RESUMEN
Plasma membrane repair is an evolutionarily conserved mechanism by which cells can seal breaches in the plasma membrane. Mutations in several proteins with putative roles in sarcolemma integrity, membrane repair, and membrane transport result in several forms of muscle disease; however, the mechanisms that are activated and responsible for sarcolemma resealing are not well understood. Using the standard assays for membrane repair, which track the uptake of FM 1-43 dye into adult skeletal muscle fibers following laser-induced sarcolemma disruption, we show that labeling of resting fibers by FM1-43 prior to membrane wounding and the induced FM1-43 dye uptake after sarcolemma wounding occurs via dynamin-dependent endocytosis. Dysferlin-deficient muscle fibers show elevated dye uptake following wounding, which is the basis for the assertion that membrane repair is defective in this model. Our data show that dynamin inhibition mitigates the differences in FM1-43 dye uptake between dysferlin-null and wild-type muscle fibers, suggesting that elevated wound-induced FM1-43 uptake in dysferlin-deficient muscle may actually be due to enhanced dynamin-dependent endocytosis following wounding, though dynamin inhibition had no effect on dysferlin trafficking after wounding. By monitoring calcium flux after membrane wounding, we show that reversal of calcium precedes the sustained, slower increase of dynamin-dependent FM1-43 uptake in WT fibers, and that dysferlin-deficient muscle fibers have persistently increased calcium after wounding, consistent with its proposed role in resealing. These data highlight a previously unappreciated role for dynamin-dependent endocytosis in wounded skeletal muscle fibers and identify overactive dynamin-dependent endocytosis following sarcolemma wounding as a potential mechanism or consequence of dysferlin deficiency.
Asunto(s)
Calcio/metabolismo , Dinaminas/genética , Disferlina/genética , Endocitosis/genética , Sarcolema/genética , Animales , Animales Modificados Genéticamente , Dimetilsulfóxido/farmacología , Dinaminas/metabolismo , Disferlina/metabolismo , Colorantes Fluorescentes/metabolismo , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hidrazonas/farmacología , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Sarcolema/efectos de los fármacos , Sarcolema/metabolismo , Sarcolema/patología , Coloración y Etiquetado/métodosRESUMEN
CRISPR/Cas9 technology is ideally suited for genome-wide screening applications due to the ease of generating guide RNAs (gRNAs) and the versatility of Cas9 or Cas9 derivatives to knockout, repress, or activate expression of target genes. Several pooled lentiviral CRISPR libraries have been developed and are now publicly available, but while using CRISPR/Cas9 for genetic experiments has become widely adopted, genome-wide screening experiments remain technically challenging. This review covers the basics of CRISPR/Cas9, describes several publicly available CRISPR libraries, and provides a general protocol for conducting genome-wide screening experiments using CRISPR/Cas9. © 2016 by John Wiley & Sons, Inc.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Biblioteca de Genes , Lentivirus/genética , Animales , Sistemas CRISPR-Cas , Marcación de Gen/métodos , Ingeniería Genética/métodos , Genoma , Humanos , Mutación , ARN Guía de Kinetoplastida/genética , Transducción Genética/métodosRESUMEN
Deficits in membrane repair may contribute to disease progression in dysferlin-deficient muscular dystrophy. Dysferlin, a type-II transmembrane phospholipid-binding protein, is hypothesized to regulate fusion of repair vesicles with the sarcolemma to facilitate membrane repair, but the dysferlin-containing compartments involved in membrane repair and the mechanism by which these compartments contribute to resealing are unclear. A dysferlin-pHluorin [dysf-pH-sensitive green fluorescent protein (pHGFP)] muscle-specific transgenic mouse was developed to examine the dynamic behavior and subcellular localization of dysferlin during membrane repair in adult skeletal muscle fibers. Live-cell confocal microscopy of uninjured adult dysf-pHGFP muscle fibers revealed that dysferlin is highly enriched in the sarcolemma and transverse tubules. Laser-wounding induced rapid recruitment of â¼30 µm of local dysferlin-containing sarcolemma, leading to formation of stable dysferlin accumulations surrounding lesions, endocytosis of dysferlin, and formation of large cytoplasmic vesicles from distal regions of the fiber. Disruption of the actin cytoskeleton decreased recruitment of sarcolemma-derived dysferlin to lesions in dysf-pHGFP fibers without affecting endocytosis and impaired membrane resealing in wild-type fibers, similar to findings in dysferlin deficiency (a 2-fold increase in FM1-43 uptake). Our data support a new mechanism whereby recruitment of sarcolemma-derived dysferlin creates an active zone of high lipid-binding activity at wounds to interact with repair vesicles and facilitate membrane resealing in skeletal muscle.
Asunto(s)
Citoesqueleto/fisiología , Proteínas de la Membrana/fisiología , Fibras Musculares Esqueléticas/metabolismo , Miocitos Cardíacos/metabolismo , Sarcolema/metabolismo , Cicatrización de Heridas/fisiología , Actinas/fisiología , Animales , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Disferlina , Endocitosis , Genes Reporteros , Genes Sintéticos , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos A , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/lesiones , Miocitos Cardíacos/ultraestructura , Proteínas Recombinantes de Fusión/metabolismo , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestructura , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Abnormalities in phosphoinositide metabolism are an emerging theme in human neurodegenerative disease. Myotubular myopathy is a prototypical disorder of phosphoinositide dysregulation that is characterized by profound muscle pathology and weakness and that is caused by mutations in MTM1, which encodes a phosphatase that targets 3-position phosphoinositides, including phosphatidylinositol 3-phosphate. Although the association between MTM1 and muscle disease has become increasingly clarified, the normal role(s) of phosphatidylinositol 3-phosphate metabolism in muscle development and homeostasis remain poorly understood. To begin to address the function of phosphatidylinositol 3-phosphate in skeletal muscle, we focused on the primary kinase responsible for its production, and created a muscle-specific conditional knockout of the class III phosphatidylinositol 3-kinase, Pik3c3. Muscle-specific deletion of Pik3c3 did not disturb embryogenesis or early postnatal development, but resulted in progressive disease characterized by reduced activity and death by 2 months of age. Histopathological analysis demonstrated changes consistent with a murine muscular dystrophy. Examination for cellular mechanism(s) responsible for the dystrophic phenotype revealed significant alterations in the autophagolysosomal pathway with mislocation of known dystrophy proteins to the lysosomal compartment. In all, we present the first analysis of Pik3c3 in skeletal muscle, and report a novel association between deletion of Pik3c3 and muscular dystrophy.
Asunto(s)
Autofagia , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/enzimología , Distrofia Muscular Animal/enzimología , Fosfatos de Fosfatidilinositol/metabolismo , Animales , Fosfatidilinositol 3-Quinasas Clase III/genética , Lisosomas/metabolismo , Lisosomas/patología , Ratones , Ratones Noqueados , Proteínas Musculares/genética , Músculo Esquelético/patología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patología , Fosfatos de Fosfatidilinositol/genéticaRESUMEN
Mutations in the dysferlin gene resulting in dysferlin-deficiency lead to limb-girdle muscular dystrophy 2B and Myoshi myopathy in humans. Dysferlin has been proposed as a critical regulator of vesicle-mediated membrane resealing in muscle fibers, and localizes to muscle fiber wounds following sarcolemma damage. Studies in fibroblasts and urchin eggs suggest that trafficking and fusion of intracellular vesicles with the plasma membrane during resealing requires the intracellular cytoskeleton. However, the contribution of dysferlin-containing vesicles to resealing in muscle and the role of the cytoskeleton in regulating dysferlin-containing vesicle biology is unclear. Here, we use live-cell imaging to examine the behavior of dysferlin-containing vesicles following cellular wounding in muscle cells and examine the role of microtubules and kinesin in dysferlin-containing vesicle behavior following wounding. Our data indicate that dysferlin-containing vesicles move along microtubules via the kinesin motor KIF5B in muscle cells. Membrane wounding induces dysferlin-containing vesicle-vesicle fusion and the formation of extremely large cytoplasmic vesicles, and this response depends on both microtubules and functional KIF5B. In non-muscle cell types, lysosomes are critical mediators of membrane resealing, and our data indicate that dysferlin-containing vesicles are capable of fusing with lysosomes following wounding which may contribute to formation of large wound sealing vesicles in muscle cells. Overall, our data provide mechanistic evidence that microtubule-based transport of dysferlin-containing vesicles may be critical for resealing, and highlight a critical role for dysferlin-containing vesicle-vesicle and vesicle-organelle fusion in response to wounding in muscle cells.