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Induction of megakaryocyte differentiation drives nuclear accumulation and transcriptional function of MKL1 via actin polymerization and RhoA activation.

ABSTRACT: How components of the cytoskeleton regulate complex cellular responses is fundamental to understanding cellular function. Megakaryoblast leukemia 1 (MKL1), an activator of serum response factor (SRF) transcriptional activity, promotes muscle, neuron, and megakaryocyte differentiation. In muscle cells, where MKL1 subcellular localization is one mechanism by which cells control SRF activity, MKL1 translocation from the cytoplasm to the nucleus in response to actin polymerization is critical for its function as a transcriptional regulator. MKL1 localization is cell-type specific; it is predominantly cytoplasmic in unstimulated fibroblasts and some muscle cell types and is constitutively nuclear in neuronal cells. In the present study, we report that in megakaryocytes, subcellular localization and regulation of MKL1 is dependent on RhoA activity and actin organization. Induction of megakaryocytic differentiation of human erythroleukemia cells by 12-O-tetradecanoylphorbol-13-acetate and primary megakaryocytes by thrombopoietin promotes MKL1 nuclear localization. This MKL1 localization is blocked by drugs inhibiting RhoA activity or actin polymerization.We also show that nuclear-localized MKL1 activates the transcription of SRF target genes. This report broadens our knowledge of the molecular mechanisms regulating megakaryocyte differentiation.

SUBMITTER: Smith EC

PROVIDER: S-EPMC3575755 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Induction of megakaryocyte differentiation drives nuclear accumulation and transcriptional function of MKL1 via actin polymerization and RhoA activation.

Smith Elenoe C EC Teixeira Alexandra M AM Chen Rachel C RC Wang Lin L Gao Yuan Y Hahn Katherine L KL Krause Diane S DS

Blood 20121214 7

How components of the cytoskeleton regulate complex cellular responses is fundamental to understanding cellular function. Megakaryoblast leukemia 1 (MKL1), an activator of serum response factor (SRF) transcriptional activity, promotes muscle, neuron, and megakaryocyte differentiation. In muscle cells, where MKL1 subcellular localization is one mechanism by which cells control SRF activity, MKL1 translocation from the cytoplasm to the nucleus in response to actin polymerization is critical for it ...[more]

PMID: 23243284

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Project description:BackgroundDendrite morphogenesis plays an essential role in establishing the connectivity and receptive fields of neurons during the development of the nervous system. To generate the diverse morphologies of branched dendrites, neurons use external cues and cell surface receptors to coordinate intracellular cytoskeletal organization; however, the molecular mechanisms of how this signaling forms branched dendrites are not fully understood.MethodsWe performed in vivo time-lapse imaging of the PVD neuron in C. elegans in several mutants of actin regulatory proteins, such as the WAVE Regulatory Complex (WRC) and UNC-34 (homolog of Enabled/Vasodilator-stimulated phosphoprotein (Ena/VASP)). We examined the direct interaction between the WRC and UNC-34 and analyzed the localization of UNC-34 in vivo using transgenic worms expressing UNC-34 fused to GFP.ResultsWe identify a stereotyped sequence of morphological events during dendrite outgrowth in the PVD neuron in C. elegans. Specifically, local increases in width ("swellings") give rise to filopodia to facilitate a "rapid growth and pause" mode of growth. In unc-34 mutants, filopodia fail to form but swellings are intact. In WRC mutants, dendrite growth is largely absent, resulting from a lack of both swelling and filopodia formation. We also found that UNC-34 can directly bind to the WRC. Disrupting this binding by deleting the UNC-34 EVH1 domain prevented UNC-34 from localizing to swellings and dendrite tips, resulting in a stunted dendritic arbor and reduced filopodia outgrowth.ConclusionsWe propose that regulators of branched and linear F-actin cooperate to establish dendritic branches. By combining our work with existing literature, we propose that the dendrite guidance receptor DMA-1 recruits the WRC, which polymerizes branched F-actin to generate "swellings" on a mother dendrite. Then, WRC recruits the actin elongation factor UNC-34/Ena/VASP to initiate growth of a new dendritic branch from the swelling, with the help of the actin-binding protein UNC-115/abLIM. Extension of existing dendrites also proceeds via swelling formation at the dendrite tip followed by UNC-34-mediated outgrowth. Following dendrite initiation and extension, the stabilization of branches by guidance receptors further recruits WRC, resulting in an iterative process to build a complex dendritic arbor.

Project description:UNLABELLED:Herpesviruses, which include important pathogens, remodel the host cell nucleus to facilitate infection. This remodeling includes the formation of structures called replication compartments (RCs) in which herpesviruses replicate their DNA. During infection with the betaherpesvirus, human cytomegalovirus (HCMV), viral DNA synthesis occurs at the periphery of RCs within the nuclear interior, after which assembled capsids must reach the inner nuclear membrane (INM) for translocation to the cytoplasm (nuclear egress). The processes that facilitate movement of HCMV capsids to the INM during nuclear egress are unknown. Although an actin-based mechanism of alphaherpesvirus capsid trafficking to the INM has been proposed, it is controversial. Here, using a fluorescently-tagged, nucleus-localized actin-binding peptide, we show that HCMV, but not herpes simplex virus 1, strongly induced nuclear actin filaments (F-actin) in human fibroblasts. Based on studies using UV inactivation and inhibitors, this induction depended on viral gene expression. Interestingly, by 24 h postinfection, nuclear F-actin formed thicker structures that appeared by super-resolution microscopy to be bundles of filaments. Later in infection, nuclear F-actin primarily localized along the RC periphery and between the RC periphery and the nuclear rim. Importantly, a drug that depolymerized nuclear F-actin caused defects in production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization near the nuclear rim, without decreasing capsid accumulation in the nucleus. Thus, our results suggest that for at least one herpesvirus, nuclear F-actin promotes capsid movement to the nuclear periphery and nuclear egress. We discuss our results in terms of competing models for these processes. IMPORTANCE:The mechanisms underlying herpesvirus nuclear egress have not been fully determined. In particular, how newly assembled capsids move to the inner nuclear membrane for envelopment is uncertain and controversial. In this study, we show that HCMV, an important human pathogen, induces actin filaments in the nuclei of infected cells and that an inhibitor of nuclear F-actin impairs nuclear egress and capsid localization toward the nuclear periphery. Herpesviruses are widespread pathogens that cause or contribute to an array of human diseases. A better understanding of how herpesvirus capsids traffic in the nucleus may uncover novel targets for antiviral intervention and elucidate aspects of the nuclear cytoskeleton, about which little is known.

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Induction of megakaryocyte differentiation drives nuclear accumulation and transcriptional function of MKL1 via actin polymerization and RhoA activation.

Publications

Induction of megakaryocyte differentiation drives nuclear accumulation and transcriptional function of MKL1 via actin polymerization and RhoA activation.

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