Scott J. Nowak

SCAR/WAVE and Arp2/3 are crucial for cytoskeletal remodeling at the site of myoblast fusion

Myoblast fusion is crucial for formation and repair of skeletal muscle. Here we show that active remodeling of the actin cytoskeleton is essential for fusion in Drosophila. Using live imaging, we have identified a dynamic F-actin accumulation (actin focus) at the site of fusion. Dissolution of the actin focus directly precedes a fusion event. Whereas several known fusion components regulate these actin foci, others target additional behaviors required for fusion. Mutations in kette/Nap1, an actin polymerization regulator, lead to enlarged foci that do not dissolve, consistent with the observed block in fusion. Kette is required to positively regulate SCAR/WAVE, which in turn activates the Arp2/3 complex. Mutants in SCAR and Arp2/3 have a fusion block and foci phenotype, suggesting that Kette-SCAR-Arp2/3 participate in an actin polymerization event required for focus dissolution. Our data identify a new paradigm for understanding the mechanisms underlying fusion in myoblasts and other tissues.

Protein Phosphatase 2A Activity Affects Histone H3 Phosphorylation and Transcription in Drosophila melanogaster

ABSTRACT Transcriptional activation of the heat shock genes during the heat shock response in Drosophila has been intimately linked to phosphorylation of histone H3 at serine 10, whereas repression of non-heat-shock genes correlates with dephosphorylation of histone H3. It is then possible that specific kinase and/or phosphatase activities may regulate histone phosphorylation and therefore transcription activation and repression, respectively. We find that treatment of cells with strong phosphatase inhibitors interferes with the genome-wide dephosphorylation of histone H3 normally observed at non-heat-shock genes during heat shock. Mutants in protein phosphatase type 2A (PP2A) also display reduced genome-wide H3 dephosphorylation, and sites of H3 phosphorylation that do not contain heat shock genes remain transcriptionally active during heat shock in PP2A mutants. Finally, the SET protein, a potent and highly selective inhibitor of PP2A activity that inhibits PP2A-mediated dephosphorylation of Ser10-phosphorylated H3, is detected at transcriptionally active regions of polytene chromosomes. These results suggest that activation and repression of gene expression during heat shock might be regulated by changes in PP2A activity controlled by the SET protein.

Nap1-mediated actin remodeling is essential for mammalian myoblast fusion

Myoblast fusion is crucial for the formation, growth, maintenance and regeneration of healthy skeletal muscle. Unfortunately, the molecular machinery, cell behaviors, and membrane and cytoskeletal remodeling events that govern fusion and myofiber formation remain poorly understood. Using time-lapse imaging approaches on mouse C2C12 myoblasts, we identify discrete and specific molecular events at myoblast membranes during fusion and myotube formation. These events include rearrangement of cell shape from fibroblast to spindle-like morphologies, changes in lamellipodial and filopodial extensions during different periods of differentiation, and changes in membrane alignment and organization during fusion. We find that actin-cytoskeleton remodeling is crucial for these events: pharmacological inhibition of F-actin polymerization leads to decreased lamellipodial and filopodial extensions and to reduced myoblast fusion. Additionally, shRNA-mediated inhibition of Nap1, a member of the WAVE actin-remodeling complex, results in accumulations of F-actin structures at the plasma membrane that are concomitant with a decrease in myoblast fusion. Our data highlight distinct and essential roles for actin cytoskeleton remodeling during mammalian myoblast fusion, provide a platform for cellular and molecular dissection of the fusion process, and suggest a functional conservation of Nap1-regulated actin-cytoskeleton remodeling during myoblast fusion between mammals and Drosophila.

Myoblast Fusion in Fly and Vertebrates: New Genes, New Processes and New Perspectives

Muscle formation and repair depends critically on the fusion of myoblasts. Despite the importance of this process, little is known about the cellular and molecular mechanisms regulating fusion. Forward genetic screens in Drosophila melanogaster have uncovered genes that, when mutated, prevent myoblast fusion. Analyses of these gene products have indicated that the actin cytoskeleton and its regulation play a central role in the fusion process. In this review, we discuss recent advances in the field, including new imaging approaches to analyze fusion as well as a description of novel genes required for fusion. In particular, we highlight what has been learned about the requirement of a specific actin structure at the site of fusion. We also place these findings from Drosophila within the context of myoblast fusion in vertebrates.

Akirin links twist-regulated transcription with the Brahma chromatin remodeling complex during embryogenesis.

The activities of developmentally critical transcription factors are regulated via interactions with cofactors. Such interactions influence transcription factor activity either directly through protein–protein interactions or indirectly by altering the local chromatin environment. Using a yeast double-interaction screen, we identified a highly conserved nuclear protein, Akirin, as a novel cofactor of the key Drosophila melanogaster mesoderm and muscle transcription factor Twist. We find that Akirin interacts genetically and physically with Twist to facilitate expression of some, but not all, Twist-regulated genes during embryonic myogenesis. akirin mutant embryos have muscle defects consistent with altered regulation of a subset of Twist-regulated genes. To regulate transcription, Akirin colocalizes and genetically interacts with subunits of the Brahma SWI/SNF-class chromatin remodeling complex. Our results suggest that, mechanistically, Akirin mediates a novel connection between Twist and a chromatin remodeling complex to facilitate changes in the chromatin environment, leading to the optimal expression of some Twist-regulated genes during Drosophila myogenesis. We propose that this Akirin-mediated link between transcription factors and the Brahma complex represents a novel paradigm for providing tissue and target specificity for transcription factor interactions with the chromatin remodeling machinery.

Novel cell-penetrating peptide-adaptors effect intracellular delivery and endosomal escape of protein cargos

ABSTRACT The use of cell-penetrating peptides (CPPs) as biomolecular delivery vehicles holds great promise for therapeutic and other applications, but development has been stymied by poor delivery and lack of endosomal escape. We have developed a CPP-adaptor system capable of efficient intracellular delivery and endosomal escape of user-defined protein cargos. The cell-penetrating sequence of HIV transactivator of transcription was fused to calmodulin, which binds with subnanomolar affinity to proteins containing a calmodulin binding site. Our strategy has tremendous advantage over prior CPP technologies because it utilizes high-affinity non-covalent, but reversible coupling between CPP and cargo. Three different cargo proteins fused to a calmodulin binding sequence were delivered to the cytoplasm of eukaryotic cells and released, demonstrating the feasibility of numerous applications in living cells including alteration of signaling pathways and gene expression. Highlighted Article: Novel cell-penetrating peptide-calmodulin adaptor proteins effect efficient delivery of user-defined cargo proteins to the cytoplasm of living mammalian cells.

Identification of a Vacuolar Proton Channel that Triggers the Bioluminescent Flash in Dinoflagellates

In 1972, J. Woodland Hastings and colleagues predicted the existence of a proton selective channel (HV1) that opens in response to depolarizing voltage across the vacuole membrane of bioluminescent dinoflagellates and conducts protons into specialized luminescence compartments (scintillons), thereby causing a pH drop that triggers light emission. HV1 channels were subsequently identified and demonstrated to have important functions in a multitude of eukaryotic cells. Here we report a predicted protein from Lingulodinium polyedrum that displays hallmark properties of bona fide HV1, including time-dependent opening with depolarization, perfect proton selectivity, and characteristic ΔpH dependent gating. Western blotting and fluorescence confocal microscopy of isolated L. polyedrum scintillons immunostained with antibody to LpHV1 confirm LpHV1’s predicted organellar location. Proteomics analysis demonstrates that isolated scintillon preparations contain peptides that map to LpHV1. Finally, Zn2+ inhibits both LpHV1 proton current and the acid-induced flash in isolated scintillons. These results implicate LpHV1 as the voltage gated proton channel that triggers bioluminescence in L. polyedrum, confirming Hastings’ hypothesis. The same channel likely mediates the action potential that communicates the signal along the tonoplast to the scintillon.

Akirin: A Context-Dependent Link Between Transcription and Chromatin Remodeling

Embryonic patterning relies upon an exquisitely timed program of gene regulation. While the regulation of this process via the action of transcription factor networks is well understood, new lines of study have highlighted the importance of a concurrently regulated program of chromatin remodeling during development. Chromatin remodeling refers to the manipulation of the chromatin architecture through rearrangement, repositioning, or restructuring of nucleosomes to either favor or hinder the expression of associated genes. While the role of chromatin remodeling pathways during tumor development and cancer progression are beginning to be clarified, the roles of these pathways in the course of tissue specification, morphogenesis and patterning remains relatively unknown. Further, relatively little is understood as to the mechanism whereby developmentally critical transcription factors coordinate with chromatin remodeling factors to optimize target gene loci for gene expression. Such a mechanism might involve direct transcription factor/chromatin remodeling factor interactions, or could likely be mediated via an unknown intermediary. Our group has identified the relatively unknown protein Akirin as a putative member of this latter group: a secondary cofactor that serves as an interface between a developmentally critical transcription factor and the chromatin remodeling machinery. This role for the Akirin protein suggests a novel regulatory mode for regulating gene expression during development.Embryonic patterning relies upon an exquisitely timed program of gene regulation. While the regulation of this process via the action of transcription factor networks is well understood, new lines of study have highlighted the importance of a concurrently regulated program of chromatin remodeling during development. Chromatin remodeling refers to the manipulation of the chromatin architecture through rearrangement, repositioning, or restructuring of nucleosomes to either favor or hinder the expression of associated genes. While the role of chromatin remodeling pathways during tumor development and cancer progression are beginning to be clarified, the roles of these pathways in the course of tissue specification, morphogenesis and patterning remains relatively unknown. Further, relatively little is understood as to the mechanism whereby developmentally critical transcription factors coordinate with chromatin remodeling factors to optimize target gene loci for gene expression. Such a mechanism might involve direct transcription factor/chromatin remodeling factor interactions, or could likely be mediated via an unknown intermediary. Our group has identified the relatively unknown protein Akirin as a putative member of this latter group: a secondary cofactor that serves as an interface between a developmentally critical transcription factor and the chromatin remodeling machinery. This role for the Akirin protein suggests a novel regulatory mode for regulating gene expression during development.

A versatile cell-penetrating peptide-adaptor system for efficient delivery of molecular cargos to subcellular destinations

Cell penetrating peptides have long held great potential for delivery of biomolecular cargos for research, therapeutic and diagnostic purposes. They allow rapid, relatively nontoxic passage of a wide variety of biomolecules through the plasma membranes of living cells. However, CPP-based research tools and therapeutics have been stymied by poor efficiency in release from endosomes and a great deal of effort has been made to solve this ‘endosomal escape problem.’ Previously, we showed that use of a reversible, noncovalent coupling between CPP and cargo using calmodulin and a calmodulin binding motif allowed efficient delivery of cargo proteins to the cytoplasm in baby hamster kidney and other mammalian cell lines. The present report demonstrates the efficacy of our CPP-adaptor scheme for efficient delivery of model cargos to the cytoplasm using a variety of CPPs and adaptors. Effective overcoming of the endosomal escape problem is further demonstrated by the delivery of cargo to the nucleus, endoplasmic reticulum and peroxisomes by addition of appropriate subcellular localization signals to the cargos. CPP-adaptors were also used to deliver cargo to myotubes, demonstrating the feasibility of the system as an alternative to transfection for the manipulation of hard-to-transfect cells.

Breaking in and busting out: Cell-penetrating peptides and the endosomal escape problem

Abstract Cell-penetrating peptides (CPPs) have long held great promise for the manipulation of living cells for therapeutic and research purposes. They allow a wide array of biomolecules from large, oligomeric proteins to nucleic acids and small molecules to rapidly and efficiently traverse cytoplasmic membranes. With few exceptions, if a molecule can be associated with a CPP, it can be delivered into a cell. However, a growing realization in the field is that CPP-cargo fusions largely remain trapped in endosomes and are eventually targeted for degradation or recycling rather than released into the cytoplasm or trafficked to a desired subcellular destination. This ‘endosomal escape problem’ has confounded efforts to develop CPP-based delivery methods for drugs, enzymes, plasmids, etc. This review provides a brief history of CPP research and discusses current issues in the field with a primary focus on the endosomal escape problem, for which several promising potential solutions have been developed. Are we on the verge of developing technologies to deliver therapeutics such as siRNA, CRISPR/Cas complexes and others that are currently failing because of an inability to get into cells, or are we just chasing after another promising but unworkable technology? We make the case for optimism.