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PubMed2006

Association of alphaB-crystallin, a small heat shock protein, with actin: role in modulating actin filament dynamics in vivo.

Disruption of cytoskeletal assembly is one of the early effects of any stress that can ultimately lead to cell death. Stabilization of cytoskeletal assembly, therefore, is a critical event that regulates cell survival under stress. alphaB-crystallin, a small heat shock protein, has been shown to associate with cytoskeletal proteins under normal and stress conditions. Earlier reports suggest that alphaB-crystallin could prevent stress-induced aggregation of actin in vitro. However, the molecular mechanisms by which alphaB-crystallin stabilizes actin filaments in vivo are not known. Using the H9C2 rat cardiomyoblast cell line as a model system, we show that upon heat stress, alphaB-crystallin preferentially partitions from the soluble cytosolic fraction to the insoluble cytoskeletal protein-rich fraction. Confocal microscopic analysis shows that alphaB-crystallin associates with actin filaments during heat stress and the extent of association increases with time. Further, immunoprecipitation experiments show that alphaB-crystallin interacts directly with actin. Treatment of heat-stressed H9C2 cells with the actin depolymerzing agent, cytochalasin B, failed to disorganize actin. We show that this association of alphaB-crystallin with actin is dependent on its phosphorylation status, as treatment of cells with MAPK inhibitors SB202190 or PD98059 results in abrogation of this association. Our results indicate that alphaB-crystallin regulates actin filament dynamics in vivo and protects cells from stress-induced death. Further, our studies suggest that the association of alphaB-crystallin with actin helps maintenance of pinocytosis, a physiological function essential for survival of cells.

Actin Cytoskeleton↗
PubMed2006

Differential arginylation of actin isoforms: the mystery of the actin N-terminus.

Actin is one of the most abundant, essential and well studied intracellular proteins, yet its regulation in vivo is still not completely understood. One of the mysteries around actin concerns the existence of multiple actin isoforms that are extremely similar to each other except for their N-termini but have been shown in multiple studies to preferentially incorporate into different actin networks and are suggested to have different roles in vivo. The mechanisms of this actin isoform segregation are unknown. My colleagues and I recently showed that beta but not gamma actin in cultured fibroblasts undergoes N-terminal arginylation, which regulates actin polymerization and lamella formation in motile cells. Here, I propose that arginylation could be a general mechanism that regulates actin isoform segregation in vivo and participates in the formation of loose beta-actin network at the leading edge of the cell.

Actin Cytoskeleton↗
PubMed1999

A quantitative analysis of G-actin binding proteins and the G-actin pool in developing chick brain.

The large G-actin pool in individual actively motile cells has been shown to be maintained primarily by the actin sequestering protein thymosin beta four (Tbeta4). It is not clear whether Tbeta4 or an isoform also plays a primary role in neural tissue containing highly motile axonal growth cones. To address this question we have made a definitive analysis of the relative contributions of all the known G-actin sequestering proteins: Tbeta4, Tbeta10, profilin, and phosphorylated (inactive) and unphosphorylated (potentially active) forms of both ADF and cofilin, in relation to the G-actin pool in developing chick brain at embryonic days 13 and 17. From our measurements we estimate the intracellular concentration of G-actin as 30-37 microM and of Tbeta4 as 50-60 microM in an 'average' brain cell in embryonic chick brain. No other beta thymosin isoforms were detected in these brain extracts. The ratio of soluble, unphosphorylated ADF to Tbeta4 is only 1:7 at 13 embryonic days, but increases to 1:4 at 17 days. Profilin and cofilin concentrations are an order of magnitude lower than Tbeta4. Combining the contributions of Tbeta4, unphosphorylated ADF and unphosphorylated cofilin, we estimate a mean G-actin critical concentration of approximately 0.45 microM and approximately 0.2 microM, respectively, in day 13 and day 17 embryonic brain extracts, suggesting a significant developmental decrease. We conclude that (a) Tbeta4 is the major actin sequestering protein in embryonic chick brain and the only beta thymosin isoform present; (b) ADF may play a significant developmental role, as its concentration changes significantly with age; (c) the known G-actin binding proteins can adequately account for the G-actin pool in embryonic chick brain.

Actin Depolymerizing Factors↗
PubMed2007

Disassembly of actin filaments by botulinum C2 toxin and actin-filament-disrupting agents induces assembly of microtubules in human leukaemia cell lines.

BACKGROUND INFORMATION: C(2) toxin produced by Clostridium botulinum types C and D ADP-ribosylates actin monomers and inactivates their polymerization activities. The disassembly of actin filaments by C(2) toxin induces a polarization of cultured human leukaemia cell lines. RESULTS: The polarization induced by C(2) toxin was temperature dependent and was prevented by nocodazole, a microtubule-disrupting agent, whereas it was promoted by paclitaxel, a microtubule-stabilizing agent. The fluorescence staining of polarized cells indicated an increase in microtubule assembly accompanying disassembly of actin filaments. Furthermore, several actin-filament-disrupting agents, other than C(2) toxin, also induced microtubule assembly and cell polarization, irrespective of their different mechanisms of action. The effects induced by some of the agents, which have lower binding affinities for actin, were reversible in response to the re-assembly of actin filaments. CONCLUSIONS: Thus the disassembly of actin filaments by C(2) toxin and actin-filament-disrupting agents induces assembly of microtubules followed by polarization of human leukaemia cell lines, indicating that the assembly/disassembly equilibrium of actin filaments influences the dynamics of microtubules, which control cell morphology and, in turn, diverse cellular processes.

Actin Cytoskeleton↗
PubMed1997

The maize actin-depolymerizing factor, ZmADF3, redistributes to the growing tip of elongating root hairs and can be induced to translocate into the nucleus with actin.

The maize actin depolymerizing factor, ZmADF3, binds G- and F-actin, and increases in vitro actin dynamics. Polyclonal antibodies have been raised against ZmADF3 and these detect a single band of approximately 17 kDa in all maize tissues examined, with the exception of pollen. In the development of root hairs, the distribution of ZmADF3 is related to actin reorganization. In the early stages of hair development, ZmADF3 is distributed throughout the cytoplasm. As the hair emerges and the microfilament bundles redirect to the outgrowth there is a simultaneous redistribution of ZmADF3 which now concentrates at the tip of the emerging hair and remains in this position as elongation proceeds. These observations show that ZmADF3 localizes to a region where actin is being remodelled during tip growth. After cytochalasin D treatment which disrupts actin filaments, short rods of ZmADF3 and actin appear in the nucleus suggesting that ZmADF3 may function by guiding actin to sites of actin polymerization.

Actin Depolymerizing Factors↗
PubMed2001

Interaction of pollen-specific actin-depolymerizing factor with actin.

We have examined the interaction of recombinant lily pollen ADF, LlADF1, with actin and found that whilst it bound both G- and F-actin, it had a much smaller effect on the polymerization and depolymerization rate constants than the maize vegetative ADF, ZmADF3. An antiserum specific to pollen ADF, antipADF, was raised and used to localize pollen ADF in daffodil--a plant in which massive reorganizations of the actin cytoskeleton have been seen to occur as pollen enters and exits dormancy. We show, for the first time, an ADF decorating F-actin in cells that did not result from artificial increase in ADF concentration. In dehydrated pollen this ADF : actin array is replaced by actin : ADF rodlets and aggregates of actin, which presumably act as a storage form of actin during dormancy. In germinated pollen ADF has no specific localization, except when an adhesion is made at the tip where actin and ADF now co-localize. These activities of pollen ADF are discussed with reference to the activities of ZmADF3 and other members of the ADF/cofilin group of proteins.

Actin Depolymerizing Factors↗
PubMed1999

XAIP1: a Xenopus homologue of yeast actin interacting protein 1 (AIP1), which induces disassembly of actin filaments cooperatively with ADF/cofilin family proteins.

We carried out affinity column chromatography using Xenopus ADF/cofilin (XAC), identified several polypeptides in oocytes specifically bound to this column with actin, and isolated a full-length cDNA clone for a 65 kDa protein in this fraction. The predicted amino acid sequence revealed that the 65 kDa protein has seven obvious WD repeats and exhibits striking homology with yeast actin interacting protein 1 (AIP1). Thus, we designated this protein Xenopus AIP1 (XAIP1). We purified XAIP1 from Xenopus oocytes, and its interaction with actin was characterized by a pelleting assay, photometrical analysis and electron microscopy. Although XAIP1 itself cosedimented with F-actin and increased unsedimented actin to some extent, it induced a rapid, drastic disassembly of actin filaments associated with XAC. Electron microscopic observation revealed that XAIP1 severs actin filaments in the presence of XAC. To elucidate the in vivo effects of XAIP1, the purified protein was injected into blastomeres at the two-cell stage. Although the localization of XAIP1 was similar to that of XAC, at the cortical cytoskeleton and diffusely in the cytoplasm, injection of a large amount of XAIP1 arrested development and abolished the strong cortical staining of both actin and XAC. From these results, we concluded that XAIP1 regulates the dynamics of the cortical actin cytoskeleton cooperatively with XAC in eggs.

Actin Depolymerizing Factors↗
PubMed2006

Rapid actin monomer-insensitive depolymerization of Listeria actin comet tails by cofilin, coronin, and Aip1.

Actin filaments in cells depolymerize rapidly despite the presence of high concentrations of polymerizable G actin. Cofilin is recognized as a key regulator that promotes actin depolymerization. In this study, we show that although pure cofilin can disassemble Listeria monocytogenes actin comet tails, it cannot efficiently disassemble comet tails in the presence of polymerizable actin. Thymus extracts also rapidly disassemble comet tails, and this reaction is more efficient than pure cofilin when normalized to cofilin concentration. By biochemical fractionation, we identify Aip1 and coronin as two proteins present in thymus extract that facilitate the cofilin-mediated disassembly of Listeria comet tails. Together, coronin and Aip1 lower the amount of cofilin required to disassemble the comet tail and permit even low concentrations of cofilin to depolymerize actin in the presence of polymerizable G actin. The cooperative activities of cofilin, coronin, and Aip1 should provide a biochemical basis for understanding how actin filaments can grow in some places in the cell while shrinking in others.

Actin Cytoskeleton↗
PubMed2005

The pleckstrin homology domain-containing protein CKIP-1 is involved in regulation of cell morphology and the actin cytoskeleton and interaction with actin capping protein.

CKIP-1 is a pleckstrin homology domain-containing protein that interacts with protein kinase CK2. To elucidate the functions of CKIP-1, we generated human osteosarcoma cell lines with tetracycline-regulated expression of Flag-CKIP-1. Flag-CKIP-1 expression resulted in distinct changes in cellular morphology. Therefore, we examined the actin profile by immunofluorescence, quantitative measurement of phalloidin binding, and immunoblot analysis. These studies demonstrate that Flag-CKIP-1 expression resulted in increases in F-actin staining and protein levels of beta-actin. To elucidate the mechanisms behind the observed phenotype, we utilized tandem affinity purification to isolate CKIP-1 interacting proteins. Mass spectrometry analysis led to the identification of the actin capping protein subunits, CPalpha and CPbeta, as novel CKIP-1 interaction partners. Interactions were confirmed by coimmunoprecipitation and by colocalization. Furthermore, we demonstrate that Ser9 of CPalpha is phosphorylated by protein kinase CK2 in vitro, that CPalpha is phosphorylated in vivo, and that treatment with a CK2-specific inhibitor results in a decrease in CPalpha phosphorylation. Finally, we demonstrate that CKIP-1 and CK2 inhibit the activity of actin capping protein at the barbed ends of actin filaments. Overall, our results are consistent with CKIP-1 playing a role in the regulation of the actin cytoskeleton through its interactions with actin capping protein.

Actin Capping Proteins↗
PubMed1991

Distribution of actin isoforms within cells of the seminiferous epithelium of the rat testis: evidence for a muscle form of actin in spermatids.

Recently, a cDNA that coded for an enteric smooth muscle gamma-actin (SMGA) that was expressed in post-meiotic mouse testicular cells was identified. To determine the cellular location(s) of the protein encoded by this cDNA, this SMGA was probed for by immunocytochemistry in the cells of the seminiferous epithelium with two different monoclonal antibodies (Mabs), B4 and HUC 1-1, known to be muscle actin selective. As a control, we also examined the immunoreactivity of a third Mab, C4, that reacts with all non-muscle and muscle vertebrate isoactins. Using light and electron microscopy, a progressive increase in immunolabeling was observed with the muscle selective HUC 1-1 Mab over a loose actin filamentous network distributed throughout the cytoplasm of steps 4-16 spermatids. Thereafter, the labeling decreased such that at step 17 spermatids, only cytoplasmic labeling in the tail of the spermatids was observed. No labeling of this network was noted with the C4 or B4 Mabs. However, myoid cells enveloping seminiferous tubules and smooth muscle cells of interstitial blood vessels demonstrated comparable intense labeling with each of the three Mabs. The C4 Mab intensely labeled actin filaments of the Sertoli-Sertoli and Sertoli-spermatid ectoplasmic specializations. Also well labeled were numerous actin filaments found in the apical Sertoli cell processes encapsulating the heads of late step 19 spermatids at stage VII of the cycle of the seminiferous epithelium. In addition, actin filamentous bundles enveloping tubulobulbar complexes of the late spermatids within the Sertoli cell apical processes were intensely labeled. The actin filaments in the Sertoli apical processes and surrounding the tubulobulbar complexes were also strongly immunolabeled with the HUC 1-1 Mab. The C4 Mab but not the B4 or HUC 1-1 Mabs, recognized actin in the subacrosomal space of steps 4-18 spermatids. This study suggests that there are muscle isoforms of actin within the cytoplasm of developing spermatids and within apical processes of Sertoli cells.

Actins↗
PubMed1989

Gel electrophoresis of native actin and the actin-deoxyribonuclease I complex.

Electrophoresis of monomeric actin (G-actin) on 8-25% acrylamide Pharmacia PhastGels was carried out using gels and agarose buffer strips preequilibrated in buffer containing adenosine triphosphate (ATP), calcium ions (Ca2+) and dithiothreitol. On these gels G-actin ran as a sharp band at an apparent molecular mass of 45 kDa relative to standard proteins which is slightly greater than its actual molecular mass of 42 kDa. Electrophoresis in the absence of these solutes led to denaturation and aggregation of the protein, as reflected by a long streak. Filamentous actin (F-actin) did not enter the gel. The actin monomer-binding protein, deoxyribonuclease I, (DNase I) forms a binary complex with G-actin. The purity and apparent molecular mass 74 kDa of this complex were determined by native gel electrophoresis. By the simple procedure of preequilibrating both gel and buffer strips with appropriate ligands, this technique could be extended to investigate interactions between actin and other G-actin-binding proteins and other proteins whose stability is ligand dependent.

Actins↗
PubMed1998

Highly divergent actin expressed in a Chlamydomonas mutant lacking the conventional actin gene.

The Chlamydomonas mutant ida5 is deficient in the conventional actin gene and its axoneme lacks a subset of inner dynein arms that contain actin as a subunit. However, this mutant retains some other inner dynein arms because a novel protein (NAP) is expressed as a substitute for actin. In this study, we show by sequence analysis that NAP is identical to a putative actin-related protein, the cDNA sequence of which has recently been reported and shown to have 64% amino acid identity with conventional actin. A polyclonal antibody raised against a synthetic polypeptide corresponding to the NH2-terminal sequence of this protein specifically reacted with the spot corresponding to NAP in two-dimensional electrophoresis patterns. NAP apparently can substitute for conventional actin in some, but not all, cellular functions, and therefore can be regarded as a highly divergent actin. This unconventional actin appears to be expressed only when conventional actin is absent.

Actins↗
PubMed2000

Acetylation at the N-terminus of actin strengthens weak interaction between actin and myosin.

The N-terminus of all actins so far studied is acetylated. Although the pathways of acetylation have been well studied, its functional importance has been unclear. A negative charge cluster in the actin N-terminal region is shown to be important for the function of actomyosin. Acetylation at the N-terminus removes a positive charge and increases the amount of net negative charges in the N-terminal region. This may augment the role of the negative charge cluster. To examine this possibility, actin with a nonacetylated N-terminus (nonacetylated actin) was produced. The nonacetylated actin polymerized and depolymerized normally. In actin-activated heavy meromyosin ATPase assays, the nonacetylated actin showed higher K(app) without significantly changing V(max), compared with those of wild-type actin. This is in contrast to the effect of the N-terminal negative charge cluster, which increases V(max) without changing K(app). These results indicate that the acetylation at the N-terminus of actin strengthens weak actomyosin interaction.

Acetylation↗
PubMed2000

Root hair formation: F-actin-dependent tip growth is initiated by local assembly of profilin-supported F-actin meshworks accumulated within expansin-enriched bulges.

Plant root hair formation is initiated when specialized elongating root epidermis cells (trichoblasts) assemble distinct domains at the plasma membrane/cell wall cell periphery complexes facing the root surface. These localities show accumulation of expansin and progressively transform into tip-growing root hair apices. Experimentation showed that trichoblasts made devoid of microtubules (MTs) were unaffected in root hair formation, whereas those depleted of F-actin by the G-actin sequestering agent latrunculin B had their root hair formation blocked after the bulge formation stage. In accordance with this, MTs are naturally depleted from early outgrowing bulges in which dense F-actin meshworks accumulate. These F-actin caps remain associated with tips of emerging and growing root hairs. Constitutive expression of the GFP-mouse talin fusion protein in transgenic Arabidopsis, which visualizes all classes of F-actin in a noninvasive mode, allowed in vivo confirmation of the presence of distinct F-actin meshworks within outgrowing bulges and at tips of young root hairs. Profilin accumulates, at both the protein and the mRNA levels, within F-actin-enriched bulges and at tips of emerging hairs. ER-based calreticulin and HDEL proteins also accumulate within outgrowing bulges and remain enriched at tips of emerging hairs. All this suggests that installation of the actin-based tip growth machinery takes place only after expansin-associated bulge formation and requires assembly of profilin-supported dynamic F-actin meshworks.

Actins↗
PubMed2000

Structural basis for the higher Ca(2+)-activation of the regulated actin-activated myosin ATPase observed with Dictyostelium/Tetrahymena actin chimeras.

Replacement of residues 228-230 or 228-232 of subdomain 4 in Dictyostelium actin with the corresponding Tetrahymena sequence (QTA to KAY replacement: half chimera-1; QTAAS to KAYKE replacement: full chimera) leads to a higher Ca(2+)-activation of the regulated acto-myosin subfragment-1 ATPase activity. The ratio of ATPase activation in the presence of tropomyosin-troponin and Ca(2+) to that without tropomyosin-troponin becomes about four times as large as the ratio for the wild-type actin. To understand the structural basis of this higher Ca(2+)-activation, we have determined the crystal structures of the 1:1 complex of Dictyostelium mutant actins (half chimera-1 and full chimera) with gelsolin segment-1 to 2.0 A and 2.4 A resolution, respectively, together with the structure of wild-type actin as a control. Although there were local changes on the surface of the subdomain 4 and the phenolic side-chain of Tyr230 displaced the side-chain of Leu236 from a non-polar pocket to a more solvent-accessible position, the structures of the actin chimeras showed that the mutations in the 228-232 region did not introduce large changes in the overall actin structure. This suggests that residues near position 230 formed part of the tropomyosin binding site on actin in actively contracting muscle. The higher Ca(2+)-activation observed with A230Y-containing mutants can be understood in terms of a three-state model for thin filament regulation in which, in the presence of both Ca(2+) and myosin heads, the local changes of actin generated by the mutation (especially its phenolic side-chain) facilitate the transition of thin filaments from a "closed" state to an "open" state. Between 394 and 469 water molecules were identified in the different structures and it was found that actin recognizes hydrated forms of the adenine base and the Ca ion in the nucleotide binding site.

Actins↗
PubMed2000

Structure of the utrophin actin-binding domain bound to F-actin reveals binding by an induced fit mechanism.

Utrophin is a large ubiquitously expressed cytoskeletal protein, homologous to dystrophin, the protein disrupted in Duchenne muscular dystrophy. The association of both proteins with the actin cytoskeleton is functionally important and is mediated by a domain at their N termini, conserved in members of the spectrin superfamily, including alpha-actinin, beta-spectrin and fimbrin. We present the structure of the actin-binding domain of utrophin in complex with F-actin, determined by cryo-electron microscopy and helical reconstruction, and a pseudo-atomic model of the complex, generated by docking the crystal structures of the utrophin domain and F-actin into the reconstruction. In contrast to the model of actin binding proposed for fimbrin, the utrophin actin-binding domain appears to associate with actin in an extended conformation. This conformation places residues that are highly conserved in utrophin and other members of the spectrin superfamily at the utrophin interface with actin, confirming the likelihood of this binding orientation. This model emphasises the importance of protein flexibility in modeling interactions and presents the fascinating possibility of a diversity of actin-binding mechanisms among related proteins.

Actins↗
PubMed2000

MARCKS-related protein binds to actin without significantly affecting actin polymerization or network structure. Myristoylated alanine-rich C kinase substrate.

Actinis a 42-kDa protein which, due to its ability to polymerize into filaments (F-actin), is one of the major constituents of the cytoskeleton. It has been proposed that MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) proteins play an important role in regulating the structure and mechanical properties of the actin cytoskeleton by cross-linking actin filaments. We have recently reported that peptides corresponding to the effector domain of MARCKS proteins promote actin polymerization and cause massive bundling of actin filaments. We now investigate the effect of MARCKS-related protein, a 20-kDa member of the MARCKS family, on both filament structure and the kinetics of actin polymerization in vitro. Our experiments document that MRP binds to F-actin with micromolar affinity and that the myristoyl chain at the N-terminus of MRP is not required for this interaction. In marked contrast to the effector peptide, binding of MRP is not accompanied by an acceleration of actin polymerization kinetics, and we also could not reliably observe an actin cross-linking activity of MRP.

Actins↗