scholarly journals Actin scaffolding by clathrin heavy chain is required for skeletal muscle sarcomere organization

2014 ◽  
Vol 205 (3) ◽  
pp. 377-393 ◽  
Author(s):  
Stéphane Vassilopoulos ◽  
Christel Gentil ◽  
Jeanne Lainé ◽  
Pierre-Olivier Buclez ◽  
Agathe Franck ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Heavy Chain ◽  
Neuromuscular Disorders ◽  
Contractile Force ◽  
Contractile Apparatus ◽  
Intracellular Membrane ◽  
Clathrin Heavy Chain ◽  
Attachment Sites ◽  
Main Component

The ubiquitous clathrin heavy chain (CHC), the main component of clathrin-coated vesicles, is well characterized for its role in intracellular membrane traffic and endocytosis from the plasma membrane (PM). Here, we demonstrate that in skeletal muscle CHC regulates the formation and maintenance of PM–sarcomere attachment sites also known as costameres. We show that clathrin forms large coated lattices associated with actin filaments and the muscle-specific isoform of α-actinin at the PM of differentiated myotubes. Depletion of CHC in myotubes induced a loss of actin and α-actinin sarcomeric organization, whereas CHC depletion in vivo induced a loss of contractile force due to the detachment of sarcomeres from the PM. Our results suggest that CHC contributes to the formation and maintenance of the contractile apparatus through interactions with costameric proteins and highlight an unconventional role for CHC in skeletal muscle that may be relevant to pathophysiology of neuromuscular disorders.

scholarly journals Characterization of a Temperature-Sensitive Vertebrate Clathrin Heavy Chain Mutant as a Tool to Study Clathrin-Dependent Events In Vivo

PLoS ONE ◽  
2010 ◽  
Vol 5 (8) ◽  
pp. e12017 ◽  
Author(s):  
Petra Neumann-Staubitz ◽  
Stephanie L. Hall ◽  
Joseph Kuo ◽  
Antony P. Jackson
Keyword(s):  
Heavy Chain ◽  
Temperature Sensitive ◽  
Clathrin Heavy Chain

scholarly journals Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor μ2 kinase

2003 ◽  
Vol 163 (2) ◽  
pp. 231-236 ◽  
Author(s):  
Antony P. Jackson ◽  
Alexander Flett ◽  
Carl Smythe ◽  
Lindsay Hufton ◽  
Frank R. Wettey ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Heavy Chain ◽  
Transferrin Receptor ◽  
Coated Pits ◽  
Permeabilized Cells ◽  
Clathrin Heavy Chain

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo–AP2 interactions occur via the μ2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for μ2 phosphorylation is in cargo recruitment because μ2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of μ2 phosphorylation. We identify clathrin as a specific activator of the μ2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of μ2 phosphorylation. Furthermore, we show that AP2 containing phospho-μ2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-μ2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-μ2 levels.

scholarly journals Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum.

1994 ◽  
Vol 126 (2) ◽  
pp. 343-352 ◽  
Author(s):  
T Ruscetti ◽  
J A Cardelli ◽  
M L Niswonger ◽  
T J O'Halloran
Keyword(s):  
Dictyostelium Discoideum ◽  
Heavy Chain ◽  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Secretory Pathway ◽  
Chain Gene ◽  
Wild Type ◽  
Clathrin Heavy Chain ◽  
Mutant Cells

The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain-deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.

scholarly journals Biomechanical Properties of the Sarcolemma and Costameres of Skeletal Muscle Lacking Desmin

2021 ◽  
Vol 12 ◽  
Author(s):  
Karla P. Garcia-Pelagio ◽  
Robert J. Bloch
Keyword(s):  
Skeletal Muscle ◽  
Lateral Force ◽  
Biomechanical Properties ◽  
Contractile Force ◽  
Contractile Apparatus ◽  
Force Transmission ◽  
Fast Twitch Muscle ◽  
Intracellular Organelles ◽  
Time Required ◽  
Fast Twitch

Intermediate filaments (IFs), composed primarily by desmin and keratins, link the myofibrils to each other, to intracellular organelles, and to the sarcolemma. There they may play an important role in transfer of contractile force from the Z-disks and M-lines of neighboring myofibrils to costameres at the membrane, across the membrane to the extracellular matrix, and ultimately to the tendon (“lateral force transmission”). We measured the elasticity of the sarcolemma and the connections it makes at costameres with the underlying contractile apparatus of individual fast twitch muscle fibers of desmin-null mice. By positioning a suction pipet to the surface of the sarcolemma and applying increasing pressure, we determined the pressure at which the sarcolemma separated from nearby sarcomeres, Pseparation, and the pressure at which the isolated sarcolemma burst, Pbursting. We also examined the time required for the intact sarcolemma-costamere-sarcomere complex to reach equilibrium at lower pressures. All measurements showed the desmin-null fibers to have slower equilibrium times and lower Pseparation and Pbursting than controls, suggesting that the sarcolemma and its costameric links to nearby contractile structures were weaker in the absence of desmin. Comparisons to earlier values determined for muscles lacking dystrophin or synemin suggest that the desmin-null phenotype is more stable than the former and less stable than the latter. Our results are consistent with the moderate myopathy seen in desmin-null muscles and support the idea that desmin contributes significantly to sarcolemmal stability and lateral force transmission.

scholarly journals Clathrin’s muscle-building regimen

2014 ◽  
Vol 205 (3) ◽  
pp. 285-285
Author(s):  
Ben Short
Keyword(s):  
Skeletal Muscle ◽  
Heavy Chain ◽  
Clathrin Heavy Chain ◽  
Membrane Scaffold

The clathrin heavy chain forms a membrane scaffold that organizes skeletal muscle sarcomeres.

scholarly journals Inactivation of clathrin heavy chain inhibits synaptic recycling but allows bulk membrane uptake

2008 ◽  
Vol 182 (5) ◽  
pp. 1007-1016 ◽  
Author(s):  
Jaroslaw Kasprowicz ◽  
Sabine Kuenen ◽  
Katarzyna Miskiewicz ◽  
Ron L.P. Habets ◽  
Liesbet Smitz ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Heavy Chain ◽  
Synaptic Activity ◽  
Synaptic Membrane ◽  
Vesicle Recycling ◽  
Clathrin Heavy Chain ◽  
Early Embryonic Lethality ◽  
Bulk Membrane ◽  
Multicellular Organisms

Synaptic vesicle reformation depends on clathrin, an abundant protein that polymerizes around newly forming vesicles. However, how clathrin is involved in synaptic recycling in vivo remains unresolved. We test clathrin function during synaptic endocytosis using clathrin heavy chain (chc) mutants combined with chc photoinactivation to circumvent early embryonic lethality associated with chc mutations in multicellular organisms. Acute inactivation of chc at stimulated synapses leads to substantial membrane internalization visualized by live dye uptake and electron microscopy. However, chc-inactivated membrane cannot recycle and participate in vesicle release, resulting in a dramatic defect in neurotransmission maintenance during intense synaptic activity. Furthermore, inactivation of chc in the context of other endocytic mutations results in membrane uptake. Our data not only indicate that chc is critical for synaptic vesicle recycling but they also show that in the absence of the protein, bulk retrieval mediates massive synaptic membrane internalization.

Mechanical stimulation improves tissue-engineered human skeletal muscle

2002 ◽  
Vol 283 (5) ◽  
pp. C1557-C1565 ◽  
Author(s):  
Courtney A. Powell ◽  
Beth L. Smiley ◽  
John Mills ◽  
Herman H. Vandenburgh
Keyword(s):  
Skeletal Muscle ◽  
Morphological Characteristics ◽  
Muscle Cells ◽  
Force Transducer ◽  
Nondestructive Method ◽  
Attachment Sites ◽  
Engineered Tissue ◽  
Applied Forces ◽  
Parallel Arrays

Human bioartificial muscles (HBAMs) are tissue engineered by suspending muscle cells in collagen/MATRIGEL, casting in a silicone mold containing end attachment sites, and allowing the cells to differentiate for 8 to 16 days. The resulting HBAMs are representative of skeletal muscle in that they contain parallel arrays of postmitotic myofibers; however, they differ in many other morphological characteristics. To engineer improved HBAMs, i.e., more in vivo-like, we developed Mechanical Cell Stimulator (MCS) hardware to apply in vivo-like forces directly to the engineered tissue. A sensitive force transducer attached to the HBAM measured real-time, internally generated, as well as externally applied, forces. The muscle cells generated increasing internal forces during formation which were inhibitable with a cytoskeleton depolymerizer. Repetitive stretch/relaxation for 8 days increased the HBAM elasticity two- to threefold, mean myofiber diameter 12%, and myofiber area percent 40%. This system allows engineering of improved skeletal muscle analogs as well as a nondestructive method to determine passive force and viscoelastic properties of the resulting tissue.

scholarly journals Human Vam6p promotes lysosome clustering and fusion in vivo

2001 ◽  
Vol 154 (1) ◽  
pp. 109-122 ◽  
Author(s):  
Steve Caplan ◽  
Lisa M. Hartnell ◽  
Rubén C. Aguilar ◽  
Naava Naslavsky ◽  
Juan S. Bonifacino
Keyword(s):  
Heavy Chain ◽  
Self Assembly ◽  
Dominant Negative ◽  
Human Protein ◽  
Clathrin Heavy Chain ◽  
Late Endosomes ◽  
Repeat Domain ◽  
Gradient Fractionation ◽  
Vacuolar Protein

Regulated fusion of mammalian lysosomes is critical to their ability to acquire both internalized and biosynthetic materials. Here, we report the identification of a novel human protein, hVam6p, that promotes lysosome clustering and fusion in vivo. Although hVam6p exhibits homology to the Saccharomyces cerevisiae vacuolar protein sorting gene product Vam6p/Vps39p, the presence of a citron homology (CNH) domain at the NH2 terminus is unique to the human protein. Overexpression of hVam6p results in massive clustering and fusion of lysosomes and late endosomes into large (2–3 μm) juxtanuclear structures. This effect is reminiscent of that caused by expression of a constitutively activated Rab7. However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7. Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein. Both the CNH and clathrin heavy chain repeat domains are required for induction of lysosome clustering and fusion. This study implicates hVam6p as a mammalian tethering/docking factor characterized with intrinsic ability to promote lysosome fusion in vivo.

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