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T. Inoue, N. P. Skiba, D. L. Epstein, P. V. Rao; Proteomic Analysis of the Trabecular Meshwork Cell Cytoskeletal Protein Fraction. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5719.
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To understand the broader role of cytoskeleton in homeostasis of aqueous humor outflow, here we characterized the cytoskeletal protein profile of trabecular meshwork (TM) cells and compared this to the profile of vascular endothelial cells.
Trabecular meshwork cells isolated from the human and porcine species, and human vascular endothelial cells (HUVEC) were cultured to confluence, and used to isolate the Triton-X-100 insoluble fraction enriched for cytoskeletal proteins. The Triton- insoluble cytoskeletal protein fraction was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent in-gel trypsin digestion of distinctly separated protein bands. The tryptic-digested peptides were identified by a combination of peptide mass finger printing analyses (MALDI-TOF-TOF MS) and MS/MS (Tandem mass spectrometry).
The cytoskeletal fraction from both human and porcine TM cells contained plectin 1, filamin A, non-muscle myosin IIA, clathrin, -actinin, vimentin, beta- and gamma-actin, and annexin A2 as major proteins. Further, the composition of the cytoskeletal protein fraction isolated from TM tissue was found to be similar to that of cultured TM cells. Moreover, the cytoskeletal composition observed for the TM cells was found to be very similar to that noted for HUVECs, with the exception of nestin being specific to HUVECs.
This study identifies plectin, filamin A, myosin II, -actinin, actin, vimentin and annexin II as predominant cytoskeletal proteins in cultured TM cells and TM tissue. Importantly these data not only confirm that the composition of the TM cell cytoskeleton is very similar to that of vascular endothelial cells, but also identifies proteins which may participate in modulating the mechanical and contractile properties of the trabecular meshwork that may be potentially involved in the regulation of aqueous humor outflow.
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