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At least 19 recordsLinked to original sources
PubMed2004

Cofilin 1 is revealed as an inhibitor of glucocorticoid receptor by analysis of hormone-resistant cells.

Significant knowledge about glucocorticoid signaling has accumulated, yet many aspects remain unknown. We aimed to discover novel factors involved in glucocorticoid receptor regulation that do not necessarily require direct receptor interaction. We achieved this by using a functional genetic screen: a stable cell line which cannot survive hormone treatment was engineered, randomly mutated, and selected in the presence of glucocorticoid. A hormone-resistant clone was analyzed by two-dimensional gel electrophoresis. Differentially expressed proteins were identified and tested as candidates for regulation of the glucocorticoid receptor. An unexpected candidate, cofilin 1, inhibited receptor activity. Cofilin is known to promote actin depolymerization and filament severing. Several experiments suggest that this feature of cofilin is involved in its inhibitory action. Both its actin depolymerization activity and its inhibitory action on the receptor are dependent on its phosphorylation state. Treatment of cells with a cytoskeleton-disrupting agent decreased receptor activity, as did overexpression of actin, particularly a mutant actin that does not polymerize. In addition, overexpression of cofilin and actin as well as chemical cytoskeleton disruption changed the subcellular receptor distribution and upregulated c-Jun, which could constitute the inhibitory mechanism of cofilin. In summary, cofilin represents a novel factor that can cause glucocorticoid resistance.

Actin Depolymerizing Factors↗
PubMed2004

Proteome analysis of hepatocellular carcinoma cell strains, MHCC97-H and MHCC97-L, with different metastasis potentials.

To better understand the mechanism underlying hepatocellular carcinoma (HCC) metastasis and to search for potential markers for HCC prognosis, differential proteome analysis on two HCC cell strains with high and low metastatic potentials, MHCC97-H and MHCC97-L, was conducted using two-dimensional (2-D) gel electrophoresis followed by matrix-assisted laser desorption/time of flight mass spectrometry and liquid chromatography ion trap mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 56 protein spots showed significant differential expression in MHCC97-H and MHCC97-L cells (Student's t-test, P < 0.05) and 4 protein spots were only detected in MHCC97-H cells. Fourteen protein spots were further identified using in-gel tryptic digestion, peptide mass fingerprinting and tandem mass spectrometry. The expressions of pyruvate kinase M2, ubiquitin carboxy-terminal hydrolase L1, laminin receptor 67 kDa, S100 calcium-binding protein A4, thioredoxin and cytokeratin 19 were elevated in MHCC97-H cells. However, manganese superoxide dismutase, calreticulin precursor, cathepsin D, lactate dehydrogenase B, non-metastatic cell protein 1, cofilin 1 and calumenin precursor were down-regulated in MHCC97-H cells. Intriguingly, most of these identified proteins have been reported to be associated with tumor metastasis. The functional implications of alterations in the levels of these proteins are discussed.

Animals↗
PubMed2005

Differential display of proteins involved in the neural differentiation of mouse embryonic carcinoma P19 cells by comparative proteomic analysis.

Mouse embryonic carcinoma P19 cell has been used extensively as a model to study molecular mechanisms of neural differentiation in vitro. After retinoic acid (RA) treatment and aggregation, P19 cells can differentiate into neural cells including neurons and glial cells. In this study, comparative proteomic analysis is utilized to approach the protein profiles associated with the RA-induced neural differentiation of P19 cells. Image analysis of silver stained two-dimensional gels indicated that 28 protein spots had significantly differential expression patterns in both quantity and quality. With mass spectrometry analysis and protein functional exploration, many proteins demonstrated an association with distinct aspects of neural differentiation. These proteins were gag polyprotein, rod cGMP-specific 3',5'-cyclic phosphodiesterase, 53 kDa BRG1-associated factor A, N-myc downstream regulated 1, Vitamin D receptor associated factor 1, stromal cell derived factor receptor 1, phosphoglycerate mutase, Ran-specific GTPase-activating protein, and retinoic acid (RA)-binding protein. While some cytoskeleton-related proteins such as beta cytoskeletal actin, gamma-actin, actin-related protein 1, tropomyosin 1, and cofilin 1 are related to cell migration and aggregation, other proteins have shown a relationship with distinct aspects of neural differentiation including energy production and utilization, protein synthesis and folding, cell signaling transduction, and self-protection. The differential expression patterns of these 28 proteins indicate their different roles during the neural differentiation of P19 cells. As an initial step toward unveiling the regulations involved in the commitment of pluripotent cells to a neural fate, information from this study may be helpful to uncover the molecular mechanisms of neural differentiation.

Animals↗
PubMed2003

Gene and protein expression profiling of human cerebral endothelial cells activated with tumor necrosis factor-alpha.

An increase in permeability of the blood-brain barrier is a critical event in the pathophysiological process of multiple sclerosis and other neurodegenerative diseases. Tumor necrosis factor alpha (TNFalpha) is known to play a crucial role in this process and is a powerful activator of endothelial cell inflammatory responses. Although many reports describe effects of TNFalpha activation in endothelial cells, the molecular mechanisms specific for activation of cerebral endothelial cells remains unclear. The objective of this study was to identify potential pharmaceutical targets for the treatment of multiple sclerosis using molecular profiling techniques. Gene expression measurements (Affymetrix Hu6800 oligonucleotide arrays) and proteomics (two-dimensional gel electrophoresis and mass spectrometry) were applied to analyze early alterations in human cerebral endothelial cells (HCEC) activated by TNFalpha. Human umbilical vein endothelial cells (HUVEC) were used as the reference system. The results presented show that HCEC and HUVEC respond similarly with respect to cell adhesion molecules, chemotaxis, apoptosis and oxidative stress molecules. However, nuclear factors NFkB1 and NFkB2, plasminogen activator inhibitor 1 and cofilin 1 are examples of cerebral specific responses. Our results indicate involvements of the urokinase plasminogen activator system and cytoskeletal rearrangements unique to TNFalpha activation of cerebral endothelial cells.

Antineoplastic Agents↗
PubMed2006

[Proteomic analysis of human ovarian cancer cell lines and their platinum-resistant clones].

OBJECTIVE: To perform comparative proteomic analysis of human ovarian cancer cell lines for detecting platinum-resistance associated proteins. METHODS: The total proteins of two sensitive (SKOV3 and A2780) and four resistant (SKOV3/CDDP, SKOV3/CBP, A2780/CDDP and A2780/CBP) human ovarian cancer cell lines were separated by two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed using image analysis software, stained with Coomassie Brilliant Blue, then identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. The mRNA and protein levels of the differentially expressed protein which was most significant in all of the four resistant cell lines were validated by RT-PCR and western blotting, respectively. RESULTS: Five proteins were found to be significant in four cell lines. Annexin A3 and destrin were up-regulated and nicotinamide-adenine dinucleotide phosphate (NADP)-dependent isocitrate dehydrogenase 1 was down-regulated in all the four resistant samples. Glutathione transferase omega 1 had an increased expression in the other three resistant cell lines except for SKOV3/CBP in which its expression was not changed. However, cofilin 1 represented a different trend. In the two resistant sublines of SKOV3, cofilin 1 had a down-regulation, but it had an up-regulation in the cell lines induced from SKOV3. The expression of annexin A3 was up-regulated by 3 - 20 fold and the results of RT-PCR and western blotting showed complete consistency with that by 2-DE. CONCLUSIONS: Proteomic techniques are useful to the identification of the resistance-associated proteins in ovarian cancer platinum-resistant cell lines and five candidates have been found. The five differential proteins might become hopeful candidate biomarkers for resistance.

Annexin A3↗
PubMed2001

Cytoskeletal changes regulated by the PAK4 serine/threonine kinase are mediated by LIM kinase 1 and cofilin.

PAK4 is the most recently identified member of the PAK family of serine/threonine kinases. PAK4 differs from other members of the PAK family in sequence and in many of its functions. Previously, we have shown that an important function of this kinase is to mediate the induction of filopodia in response to the Rho GTPase Cdc42. Here we show that PAK4 also regulates the activity of the protein kinase LIM kinase 1 (LIMK1). PAK4 was shown to interact specifically with LIMK1 in binding assays. Immune complex kinase assays revealed that both wild-type and constitutively active PAK4 phosphorylated LIMK1 even more strongly than PAK1, and activated PAK4 stimulated LIMK1's ability to phosphorylate cofilin. Immunofluorescence experiments revealed that PAK4 and LIMK1 cooperate to induce cytoskeletal changes in C2C12 cells. Furthermore, dominant negative LIMK1 and a mutant cofilin inhibited the specific cytoskeletal and cell shape changes that were induced in response to a recently characterized constitutively activated PAK4 mutant.

Actin Depolymerizing Factors↗
PubMed2005

Regulation of LIM-kinase 1 and cofilin in thrombin-stimulated platelets.

Cofilin is a regulator of actin filament dynamics. We studied whether during platelet activation Rho kinase stimulates LIM kinase (LIMK) leading to subsequent phosphorylation and inactivation of cofilin. Platelet shape change and aggregation/secretion were induced by low and high concentrations of thrombin, respectively. We found that during these platelet responses Rho kinase activation was responsible for mediating rapid Thr508 phosphorylation and activation of LIMK-1 and for the F-actin increase during shape change and, in part, during secretion. Surprisingly, during shape change cofilin phosphorylation was unaltered, and during aggregation/secretion cofilin was first rapidly dephosphorylated by an okadaic acid-insensitive phosphatase and then slowly rephosphorylated by LIMK-1. LIMK-1 phosphorylation and cofilin dephosphorylation and rephosphorylation during aggregation were independent of integrin alpha(IIb)beta(3) engagement. Cofilin phosphorylation did not regulate cofilin association with F-actin and was unrelated to the F-actin increase in thrombin-activated platelets. Our study identifies LIMK-1 as being activated by Rho kinase in thrombin-stimulated platelets. Two counteracting pathways, a cofilin phosphatase and LIMK-1, are activated during platelet aggregation/secretion regulating cofilin phosphorylation sequentially and independently of integrin alpha(IIb)beta(3) engagement. Rho kinase-mediated F-actin increase during platelet shape change and secretion involves a mechanism other than LIMK-1-mediated cofilin phosphorylation, raising the possibility of another LIMK substrate regulating platelet actin assembly.

Actin Depolymerizing Factors↗
PubMed1996

A role of cofilin/destrin in reorganization of actin cytoskeleton in response to stresses and cell stimuli.

1. Cofilin is an essential actin-regulating protein widely distributed in all eucaryotes. The structure and function of cofilin are conserved during evolution. 2. Cofilin depolymerizes F-actin in vitro at alkaline pH and severs F-actin in vitro at pH lower than 7.3. Overexpression of cofilin in viable cells induced bundles of actin filaments suggesting that the severing activity rather than the actin-depolymerizing or monomeric actin-sequestering activity is physiologically significant in vivo. 3. The actin bundle formation induced by overexpression of cofilin is accompanied with an increase in cell motility of Dictyostelium cells. 4. In higher vertebrates, the actin-binding activity of cofilin is negatively regulated by phosphorylation on its Ser-3 residue. The actin-binding activity is essential for yeast cells to grow. 5. Stresses and various cell stimuli activate cofilin by inducing dephosphorylation of cofilin in resting vertebrate cells. 6. Cofilin has an nuclear localization signal sequence and translocates into the nucleus together with actin in response to various stresses. Functional roles of cofilin/actin in the nucleus remain to be elucidated. 7. Tertiary structure of destrin (cofilin) resembles that of gelsolin segment 1 and well explains its functions such as Ca(2+)-independent actin binding activity.

Actin Depolymerizing Factors↗
PubMed2005

Description of a PCR-based technique for DNA splicing and mutagenesis by producing 5' overhangs with run through stop DNA synthesis utilizing Ara-C.

BACKGROUND: Splicing of DNA molecules is an important task in molecular biology that facilitates cloning, mutagenesis and creation of chimeric genes. Mutagenesis and DNA splicing techniques exist, some requiring restriction enzymes, and others utilize staggered reannealing approaches. RESULTS: A method for DNA splicing and mutagenesis without restriction enzymes is described. The method is based on mild template-dependent polymerization arrest with two molecules of cytosine arabinose (Ara-C) incorporated into PCR primers. Two rounds of PCR are employed: the first PCR produces 5' overhangs that are utilized for DNA splicing. The second PCR is based on polymerization running through the Ara-C molecules to produce the desired final product. To illustrate application of the run through stop mutagenesis and DNA splicing technique, we have carried out splicing of two segments of the human cofilin 1 gene and introduced a mutational deletion into the product. CONCLUSION: We have demonstrated the utility of a new PCR-based method for carrying out DNA splicing and mutagenesis by incorporating Ara-C into the PCR primers.

Alternative Splicing↗
PubMed2002

Stromal cell-derived factor 1alpha activates LIM kinase 1 and induces cofilin phosphorylation for T-cell chemotaxis.

Stromal cell-derived factor 1 alpha (SDF-1alpha), the ligand for G-protein-coupled receptor CXCR4, is a chemotactic factor for T lymphocytes. LIM kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing and -severing protein, at Ser-3 and regulates actin reorganization. We investigated the role of cofilin phosphorylation by LIMK1 in SDF-1alpha-induced chemotaxis of T lymphocytes. SDF-1alpha significantly induced the activation of LIMK1 in Jurkat human leukemic T cells and peripheral blood lymphocytes. SDF-1alpha also induced cofilin phosphorylation, actin reorganization, and activation of small GTPases, Rho, Rac, and Cdc42, in Jurkat cells. Pretreatment with pertussis toxin inhibited SDF-1alpha-induced LIMK1 activation, thus indicating that Gi protein is involved in LIMK1 activation. Expression of dominant negative Rac (DN-Rac), but not DN-Rho or DN-Cdc42, blocked SDF-1alpha-induced activation of LIMK1, which means that SDF-1alpha-induced LIMK1 activation is mediated by Rac but not by Rho or Cdc42. We used a cell-permeable peptide (S3 peptide) that contains the phosphorylation site (Ser-3) of cofilin to inhibit the cellular function of LIMK1. S3 peptide inhibited the kinase activity of LIMK1 in vitro. Treatment of Jurkat cells with S3 peptide inhibited the SDF-1alpha-induced cofilin phosphorylation, actin reorganization, and chemotactic response of Jurkat cells. These results suggest that the phosphorylation of cofilin by LIMK1 plays a critical role in the SDF-1alpha-induced chemotactic response of T lymphocytes.

Actin Depolymerizing Factors↗
PubMed2003

Functional proteomics of neurokinin B in the placenta indicates a novel role in regulating cytotrophoblast antioxidant defences.

Neurokinin B (NKB) has recently been demonstrated to be secreted from the placenta in abnormally high amounts in preeclampsia (PE) and to cause hypertension in rats, suggesting it may be a mediator of some pathophysiological features of PE. It is also known that NKB receptors exist in the placenta. To determine the effect of high levels of NKB on the placenta, we have performed proteomics on five separate preparations of cultured purified human term cytotrophoblast cells. The results showed a statistically significant decrease in 20 proteins, of which five were unknown proteins. Proteins important in antioxidant defenses that decreased were thioredoxin, cyclophilin A, cytokeratin 1, and peroxiredoxin 5. Two proteins that inhibit intravascular anticoagulation, cytokeratin 1 and annexin 11 were also decreased. Pathways involving pro-inflammatory cytokine activation of NF-kappa B are opposed by Raf kinase inhibitor protein, which was also decreased. Cofilin 1, a protein involved in defense against bacteria, was also decreased. Among other proteins that were suppressed by NKB were proteasome proteins, desmoplakin, and calgizzarin. Western blots confirmed the decrease in cytokeratin 1 and cyclophilin A protein after NKB exposure. In PE, there is reduced antioxidant activity and increased intravascular coagulation. The findings that high levels of NKB, similar to those observed in PE, can impair these two classes of activity support the hypothesis that high NKB levels may contribute to the pathogenesis of PE.

Annexins↗
PubMed2005

Proteomic analysis reveals changes in the liver protein pattern of rats exposed to dietary folate deficiency.

Epidemiologic and experimental studies showed that folate deficiency is associated with increased risk of degenerative diseases by enhancing abnormal one-carbon metabolism. We studied the changes in the proteome of liver, the main tissue of folate storage and metabolism, in a rat model of dietary folate depletion. Four-month-old rats were fed for 4 wk an amino acid-defined diet without folate and compared with pair-fed rats given the same diet adequately supplemented with folic acid. Folate deprivation decreased plasma and hepatic folate concentrations dramatically, while increasing homocysteinemia significantly. Using 2-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight MS, we identified 9 spots corresponding to differentially expressed proteins in the liver of folate-deficient rats compared with controls. Among those spots, 4 had a significantly increased volume, whereas the volume of the 5 other spots was decreased. Upregulated proteins included glutathione peroxidase (GPx) 1 and peroxiredoxin 6, 2 enzymes involved in the response to oxidative stress, and MAWD binding protein (MAWDBP), which has been associated with cancer. MAWDBP was simultaneously identified as a second spot with a lower isoelectric point (pI) that vanished almost completely after folate deficiency. Decreased abundance was also observed for cofilin 1, a protein linked to tumorigenesis, and for the GRP 75 precursor and preproalbumin, both of which are responsive to oxidative stress and/or inflammation. Moreover, an enzyme activity assay and/or Western blot analysis of GPx-1 and MAWDBP confirmed the proteomic findings. Our results show that folate deficiency modifies the abundance of several liver proteins consistently with adaptive tissue responses to oxidative and degenerative processes.

Amino Acids↗
PubMed2006

Proteomic identification of alterations in metabolic enzymes and signaling proteins in hypokalemic nephropathy.

Hypokalemic nephropathy caused by prolonged K(+) deficiency is associated with metabolic alkalosis, polydipsia, polyuria, growth retardation, hypertension, and progressive tubulointerstitial injury. Its pathophysiology, however, remains unclear. We performed gel-based, differential proteomics analysis of kidneys from BALB/c mice fed with high-normal-K(+) (HNK), low-normal-K(+) (LNK), or K(+)-depleted diet for 8 wk (n = 6 in each group). Plasma K(+) levels were 4.62 +/- 0.35, 4.46 +/- 0.23, and 1.51 +/- 0.21 mmol/L for HNK, LNK, and KD mice, respectively (p < 0.0001; KD vs. others). With comparable amounts of food intake, the KD mice drank significantly more water than the other two groups and had polyuria. Additionally, the KD mice had growth retardation, metabolic alkalosis, markedly enlarged kidneys, renal tubular dilation, intratubular deposition of amorphous and laminated hyaline materials, and tubular atrophy. A total of 33 renal proteins were differentially expressed between the KD mice and others, whereas only eight proteins were differentially expressed between the HNK and LNK groups, as determined by quantitative intensity analysis and ANOVA with Tukey's post hoc multiple comparisons. Using MALDI-MS and/or quadrupole-TOF MS/MS, 30 altered proteins induced by K(+)-depletion were identified as metabolic enzymes (e.g., carbonic anhydrase II, aldose reductase, glutathione S-transferase GT41A, etc.), signaling proteins (14-3-3 epsilon, 14-3-3 zeta, and cofilin 1), and cytoskeletal proteins (gamma-actin and tropomyosin). Some of these altered proteins, particularly metabolic enzymes and signaling proteins, have been demonstrated to be involved in metabolic alkalosis, polyuria, and renal tubular injury. Our findings may lead to a new road map for research on hypokalemic nephropathy and to better understanding of the pathophysiology of this medical disease when the functional and physiological significances of these altered proteins are defined.

Animals↗
PubMed2006

Regulation of the actin cytoskeleton in cancer cell migration and invasion.

Malignant cancer cells utilize their intrinsic migratory ability to invade adjacent tissues and the vasculature, and ultimately to metastasize. Cell migration is the sum of multi-step processes initiated by the formation of membrane protrusions in response to migratory and chemotactic stimuli. The driving force for membrane protrusion is localized polymerization of submembrane actin filaments. Recently, several studies revealed that molecules that link migratory signals to the actin cytoskeleton are upregulated in invasive and metastatic cancer cells. In this review, we summarize recent progress on molecular mechanisms of formation of invasive protrusions used by tumor cells, such as lamellipodia and invadopodia, with regard to the functions of key regulatory proteins of the actin cytoskeleton; WASP family proteins, Arp2/3 complex, LIM-kinase, cofilin, and cortactin.

Actins↗
PubMed2004

Stretch of the vascular wall induces smooth muscle differentiation by promoting actin polymerization.

Stretch of the vascular wall by the intraluminal blood pressure stimulates protein synthesis and contributes to the maintenance of the smooth muscle contractile phenotype. The expression of most smooth muscle specific genes has been shown to be regulated by serum response factor and stimulated by increased actin polymerization. Hence we hypothesized that stretch-induced differentiation is promoted by actin polymerization. Intact mouse portal veins were cultured under longitudinal stress and compared with unstretched controls. In unstretched veins the rates of synthesis of several proteins associated with the contractile/cytoskeletal system (alpha-actin, calponin, SM22alpha, tropomyosin, and desmin) were dramatically lower than in stretched veins, whereas other proteins (beta-actin and heat shock proteins) were synthesized at similar rates. The cytoskeletal proteins gamma-actin and vimentin were weakly stretch-sensitive. Inhibition of Rho-associated kinase by culture of stretched veins with Y-27632 produced similar but weaker effects compared with the absence of mechanical stress. Induction of actin polymerization by jasplakinolide increased SM22alpha synthesis in unstretched veins to the level in stretched veins. Stretch stimulated Rho activity and phosphorylation of the actin-severing protein cofilin-2, although both effects were slow in onset (Rho-GTP, >15 min; cofilin-P, >1 h). Cofilin-2 phosphorylation of stretched veins was inhibited by Y-27632. The F/G-actin ratio after 24 h of culture was significantly greater in stretched than in unstretched veins, as shown by both ultracentrifugation and confocal imaging with phalloidin/DNase I labeling. The results show that stretch of the vascular wall stimulates increased actin polymerization, activating synthesis of smooth muscle-specific proteins. The effect is partially, but probably not completely, mediated via Rho-associated kinase and cofilin downstream of Rho.

Actin Depolymerizing Factors↗
PubMed1996

Xenopus laevis actin-depolymerizing factor/cofilin: a phosphorylation-regulated protein essential for development.

Two cDNAs, isolated from a Xenopus laevis embryonic library, encode proteins of 168 amino acids, both of which are 77% identical to chick cofilin and 66% identical to chick actin-depolymerizing factor (ADF), two structurally and functionally related proteins. These Xenopus ADF/cofilins (XADs) differ from each other in 12 residues spread throughout the sequence but do not differ in charge. Purified GST-fusion proteins have pH-dependent actin-depolymerizing and F-actin-binding activities similar to chick ADF and cofilin. Similarities in the developmental and tissue specific expression, embryonic localization, and in the cDNA sequence of the noncoding regions, suggest that the two XACs arise from allelic variants of the pseudotetraploid X. laevis. Immunofluorescence localization of XAC in oocyte sections with an XAC-specific monoclonal antibody shows it to be diffuse in the cortical cytoplasm. After fertilization, increased immunostaining is observed in two regions: along the membrane, particularly that of the vegetal hemisphere, and at the interface between the cortical and animal hemisphere cytoplasm. The cleavage furrow and the mid-body structure are stained at the end of first cleavage. Neuroectoderm derived tissues, notochord, somites, and epidermis stain heavily either continuously or transiently from stages 18-34. A phosphorylated form of XAC (pXAC) was identified by 2D Western blotting, and it is the only species found in oocytes. Dephosphorylation of >60% of the pXAC occurs within 30 min after fertilization. Injection of one blastomere at the 2 cell stage, either with constitutively active XAC or with an XAC inhibitory antibody, blocked cleavage of only the injected blastomere in a concentration-dependent manner without inhibiting nuclear division. The cleavage furrow of eggs injected with constitutively active XAC completely regressed. Blastomeres injected with neutralized antibody developed normally. These results suggest that XAC is necessary for cytokinesis and that its activity must be properly regulated for cleavage to occur.

Actin Depolymerizing Factors↗
PubMed1997

Xenopus actin depolymerizing factor/cofilin (XAC) is responsible for the turnover of actin filaments in Listeria monocytogenes tails.

In contrast to the slow rate of depolymerization of pure actin in vitro, populations of actin filaments in vivo turn over rapidly. Therefore, the rate of actin depolymerization must be accelerated by one or more factors in the cell. Since the actin dynamics in Listeria monocytogenes tails bear many similarities to those in the lamellipodia of moving cells, we have used Listeria as a model system to isolate factors required for regulating the rapid actin filament turnover involved in cell migration. Using a cell-free Xenopus egg extract system to reproduce the Listeria movement seen in a cell, we depleted candidate depolymerizing proteins and analyzed the effect that their removal had on the morphology of Listeria tails. Immunodepletion of Xenopus actin depolymerizing factor (ADF)/cofilin (XAC) from Xenopus egg extracts resulted in Listeria tails that were approximately five times longer than the tails from undepleted extracts. Depletion of XAC did not affect the tail assembly rate, suggesting that the increased tail length was caused by an inhibition of actin filament depolymerization. Immunodepletion of Xenopus gelsolin had no effect on either tail length or assembly rate. Addition of recombinant wild-type XAC or chick ADF protein to XAC-depleted extracts restored the tail length to that of control extracts, while addition of mutant ADF S3E that mimics the phosphorylated, inactive form of ADF did not reduce the tail length. Addition of excess wild-type XAC to Xenopus egg extracts reduced the length of Listeria tails to a limited extent. These observations show that XAC but not gelsolin is essential for depolymerizing actin filaments that rapidly turn over in Xenopus extracts. We also show that while the depolymerizing activities of XAC and Xenopus extract are effective at depolymerizing normal filaments containing ADP, they are unable to completely depolymerize actin filaments containing AMPPNP, a slowly hydrolyzible ATP analog. This observation suggests that the substrate for XAC is the ADP-bound subunit of actin and that the lifetime of a filament is controlled by its nucleotide content.

Actin Cytoskeleton↗
PubMed2002

The three mouse actin-depolymerizing factor/cofilins evolved to fulfill cell-type-specific requirements for actin dynamics.

Actin-depolymerizing factor (ADF)/cofilins are essential regulators of actin filament turnover. Several ADF/cofilin isoforms are found in multicellular organisms, but their biological differences have remained unclear. Herein, we show that three ADF/cofilins exist in mouse and most likely in all other mammalian species. Northern blot and in situ hybridization analyses demonstrate that cofilin-1 is expressed in most cell types of embryos and adult mice. Cofilin-2 is expressed in muscle cells and ADF is restricted to epithelia and endothelia. Although the three mouse ADF/cofilins do not show actin isoform specificity, they all depolymerize platelet actin filaments more efficiently than muscle actin. Furthermore, these ADF/cofilins are biochemically different. The epithelial-specific ADF is the most efficient in turning over actin filaments and promotes a stronger pH-dependent actin filament disassembly than the two other isoforms. The muscle-specific cofilin-2 has a weaker actin filament depolymerization activity and displays a 5-10-fold higher affinity for ATP-actin monomers than cofilin-1 and ADF. In steady-state assays, cofilin-2 also promotes filament assembly rather than disassembly. Taken together, these data suggest that the three biochemically distinct mammalian ADF/cofilin isoforms evolved to fulfill specific requirements for actin filament dynamics in different cell types.

Actin Depolymerizing Factors↗