MUSCLE BIOLOGY OVERVIEW OF THE SKELETAL MUSCLE CYTOSKELETON

Transcription

1 MUSCLE BIOLOGY OVERVIEW OF THE SKELETAL MUSCLE CYTOSKELETON Ted Huiatt, Ph.D. Associate Professor, Department of Animal Science and Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University

2 Richard M. Robson Long time member of AMSA Received the AMSA Distinguished Research Award, 1984 Served on many AMSA Committees Driving force for study of intermediate filaments and cytoskeletal attachments in the Muscle Biology Group at Iowa State University

3 Skeletal Muscle The most beautiful cells in the animal kingdom Why? What structures are responsible for maintaining the structural integrity of these very highly organized cells?

4 The Skeletal Muscle Cytoskeleton Integrins Extracellular Matrix Sarcolemma Intermediate Filaments Z-disk

5 The Attachments Inside Myofibrils = the sarcolemmal cytoskeleton Elements: Titin Nebulin Z-disk M-line

6 Titin Z-line M-line Z-line Largest known protein (3,700 kda) Extends over ½ of sarcomere (M-line to Z-disk) Bound to thick filament in A-band Elastic region in I band Extends into Z-line Rapidly degraded postmortem

7 Possible Functions of Titin Passive tension Restoration of sarcomere length during relaxation Maintains thick filaments in center of sarcomere during contraction Molecular ruler to regulate thick filament length Assembly of myofibrils during myogenesis Molecular blueprint for sarcomere Signaling of mechanical stretch/strain; protein turnover Mechanical integrity of entire myofibril Other functions??

8 Nebulin Firmly attached to thin filament Molecular ruler for F-actin/thin filament length Stabilizes thin filament Functions in attachment of thin filament to Z-line Nemaline myopathy Nebulin mutation Disrupts Z-iline structure Knockout in mice alters contractility, disrupts Z-line

9 Z-disk Transmits contractile force from actin to rest of cell and rest of muscle Complex structure Actin filaments overlap in Z-disk No TM or TN in Z-disk, but nebulin continues into Z CapZ Binds barbed end of F-actin (caps filament) Binds to region of nebulin located at edge of Z-disk - could link to thin filament from opposing sarcomere Titin associates with actin at edge of Z-disk, extends into Z # of Z-disk repeats correlate with Z-disk width in different muscles

10 More on Z-disk α-actinin crosslinks actin from opposing sarcomeres also binds titin # of α-actinin crosslinks correlates with # of Z-disk titin domains Titin crosslinked by telethonin Many other proteins associated with Z-disk - myotilin - myopallidin - archvillin - cypher/zasp - MURF-1 - muscle LIM protein Loss of Z-disks postmorten due to titin & nebulin degradation?

11 M-line Links thick filaments Proteins common to all vertebrate skeletal muscle M-lines: Myosin Titin Myomesin Obscurin/Obscurin-like protein 1 (obsl1) Obscurin at periphery, obsl1 integral to M-line Creatine kinase M-protein only in fast, not considered essential to all M-lines Attachments from complex of titin, myomesin and obsl1 Obscurin involved in SR attachment with ankyrin isoform (sank1)

12 The Skeletal Muscle Cytoskeleton Integrins Extracellular Matrix Sarcolemma Intermediate Filaments Z-disk

13 Attachment of Myofibrils to Each Other and to the Sarcolemma Intermediate filaments (IFs) primarily responsible Desmin major IF protein in muscle Desmin localized at periphery of Z-disk IFs surround Z-disks and run to attachment sites on sarcolemma Other IF proteins in muscle: synemin, cytokeratins, nestin (paranemin), syncoilin

14 How are IFs attached to Z-disks? Synemin Robson Group Originally identified as an IF-associated protein (Granger & Lazarides, 1980) Contains characteristic IF α-helical domain Large IF protein: α-synemin, 173 kda; β-synemin, 140 kda (compare to 55 kda desmin Long C-terminal tail Does not form IFs alone, but forms heteropolymers with desmin Tail binds α-actinin Colocalized with desmin at periphery of Z-disks Highly susceptible to proteolysis

15 IF Attachment to Z-disks, continued Plectin the universal cytoskeletal crosslinker (G Wiche and coworkers) Large protein, ~500 kda, several isoforms Binds IFs (desmin, vimentin, keratins ); also F-actin and microtubules Colocalized with desmin at Z-disk periphery Knockouts result in IF aggregation, disruption of MF organization, Mechanism for attachment to Z-disk not characterized Z-disk region of nebulin interacts with α-helical region of desmin (Bang et al., 2002)

16 Costameres: Sarcolemmal attachments First identified by immunofluorescence localization of vinculin, an attachment site protein (Pardo et al., 1983). Located on inside of sarcolemma, around cell ( rib-like ), level of myofibril Z-lines Contain 3 types of cytoskeletal-sarcolemma-extracellular matrix attachments Focal adhesion Dystrophin-associated protein complex (DAPC) Spectrin-ankyrin attachments

17 Cytoskeletal-Membrane-ECM Attachments

18 Potential Mechanisms for Myofibril-Sarcolemmal Attachment Focal Adhesions α-synemin tail interacts with both vinculin and talin attach heteropolymeric desmin/synemin IFs to focal adhesions Linkage of actin in membrane skeleton linkage to Z-line? DAPC Tail of α-synemin can bind dystrophin Rod region of synemin can bind dystrophin and α-dystrobrevin Plectin binds β-dystroglycan in DAPC and dystrophin. Unusual IF protein syncoilin binds desmin and α-dystrobrevin Ankyrin-spectrin interactions with the membrane skeleton

19 Conclusions Cytoskeletal attachment sites are of vital importance in maintaining the integrity of skeletal muscle cells Each of the attachment sites is complex, and each appears to utilize more than one mechanism to carry out their function All of these attachments have roles in muscle disease Postmortem degradion of these cytoskeletal attachments has important consequences for meat quality

20 Acknowledgements Contributors to the work from ISU described in this presentation. Bruno Becker Stephen Bilak Robert Bellin Rahul Bhosle Susan Sernett Ning Sun Collaborators for the work on synemin were: Kevin P. Campbell Yassemi Capetanaki David R. Critchley Denise Paulin Wallace Ip This research was supported in part by: American Heart Association Iowa Agriculture Experiment Station Muscular Dystrophy Association National Live Stock and Meat Board USDA National Research Initiative Competitive Grants Program

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