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Intermediate Filaments and Myogenesis in vitro

Citation

Gard, David Lynn (1982) Intermediate Filaments and Myogenesis in vitro. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/e5gk-4c77. https://resolver.caltech.edu/CaltechTHESIS:05142018-174410594

Abstract

This thesis describes my investigations into the composition and function of intermediate filaments (IF) during myogenesis in vitro. I have found that avian embryonic myotubes cultured in vitro contain two intermediate filament subunits, desmin and vimentin. Prior to myoblast fusion vimentin is the sole IF subunit protein detectable by electrophoretic and immunological techniques. The onset of desmin synthesis and its cytoplasmic accumulation appear to coincide with fusion of myoblasts into multinucleate myotubes. Immunofluorescence reveals dense networks of desmin- and vimentin-containing filaments in the sarcoplasm of immature myotubes; however, late in myogenesis antisera to both desmin and vimentin are observed to stain the Z-lines of myofibrils. Double immunofluorescence microscopy using antisera to α-actinin and desmin revealed that this association occurs after the assembly of α-actinin into Z-lines, at a time when individual myofibrils are being organized into bundles. Phosphorylation of intermediate filament proteins in muscle has been previously reported (O'Connor et al., Proc. Natl. Acad. Sci. U.S.A. 76: 819-823, 1979). Using two-dimensional tryptic analysis I have found that desmin from embryonic myotubes is phosphorylated at multiple sites which correspond to sites phosphorylated by cAMP-dependent protein kinase in vitro. I have observed phosphorylation of desmin and vimentin in intact myotubes at all stages of myogenesis. However, treatment of mature (7 day and older) myotubes with 8-BrcAMP or isoproterenol results in a specific 2-3 fold increase in phosphorylation of these proteins, with a corresponding increase in 32PO4 incorporation into one major phosphopeptide of desmin. Preliminary evidence indicates that treatment of 6-8 day myotubes with 8-BrcAMP or isoproterenol significantly inhibits the transition of intermediate filaments to the Z-line which occurs during normal myogenesis. These observations suggest that intermediate filaments containing desmin and vimentin are responsible for the organization of skeletal muscle myofibrils into an integral contractile machinery, and that cAMP-dependent phosphorylation of desmin and vimentin plays an important role in the regulation of this function.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Cell Biology
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Biology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Lazarides, Elias
Thesis Committee:
  • Lazarides, Elias (chair)
  • Brokaw, Charles J.
  • Kennedy, Mary B.
  • Mitchell, Herschel K.
  • Revel, Jean-Paul
Defense Date:29 October 1981
Funders:
Funding AgencyGrant Number
Jean Weigle Memorial FundUNSPECIFIED
NSFUNSPECIFIED
NIHUNSPECIFIED
Record Number:CaltechTHESIS:05142018-174410594
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:05142018-174410594
DOI:10.7907/e5gk-4c77
Related URLs:
URLURL TypeDescription
https://doi.org/10.1073/pnas.76.8.3894DOIArticle adapted for Ch. 2
https://doi.org/10.1016/0092-8674(80)90408-0DOIArticle adapted for Ch. 3
https://doi.org/10.1016/0092-8674(81)90278-6DOIArticle adapted for Ch. 4
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10886
Collection:CaltechTHESIS
Deposited By: Melissa Ray
Deposited On:15 May 2018 18:47
Last Modified:19 Apr 2021 22:32

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