Obscurin modulates the assembly and organization of sarcomeres and the sarcoplasmic reticulum.
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.
Kontrogianni-Konstantopoulos
Aikaterini
A
Department of Physiology, University of Maryland Baltimore, School of Medicine, Baltimore, MD 21201, USA. akons001@umaryland.edu
Catino
Dawn H
DH
Strong
John C
JC
Sutter
Sarah
S
Borisov
Andrei B
AB
Pumplin
David W
DW
Russell
Mark W
MW
Bloch
Robert J
RJ
D000818:Animals; D017487:Ankyrins; D002478:Cells, Cultured; D064211:Connectin; D020662:Guanine Nucleotide Exchange Factors / Q000172:deficiency / Q000502:physiology*; D024510:Muscle Development; D018485:Muscle Fibers, Skeletal / Q000166:cytology* / Q000648:ultrastructure; D009124:Muscle Proteins / Q000172:deficiency / Q000502:physiology*; D009218:Myosins; D051381:Rats; D012518:Sarcomeres / Q000378:metabolism / Q000648:ultrastructure*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure*
D017487:Ankyrins; D064211:Connectin; D020662:Guanine Nucleotide Exchange Factors; D009124:Muscle Proteins; C503780:Obscn protein, rat; D009218:Myosins