Small ankyrin 1 (sAnk1; Ank1.5) is a ~20 kDa protein of striated muscle that concentrates in the network compartment of the sarcoplasmic reticulum (nSR). We used siRNA targeted to sAnk1 to assess its role in organizing the sarcoplasmic reticulum (SR) of skeletal myofibers in vitro. siRNA reduced sAnk1 mRNA and protein levels and disrupted the organization of the remaining sAnk1. Sarcomeric proteins were unchanged, but two other proteins of the nSR, SERCA and sarcolipin, decreased significantly in amount and segregated into distinct structures containing sarcolipin and sAnk1, and SERCA, respectively. Exogenous sAnk1 restored SERCA to its normal distribution. Ryanodine receptors and calsequestrin in the junctional SR, and L-type Ca2+ channels in the transverse tubules were not reduced, although their striated organization was mildly altered. Consistent with the loss of SERCA, uptake and release of Ca2+ were significantly inhibited. Our results show that sAnk1 stabilizes the nSR and that its absence causes the nSR to fragment into distinct membrane compartments.
Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. Our previous results show that the tibialis anterior muscles (TAs) of mice lacking keratin 19 (K19) lose costameres, accumulate mitochondria under the sarcolemma, and generate lower specific tension than controls. PURPOSE: Here we compare the physiology and morphology of TA muscles of mice lacking K19 with muscles lacking desmin or both proteins (DKO). METHODS: The TA was isolated in situ to measure contractile characteristics (i.e., twitch, tetany, and fatigue). Dorsiflexor torque was measured before and at several time points after animals sustained an injury induced by lengthening contractions. We evaluated membrane damage by measuring serum CK and counting Evans blue dye labeled fibers, assessed structural changes with confocal and electron microscopy, and determined functional changes on a treadmill. RESULTS: The absence of desmin caused a larger change in specific tension (40%) than the absence of K19 (19%), and played the predominant role in the DKO (40%). By contrast, the absence of both proteins was required to obtain a significantly greater loss of contractile torque after injury (48%), compared to wild type (39%), or a significant decrease in the tolerance to exercise. Although the loss of organization of costameres was much greater in the desmin-null TA muscle, the absence of both K19 and desmin was required for near complete disruption of costameres. By contrast, the subsarcolemmal accumulation of mitochondria was only seen in K19-null TA muscles, and the absence of both K19 and desmin yielded a milder phenotype. CONCLUSIONS: Our results suggest that keratin filaments containing K19 and desmin-based intermediate filaments play distinct but complementary roles in the physiology and morphology of fast-twitch skeletal muscle. Supported by grants form the Muscular Dystrophy association (MDA, grant 4278) and NIH-NIAMS (K01AR053235) to RML and grants from the MDA and the NIH (RO1AR055928) to RJB
Obscurin is a giant sarcomeric protein composed of adhesion modules and signaling domains. It surrounds myofibrils at the level of the Z disk and the M line. To study the role of obscurin during myofibrillogenesis, we used adenovirus-mediated gene delivery to overexpress part of its COOH terminus in primary cultures of postnatal day 1 (P1) skeletal myotubes. Examination of the subcellular distribution of a number of sarcomeric proteins revealed that the organization of myosin into A bands was dramatically reduced. Myosin assembled into A bands normally in mock- or control-infected P1 myotubes. Overexpression of the COOH terminus of obscurin did not affect the organization of other sarcomeric markers, including actin, alpha-actinin, titin, and myomesin. Assembly of myomesin into nascent M lines in treated myotubes suggests that these structures can form independently of A bands. Immunoblot analysis indicated that there was a small ( approximately 20%) but consistent decrease in the amount of myosin expressed in cells infected with the COOH terminus of obscurin. Coimmunoprecipitation experiments in which we used adult skeletal muscle homogenates demonstrated that obscurin exists in a complex with myosin. Thus our findings suggest that the COOH-terminal region of obscurin interacts with sarcomeric myosin and may play a critical role in its ability to assemble into A bands in striated muscle.
Abstract We have made several technical improvements for quick‐freeze, deep‐etch replication of monolayers of cells grown on, or attached to, glass coverslips. Cells studied include muscle cells of rat and Xenopus cultured on glass coverslips, and erythrocytes attached to coverslips coated with poly‐L‐lysine. We describe methods for identifying particular areas of cultures, e.g., clusters of acetylcholine receptors on muscle cells, by light microscopy and then relocating these areas after replication. For good preservation of structure by quick‐freezing, it is necessary to ensure that the surface to be frozen is covered by a minimum depth of water (< 10 μm). Insufficient or excess water left on the sample during freezing causes recognizable artifacts in its replica. We describe two ways to control the water table–one by improving visual control of water removal, the other by blowing excess water off the sample surface with a jet of nitrogen applied during its descent to the freezing block. Finally, we describe a new specimen holder that allows us to etch and replicate six samples at once with good thermal contact between the stage and samples.
Journal Article Studying Membrane Trafficking in Toxoplasma gondii Using Correlative Light and Electron Microscopy (CLEM) Get access Ru-ching Hsia, Ru-ching Hsia Electron Microscopy Core Imaging Facility, University of Maryland Baltimore, Baltimore, USADepartment of Neural and Pain Sciences, University of Maryland Dental School, Baltimore, USA Search for other works by this author on: Oxford Academic Google Scholar John Strong, John Strong Electron Microscopy Core Imaging Facility, University of Maryland Baltimore, Baltimore, USA Search for other works by this author on: Oxford Academic Google Scholar Julia Romano, Julia Romano Department of Molecular Microbiology and Immunology, Johns Hopkins University, Baltimore, USA Search for other works by this author on: Oxford Academic Google Scholar Isabelle Coppens Isabelle Coppens Department of Molecular Microbiology and Immunology, Johns Hopkins University, Baltimore, USA Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 21, Issue S3, 1 August 2015, Pages 535–536, https://doi.org/10.1017/S1431927615003475 Published: 23 September 2015
Obscurin (approximately 800 kDa) in striated muscle closely surrounds sarcomeres at the level of the M-band and Z-disk where, we hypothesize, it participates in the assembly of the contractile apparatus and membrane systems required for Ca2+ homeostasis. In this study, we used small inhibitory RNA (siRNA) technology to reduce the levels of obscurin in primary cultures of skeletal myotubes to study its role in myofibrillogenesis and the organization of the sarcoplasmic reticulum (SR). siRNA-treated myotubes showed a specific and dramatic reduction in the approximately 800 kDa form of obscurin by reverse transcription-polymerase chain reaction, immunoblotting, and immunofluorescence. M-bands and A-bands, but not Z-disks or I-bands, were disrupted when the synthesis of obscurin was inhibited. Small ankyrin 1, an integral protein of the network SR that binds to obscurin, also failed to align around developing sarcomeres in treated myotubes. Myosin and myomesin levels were significantly reduced in treated myotubes but alpha-actinin was not, suggesting that down-regulation of obscurin destabilizes proteins of the M-band and A-band but not of the Z-disk. Our findings suggest that obscurin is required for the assembly of the M-band and A-band and for the regular alignment of the network SR around the contractile apparatus.
