dArp6 that localizes with HP1) exhibit early flowering and dwarf phenotypes (R.B. Deal, unpublished). There are orthologs of HP1 in plants and mutations in the Arabidopsis AtHP1 gene also show early-flowering phenotypes [74]. Because dARP6 is suggested to play a role in higher-order chromatin structure in Drosophila [1,71], further studies of the conserved ARP6 protein in plants might shed light on the molecular mechanisms of heterochromatin organization. Moreover, plants contain many homologs of SWI/SNF ATPases (e.g. PKL [75] and SYD [76]) that are thought to be involved in the chromatinmediated control of gene expression during plant development. The functional characterization by genetic studies of the various chromatin remodeling factors is the subject of several recent reviews [77– 80]. Because we are concerned here mainly with actin-related proteins, we do not discuss other remodeling factors. However, it would be interesting to explore whether or not there are differences between plant and animal chromatin remodeling machines, and whether or not plant complexes contain ARPs as components as their counterparts in other eukaryotes do. Conclusion and future directions The past four years have witnessed the discovery of the whole ARP gene family of Arabidopsis, containing six known and two novel ARP classes. The rice genome contains homologs for all eight classes of Arabidopsis ARPs, demonstrating their sequence conservation and hence functional significance to higher plants. However, we lack an understanding of how these ancient ARP proteins and their presumed protein partners in various ARP-containing complexes work in plants. It is obvious that plants lack ARP1 and ARP10, and possibly the whole dynactin complex. Hence, it will be interesting to determine at what stage in vascular plant evolution these genes were lost and how their functions are fulfilled in higher plants. Studies of ARP gene families in lower plants might provide answers to these evolutionarily significant questions. Additionally, functional analysis of the ARP2/3 complex, which has been shown to control the organization of actin cytoskeleton in many eukaryotes, and its regulators in tip growing plant cells such as pollen tubes and root hairs would enhance our understanding of nucleation and dynamics of actin in plants. Studies in other eukaryotes, especially yeast, show that the divergent nuclear ARPs play significant, exciting roles in chromatin remodeling and transcriptional regulation. Identifying the various nuclear ARP-containing complexes and unraveling their molecular mechanisms would help us to understand and appreciate the role of divergent ARPs in plant gene regulation, DNA repair and development. Plants are extremely suitable for genetic analysis of chromatin remodeling factors because they are relatively tolerant of gene disruptions and many mutants that are lethal in animals are viable but show clear developmental defects. Therefore, mutant plants can reveal the function of ARPs www.sciencedirect.com
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and other chromatin regulators throughout the life of the plant. Because Arabidopsis has a large collection of T-DNA insertion mutants, it will serve as a good model genetic system to characterize the function of ARPs in plants. Also, the successful use of RNA interference specifically to downregulate the expression of different genes in plants is an added advantage when knockout mutants are lethal. Acknowledgements We thank Gay Gragson for editing the manuscript. Our research was supported by funds from National Institutes of Health (GM 36397 – 14) and NIH training grant (GM 07103 – 29).
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Plant actin-related proteins Muthugapatti K. Kandasamy, Roger B. Deal, Elizabeth C. McKinney and Richard B. Meagher Department of Genetics, University of Georgia, Athens, GA 30602, USA
Actin-related proteins (ARPs) constitute a family of divergent and evolutionarily ancient eukaryotic proteins whose primary sequences display homology to conventional actins. Whereas actins play well-characterized cytoskeletal roles, the ARPs are implicated in various cellular functions in both the cytoplasm and in the nucleus. Cytoplasmic ARPs, for example, are known to participate in the assembly of branched actin filaments and dynein-mediated movement of vesicles in many eukaryotes. Nuclear ARPs, by contrast, are enigmatic components of various chromatin-modifying complexes involved in transcriptional regulation. Here, we review homologs to several known classes of ARPs and two distinct ARP classes in plants, and summarize recent work elucidating the biological functions of ARPs in eukaryotes. Actin-related proteins (ARPs), a novel family of highly conserved proteins that exhibit moderate sequence identity (17 – 60%) to conventional actins, have been found in all eukaryotic kingdoms [1– 3]. The ARPs and actins possess a common tertiary structure centered on the nucleotide-binding pocket known as the actin fold [4]. This fold undergoes major conformational changes in response to the 50 phosphorylation state of the adenine-containing nucleotide, and such changes are thought to be central to the function of the actin superfamily members, including ARPs. However, the various ARPs have divergent surface features caused by insertions, deletions and point mutations, suggesting that they are functionally distinct from actins and from each other [5]. Studies over the past decade have implicated ARPs in several roles in the cytoplasm, notably modulation of actin assembly and the microtubule-based motility of vesicles [2,6]. Recently, ARPs and actins have been discovered in the nucleus as integral components of many chromatin remodeling and histone acetyltransferase (HAT) complexes [7– 12]. These multiprotein complexes are involved in the modulation of chromatin structure, transcription and possibly DNA repair. The ARPs are grouped into several classes or subfamilies that are highly conserved in a wide range of eukaryotes, from yeast to plants and humans [13]. Each class or subfamily is distinguished by its degree of similarity to conventional actin. For example, the ten ARPs of budding yeast are classified into subfamilies 1 – 10, where ARP1 is the most and ARP10 is the least similar to yeast actin [14]. This yeast classification system Corresponding author: Muthugapatti K. Kandasamy ([email protected]).
is used to group the plant and human ARP sequences into various classes (Figure 1, neighbor joining tree). The ARPs exhibit structural and functional diversity, and different subcellular distribution (Table 1). ARP1 – ARP3 and ARP10 in yeast, and their homologs in other organisms ScARP1 ARP1 HsARP1 ScARP2 ARP2 HsARP2 OsARP2 AtARP2 ScARP3 HsARP3 OsARP3 AtARP3
ARP3 AtARP4A AtARP4 OsARP4 HsBAF53A ScARP4
Actin ScACT1 HsACTB AtACT2
ARP4 ARP5 OsARP5 AtARP5 HsARP5 ScARP5 OsARP6 ARP6 AtARP6 HsARP6X ScARP6
OsARP8 AtARP8
OsRAC1
Plant ARP8
ARP8, ARP9, ARP10 OsARP9 AtARP9 HsARP8 ScARP8 ScARP10 ScARP9 HsARP11 ARP7 ScARP7
100 changes
OsARP7 AtARP7
Plant ARP7 TRENDS in Plant Science
Figure 1. Phylogenetic relationships of actin and actin-related proteins. Arabidopsis, rice, human and yeast ARP sequences are compared in a neighbor-joining (NBJ) tree. Clades of ARP sequences are named based primarily on the nomenclature of yeast ARPs or distinct plant ARPs. The tree is rooted to highly conserved conventional actins. Because branch length is sensitive to the degree of sequence divergence in the NBJ tree, the accelerated rate of divergence among ARPs relative to actins is supported by the long horizontal distances among ARPs compared with the short distances among actins. The tree shows that Arabidopsis and rice contain eight ancient and highly divergent classes of ARPs, six classes that have homologs in yeast and human (ARP2– ARP6 and ARP9), and two classes that are specific to plants (ARP7 and ARP8). The accession numbers for the protein sequences used in the tree are as follows. Human: HsACTB, P02570; HsARP1, S29089; HsARP2A, AB64187; HsARP3, P32391; HsBAF53A, 096019; HsARP5, CAD37358; HsARP6X, Q9GZN1; HsARP8, Q9H981; HsARP11, Q9NZ32. Yeast: ScACT1, P02579; ScARP1, P38696; ScARP2, P32381; ScARP3, P47117; ScARP4, P80428; ScARP5, P53946; ScARP6, Q12509; ScARP7, Q12406; ScARP8, Q12386; ScARP9, Q055123; ScARP10, Q04549. Arabidopsis: AtACT2, Q96292 (for ARPs, see Table 2). Rice: OsRAC1, AA038821 (for ARPs, see Table 2). The various yeast, human and plant ARPs and actin are shown in black, purple and green, respectively. The major ARP classes are indicated adjacent to each clade of ARPs; the two novel plant-specific ARP classes are in green.
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Table 1. Summary of properties of Arabidopsis ARPs and those in other kingdoms Arabidopsis ARP – AtARP2 AtARP3 AtARP4 AtARP4A AtARP5 AtARP6 AtARP7 AtARP8 – AtARP9b – –
ARP class ARP1 ARP2 ARP3 ARP4 ARP4 ARP5 ARP6 Novel Novel ARP7 ARP8 ARP9 ARP10
Essential in yeast Yes Yes Yes Yes – Yes No – – Yes No Yes N/A
Localizationa MFY
Cytoplasm CytoplasmMFY CytoplasmMFY NucleusMYA N/A NucleusY NucleusFY NucleusA N/A NucleusY NucleusY NucleusY CytoplasmMY
Complexa DynactinMFY ARP2/3MFYA ARP2/3MFYA SWI/SNFM, INO80Y, NuA4Y, HATMY ? INO80Y Localized to heterochromatinF ? ? SWI/SNFY, RSCY INO80Y SWI/SNFY, RSCY DynactinM
Abbreviations: N/A, data not available; – , not represented in the genome; ?, unknown. a Superscripts (M,F,Y,A) refer to the type of organism (mammal, fly, yeast, Arabidopsis, respectively) in which the observation was made. This table is constructed from information from Refs [3,13]. b AtARP9 groups weakly with the ARP8 class.
are localized to the cytoplasm. These ARPs have functions distinct from those of actin, and each subclass has a similar role across phyla. By contrast, the divergent members of ARP subfamilies 4 –9 are localized to the nucleus in Saccharomyces cerevisiae [9]. Functional analyses of the ARP classes reveal that they, with other proteins, assemble into stable heteromultimeric protein complexes (Table 1). Few, if any, ARPs are known to function outside such complexes. ARP1 and ARP10 are major components of the dynactin complex, which is required for the orientation of mitotic spindle and nuclear migration [2]. ARP2 and ARP3, together with five other novel proteins, make up the ARP2/3 complex, which is implicated in capping, nucleation and branching of actin filaments [6,15,16]. Several of the divergent nuclear ARPs, such as ARP4, ARP5 and ARP7–ARP9, are components of different chromatin remodeling complexes (e.g. SWI/SNF, INO80, RSC, and NuA4) that are required for altering chromatin structure and/or transcriptional regulation [7,8,17,18]. These nuclear multiprotein complexes alter the nucleosome architecture either by covalent modification of the N-terminal histone tails (e.g. the HATs) or by ATP-dependent perturbations of histone-DNA interactions (e.g. SWI/SNF family of protein complexes) [19–21]. Thus, the biological functions of ARPs should be considered in light of their roles as components of larger macromolecular machines. Significant progress has been made in understanding the role of the highly conserved ARPs of classes 1–3 in many eukaryotes [2,22–24]. However, characterization of the function of highly divergent ARPs of animals and yeast has received relatively little attention [11,25–27]. In plants, which contain almost the full complement of the ARP gene family [3], we are just beginning to understand and appreciate the intriguing roles of ARPs in various cellular processes. Structure and evolution of the actin superfamily Actins and ARPs represent two ancient branches of a highly diverse superfamily of ATPases that also includes heat-shock proteins (HSPs) and their cognates (e.g. HSP60, HSC70), sugar kinases (e.g. hexokinase) of both prokaryotes and eukaryotes, and a range of bacterial ATP-binding proteins (e.g. MreB, FtsA, StbA) [1,23]. Although the actin and ARP proteins share no sequence homology with these www.sciencedirect.com
other proteins, all the actin superfamily members contain a common structural motif: the actin fold. This fold has been shown for many of these diverse proteins to bind ATP and/or ADP, and this binding allows major conformational changes in the protein. This reversible structural shift is thought to be crucial to the function of various actin family members (e.g. control of polymerization of F-actin and initiation of protein complex formation for the ARPs and HSPs). Also, the divergent nature of molecular surfaces for each protein is thought to indicate unique functions specific for each family member [1,23]. All the plant actins seem to have originated from a single green-algal ancestral actin gene [28]. Subsequent gene duplications and divergence in vascular plants have resulted in two classes, vegetative and reproductive, and at least five ancient subclasses of actin that pre-date the monocot– dicot separation. In the Crucifer Arabidopsis, further divergence resulted in a total of eight functional actin genes [29]. Both the vegetative and reproductive classes of actins in plants show distinct spatial and temporal patterns of expression. The vegetative actins are strongly expressed in all vegetative organs and cell types, and the reproductive actins are expressed predominantly in the mature pollen, the ovule and, in some cases, the embryo [29]. However, the ARPs of plants have an entirely different phylogeny from the actins and have a much older gene family structure. The ARP protein sequence tree in Figure 1 compares all the known or predicted ARP genes from the completed genomic sequences of Arabidopsis and the available sequences from rice [30,31] with those from human and yeast. There appear to be eight distinct classes of ARPs in these two distant plant genomes. Unlike actin isovariants, which predominate either in vegetative or reproductive tissues, most ARPs in plants are constitutively expressed in all organs and tissues [3,32]. The plant ARP genes are predicted to encode proteins ranging from 146 to 717 amino acids in length, whereas plant actin genes encode proteins containing 377 amino acids [3,32]. ARP gene family in plants Examination of the completed genome sequence of Arabidopsis revealed nine highly divergent ARP genes
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Table 2. Relationships of Arabidopsis and rice ARP homologs Arabidopsisa
Accession number
Riceb
Clone number
Percentage identity
AtARP2 AtARP3 AtARP4 AtARP4A AtARP5 AtARP6 AtARP7 AtARP8 AtARP9
BK000428 BK000427 BK000422 BK000424 AY052346 BK000425 BK000423 BK000426 NM_123716
OsARP2 OsARP3 OsARP4 – OsARP5 OsARP6 OsARP7 OsARP8 OsARP9
OSJNBa0091C18 OJ1568_B05 OJ1613_604 – P0024G09 P0667A10 OSJNBa0091J19 B0811B10 OSJNBa0096F01
87 82 67 – 63 65 73 63 52
Abbreviation: – , not applicable. a TAIR (The Arabidopsis Information Resource) Database (http://www.arabidopsis.org/). b TIGR (The Institute for Genomic Research) Rice Genome Project (http://www.tigr.org/tdb/e2k1/osa1/).
that fall into eight ancient classes (Figure 1). Except for the ARP4 class, which represents two closely related genes (AtARP4 and AtARP4A), all classes of Arabidopsis ARPs (ARP2, ARP3, ARP5 – ARP9) are encoded by single genes. Comparison of Arabidopsis ARP gene sequences with the available rice genome database showed the existence of a closely related homolog for each of the eight Arabidopsis ARP classes. The relationships between these two plants ARP classes are illustrated by various tree-building methods (not shown) and are summarized in Figure 1 and Table 2. Because the monocot rice and dicot Arabidopsis have not shared a common ancestor for 200 million years, these eight ARP sequence clades are probably conserved in all higher plants. Also, six of the eight plant ARP classes (ARP2 – ARP6 and APR9) have orthologs in yeast and humans. However, the plant ARP7 and ARP8 classes represent novel ARPs that are specific to plants. Surprisingly, the yeast ARP1 and ARP10 classes are completely absent from Arabidopsis, rice and possibly other plants [3,13]. Moreover, we do not find homologs for yeast ScARP7 and ScARP9 in plants, yet the plant ARP9 groups with yeast ARP8 in the neighbor joining tree shown in Figure 1. The relationships among the most divergent ARPs (ScARP8 – ScARP10), which are only 17 – 30% identical to conventional actin, are difficult to resolve in the ARP sequence tree owing to low degrees of homology and numerous insertions and deletions. The nine Arabidopsis ARP genes are dispersed on three of the five chromosomes (AtARP2, AtARP5, AtARP6 and AtARP7 on chromosome 3, AtARP3, AtARP4 and AtARP4A on chromosome 1, and AtARP8 and AtARP9 on chromosome 5). Reverse-transcription PCR analysis reveals that seven of the nine Arabidopsis ARP genes (AtARP2, AtARP3, AtARP4, AtARP4A, AtARP6, AtARP7 and AtARP8) are expressed at 10 – 100 times lower levels than actin in most plant organs [3]. In addition, transcripts for AtARP5 and AtARP9 are represented in the expressed sequence tag database of Arabidopsis, suggesting that all nine ARP genes are expressed and are probably functional. In rice, the eight ARP genes are dispersed on five of the 12 chromosomes (OsARP2 and OsARP4 on chromosome 8, OsARP3 on chromosome 2, OsARP5 and OsARP6 on chromosome 1, OsARP7 on chromosome 3, and OsARP8 and OsARP9 on chromosome 4) and nothing is yet known about their cellular expression. www.sciencedirect.com
Properties of ancient ARP classes The existence of the various classes of actin-related proteins in widely divergent eukaryotic organisms and the predominant localization of ARPs to the cytoplasm or nuclear compartment suggests that they have conserved cytoplasmic and nuclear functions across phyla. Because ARPs exist mostly as components of multiprotein complexes, most of our understanding of the roles of various ARPs is based on knowledge of the function of different ARP-containing complexes. However, there is currently no reported evidence that plant ARPs form complexes. Moreover, our understanding of the functional hallmarks of ARPs in plants is still in its infancy and thus we here consider results mainly from yeast and other eukaryotes concerning ARP functions. Cytoplasmic actin-related proteins ARP1 and ARP10 ARP1, also called centractin, was first identified in vertebrate cells as a major component of the dynactin complex [33,34]. Since then, it has been cloned and characterized in a wide range of organisms, from yeast to humans [2,22]. Yeast has a single gene in the ARP1 class, whereas the higher eukaryotes have at least two and perhaps three isoforms of ARP1 (centractin a, b and g) [35]. ARP1 proteins are generally 376 amino acids long in vertebrates and vary from 380 to 385 amino acids in other species, and they are 55 – 60% identical to conventional actins [22]. However, ARP1 orthologs are much closer to each other than to any true actin. A remarkable feature of the ARP1 protein is that it is the only actin-related protein known to polymerize into filaments. On the basis of ultrastructural analysis and stoichiometry measurements, the ARP1 minifilaments within dynactin are predicted to contain only eight to ten monomers each [2,22]. Like ARP1, a second actin-related protein is also found in the dynactin complex – ARP10 (yeast) or ARP11 (vertebrates and fly) – along with nine other protein subunits. The ARP10 class protein is believed to play distinct roles in dynactin. Its predicted structure suggests it might cap the ARP1 filament to disallow further subunit addition or filament annealing [36]. The dynactin complex is suggested to promote dynein-mediated movement of membrane vesicles along microtubules [2] and breakdown of the nuclear envelope during open mitosis [37]. Dynactin structure and
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function in yeast and other organisms are the subject of several reviews [2,38 – 40]. Analysis of the Arabidopsis and rice genomes with yeast or human ARP1 as the query sequence revealed the absence of an ARP1 homolog in both of these highly divergent plants. From our search, it was interesting that ARP10 (the other ARP component of the dynactin complex) was also not obviously present in Arabidopsis [3] or rice. Are members of these subfamilies totally missing or just unrecognizable in flowering plants? Given that Arabidopsis also appears to lack cytoplasmic dynein [41] and that dynactin is an activator of cytoplasmic dynein, it is tempting to speculate that plants lack dynactin and therefore lack the dynactin-associated ARPs (ARP1 and ARP10). The loss of these ARPs is predicted to correlate with the loss of motile sperm in pre-angiosperms [41]. ARP2 and ARP3 ARP2 and ARP3 are present in all eukaryotes and they are probably the best-characterized ARP family members. They are closely related to actin, with 40–50% and 30–40% sequence identity, respectively. However, unlike ARP1, they are incapable of forming actin-like filaments. In animals, protists and yeast, ARP2 and ARP3 are stable components of a seven-subunit ARP2/3 complex that contains a WD repeat protein (ARPC1, also known as p40 or Sop2) and four novel proteins (ARPC2– ARPC5), suggesting that the complex is highly conserved throughout eukaryotic evolution. The ARP2/3 complex was first isolated from Acanthamoeba castellanii based on its affinity for the actin monomer binding protein profilin [15]. In Acanthamoeba, the Arp2/3 complex is abundant, being present at concentrations of ,2 mM in the cytoplasm or 1/100 of the total concentration of actin [6]. This complex was subsequently purified from several organisms including humans [42] and the yeast S. cerevisiae [43]. In yeast cells, the ARP2/3 complex is concentrated in the actin patches, whereas, in migrating animal cells, it is localized to the lamellipodia and actin-rich spots [42,44 – 46]. In Listeria monocytogenes, the Arp2/3 complex localizes along the whole actin tail [47], suggesting that it remains attached to the growing actin filament network after actin nucleation. The Arp2/3 complex has been shown to play a key role in actin filament nucleation and branching, and force production by actin polymerization at the leading edge of motile cells [6,48]. The structure of this complex was ˚ resolution, providing atomic recently determined at 2-A scale insight into its functions [49]. The Arp2/3 complex promotes formation of actin filaments as 708 branches on the sides of older, pre-existing filaments. Molecular models suggest that ARP2 and ARP3 proteins form a stable dimer with a capacity to bind the slow growing ‘pointed’ end of an actin filament and hence to nucleate filaments that grow at the fast, ‘barbed’ end direction [16]. The ARP2/3 complex is inactive until stimulated by activating proteins such as ActA [50], WASP (Wiskott – Aldrich syndrome protein) [51], cortactin [52] or ABP1p [53]. Among these activators, WASP/Scar family proteins are the prominent cellular nucleation factors, but their homologs have not been found in plants. They bring together an actin monomer and the www.sciencedirect.com
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Arp2/3 complex in solution or on the side of an existing actin filament to initiate a new filament that grows in the barbed end direction [46]. Not surprisingly, genetic experiments have indicated a central role for the Arp2/3 complex in actin cytoskeletal function. In yeast, deletion of Arp2/3 complex subunits causes severe growth defects or lethality [44,54,55]. A flurry of new contributions using fruit fly as a model system have recently shown that the Arp2/3 complex is crucial for a diverse range of developmental processes [56,57], including the growth of ring canals during oogenesis, furrow formation during divisions of the syncytial blastoderm, formation of the central nervous system and morphogenesis of the eye and sensory bristles [58]. Helene Gournier et al. [59] reconstituted a recombinant human Arp2/3 complex using a baculovirus expression system to define the biochemical function of its subunits. Analysis of subcomplexes lacking one or more subunits revealed that the filament nucleating and organizing functions of Arp2/3 complex subunits are separable. Genes encoding ARP2 and ARP3 homologs in Arabidopsis have been cloned [3,60]. AtARP2 and AtARP3 are, respectively, , 45% and 37% identical to the Arabidopsis vegetative actin AtACT2, and they group with the yeast and human ARP2 and ARP3 classes (Figure 1). Rice also has single genes encoding ARP2 and ARP3 homologs that are 87% and 82% identical to the Arabidopsis AtARP2 and AtARP3, respectively (Table 2). Recent immunological studies suggest the presence of ARP3 homologs in other plants such as tobacco and maize [61]. In addition to ARP2 and ARP3, the five other subunits of the ARP2/3 complex have also been recently identified in the Arabidopsis sequence database [62,63]. Although the promoter of AtARP2 has been reported to be active only in pollen and a subset of vascular tissues [60], reverse-transcription PCR analysis revealed ubiquitous expression of AtARP2 and AtARP3, as well as the genes encoding other subunits, in most plant tissues [3,62]. However, relative to conventional actin and the other ARPs, AtARP2 and AtARP3 transcripts are present at low levels in all organs of Arabidopsis [3]. Recently the research groups of Elizabeth Lord (University of California, Riverside; CA, USA) [62], Daniel Szymanski (Purdue University, IN, USA) [64] and Martin Hulskamp (University of Koln, Germany) [65], using genetic approaches, have independently demonstrated that, in Arabidopsis, the putative Arp2/3 complex controls cell morphogenesis through its roles in cell expansion and establishing cell polarity. Their studies suggest a function for the complex in the modulation of the spatial distribution of cortical F-actin in specific plant cells. Mutations in the Arabidopsis AtARP2 (WURM) and AtARP3 (DISTORTED) genes cause severe growth defects in the epidermal cells, especially the trichomes [62,64,65]. The potential parallels in the action of ARP2/3 complex in controlling the polymerization and organization of actin in plants and animals have recently been summarized [66]. Thus, the ARP2/3 complex appears to be essential for several actin-mediated cellular and developmental functions in eukaryotes, including plants. A necessary next step is to demonstrate that ARP2 and ARP3, along with
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other protein components, form a functional complex in plants. Nuclear actin-related proteins: ARP4 –ARP9 These divergent actin-related proteins share only 17 –45% sequence homology with actin, yet they are predicted to be structurally similar. Interestingly, all these ARPs are localized to the nucleus in yeast [9,67], and many of their homologs in other organisms such as humans [e.g. HsArpNa and HsArpNb/BAF53 of ARP4 class HsBAF53A (Figure 1) [68]], mouse (e.g. MmArpNa [69]) and flies (e.g. DmArp6 of ARP6 class [70]) are also found in the nucleus. In Drosophila, DmArp6 is localized to heterochromatin together with heterochromatin protein 1 (HP1) [70,71]. Most of the nuclear ARPs are essential components of large multiprotein chromatinmodifying complexes (Table 1). For example, yeast ScARP7 and ScARP9 are important subunits of the related ATP-dependent chromatin remodeling complexes SWI/SNF and RSC. The human SWI/SNF and Drosophila Brm complexes contain one ARP (HsBAF53 in human and DmBAP55 in Drosophila) and one molecule of b-actin. Remarkably, yeast INO80 complex contains three ARPs (ARP4, ARP5 and ARP8) and b-actin [10,26]. ARP4 and b-actin are also present in HAT complexes such as the essential yeast H4 HAT complex NuA4 [11] and human TIP60 [72], which has roles in apoptosis and DNA repair. Although we understand little about the function of the nuclear ARPs within various multiprotein complexes, genetic and biochemical studies point to a structural or direct enzymatic role for ARPs in chromatin-mediated gene regulation. For instance, phenotypic analysis of yeast arp4 mutants revealed epigenetic transcriptional defects consistent with a function for ARPs in transcriptional regulation and chromatin structure [11,25], and purified ARP4 protein has been shown to bind to all four core histones in vitro [73]. Also in yeast, mutations in ARP7 or ARP9 display a swi/snf phenotype, suggesting that they are required for the function of the SWI/SNF complex. However, mutagenesis of residues that were predicted to mediate ATP binding or hydrolysis did not affect ARP7 or ARP9 functions in vivo. Moreover, RSC complex isolated from yeast mutants lacking ARP7 and ARP9 proteins nevertheless displays robust nucleosome remodeling activity. These studies indicate that these ARP proteins provide a structural, rather than an enzymatic, function in the complex [8,27]. However, a screen for suppressors of arp7 and arp9 mutations yielded the DNA bending, architectural transcription factor Nhp6, which interacts physically and functionally with RSC and shows facilitated binding to nucleosomes by RSC [8,27]. This finding suggests that ARP7 and ARP9 connect the RSC complex to interacting proteins or other complexes, allowing functionality in vivo. Moreover, when mutant INO80 complexes lacking ARP5 and APR8 are purified, they are deficient for ARP4 and actin, the other components of the complex. Also, the mutant complexes are compromised for INO80 ATPase activity, DNA binding and nucleosome mobilization. These studies of yeast INO80 complex suggest direct functions for ARPs in chromatin remodeling [26]. www.sciencedirect.com
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In Arabidopsis and rice, four divergent ARP classes (ARP4 –ARP6 and ARP9) are sequence homologs of ARPs that are nuclear in animals and fungi (Figure 1). In addition, plants have two novel ARP classes (ARP7 and ARP8), one of which (ARP7) is also localized to the nucleus during interphase in Arabidopsis. However, in mitotic plant cells lacking a nuclear envelope (e.g. metaphase, anaphase and early telophase stages), AtARP7 is dispersed throughout the cytoplasm [32]. Arabidopsis AtARP4 protein also exhibits similar cell-cycle-dependent subcellular distribution (Figure 2). AtARP4 is likely to be involved in transcriptional regulation via chromatin remodeling, because of its high sequence homology to human BAF53 and yeast ARP4 and its nuclear localization.
(a)
AtARP4 (b)
DNA (c)
AtARP7 (d)
DNA (e)
(f) C T
AtARP4
M
DNA
Figure 2. Nuclear localization of Arabidopsis AtARP4 and AtARP7. Root cells labeled with a monoclonal antibody (mAb) against AtARP4 (a,e) or AtARP7 (c) are shown in green. DAPI (4,6-diamidino-2-phenylindole) staining of DNA is shown in red (b,d,f). Both AtARP4 and AtARP7 are concentrated in the nucleoplasm of interphase cells (a,c). In dividing cells (e), AtARP4 is not associated with the chromosomes but is dispersed throughout the cytoplasm. Abbreviations: C, cytokinesis; M metaphase; T, telophase. AtARP7 also exhibits a similar pattern of subcellular distribution in mitotic cells [32].
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Consistent with this proposed function, knockdown of ARP4 protein by RNA interference or T-DNA insertion results in severe defects in overall plant and flower development (M.K. Kandasamy et al., unpublished). Similarly, knockout mutations in AtARP6 (a homolog of Drosophila dArp6 that localizes with HP1) exhibit early flowering and dwarf phenotypes (R.B. Deal, unpublished). There are orthologs of HP1 in plants and mutations in the Arabidopsis AtHP1 gene also show early-flowering phenotypes [74]. Because dARP6 is suggested to play a role in higher-order chromatin structure in Drosophila [1,71], further studies of the conserved ARP6 protein in plants might shed light on the molecular mechanisms of heterochromatin organization. Moreover, plants contain many homologs of SWI/SNF ATPases (e.g. PKL [75] and SYD [76]) that are thought to be involved in the chromatinmediated control of gene expression during plant development. The functional characterization by genetic studies of the various chromatin remodeling factors is the subject of several recent reviews [77– 80]. Because we are concerned here mainly with actin-related proteins, we do not discuss other remodeling factors. However, it would be interesting to explore whether or not there are differences between plant and animal chromatin remodeling machines, and whether or not plant complexes contain ARPs as components as their counterparts in other eukaryotes do. Conclusion and future directions The past four years have witnessed the discovery of the whole ARP gene family of Arabidopsis, containing six known and two novel ARP classes. The rice genome contains homologs for all eight classes of Arabidopsis ARPs, demonstrating their sequence conservation and hence functional significance to higher plants. However, we lack an understanding of how these ancient ARP proteins and their presumed protein partners in various ARP-containing complexes work in plants. It is obvious that plants lack ARP1 and ARP10, and possibly the whole dynactin complex. Hence, it will be interesting to determine at what stage in vascular plant evolution these genes were lost and how their functions are fulfilled in higher plants. Studies of ARP gene families in lower plants might provide answers to these evolutionarily significant questions. Additionally, functional analysis of the ARP2/3 complex, which has been shown to control the organization of actin cytoskeleton in many eukaryotes, and its regulators in tip growing plant cells such as pollen tubes and root hairs would enhance our understanding of nucleation and dynamics of actin in plants. Studies in other eukaryotes, especially yeast, show that the divergent nuclear ARPs play significant, exciting roles in chromatin remodeling and transcriptional regulation. Identifying the various nuclear ARP-containing complexes and unraveling their molecular mechanisms would help us to understand and appreciate the role of divergent ARPs in plant gene regulation, DNA repair and development. Plants are extremely suitable for genetic analysis of chromatin remodeling factors because they are relatively tolerant of gene disruptions and many mutants that are lethal in animals are viable but show clear developmental defects. Therefore, mutant plants can reveal the function of ARPs www.sciencedirect.com
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and other chromatin regulators throughout the life of the plant. Because Arabidopsis has a large collection of T-DNA insertion mutants, it will serve as a good model genetic system to characterize the function of ARPs in plants. Also, the successful use of RNA interference specifically to downregulate the expression of different genes in plants is an added advantage when knockout mutants are lethal. Acknowledgements We thank Gay Gragson for editing the manuscript. Our research was supported by funds from National Institutes of Health (GM 36397 – 14) and NIH training grant (GM 07103 – 29).
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