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PubMed1988

Probing actin polymerization by intermolecular cross-linking.

We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an apparent molecular mass of 86 kD by SDS-PAGE [LD]) is attenuated, while an actin filament dimer ("upper" dimer with an apparent molecular mass of 115 kD by SDS-PAGE [UD] as characterized [Elzinga, M., and J. J. Phelan. 1984. Proc. Natl. Acad. Sci. USA. 81:6599-6602]) is formed. This shift from LD to UD occurs concomitant with formation of filaments as assayed by N-(1-pyrenyl)iodoacetamide fluorescence enhancement and electron microscopy. Isolated cross-linked LD does not form filaments, while isolated cross-linked UD will assemble into filaments indistinguishable from those polymerized from unmodified G-actin under typical filament-forming conditions. The presence of cross-linked LD does not effect the kinetics of polymerization of actin monomer, whereas cross-linked UD shortens the "lag phase" of the polymerization reaction in a concentration-dependent fashion. Several converging lines of evidence suggest that, although accounting for a significant oligomeric species formed during early polymerization, the LD is incompatible with the helical symmetry defining the mature actin filament; however, it could represent the interfilament dimer found in paracrystalline arrays or filament bundles. Furthermore, the LD is compatible with the unit cell structure and symmetry common to various types of crystalline actin arrays (Aebi, U., W. E. Fowler, G. Isenberg, T. D. Pollard, and P. R. Smith. 1981. J. Cell Biol. 91:340-351) and might represent the major structural state in which a mutant beta-actin (Leavitt, J., G. Bushar, T. Kakunaga, H. Hamada, T. Hirakawa, D. Goldman, and C. Merril. 1982. Cell. 28:259-268) is arrested under polymerizing conditions.

Actin Cytoskeleton↗
PubMed2024

SUN2 mediates calcium-triggered nuclear actin polymerization to cluster active RNA polymerase II.

The nucleoskeleton is essential for nuclear architecture as well as genome integrity and gene expression. In addition to lamins, titin or spectrins, dynamic actin filament polymerization has emerged as a potential intranuclear structural element but its functions are less well explored. Here we found that calcium elevations trigger rapid nuclear actin assembly requiring the nuclear membrane protein SUN2 independently of its function as a component of the LINC complex. Instead, SUN2 colocalized and associated with the formin and actin nucleator INF2 in the nuclear envelope in a calcium-regulated manner. Moreover, SUN2 is required for active RNA polymerase II (RNA Pol II) clustering in response to calcium elevations. Thus, our data uncover a SUN2-formin module linking the nuclear envelope to intranuclear actin assembly to promote signal-dependent spatial reorganization of active RNA Pol II.

RNA Polymerase II↗
PubMed1992

The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization.

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen capable of rapid movement through the host cell cytoplasm. The biophysical basis of the motility of L. monocytogenes is an interesting question in its own right, the answer to which may shed light on the general processes of actin-based motility in cells. Moving intracellular bacteria display phase-dense 'comet tails' made of actin filaments, the formation of which is required for bacterial motility. We have investigated the dynamics of the actin filaments in the comet tails using the technique of photoactivation of fluorescence, which allows monitoring of the movement and turnover of labelled actin filaments after activation by illumination with ultraviolet light. We find that the actin filaments remain stationary in the cytoplasm as the bacterium moves forward, and that length of the comet tails is linearly proportional to the rate of movement. Our results imply that the motile mechanism involves continuous polymerization and release of actin filaments at the bacterial surface and that the rate of filament generation is related to the rate of movement. We suggest that actin polymerization provides the driving force for bacterial propulsion.

Actins↗
PubMed1984

[Effects of pH and ATP on the cytoplasm of Amoeba proteus: relation between cytoplasm motility and actin polymerization].

The effect of pH and ATP was studied on isolated cytoplasm of Amoeba proteus. These two parameters were shown to influence both the motility and the organization of actin filaments in the isolated cytoplasm. Furthermore, our results demonstrate that there is a relationship between the motility and the polymeric state of actin. When the isolated cytoplasm is non-motile, actin is highly polymerized into long filaments arranged parallel in bundles. When this cytoplasm is motile, however, actin can either be weakly polymerized, i.e. observed as few short filaments, or can be polymerized in long branched filaments forming a loose network.

Actins↗
PubMed2001

Actin polymerization is essential for pollen tube growth.

Actin microfilaments, which are prominent in pollen tubes, have been implicated in the growth process; however, their mechanism of action is not well understood. In the present work we have used profilin and DNAse I injections, as well as latrunculin B and cytochalasin D treatments, under quantitatively controlled conditions, to perturb actin microfilament structure and assembly in an attempt to answer this question. We found that a approximately 50% increase in the total profilin pool was necessary to half-maximally inhibit pollen tube growth, whereas a approximately 100% increase was necessary for half-maximal inhibition of cytoplasmic streaming. DNAse I showed a similar inhibitory activity but with a threefold more pronounced effect on growth than streaming. Latrunculin B, at only 1--4 nM in the growth medium, has a similar proportion of inhibition of growth over streaming to that of profilin. The fact that tip growth is more sensitive than streaming to the inhibitory substances and that there is no correlation between streaming and growth rates suggests that tip growth requires actin assembly in a process independent of cytoplasmic streaming.

Actin Cytoskeleton↗
PubMed2006

Thyroid hormone induction of actin polymerization in somatotrophs of hypothyroid rats: potential repercussions in growth hormone synthesis and secretion.

Thyroid hormone was shown to induce actin cytoskeleton polymerization in hypothyroid astrocytes and osteoblastic cells by a nongenomic mechanism. Polyadenylation of GH mRNA, a process that depends on cytoskeleton-associated proteins, was also shown to be regulated by thyroid hormone. Here we investigated by histochemistry and immunohistochemistry whether acute (100 microg per 100 g body weight, iv, for 30 min) or chronic (5 microg per 100 g body weight, ip, 5 d) administration of T3 to thyroidectomized (Tx) and sham-operated rats affects the somatotrophs F-actin cytoskeleton arrangement and its potential repercussion on GH synthesis and secretion. Thyroidectomy dramatically decreased the amount of somatotrophs F-actin content and induced the disassembly of the actin cytoskeleton. These effects were reversed by acute and chronic administration of T3. In addition, in Tx rat somatotrophs, GH labeling was detected mostly at the cell periphery. After 30 min of T3 administration, GH labeling decreased at periphery and increased in the perinuclear region, suggesting that GH secretion and synthesis were stimulated by T3. No differences were detected in the total actin protein content, although a decrease in the F- and increase in G-actin contents were detected in Tx rat pituitaries, a panorama that was reversed by acute T3 treatment, as shown by Western blotting analysis. The sham-operated animals' somatotrophs were only mildly affected by acute T3 administration. The results indicate that the T3-induced rapid alterations on somatotroph actin cytoskeleton and GH cellular distribution resulted from actin filaments rearrangement, which characterizes a nongenomic action.

Actin Cytoskeleton↗
PubMed1990

Flow cytometric studies on actin polymerization in PMN cells from chronic myeloid leukemia (CML) patients.

Studies in our laboratory have shown that polymorphonuclear leucocytes (PMNL) from chronic myeloid leukemia (CML) patients are defective in chemotaxis towards a synthetic peptide, n-formyl-methionyl-leucyl-phenylalanine (FMLP), during the active phases of the disease and in remission. Actin plays a major role in cellular movements and binding of chemo-attractant to cells induces polymerization of G-actin to F-actin. We have, therefore, compared polymerization of actin in FMLP stimulated PMNL from CML patients with those from normal subjects by fluorescence microscopy and flow cytometry, using F-actin specific probe, NBD-phallacidin. Our results show that binding of FMLP to normal PMNL induces rapid conversion of G-actin to F-actin followed by depolymerization to some extent. In CML PMNL, such a biphasic response is not seen. Conversion of G-actin to F-actin is slower and F-actin content is significantly lower than that in normal PMNL. Moreover, organization of F-actin is different in CML PMNL as compared to that in normal PMNL.

Actins↗
PubMed1984

Actin polymerization induced by pulsed electric stimulation of bone cells in vitro.

Electric field pulses, capacitively applied to tissue cultures of embryonic bone cells, were shown to induce changes in the state of cellular actin. Three actin states could be defined by DNAase I inhibition. A rapidly (20-30 s) inhibiting fraction, attributed to monomeric G-actin, amounts to 55% of total actin in nonstimulated cells. An additional fraction of 8% required approx. 20 min to reach full inhibition and was tentatively defined as polymeric 'F'-actin. The remaining 37% could be detected only after treatment of the cells with 0.75 M guanidine hydrochloride, which dissociates actin from all its protein interactions. This fraction, N-actin (network actin) is believed to represent F-actin integrated into some supramolecular structure, where it is not accessible to DNAase I. Upon short electric stimulation the distribution changed to 40% G-actin, 12% F-actin and 48% N-actin. 3-Isobutyl-1-methylxanthine (IBMX; an inhibitor of cAMP phosphodiesterase), depletion of extracellular calcium, and calmodulin inhibitors abolished this field effect.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Actin polymerization induced by calspectin, a calmodulin-binding spectrin-like protein.

We have purified from a membrane fraction of bovine brain a calmodulin-binding protein (calspectin) that shares a number of properties with erythrocyte spectrin: It has a heterodimeric structure with Mr 240 000 and 235 000 and binds to (dimeric form) or crosslinks (tetrameric form) F-actin. We show that calspectin (tetramer) is capable of inducing the polymerization of G-actin to actin filaments by increasing nucleation under conditions where actin alone polymerizes at a much slower rate. Thus, brain calspectin behaves in the same manner as erythrocyte spectrin, supporting the idea that, in conjunction with actin oligomers it comprises the cytoskeletal meshwork underlying the cytoplasmic surface of the nerve cell.

Actins↗
PubMed1976

Actin polymerization and interaction with other proteins in temperature-induced gelation of sea urchin egg extracts.

The gel which forms on warming the extracts of the cytoplasmic proteins of sea urchin eggs has been separated into two fractions, one containing F-actin and the other containing two proteins of 58,000 and 22,000 mol wt. When combined in 0.1 M KCl, even at 0 degrees C, these components will form gel material identical to that formed by warming extracts. This gel is a network of laterally aggregated F-actin filaments which are in register and which display a complex cross-banding pattern generated by the presence of the other two proteins. Low concentrations of calcium block the assembly of these proteins to form this complex structure, which may play some cytoskeletal role in the cytoplasm. This association of F-actin with the other proteins to form a gel is very likely the last step fo the process occurring in warmed extracts. At low temperatures, gelation of extracts is limited by the relative absence of F-actin, as demonstrated by the inability to sediment it at 100,000 g and also by the fact that gelation occurs immediately if exogenous F-actin is added to cold extracts. The transformation of the G-actin present in the extract to the F-form is apparently repressed at low temperatures. This is shown directly by the failure of added G-actin to polymerize at low temperatures in the presence of extract. These observations resemble those which have been reported on preparations from amoeboid cells and may be significant in the involvement of actin and these other proteins in cell division and later developmental processes.

Actins↗
PubMed2003

AIF-1 is an actin-polymerizing and Rac1-activating protein that promotes vascular smooth muscle cell migration.

Development of vascular restenosis is a multifaceted process characterized by migration and proliferation of vascular smooth muscle cells (VSMCs), resulting in loss of lumen diameter. Characterization of proteins that mediate this process is essential in our understanding of the pathogenesis of arterial injury. Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic, calcium-binding protein that is expressed in VSMCs by allograft and balloon angioplasty injury. AIF-1 is not present in cultured human VSMCs but is induced by cytokines, and overexpression of AIF-1 results in increased VSMC growth and cell-cycle gene expression. To characterize AIF-1 modulatory effects in primary human VSMCs, AIF-1-interacting proteins were identified by an AIF-1/glutathione S transferase fusion protein affinity assay. MALDI-TOF mass spectrophotometric amino analysis identified actin as an AIF-1 interacting protein. This interaction was verified by coimmunoprecipitation. This is a functional interaction, because AIF-1 binds to and polymerizes F-actin in vitro. In unstimulated VSMCs, AIF-1 colocalizes with F-actin but translocates to lamellipodia on stimulation with platelet-derived growth factor. VSMCs stably transduced with AIF-1 retrovirus migrate 2.6-fold more rapidly (85.1+/-2.9 versus 225.5+/-16.6; P<0.001) in response to platelet-derived growth factor versus control cells. AIF-1 colocalizes with Rac1, and AIF-1-transduced VSMCs show a constitutive and enhanced activation of Rac1, providing a mechanism for the increased migration. These data indicate that AIF-1 binds and polymerizes F actin and also regulates Rac1 activity and VSMC migration. Considering the AIF-1 expression pattern in injured arteries, this suggests that AIF-1 may be involved in the cytoskeletal signaling network leading to vascular remodeling.

Actins↗
PubMed2002

Increased actin polymerization reduces the inhibition of serum response factor activity by Yin Yang 1.

Recent evidence has implicated CC(A/T(richG))GG (CArG) boxes, binding sites for serum response factor (SRF), in the regulation of expression of a number of genes in response to changes in the actin cytoskeleton. In many cases, the activity of SRF at CArG boxes is modulated by transcription factors binding to overlapping (e.g. Yin Yang 1, YY1) or adjacent (e.g. ets) binding sites. However, the mechanisms by which SRF activity is regulated by the cytoskeleton have not been determined. To investigate these mechanisms, we screened for cells that did or did not increase the activity of a fragment of the promoter for a smooth-muscle (SM)-specific gene SM22alpha, in response to changes in actin cytoskeletal polymerization induced by LIM kinase. These experiments showed that vascular SM cells (VSMCs) and C2C12 cells increased the activity of promoters containing at least one of the SM22alpha CArG boxes (CArG near) in response to LIM kinase, whereas P19 cells did not. Bandshift assays using a probe to CArG near showed that P19 cells lacked detectable YY1 DNA binding to the CArG box in contrast with the other two cell types. Expression of YY1 in P19 cells inhibited SM22alpha promoter activity and conferred responsiveness to LIM kinase. Mutation of the CArG box to inhibit YY1 or SRF binding indicated that both factors were required for the LIM kinase response in VSMCs and C2C12 cells. The data indicate that changes in the actin cytoskeletal organization modify SRF activity at CArG boxes by modulating YY1-dependent inhibition.

Actins↗