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1.
Mol Biol Cell ; 27(14): 2272-85, 2016 07 15.
Article in English | MEDLINE | ID: mdl-27226483

ABSTRACT

Cells rapidly reseal after damage, but how they do so is unknown. It has been hypothesized that resealing occurs due to formation of a patch derived from rapid fusion of intracellular compartments at the wound site. However, patching has never been directly visualized. Here we study membrane dynamics in wounded Xenopus laevis oocytes at high spatiotemporal resolution. Consistent with the patch hypothesis, we find that damage triggers rampant fusion of intracellular compartments, generating a barrier that limits influx of extracellular dextrans. Patch formation is accompanied by compound exocytosis, local accumulation and aggregation of vesicles, and rupture of compartments facing the external environment. Subcellular patterning is evident as annexin A1, dysferlin, diacylglycerol, active Rho, and active Cdc42 are recruited to compartments confined to different regions around the wound. We also find that a ring of elevated intracellular calcium overlaps the region where membrane dynamics are most evident and persists for several minutes. The results provide the first direct visualization of membrane patching during membrane repair, reveal novel features of the repair process, and show that a remarkable degree of spatial patterning accompanies damage-induced membrane dynamics.


Subject(s)
Wound Healing/physiology , Animals , Annexin A1/metabolism , Calcium/metabolism , Calcium Signaling , Cell Membrane/physiology , Dysferlin , Exocytosis/physiology , Female , Humans , Intracellular Fluid/metabolism , Lysosomes/metabolism , Membrane Proteins/metabolism , Muscle Proteins/metabolism , Oocytes/metabolism , Optical Imaging/methods , Organelles/metabolism , Xenopus laevis
2.
Annu Rev Cell Dev Biol ; 27: 237-63, 2011.
Article in English | MEDLINE | ID: mdl-21721944

ABSTRACT

The importance of wound healing to medicine and biology has long been evident, and consequently, wound healing has been the subject of intense investigation for many years. However, several relatively recent developments have added new impetus to wound repair research: the increasing application of model systems; the growing recognition that single cells have a robust, complex, and medically relevant wound healing response; and the emerging recognition that different modes of wound repair bear an uncanny resemblance to other basic biological processes such as morphogenesis and cytokinesis. In this review, each of these developments is described, and their significance for wound healing research is considered. In addition, overlapping mechanisms of single-cell and multicellular wound healing are highlighted, and it is argued that they are more similar than is often recognized. Based on this and other information, a simple model to explain the evolutionary relationships of cytokinesis, single-cell wound repair, multicellular wound repair, and developmental morphogenesis is proposed. Finally, a series of important, but as yet unanswered, questions is posed.


Subject(s)
Extracellular Matrix/metabolism , Models, Biological , Wound Healing/physiology , Animals , Cell Membrane/metabolism , Cell Movement , Cell Proliferation , Cytoskeleton/metabolism , Embryo, Mammalian/physiology , Endocytosis/physiology , Hemostasis/physiology , Humans , Inflammation/metabolism , Membrane Fusion/physiology , Signal Transduction/physiology
3.
PLoS Genet ; 6(10): e1001158, 2010 Oct 14.
Article in English | MEDLINE | ID: mdl-20976199

ABSTRACT

Hair cell stereocilia structure depends on actin filaments composed of cytoplasmic ß-actin and γ-actin isoforms. Mutations in either gene can lead to progressive hearing loss in humans. Since ß-actin and γ-actin isoforms are 99% identical at the protein level, it is unclear whether each isoform has distinct cellular roles. Here, we compared the functions of ß-actin and γ-actin in stereocilia formation and maintenance by generating mice conditionally knocked out for Actb or Actg1 in hair cells. We found that, although cytoplasmic actin is necessary, neither ß-actin nor γ-actin is required for normal stereocilia development or auditory function in young animals. However, aging mice with ß-actin- or γ-actin-deficient hair cells develop different patterns of progressive hearing loss and distinct pathogenic changes in stereocilia morphology, despite colocalization of the actin isoforms. These results demonstrate overlapping developmental roles but unique post-developmental functions for ß-actin and γ-actin in maintaining hair cell stereocilia.


Subject(s)
Actins/metabolism , Hair Cells, Auditory/metabolism , Organ of Corti/metabolism , Actins/genetics , Animals , Auditory Threshold , Blotting, Western , Cadherins/genetics , Cadherins/metabolism , Cilia/genetics , Cilia/metabolism , Cilia/pathology , Female , Hair Cells, Auditory/cytology , Hearing Loss/genetics , Hearing Loss/metabolism , Immunohistochemistry , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron, Scanning , Organ of Corti/growth & development , Organ of Corti/ultrastructure
4.
Proc Natl Acad Sci U S A ; 106(24): 9703-8, 2009 Jun 16.
Article in English | MEDLINE | ID: mdl-19497859

ABSTRACT

Beta(cyto)-actin and gamma(cyto)-actin are ubiquitous proteins thought to be essential building blocks of the cytoskeleton in all non-muscle cells. Despite this widely held supposition, we show that gamma(cyto)-actin null mice (Actg1(-/-)) are viable. However, they suffer increased mortality and show progressive hearing loss during adulthood despite compensatory up-regulation of beta(cyto)-actin. The surprising viability and normal hearing of young Actg1(-/-) mice means that beta(cyto)-actin can likely build all essential non-muscle actin-based cytoskeletal structures including mechanosensory stereocilia of hair cells that are necessary for hearing. Although gamma(cyto)-actin-deficient stereocilia form normally, we found that they cannot maintain the integrity of the stereocilia actin core. In the wild-type, gamma(cyto)-actin localizes along the length of stereocilia but re-distributes to sites of F-actin core disruptions resulting from animal exposure to damaging noise. In Actg1(-/-) stereocilia similar disruptions are observed even without noise exposure. We conclude that gamma(cyto)-actin is required for reinforcement and long-term stability of F-actin-based structures but is not an essential building block of the developing cytoskeleton.


Subject(s)
Actins/physiology , Cytoskeleton/physiology , Actins/genetics , Animals , Hearing Loss/genetics , Mice , Mice, Knockout , Microscopy, Electron, Scanning , Microscopy, Fluorescence
5.
PLoS Med ; 6(5): e1000083, 2009 May 26.
Article in English | MEDLINE | ID: mdl-19478831

ABSTRACT

BACKGROUND: The loss of dystrophin compromises muscle cell membrane stability and causes Duchenne muscular dystrophy and/or various forms of cardiomyopathy. Increased expression of the dystrophin homolog utrophin by gene delivery or pharmacologic up-regulation has been demonstrated to restore membrane integrity and improve the phenotype in the dystrophin-deficient mdx mouse. However, the lack of a viable therapy in humans predicates the need to explore alternative methods to combat dystrophin deficiency. We investigated whether systemic administration of recombinant full-length utrophin (Utr) or DeltaR4-21 "micro" utrophin (muUtr) protein modified with the cell-penetrating TAT protein transduction domain could attenuate the phenotype of mdx mice. METHODS AND FINDINGS: Recombinant TAT-Utr and TAT-muUtr proteins were expressed using the baculovirus system and purified using FLAG-affinity chromatography. Age-matched mdx mice received six twice-weekly intraperitoneal injections of either recombinant protein or PBS. Three days after the final injection, mice were analyzed for several phenotypic parameters of dystrophin deficiency. Injected TAT-muUtr transduced all tissues examined, integrated with members of the dystrophin complex, reduced serum levels of creatine kinase (11,290+/-920 U versus 5,950+/-1,120 U; PBS versus TAT), the prevalence of muscle degeneration/regeneration (54%+/-5% versus 37%+/-4% of centrally nucleated fibers; PBS versus TAT), the susceptibility to eccentric contraction-induced force drop (72%+/-5% versus 40%+/-8% drop; PBS versus TAT), and increased specific force production (9.7+/-1.1 N/cm(2) versus 12.8+/-0.9 N/cm(2); PBS versus TAT). CONCLUSIONS: These results are, to our knowledge, the first to establish the efficacy and feasibility of TAT-utrophin-based constructs as a novel direct protein-replacement therapy for the treatment of skeletal and cardiac muscle diseases caused by loss of dystrophin.


Subject(s)
Dystrophin/deficiency , Muscular Dystrophy, Animal/drug therapy , Muscular Dystrophy, Duchenne/drug therapy , Recombinant Fusion Proteins/therapeutic use , Utrophin/therapeutic use , Animals , Creatine Kinase/blood , Dystrophin/genetics , Gene Products, tat/genetics , Mice , Mice, Inbred mdx , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Muscular Dystrophy, Animal/pathology , Muscular Dystrophy, Duchenne/pathology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Utrophin/genetics , Utrophin/metabolism
6.
FASEB J ; 23(7): 2205-14, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19279140

ABSTRACT

We generated transgenic mice that overexpressed gamma-(cyto) actin 2000-fold above wild-type levels in skeletal muscle. gamma-(cyto) actin comprised 40% of total actin in transgenic skeletal muscle, with a concomitant 40% decrease in alpha-actin. Surprisingly, transgenic muscle was histologically and ultrastructurally identical to wild-type muscle despite near-stoichiometric incorporation of gamma-(cyto) actin into sarcomeric thin filaments. Furthermore, several parameters of muscle physiological performance in the transgenic animals were not different from wild type. Given these surprising results, we tested whether overexpression of gamma-(cyto) actin could rescue the early postnatal lethality in alpha-(sk) actin-null mice (Acta1(-/-)). By quantitative Western blot analysis, we found total actin levels were decreased by 35% in Acta1(-/-) muscle. Although transgenic overexpression of gamma-(cyto) actin on the Acta1(-/-) background restored total actin levels to wild type, resulting in thin filaments composed of 60% gamma-(cyto) actin and a 40% mixture of cardiac and vascular actin, the life span of transgenic Acta1(-/-) mice was not extended. These results indicate that sarcomeric thin filaments can accommodate substantial incorporation of gamma-(cyto) actin without functional consequences, yet gamma-(cyto) actin cannot fully substitute for alpha-(sk) actin.


Subject(s)
Actins/genetics , Muscle, Skeletal/chemistry , Actins/analysis , Animals , Cytoplasm/chemistry , Endothelium, Vascular/chemistry , Longevity , Mice , Mice, Knockout , Mice, Transgenic , Muscle, Skeletal/ultrastructure , Myocardium/chemistry , Sarcomeres/chemistry , Sarcomeres/ultrastructure
7.
Int Rev Cytol ; 265: 191-225, 2008.
Article in English | MEDLINE | ID: mdl-18275889

ABSTRACT

Since its first description in 1990, the dystrophin-glycoprotein complex has emerged as a critical nexus for human muscular dystrophies arising from defects in a variety of distinct genes. Studies in mammals widely support a primary role for the dystrophin-glycoprotein complex in mechanical stabilization of the plasma membrane in striated muscle and provide hints for secondary functions in organizing molecules involved in cellular signaling. Studies in model organisms confirm the importance of the dystrophin-glycoprotein complex for muscle cell viability and have provided new leads toward a full understanding of its secondary roles in muscle biology.


Subject(s)
Dystrophin/physiology , Glycoproteins/physiology , Muscle, Striated/physiology , Signal Transduction/physiology , Animals , Cell Survival/physiology , Humans , Muscle, Striated/cytology
8.
Dev Cell ; 11(3): 387-97, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16950128

ABSTRACT

Nonmuscle gamma(cyto)-actin is expressed at very low levels in skeletal muscle but uniquely localizes to costameres, the cytoskeletal networks that couple peripheral myofibrils to the sarcolemma. We generated and analyzed skeletal muscle-specific gamma(cyto)-actin knockout (Actg1-msKO) mice. Although muscle development proceeded normally, Actg1-msKO mice presented with overt muscle weakness accompanied by a progressive pattern of muscle fiber necrosis/regeneration. Functional deficits in whole-body tension and isometric twitch force were observed, consistent with defects in the connectivity between muscle fibers and/or myofibrils or at the myotendinous junctions. Surprisingly, gamma(cyto)-actin-deficient muscle did not demonstrate the fibrosis, inflammation, and membrane damage typical of several muscular dystrophies but rather presented with a novel progressive myopathy. Together, our data demonstrate an important role for minimally abundant but strategically localized gamma(cyto)-actin in adult skeletal muscle and describe a new mouse model to study the in vivo relevance of subcellular actin isoform sorting.


Subject(s)
Actins/genetics , Actins/physiology , Cytoplasm/metabolism , Muscle, Skeletal/embryology , Muscular Diseases/metabolism , Animals , Cell Membrane/metabolism , Dystrophin/metabolism , Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Muscle, Skeletal/metabolism , Muscular Diseases/pathology , Organogenesis
9.
J Biol Chem ; 281(15): 9996-10001, 2006 Apr 14.
Article in English | MEDLINE | ID: mdl-16478721

ABSTRACT

This study was designed to define the molecular epitopes of dystrophin-actin interaction and to directly compare the actin binding properties of dystrophin and utrophin. According to our data, dystrophin and utrophin both bound alongside actin filaments with submicromolar affinities. However, the molecular epitopes involved in actin binding differed between the two proteins. In utrophin, the amino-terminal domain and an adjacent string of the first 10 spectrin-like repeats more fully recapitulated the activities measured for full-length protein. The homologous region of dystrophin bound actin with low affinity and near 1:1 stoichiometry as previously measured for the isolated amino-terminal, tandem (CH) domain. In contrast, a dystrophin construct including a cluster of basic spectrin-like repeats and spanning from the amino terminus through repeat 17, bound actin with properties most similar to full-length dystrophin. Dystrophin and utrophin both stabilized preformed actin filaments from forced depolymerization with similar efficacies but did not appear to compete for binding sites on actin. We also found that dystrophin binding to F-actin was markedly sensitive to increasing ionic strength, although utrophin binding was unaffected. Although dystrophin and utrophin are functionally homologous actin-binding proteins, these results indicate that their respective modes of contact with actin filaments are markedly different. Finally, we reassessed the abundance of dystrophin in striated muscle using full-length protein as the standard and measured greater than 10-fold higher values than previously reported.


Subject(s)
Actins/chemistry , Dystrophin/physiology , Utrophin/physiology , Animals , Binding Sites , Dose-Response Relationship, Drug , Dystrophin/metabolism , Electrophoresis, Polyacrylamide Gel , Epitopes/chemistry , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muscle, Skeletal/metabolism , Myocardium/metabolism , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Time Factors , Utrophin/chemistry , Utrophin/metabolism
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