• Journal of Life Science
• /
• v.17 no.2 s.82
• /
• pp.204-210
• /
• 2007

Previous reports indicated that the carboxyl terminal residues, glutamine276-threonine277 in particular, were important for actin affinity of the unacetylated smooth ${\alpha}-tropomyosin$. To determine the role of the glutamine and threonine residues in C-terminal region in actin binding, we constructed mutant striated muscle ${\alpha}-tropomyosin$ (TMs), in which these two residues were individually substituted. These mutant tropomyosins, designated TM18 (HT) and TM19 (QA), were overexpressed in E. coli as an either unacetylated form or Ala-Ser. (AS) dipeptide fusion form, and were analyzed F-actin affinity by cosedimentation. Unacetylated TM19 (QA) bound to actin approximately three times stronger than TM18 (HT) and much stronger than ST (HA). AS/TM19 (QA) showed four times stronger, in actin affinity than AS/ST (HA) while AS/TM14 (QT) bound to actin stronger to some extent than AS/TM18 (HT). These results suggested that the presence of Gln residue at 276 be primarily attributed to higher actin affinity of smooth ${\alpha}-tropomyosin$.

• Molecules and Cells
• /
• v.41 no.5
• /
• pp.436-443
• /
• 2018

The actin cytoskeleton plays a key role in the entry of mitosis as well as in cytokinesis. In a previous study, we showed that actin disruption delays mitotic entry at G2/M by sustained activation of extracellular signal-related kinase 1/2 (ERK1/2) in primary cells but not in transformed cancer cell lines. Here, we examined the mechanism of cell cycle delay at G2/M by actin dysfunction in IMR-90 normal human fibroblasts. We observed that de-polymerization of actin with cytochalasin D (CD) constitutively activated ribosomal S6 kinase (RSK) and induced inhibitory phosphorylation of Cdc2 (Tyr 15) in IMR-90 cells. In the presence of an actin defect in IMR-90 cells, activating phosphorylation of Wee1 kinase (Ser 642) and inhibitory phosphorylation of Cdc25C (Ser 216) was also maintained. However, when kinase-dead RSK (DN-RSK) was overexpressed, we observed sustained activation of ERK1/2, but no delay in the G2/M transition, demonstrating that RSK functions downstream of ERK in cell cycle delay by actin dysfunction. In DN-RSK overexpressing IMR-90 cells treated with CD, phosphorylation of Cdc25C (Ser 216) was blocked and phosphorylation of Cdc2 (Tyr 15) was decreased, but the phosphorylation of Wee1 (Ser 642) was maintained, demonstrating that RSK directly controls phosphorylation of Cdc25C (Ser 216), but not the activity of Wee1. These results strongly suggest that actin dysfunction in primary cells activates ERK1/2 to inhibit Cdc2, delaying the cell cycle at G2/M by activating downstream RSK, which phosphorylates and blocks Cdc25C, and by directly activating Wee1.

• IMMUNE NETWORK
• /
• v.9 no.6
• /
• pp.274-284
• /
• 2009

Background: Swiprosin-1 was identified in human CD8+ lymphocytes, mature B cells and non-lymphonoid tissue. We have recently reported that swiprosin-1 is expressed in mast cells and up-regulated in both in vitro and in vivo. Methods: The expression of cytokines and swiprosin-1 were determined by by real time PCR and conventional PCR. Pharmacological inhibitors were treated to investigate potential mechanism of swiprosin-1 in mast cell activation. Actin content was evaluated by confocal microscopy and flow cytometry. Results: The swiprosin-1 augmented PMA/A23187-induced expression of cytokines and release of histamine. However, knock-down of swiprosin-1 showed only a modest effect on PMA/A23187-induced cytokine expression, suggesting that swiprosin-1 has gain-of-function characteristics. Swiprosin-1 was found in microvilli-like membrane protrusions and highly co-localized with F-actin. Importantly, either disruption of actin by cytochalasin B or inhibition of PI3 kinase, an enzyme involved in actin remodeling, by wortmannin blocked cytokine expression only in swiprosin-1-overexpressing cells. Conclusion: These results suggest that swiprosin-1 modulates mast cell activation potentially through actin regulation.

• ALGAE
• /
• v.25 no.4
• /
• pp.197-204
• /
• 2010

Cytoskeletal changes were observed during cell division of the green alga Zygnema cruciatum using flourescein isothiocynate (FITC)-conjugated phallacidin for F-actin staining and FITC-anti-$\alpha$-tubulin for microtubule staining. Z. cruciatum was uninucleate with two star-shaped chloroplasts. Nuclear division and cell plate formation occurred prior to chloroplast division. Actin filaments appeared on the chromosome and nuclear surface during prophase, and the F-actin ring appeared as the cleavage furrow developed. FITC-phallacidin revealed that actin filaments were attached to the chromosomes during metaphase. The F-actin ring disappeared at late metaphase. At telophase, FITC-phallacidin staining of actin filaments disappeared. FITC-anti-$\alpha$-tubulin staining revealed that microtubules were arranged beneath the protoplasm during interphase and then localized on the nuclear region at prophase, and that the mitotic spindle was formed during metaphase. The microtubules appeared between dividing chloroplasts. The results indicate that a coordination of actin filaments and microtubules might be necessary for nuclear division and chromosome movement in Z. cruciatum.

• Parasites, Hosts and Diseases
• /
• v.34 no.4
• /
• pp.215-224
• /
• 1996

actin and some actin binding proteins such as tropomyosin, α-actinin and troponin T were localized by simultaneous double immunogold labeling in several developmental stages of Cryptosporidium parvum. All of the observed developmental stages have many paricles of tropomyosin and actin around pellicle and cytoplasm. Tropomyosin was labeled much more than the actin when these two proteins were labeled simultaneously. And α-actinin was labeled mostly in the pellicle, but troponin T labeling weas very rarely observed. From this study it was suggested that tropomyosin seemed to be one of the major proteins of C. parvum, so it must be playing important roles in C. parvum.

• Fisheries and Aquatic Sciences
• /
• v.12 no.2
• /
• pp.98-103
• /
• 2009

The potential utility of the rockbream (Oplegnathus fasciatus) $\beta$-actin 5'-upstream sequence as a regulatory element for DNA vaccination was evaluated based on in vitro and in vivo heterologous expression assays. In the in vitro transfection experiment, the efficacy of the rockbream $\beta$-actin promoter to drive the expression of a downstream lacZ gene was significantly higher (more than fourfold) than that of the human cytomegalovirus (hCMV) promoter in two fish cell lines (grunt Haemulon plumierii fin and bluegill Lepomis macrochirus fry cell lines). In contrast, the functional activity of the rockbream $\beta$-actin promoter was hardly detectable in a mammalian mouse embryonic fibroblast cell line. Rockbream skeletal muscles injected in vivo with a GFP reporter construct driven by the $\beta$-actin promoter displayed the significantly higher expression of a GFP protein (more than threefold) than did those injected with hCMV promoter driven construct. Data from this study suggest that the homologous rockbream $\beta$-actin promoter could be used as a potential regulator for DNA vaccination in this species.

• Fisheries and Aquatic Sciences
• /
• v.14 no.4
• /
• pp.303-310
• /
• 2011

We characterized a cytoplasmic actin gene locus in greenling Hexagrammos otakii (Scorpaeniformes). Genomic clones isolated from the greenling DNA library contained two homologous cytoplasmic actin gene copies (HObact2.1 and HObact2.2) in a tail-to-head orientation. Their gene structure is characterized by six translated exons and one non-translated exon. Exon-intron organization and the nucleotide sequences of the two actin gene isoforms are very similar. However, only the HObact2.1 isoform contains microsatellite-like, dinucleotide repeats in the 5'-flanking region (named HOms2002) and intron 1 following the non-translated exon 1 (named HOms769). One microsatellite locus (HOms769) was highly polymorphic while the other (HOms2002) was not. Based on bioinformatic analysis, different transcription factor binding motifs are related to stress and immune responses in the two actin isoforms. Semiquantitative and real-time reverse transcription-PCR assays showed that both isoform transcripts were detectable ubiquitously in all the tissues examined. However, the basal expression levels of each isoform varied across tissues. Overall, the two isoforms showed a similar, but not identical, expression pattern. Our data suggest that the cytoplasmic actin genes may be the result of a recent duplication event in the greenling genome, which has not experienced significant subfunctionalization in their housekeeping roles.

• Proceedings of the Korean Society for Food Science of Animal Resources Conference
• /
• 2004.05a
• /
• pp.198-201
• /
• 2004

To investigate hydrostatic pressure (HP) effect on myofibrillar protein (Mf) extracted from bovine Semitendinosus muscle, Ca- and Mg-ATPase activities to evaluate denaturation of myosin and actin, and soluble protein contents were observed. In Mf treated with 100 MPa for 5 min was not observed denaturation of myosin and actin. In Mf treated with 200 MPa for 5 min, denaturation of myosin and actin were observed but inactivation rate was low (0.0136 $min^{-1}$). Inactivation rate of myosin and actin was dramatically increased above 300 MPa treatment. However denaturation of myosin and actin was not that critical with duration time. By increasing pressure size, the amount of myosin and actin in soluble protein eluted in 20 mM potassium phosphate buffer (pH 7.0) containing 0.6 M NaCl were decreased. SDS-PAGE of soluble protein released from Mf suspension in 0.1 M NaCl buffer (pH 7.0) showed that low molecular weight proteins (15${\sim}$36 KDa) were released by HP treatment above 200 MPa. From the results, denaturation of myosin and actin, and release of light molecule proteins of Mf were observed by HP treatment over 200 MPa.

• BMB Reports
• /
• v.54 no.12
• /
• pp.614-619
• /
• 2021

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC), which is a highly aggressive cancer. HBV X protein (HBx), one of four HBV gene products, plays pivotal roles in the development and metastasis of HCC. It has been reported that HBx induces liver cancer cell migration and reorganizes actin cytoskeleton, however the molecular basis for actin cytoskeleton reorganization remains obscure. In this study, we for the first time report that HBx promotes actin polymerization and liver cancer cell migration by regulating calcium modulated protein, calmodulin (CaM). HBx physically interacts with CaM to control the level of phosphorylated cofilin, an actin depolymerizing factor. Mechanistically, HBx interacts with CaM, liberates Hsp90 from its inhibitory partner CaM, and increases the activity of Hsp90, thus activating LIMK1/cofilin pathway. Interestingly, the interaction between HBx and CaM is calcium-dependent and requires the CaM binding motif on HBx. These results indicate that HBx modulates CaM which plays a regulatory role in Hsp90/LIMK1/cofilin pathway of actin reorganization, suggesting a new mechanism of HBV-induced HCC metastasis specifically derived by HBx.

• Journal of Life Science
• /
• v.19 no.5
• /
• pp.573-580
• /
• 2009

It has been previously reported that the carboxyl terminal residues 276 and 277 of ${\alpha}$-tropomyosin are important for actin affinity. In order to investigate actin affinities of these two residues of skeletal (HA) and smooth (QT) muscle ${\alpha}$-tropomyosins, a series of mutant tropomyosins were constructed in which residues at either 276 or 277 were individually replaced with a cysteine residue for chemical modification. These mutants were overexpressed in E. coli as unacetylated and Ala-Ser (AS) dipeptide fusion forms. While actin affinities of unacetylated tropomyosins were considerably low, those of AS/TMs were remarkably higher than those of corresponding unacetylated tropomyosins. However, actin affinities of AS/TM24 (QC) and AS/TM29 (HC) were dramatically lower than those of other AS/TMs and were close to those of unacetylated tropomyosins. In addition, actin affinities of unacetylated TM24 (QC) and TM29 (HC) failed to be restored in the presence of troponin, unlike unacetylated TM10 (HA) and TM23 (CA). These results indicated that the presence of a cysteine residue at 277 caused a drastic decrease in actin affinity, and also that the residue 277 is important for actin affinity of ${\alpha}$-tropomyosin. Since TM23 (CA) showed high actin affinity, it may serve as a valuable tool for chemical modification studies for investigating the interaction of the carboxyl terminal residues of ${\alpha}$-tropomyosin with actin and/or troponin.