Inhibiting myostatin reverses muscle fibrosis through apoptosis

Advance Online Publication June 8, 2012 doi: 10.1242/?jcs.090365 Zhao Bo Li, Jiangyang Zhang and Kathryn R. Wagner*?*Communicating Author: Kathryn R. Wagner, M.D., Ph.D., Center for Genetic Muscle Disorders, The Kennedy Krieger Institute, The Johns Hopkins School of Medicine, 707 North Broadway, Baltimore, MD 21205, 443-923-9525 (t), 443-923-9545 (f), wagnerk{at}kennedykrieger.orgSkeletal muscle fibrosis is a defining feature of the muscular dystrophies in which contractile myofibers are replaced by fibroblasts, adipocytes and extracellular matrix. This maladaptive response of muscle to repetitive injury is progressive, self-perpetuating and thus far, has been considered irreversible. We have previously shown that myostatin, a known endogenous modulator of muscle growth, stimulates normal muscle fibroblasts to proliferate. Here, we demonstrate that myostatin also regulates the proliferation of dystrophic muscle fibroblasts, and increases resistance of fibroblasts to apoptosis through Smad and MAPK signaling. Inhibiting myostatin signaling pathways with a soluble activin IIB receptor (ActRIIB.Fc), reduces resistance of muscle fibroblasts to apoptosis in vitro. Systemic administration of ActRIIB.Fc in senescent mdx mice, a model of muscular dystrophy, significantly increases the number of muscle fibroblasts undergoing apoptosis. This leads to the reversal of pre-existed muscle fibrosis as determined by histological, biochemical and radiographical criteria. These results demonstrate that skeletal muscle fibrosis can be pharmacologically reversed through induction of fibroblast apoptosis.

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Cofilin Nuclear-Cytoplasmic Shuttling Affects Cofilin-Actin Rod Formation During Stress

Advance Online Publication May 23, 2012 doi: 10.1242/?jcs.097667 Cofilin protein is involved in regulating the actin cytoskeleton during typical steady state conditions, as well as during cell stress conditions where cofilin saturates F-actin forming cofilin-actin rods. Cofilin can enter the nucleus through an active nuclear localization signal (NLS), accumulating in nuclear actin rods during stress. Here, we characterize the active nuclear export of cofilin through a leptomycin-B sensitive, CRM1-dependent, nuclear export signal (NES). We also redefine the NLS of cofilin as a bipartite NLS, with an additional basic epitope required for nuclear localization. Using fluorescence lifetime imaging microscopy (FLIM) and Förster resonant energy transfer (FRET) between cofilin moieties and actin, as well as automated image analysis in live cells, we have defined subtle mutations in the cofilin NLS that allow cofilin to bind actin in vivo and affect cofilin dynamics during stress. We further define the requirement of cofilin-actin rod formation in a system of cell stress by temporal live cell imaging. We propose that cofilin nuclear shuttling is critical for the cofilin-actin rod stress response with cofilin dynamically communicating between the nucleus and cytoplasm during cell stress.

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Alternative translation initiation gives rise to two isoforms of orai1 with distinct plasma membrane mobilities

Advance Online Publication May 28, 2012 doi: 10.1242/?jcs.104919 Store-operated calcium entry is a nearly ubiquitous signaling pathway in eukaryotic cells. The plasma membrane store-operated channels are comprised of subunits of the recently discovered Orai proteins, the major one being Orai1.We have discovered that native Orai1 as well as expressed Orai1 exists in two forms in similar quantities: a longer form (Orai1a) of approximately 33 kDa, and a shorter form (Orai1ß) of approximately 23 kDa. The second Orai1ß form arises from alternative translation initiation from a methionine at position 64, and possibly also 71, in the longer, Orai1a form. In the sequence upstream of the initiation site of Orai1ß, there is a poly-arginine sequence previously suggested to be involved in interaction of Orai1 with plasma membrane phosphatidylinositol 4,5-bisphosphate. The loss of this phospholipid binding domain would be expected to influence the mobility of Orai1 protein in the plasma membrane. Indeed, experiments utilizing fluorescence recovery after photobleaching (FRAP) revealed that the recovery half-time for Orai1ß was significantly faster than for Orai1a. Since Orai1 must diffuse to sites of interaction with the Ca2+ sensor, STIM1, these two mobilities might provide for efficient recruitment of Orai1 subunits to sites of store-operated Ca2+ entry during agonist-induced Ca2+ signaling.

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ATF2 - at the crossroad of nuclear and cytosolic functions

Advance Online Publication June 8, 2012 doi: 10.1242/?jcs.095000 An increasing number of transcription factors have been shown to elicit oncogenic and tumor suppressor activities, depending on the tissue and cell context. Activating transcription factor 2 (ATF2; also known as cAMP-dependent transcription factor ATF-2) has oncogenic activities in melanoma and tumor suppressor activities in non-malignant skin tumors and breast cancer. Recent work has shown that the opposing functions of ATF2 are associated with its subcellular localization. In the nucleus, ATF2 contributes to global transcription and the DNA damage response, in addition to specific transcriptional activities that are related to cell development, proliferation and death. ATF2 can also translocate to the cytosol, primarily following exposure to severe genotoxic stress, where it impairs mitochondrial membrane potential and promotes mitochondrial-based cell death. Notably, phosphorylation of ATF2 by the epsilon isoform of protein kinase C (PKCe) is the master switch that controls its subcellular localization and function. Here, we summarize our current understanding of the regulation and function of ATF2 in both subcellular compartments. This mechanism of control of a non-genetically modified transcription factor represents a novel paradigm for ‘oncogene addiction’.

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The Nup155-mediated Organization of Inner Nuclear Membrane Proteins is Independent of Nup155 Anchoring to the Metazoan Nuclear Pore Complex

Advance Online Publication June 20, 2012 doi: 10.1242/?jcs.105809 The nuclear envelope (NE), an important barrier between the nucleus and the cytoplasm, is composed of three structures: The outer nuclear membrane (ONM), which is continuous with the ER, the inner nuclear membrane (INM), which interfaces with chromatin, and nuclear pore complexes (NPCs), which are essential for the exchange of macromolecules between the two compartments. The NPC protein Nup155 has an evolutionarily conserved role in the metazoan NE formation; but the in vivo analysis of Nup155 has been severely hampered by the essential function of this protein in cell viability. Here, we take advantage of the hypomorphicity of RNAi systems and use a combination of protein binding and rescue assays to map the interaction sites of two neigbouring NPC proteins Nup93 and Nup53 on Nup155, and to define the requirement of these interactions in INM protein organization. We show that different parts of Drosophila Nup155 have distinct functions: The Nup155 ß-propeller anchors the protein to the NPC, while the a-solenoid part of Nup155 is essential for the correct localization of INM proteins LBR and otefin. Using chromatin extracts from semi-synchronized cells, we also provide evidence that the Nup155 a-solenoid has a chromatin-binding activity that is stronger at the end of mitosis. Our results argue that the role of Nup155 in INM protein localization is not mediated through the NPC anchoring activity of the protein and suggest that regions other than Nup155 ß-propeller are necessary for the targeting of proteins to the INM.

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Advances in high-resolution imaging - techniques for three-dimensional imaging of cellular structures

Advance Online Publication June 8, 2012 doi: 10.1242/?jcs.090027 A fundamental goal in biology is to determine how cellular organization is coupled to function. To achieve this goal, a better understanding of organelle composition and structure is needed. Although visualization of cellular organelles using fluorescence or electron microscopy (EM) has become a common tool for the cell biologist, recent advances are providing a clearer picture of the cell than ever before. In particular, advanced light-microscopy techniques are achieving resolutions below the diffraction limit and EM tomography provides high-resolution three-dimensional (3D) images of cellular structures. The ability to perform both fluorescence and electron microscopy on the same sample (correlative light and electron microscopy, CLEM) makes it possible to identify where a fluorescently labeled protein is located with respect to organelle structures visualized by EM. Here, we review the current state of the art in 3D biological imaging techniques with a focus on recent advances in electron microscopy and fluorescence super-resolution techniques.

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