Characterization of cytoskeleton-enriched protein fraction of the trabecular meshwork and ciliary muscle cells.
OBJECTIVE
To understand the molecular basis for the known distinct contractile characteristics of trabecular meshwork (TM) and ciliary muscle (CM) cells, the cytoskeleton-enriched protein fractions of the TM and CM cells were isolated and characterized.


METHODS
The nonionic surfactant insoluble fraction enriched for cytoskeletal proteins was isolated from human and porcine TM tissue and cells and from CM cells and was characterized by SDS-PAGE, mass spectrometry, and immunoblotting techniques.


RESULTS
The cytoskeleton-enriched protein fraction derived from both human and porcine TM cells contained Plectin 1, Filamin A, non-muscle myosin IIA, clathrin, α-actinin, vimentin, actin, caldesmon, myosin IC, and annexin A2 as major proteins and was noted to exhibit compositional similarity with the cytoskeletal protein fraction isolated from TM tissue. Importantly, the cytoskeletal protein composition of the TM cells was also found to be similar to that noted for CM and vascular endothelial cells. Although the activity of myosin II, a crucial regulator of cellular contraction and a major component of the cytoskeletal protein fraction in TM and CM cells, was regulated predominantly by Rho kinase in both cell types, myosin light chain kinase (MLCK) also appeared to control myosin II activity in CM cells.


CONCLUSIONS
These data reveal that the activity of non-muscle myosin II, a critical molecule of cellular contraction, was found to be regulated differentially in TM and CM cells by the Rho kinase and the MLCK pathways despite their compositional similarity in cytoskeletal protein profile.
Inoue
Toshihiro
T
Department of Ophthalmology, Duke University School of Medicine, Durham, NC 27710, USA.
Pecen
Paula
P
Maddala
Rupalatha
R
Skiba
Nikolai P
NP
Pattabiraman
Padmanabhan P
PP
Epstein
David L
DL
Rao
P Vasantha
PV
D000293:Adolescent; D000328:Adult; D000818:Animals; D018929:Cell Culture Techniques; D002469:Cell Separation; D002924:Ciliary Body / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D013058:Mass Spectrometry; D008875:Middle Aged; D009130:Muscle, Smooth / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D024507:Nonmuscle Myosin Type IIA / Q000378:metabolism; D010766:Phosphorylation; D013552:Swine; D014129:Trabecular Meshwork / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism
D003598:Cytoskeletal Proteins; D018994:Myosin Light Chains; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase; D024507:Nonmuscle Myosin Type IIA