Process elongation of oligodendrocytes is promoted by the Kelch-related actin-binding protein Mayven

Rearrangement of the cytoskeleton leading to the extension of cellular processes is essential for the myelination of axons by oligodendrocytes. We observed that the actin-binding protein, Mayven, is expressed during all stages of the oligodendrocyte lineage, and that its expression is up-regulated during oligodendrocyte differentiation. Mayven is localized in the cytoplasm and along the cell processes. Mayven also binds actin, and is involved in the cytoskeletal reorganization in oligodendrocyte precursor cells (O-2A cells) that leads to process elongation. Mayven overexpression resulted in an increase in the process outgrowth of O-2A cells and in the lengths of the processes, while microinjection of Mayven-specific antibodies inhibited process extension in these cells. Furthermore, O-2A cells transduced with recombinant retroviral sense Mayven (pMIG-W-Mayven) showed an increase in the number of oligodendrocyte processes with outgrowth, while recombinant retroviral antisense Mayven (pMIG-W-Mayven-AS) blocked O-2A process extension. Interestingly, co-localization and association of Mayven with Fyn kinase were found in O-2A cells, and these interactions were increased during the outgrowth of oligodendrocyte processes. This association was mediated via the SH3 domain ligand (a.a. 1-45) of Mayven and the SH3 domain of Fyn, suggesting that Mayven may act as a linker to bind Fyn, via its N-terminus. Thus, Mayven plays a role in the dynamics of cytoskeletal rearrangement leading to the process extension of oligodendrocytes.     

The actin-binding and bundling protein,EPLIN, is required for cytokinesis

Cytokinesis involves two phases: 1) membrane ingression followed by 2) membrane abscission. The ingression phase generates a cleavage furrow and this requires co-operative function of the actin-myosin II contractile ring and septin filaments. We demonstrate that the actin-binding protein, EPLIN, locates to the cleavage furrow during cytokinesis and this is possibly via association with the contractile ring components, myosin II, and the septin, Sept2. Depletion of EPLIN results in formation of multinucleated cells and this is associated with inefficient accumulation of active myosin II (MRLCS19) and Sept2 and their regulatory small GTPases, RhoA and Cdc42, respectively, to the cleavage furrow during the final stages of cytokinesis. We suggest that EPLIN may function during cytokinesis to maintain local accumulation of key cytokinesis proteins at the furrow.     

Shroom, a PDZ domain-containing actin-binding protein, is required for neural tube morphogenesis in mice

Using gene trap mutagenesis, we have identified a mutation in mice that causes exencephaly, acrania, facial clefting, and spina bifida, all of which can be attributed to failed neural tube closure. This mutation is designated shroom (shrm) because the neural folds "mushroom" outward and do not converge at the dorsal midline. shrm encodes a PDZ domain protein that is involved at several levels in regulating aspects of cytoarchitecture. First, endogenous Shrm localizes to adherens junctions and the cytoskeleton. Second, ectopically expressed Shrm alters the subcellular distribution of F-actin. Third, Shrm directly binds F-actin. Finally, cytoskeletal polarity within the neuroepithelium is perturbed in mutant embryos. In concert, these observations suggest that Shrm is a critical determinant of the cellular architecture required for proper neurulation.     

Predominant induction of gelsolin and actin-binding protein during myeloid differentiation

Three actin-associated proteins, actin-binding protein, gelsolin, and profilin, influence gelation, solation, and polymerization, respectively, of actin in vitro. As assessed with specific cDNA probes and immunoaffinity reagents, a 7-50-fold increase in gelsolin, 3-5-fold increase in actin-binding protein, and less than 2-fold increases in actin and profilin protein and mRNA levels accompanied tetradecanoylphorbolacetate-induced differentiation of the myeloid cell lines U937 and HL60 into macrophage-like cells. Such induction in actin-binding protein or gelsolin did not occur in K562 cells, which respond minimally to tetradecanoylphorbolacetate, or following 1,25-dihydroxyvitamin D3-induced monocyte-like differentiation of U937, which results in a less motile phenotype. These observations suggest that increases in gelsolin and actin-binding protein are essential to the expression of many regulated motile functions which takes place during differentiation of myeloid cells.     

DIXDC1 isoform, l-DIXDC1, is a novel filamentous actin-binding protein

Ccd1, a DIX domain containing Zebrafish protein involved in neural patterning, is a positive regulator of the Wnt signaling pathway. DIXDC1, the human homolog of Ccd1, has two predominant isoforms. The short form (s-DIXDC1) has a similar amino acid sequence compared with Ccd1, while the long form (l-DIXDC1) contains an extra N-terminal sequence containing a calponin-homology (CH) domain, suggesting additional interaction with actin that we have performed detailed analysis in this report. We show that mRNA expression of both DIXDC1 isoforms can be detected in various adult tissues by Northern blot analysis and is most abundant in cardiac and skeletal muscles. Both endogenous and ectopically expressed l-DIXDC1, but not s-DIXDC1, in cultured mammalian cells is localized to actin stress fibers at the filament ends in focal adhesion plaques. More importantly, l-DIXDC1 can directly bind to filamentous actin both in vitro and in vivo and the binding is mediated via a novel actin-binding domain (ABD) from amino acid 127 to 300. Thus, our data provide the first evidence that l-DIXDC1 may act as a novel branching component in the Wnt signaling pathway targeting both beta-catenin-TCF complex for gene expression and cytoskeleton for regulating dynamics of actin filaments.     

p38MAP Kinase activity is required for human primary adipocyte differentiation

Little is known about the role of p38MAPK in human adipocyte differentiation. Here we showed that p38MAPK activity increases during human preadipocytes differentiation. Pharmacological inhibition of p38MAPK during adipocyte differentiation of primary human preadipocytes markedly reduced triglycerides accumulation and adipocyte markers expression. Cell cycle arrest or proliferation was not affected by p38MAPK inhibition. Although induction of C/EBPbeta was not altered by the p38MAPK inhibitor, its phosphorylation on Threonine(188) was decreased as well as PPARgamma expression. These results indicate that p38MAPK plays a positive role in human adipogenesis through regulation of C/EBPbeta and PPARgamma factors.     

Paralemmin-1, a modulator of filopodia induction is required for spine maturation

Dendritic filopodia are thought to participate in neuronal contact formation and development of dendritic spines; however, molecules that regulate filopodia extension and their maturation to spines remain largely unknown. Here we identify paralemmin-1 as a regulator of filopodia induction and spine maturation. Paralemmin-1 localizes to dendritic membranes, and its ability to induce filopodia and recruit synaptic elements to contact sites requires protein acylation. Effects of paralemmin-1 on synapse maturation are modulated by alternative splicing that regulates spine formation and recruitment of AMPA-type glutamate receptors. Paralemmin-1 enrichment at the plasma membrane is subject to rapid changes in neuronal excitability, and this process controls neuronal activity-driven effects on protrusion expansion. Knockdown of paralemmin-1 in developing neurons reduces the number of filopodia and spines formed and diminishes the effects of Shank1b on the transformation of existing filopodia into spines. Our study identifies a key role for paralemmin-1 in spine maturation through modulation of filopodia induction.     

Elongation factor 1beta is an actin-binding protein

A 17 kDa polypeptide found in association with actin in cellular extracts of Dictyostelium discoideum was identified as a proteolytic fragment of eEF1beta. Antibody elicited against the 17 kDa protein reacted with a single 29 kDa polypeptide in Dictyostelium, indicating that the 17 kDa peptide arises from degradation of a larger precursor. The cDNA isolated from a Dictyostelium library using this antibody as a probe encodes Dictyostelium elongation factor 1beta. Amino acid degradation of the 17 kDa protein fragment confirmed the identity of the protein as eEF1beta. Direct interaction of eEF1beta with actin in vitro was further demonstrated in mixtures of actin with the 17 kDa protein fragment of Dictyostelium eEF1beta, recombinant preparations of Dictyostelium eEF1beta expressed in Escherichia coli, and the intact eEF1betagamma complex purified from wheat germ. Localization of eEF1beta in Dictyostelium by immunofluorescence microscopy reveals both diffuse cytoplasmic staining, and some concentration in the cortical and hyaline cytoplasm. The results support the existence of physical and functional interactions of the translation apparatus with the cytoskeleton, and suggest that eEF1beta may function in a dual role both to promote the elongation phase of protein synthesis, and to interact with cytoplasmic actin.     

Notch signaling is required for lateral induction of Jagged1 during FGF-induced lens fiber differentiation

Previous studies of the developing lens have shown that Notch signaling regulates differentiation of lens fiber cells by maintaining a proliferating precursor pool in the anterior epithelium. However, whether Notch signaling is further required after the onset of fiber cell differentiation is not clear. This work investigates the role of Notch2 and Jagged1 (Jag1) in secondary fiber cell differentiation using rat lens epithelial explants undergoing FGF-2 dependent differentiation in vitro. FGF induced Jag1 expression and Notch2 signaling (as judged by the appearance of activated Notch2 Intracellular Domain (N2ICD)) within 12-24 h. These changes were correlated with induction of the Notch effector, Hes5, upregulation of N-cadherin (N-cad), and downregulation of E-cadherin (E-cad), a cadherin switch characteristic of fiber cell differentiation. Induction of Jag1 was efficiently blocked by U0126, a specific inhibitor of MAPK/ERK signaling, indicating a requirement for signaling through this pathway downstream of the FGF receptor. Other growth factors that activate MAPK/ERK signaling (EGF, PDGF, IGF) did not induce Jag1. Inhibition of Notch signaling using gamma secretase inhibitors DAPT and L-685,458 or anti-Jag1 antibody markedly decreased FGF-dependent expression of Jag1 demonstrating Notch-dependent lateral induction. In addition, inhibition of Notch signaling reduced expression of N-cad, and the cyclin dependent kinase inhibitor, p57Kip2, indicating a direct role for Notch signaling in secondary fiber cell differentiation. These results demonstrate that Notch-mediated lateral induction of Jag1 is an essential component of FGF-dependent lens fiber cell differentiation.     

A plasma kallikrein-dependent plasminogen cascade required for adipocyte differentiation

Here we show that plasma kallikrein (PKal) mediates a plasminogen (Plg) cascade in adipocyte differentiation. Ecotin, an inhibitor of serine proteases, inhibits cell-shape change, adipocyte-specific gene expression, and lipid accumulation during adipogenesis in culture. Deficiency of Plg, but not of urokinase or tissue-type plasminogen activator, suppresses adipogenesis during differentiation of 3T3-L1 cells and mammary-gland involution. PKal, which is inhibited by ecotin, is required for adipose conversion, Plg activation and 3T3-L1 differentiation. Human plasma lacking PKal does not support differentiation of 3T3-L1 cells. PKal is therefore a physiological regulator that acts in the Plg cascade during adipogenesis. We propose that the Plg cascade fosters adipocyte differentiation by degradation of the fibronectin-rich preadipocyte stromal matrix.     

ADAR1 is required for differentiation and neural induction by regulating microRNA processing in a catalytically independent manner

Adenosine deaminases acting on RNA (ADARs) are involved in adenosine-to-inosine RNA editing and are implicated in development and diseases. Here we observed that ADAR1 deficiency in human embryonic stem cells (hESCs) significantly affected hESC differentiation and neural induction with widespread changes in mRNA and miRNA expression, including upregulation of self-renewal-related miRNAs, such as miR302s. Global editing analyses revealed that ADAR1 editing activity contributes little to the altered miRNA/mRNA expression in ADAR1-deficient hESCs upon neural induction. Genome-wide iCLIP studies identified that ADAR1 binds directly to pri-miRNAs to interfere with miRNA processing by acting as an RNA-binding protein. Importantly, aberrant expression of miRNAs and phenotypes observed in ADAR1-depleted hESCs upon neural differentiation could be reversed by an enzymatically inactive ADAR1 mutant, but not by the RNA-binding-null ADAR1 mutant. These findings reveal that ADAR1, but not its editing activity, is critical for hESC differentiation and neural induction by regulating miRNA biogenesis via direct RNA interaction.     

Heat shock protein 60: Evidence for receptor-mediated induction of proinflammatory mediators during adipocyte differentiation

Adipocytes play important roles in lipid metabolism but also in the control of inflammatory processes. Based on our previous findings of heat shock protein (Hsp) 60-induced activation of preadipocytes we investigated whether the capacity of heat shock protein 60 (Hsp60) to interact with adipocytes and to stimulate their proinflammatory activity is determined by the differentiation state of the cells. Hsp60 bound to adipocytes and stimulated the release of inflammatory mediators independent of their differentiation state. Hsp60-adipocyte interactions revealed basic characteristics of a receptor-mediated process. Our findings characterize Hsp60 binding and Hsp60-induced release of proinflammatory mediators as fundamental properties of adipocytes independent of their differentiation state.