scholarly journals Perturbation of PTEN-PI3K/AKT Signalling Impaired Autophagy Modulation in Dystrophin-Deficient Myoblasts

2017 ◽  
Author(s):  
Muhammad Dain Yazid ◽  
Janet Smith
Keyword(s):  
Plasma Membrane ◽  
Immunoblot Analysis ◽  
Autophagic Flux ◽  
Protein Regulation ◽  
Serine Threonine Kinase ◽  
Fast Myosin ◽  
Phosphorylated Akt ◽  
Fast Myosin Heavy Chain ◽  
Immunoblot Technique ◽  
Protein Transcription

AbstractAlteration of single protein regulation has given a massive implication in Muscular Dystrophy pathogenesis. Herein, we investigated the contribution of defected dystrophin that has impaired PI3K/Akt signalling and subsequently reduced autophagy in dystrophin-deficient myoblasts. In this study, dfd13 (dystrophin-deficient) and C2C12 (non-dystrophic) myoblasts were cultured in low mitogen condition for 10 days to induce differentiation. Analyses of protein expression has been done by using immunoblot technique, immunofluorescence and flow cytometry. In our myoblasts differentiation system, the dfd13 myoblasts did not achieved terminal differentiation as fewer myotube formation and fast-myosin heavy chain expression almost not detected. Immunoblot analysis showed that PTEN expression is profoundly increased in dfd13 myoblasts throughout the differentiation day. As a result, the PI3K activity is decreased and has caused serine/threonine kinase Akt inactivation. Both residues; Thr308 and Ser473, on Akt were found not phosphorylated. The mTOR activation by Ser2448 phosphorylation was decreased indicates an impairment for raptor and rictor binding. Unable to form complexes; mTORC1 target protein, p70S6K1 activation was found reduced at the same time explained un-phosphorylated-Akt at Ser473 by rictor-mTORC2. As one of Akt downstream protein, transcription factor FoxO3 regulation was found impaired as it was highly expressed and highly mainly localised in the nucleus in dfd13 towards the end of the differentiation day. This occurrence has caused higher activation of autophagy related genes; Beclin1, Atg5, Atg7, in dfd13 myoblasts. Autophagosome formation was increased as LC3B-I/II showed accumulation upon differentiation. However, ratio of LC3B lipidation and autophagic flux were shown decreased which exhibited dystrophic features. As a conclusion, destabilisation of plasma membrane owing to dystrophin mutation has caused the alteration of plasma membrane protein regulation particularly PTEN-PI3K, thus impaired autophagy modulation that critical for myoblasts development.

scholarly journals Ubl4A is required for insulin-induced Akt plasma membrane translocation through promotion of Arp2/3-dependent actin branching

2015 ◽  
Vol 112 (31) ◽  
pp. 9644-9649 ◽  
Author(s):  
Yu Zhao ◽  
Yuting Lin ◽  
Honghong Zhang ◽  
Adriana Mañas ◽  
Wenwen Tang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glycogen Synthesis ◽  
Membrane Translocation ◽  
Insulin Stimulation ◽  
Serine Threonine Kinase ◽  
Newborn Mice ◽  
Threonine Kinase ◽  
Cell Mobility ◽  
Close Proximity ◽  
Extracellular Stimuli

The serine-threonine kinase Akt is a key regulator of cell proliferation and survival, glucose metabolism, cell mobility, and tumorigenesis. Activation of Akt by extracellular stimuli such as insulin centers on the interaction of Akt with PIP3 on the plasma membrane, where it is subsequently phosphorylated and activated by upstream protein kinases. However, it is not known how Akt is recruited to the plasma membrane upon stimulation. Here we report that ubiquitin-like protein 4A (Ubl4A) plays a crucial role in insulin-induced Akt plasma membrane translocation. Ubl4A knockout newborn mice have defective Akt-dependent glycogen synthesis and increased neonatal mortality. Loss of Ubl4A results in the impairment of insulin-induced Akt translocation to the plasma membrane and activation. Akt binds actin-filaments and colocalizes with actin-related protein 2 and 3 (Arp2/3) complex in the membrane ruffles and lamellipodia. Ubl4A directly interacts with Arp2/3 to accelerate actin branching and networking, allowing Akt to be in close proximity to the plasma membrane for activation upon insulin stimulation. Our finding reveals a new mechanism by which Akt is recruited to the plasma membrane for activation, thereby providing a missing link in Akt signaling.

The relationship between slow and fast myosin heavy chain content, calpastatin and meat tenderness in different ovine skeletal muscles

Meat Science ◽  
2005 ◽  
Vol 69 (1) ◽  
pp. 17-25 ◽  
Author(s):  
A.Q. Sazili ◽  
T. Parr ◽  
P.L. Sensky ◽  
S.W. Jones ◽  
R.G. Bardsley ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Skeletal Muscles ◽  
Meat Tenderness ◽  
Fast Myosin ◽  
Fast Myosin Heavy Chain ◽  
The Relationship

Effect of unilateral denervation on maximum specific force in rat diaphragm muscle fibers

2001 ◽  
Vol 90 (4) ◽  
pp. 1196-1204 ◽  
Author(s):  
Paige C. Geiger ◽  
Mark J. Cody ◽  
Rebecca L. Macken ◽  
Megan E. Bayrd ◽  
Gary C. Sieck
Keyword(s):  
Diaphragm Muscle ◽  
Specific Force ◽  
Rat Diaphragm ◽  
Standard Curve ◽  
Fast Myosin ◽  
Sds Page ◽  
Fast Myosin Heavy Chain ◽  
Cross Bridges ◽  
Triton X ◽  
Permeabilized Fibers

We hypothesize that 1) the effect of denervation (DNV) is more pronounced in fibers expressing fast myosin heavy chain (MHC) isoforms and 2) the effect of DNV on maximum specific force reflects a reduction in MHC content per half sarcomere or the number of cross bridges in parallel. Studies were performed on single Triton X-100-permeabilized fibers activated at a pCa (−log Ca2+ concentration) of 4.0. MHC content per half sarcomere was determined by densitometric analysis of SDS-PAGE gels and comparison to a standard curve of known MHC concentrations. After 2 of wk DNV, the maximum specific force of fibers expressing MHC2X was reduced by ∼40% (MHC2Bexpression was absent), whereas the maximum specific force of fibers expressing MHC2A and MHCslow decreased by only ∼20%. DNV also reduced the MHC content in fibers expressing MHC2X, with no effect on fibers expressing MHC2A and MHCslow. When normalized for MHC content per half sarcomere, force generated by DNV fibers expressing MHC2X and MHC2A was decreased compared with control fibers. These results suggest the force per cross bridge is also affected by DNV.

scholarly journals Slow and fast myosin heavy chain content defines three types of myotubes in early muscle cell cultures.

1985 ◽  
Vol 101 (5) ◽  
pp. 1643-1650 ◽  
Author(s):  
J B Miller ◽  
M T Crow ◽  
F E Stockdale
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Pectoral Muscle ◽  
Myosin Heavy Chain Isoforms ◽  
Fiber Formation ◽  
Single Type ◽  
Fast Myosin ◽  
Slow Myosin Heavy Chain ◽  
Fast Myosin Heavy Chain ◽  
Different Populations

We prepared monoclonal antibodies specific for fast or slow classes of myosin heavy chain isoforms in the chicken and used them to probe myosin expression in cultures of myotubes derived from embryonic chicken myoblasts. Myosin heavy chain expression was assayed by gel electrophoresis and immunoblotting of extracted myosin and by immunostaining of cultures of myotubes. Myotubes that formed from embryonic day 5-6 pectoral myoblasts synthesized both a fast and a slow class of myosin heavy chain, which were electrophoretically and immunologically distinct, but only the fast class of myosin heavy chain was synthesized by myotubes that formed in cultures of embryonic day 8 or older myoblasts. Furthermore, three types of myotubes formed in cultures of embryonic day 5-6 myoblasts: one that contained only a fast myosin heavy chain, a second that contained only a slow myosin heavy chain, and a third that contained both a fast and a slow heavy chain. Myotubes that formed in cultures of embryonic day 8 or older myoblasts, however, were of a single type that synthesized only a fast class of myosin heavy chain. Regardless of whether myoblasts from embryonic day 6 pectoral muscle were cultured alone or mixed with an equal number of myoblasts from embryonic day 12 muscle, the number of myotubes that formed and contained a slow class of myosin was the same. These results demonstrate that the slow class of myosin heavy chain can be synthesized by myotubes formed in cell culture, and that three types of myotubes form in culture from pectoral muscle myoblasts that are isolated early in development, but only one type of myotube forms from older myoblasts; and they suggest that muscle fiber formation probably depends upon different populations of myoblasts that co-exist and remain distinct during myogenesis.

Absence of a growth hormone effect on rat soleus atrophy during a 4-day spaceflight

1993 ◽  
Vol 74 (2) ◽  
pp. 527-531 ◽  
Author(s):  
B. Jiang ◽  
R. R. Roy ◽  
C. Navarro ◽  
V. R. Edgerton
Keyword(s):  
Growth Hormone ◽  
Succinate Dehydrogenase ◽  
Cross Sections ◽  
Fiber Type ◽  
Cross Sectional Area ◽  
Sectional Area ◽  
Cross Sectional ◽  
Fast Myosin ◽  
Hormone Effect ◽  
Fast Myosin Heavy Chain

The objectives of the present study were to determine the size and enzyme properties of soleus fibers of rats subjected to a 4-day spaceflight (National Aeronautics and Space Administration, STS-41) and the effects of exogenous growth hormone (GH) on the atrophic response of the muscle. Four groups of rats were studied: 1) control (Con), 2) Con plus GH treated (Con + GH), 3) flight (Fl), and 4) F1 plus GH treated (Fl + GH). Cross-sectional area and the activities of succinate dehydrogenase and myofibrillar adenosinetriphosphatase (ATPase) were determined in fibers identified in frozen serial cross sections. Fibers were categorized immunohistochemically as slow, fast, or slow-fast on the basis of their reaction with slow and fast myosin heavy-chain (MHC) monoclonal antibodies. Fibers also were categorized as light or dark on the basis of their staining for ATPase at pH 8.6. After the 4-day flight, mean body weight was significantly decreased compared with control. The absolute and relative (muscle wt/body wt) soleus weights were significantly smaller in the Fl and Fl + GH rats compared with their respective ground-based controls. In both flight groups, the cross-sectional area of the light ATPase fibers was significantly smaller (approximately 30%) than control. Three of 11 flight rats had a higher proportion of fibers expressing both slow and fast MHCs than expected on the basis of the fiber type distribution in the 11 control rats. Mean fiber succinate dehydrogenase and ATPase activities were similar among the four groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Reconstitution of water channel function of aquaporins 1 and 2 by expression in yeast secretory vesicles

1998 ◽  
Vol 274 (1) ◽  
pp. F34-F42 ◽  
Author(s):  
Larry A. Coury ◽  
John C. Mathai ◽  
G. V. Ramesh Prasad ◽  
Jeffrey L. Brodsky ◽  
Peter Agre ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Water Flow ◽  
Water Channel ◽  
Immunoblot Analysis ◽  
High Water ◽  
Activation Energies ◽  
Secretory Vesicles ◽  
Channel Function ◽  
Other Substances

Aquaporins 1 (AQP1) and 2 (AQP2) were expressed in the yeast secretory mutant sec6-4. The mutant accumulates post-Golgi, plasma membrane-targeted vesicles and may be used to produce large quantities of membrane proteins. AQP1 or AQP2 were inducibly expressed in yeast and were localized within isolated sec6-4 vesicles by immunoblot analysis. Secretory vesicles containing AQP1 and AQP2 exhibited high water permeabilities and low activation energies for water flow, indicating expression of functional AQP1 and AQP2. AQP1 solubilized from secretory vesicles was successfully reconstituted into proteoliposomes, demonstrating the ability to use the yeast system to express aquaporins for reconstitution studies. The AQP2-containing secretory vesicles showed no increased permeability toward formamide, urea, glycerol, or protons compared with control vesicles, demonstrating that AQP2 is highly selective for water over these other substances. We conclude that the expression of aquaporins in yeast sec6 vesicles is a valid system to further study mammalian water channel function.

scholarly journals Proteomics Analysis of Plasma Membrane Fractions of the Root, Leaf, and Flower of Rice

2020 ◽  
Vol 21 (19) ◽  
pp. 6988
Author(s):  
Yukimoto Iwasaki ◽  
Takafumi Itoh ◽  
Yusuke Hagi ◽  
Sakura Matsuta ◽  
Aki Nishiyama ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Plasma Membranes ◽  
Cell Function ◽  
Immunoblot Analysis ◽  
Leaf Sheath ◽  
Green Leaf ◽  
Organ Specificity ◽  
Plasma Membrane Protein ◽  
Proteomics Data

The plasma membrane regulates biological processes such as ion transport, signal transduction, endocytosis, and cell differentiation/proliferation. To understand the functional characteristics and organ specificity of plasma membranes, plasma membrane protein fractions from rice root, etiolated leaf, green leaf, developing leaf sheath, and flower were analyzed by proteomics. Among the proteins identified, 511 were commonly accumulated in the five organs, whereas 270, 132, 359, 146, and 149 proteins were specifically accumulated in the root, etiolated leaf, green leaf, developing leaf sheath, and developing flower, respectively. The principle component analysis revealed that the functions of the plasma membrane in the root was different from those of green and etiolated leaves and that the plasma membrane protein composition of the leaf sheath was similar to that of the flower, but not that of the green leaf. Functional classification revealed that the root plasma membrane has more transport-related proteins than the leaf plasma membrane. Furthermore, the leaf sheath and flower plasma membranes were found to be richer in proteins involved in signaling and cell function than the green leaf plasma membrane. To validate the proteomics data, immunoblot analysis was carried out, focusing on four heterotrimeric G protein subunits, Gα, Gβ, Gγ1, and Gγ2. All subunits could be detected by both methods and, in particular, Gγ1 and Gγ2 required concentration by immunoprecipitation for mass spectrometry detection.

scholarly journals A new congenital multicore titinopathy associated with fast myosin heavy chain deficiency

2020 ◽  
Vol 7 (5) ◽  
pp. 846-854
Author(s):  
Aurélien Perrin ◽  
Corinne Metay ◽  
Marcello Villanova ◽  
Robert‐Yves Carlier ◽  
Elena Pegoraro ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Fast Myosin ◽  
Fast Myosin Heavy Chain

scholarly journals Myosin Heavy Chains IIa and IId Are Functionally Distinct in the Mouse

1998 ◽  
Vol 141 (4) ◽  
pp. 943-953 ◽  
Author(s):  
Carol A. Sartorius ◽  
Brian D. Lu ◽  
Leslie Acakpo-Satchivi ◽  
Renee P. Jacobsen ◽  
William C. Byrnes ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid Level ◽  
Spinal Curvature ◽  
Developmental Expression ◽  
Cross Sectional ◽  
Fast Myosin ◽  
Fast Myosin Heavy Chain ◽  
Fiber Number ◽  
Murine Skeletal Muscle ◽  
And Function

Myosin in adult murine skeletal muscle is composed primarily of three adult fast myosin heavy chain (MyHC) isoforms. These isoforms, MyHC-IIa, -IId, and -IIb, are >93% identical at the amino acid level and are broadly expressed in numerous muscles, and their genes are tightly linked. Mice with a null mutation in the MyHC-IId gene have phenotypes that include growth inhibition, muscle weakness, histological abnormalities, kyphosis (spinal curvature), and aberrant kinetics of muscle contraction and relaxation. Despite the lack of MyHC-IId, IId null mice have normal amounts of myosin in their muscles because of compensation by the MyHC-IIa gene. In each muscle examined from IId null mice, there was an increase in MyHC-IIa– containing fibers. MyHC-IIb content was unaffected in all muscles except the masseter, where its expression was extinguished in the IId null mice. Cross-sectional fiber areas, total muscle cross-sectional area, and total fiber number were affected in ways particular to each muscle. Developmental expression of adult MyHC genes remained unchanged in IId null mice. Despite this universal compensation of MyHC-IIa expression, IId null mice have severe phenotypes. We conclude that despite the similarity in sequence, MyHC-IIa and -IId have unique roles in the development and function of skeletal muscle.

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