Abstract
Abstract: :
Purpose: To characterize the molecular composition of CLANs observed in dexamethasone (DEX)–treated and glaucomatous HTM cells in order to understand the regulation of CLAN formation. Methods: Immunofluorescence microscopy was used to examine CLAN composition in normal and glaucomatous HTM cells. Cells were allowed to spread on coverslips coated with fibronectin (FN), type IV collagen (CIV) or vitronectin (VN) for 3hr. Cells were labeled with phalloidin or antibodies against F–actin, α–actinin, syndecan–4, phosphatidylinositol–4,5–bisphosphate (PIP–2), protein kinase Cα (PKCα), Arp3 or cortactin. In some experiments, cells were treated with protein kinase C inhibitors (hispidin, Gö 6976, Ro–31–7549 or Ro–31–8220) or wortmannin (a phosphatidylinositol 3–kinase (PI–3K) inhibitor) prior to plating onto coverslips. Alternatively, cells were pretreated with DEX for 10–12 days prior to plating them onto different substrates. Results:CLANs were observed in cells spread on FN, CIV or VN. The vertices of CLANs ("vertisomes") labeled positively for α–actinin, syndecan–4 and PIP2 when cells were allowed to spread on all three substrates. PKCα staining was also present in vertisomes as well as along actin filaments in CLANs. Vertisomes and CLANs failed to stain for Arp3 or cortactin, two proteins typically found in branched actin structures. Neither PKC inhibitors nor wortmannin effected the formation of CLANs in spreading cells. DEX treatment enhanced the rate of CLAN formation in cells but did not increase the number of CLAN–positive cells. Conclusions: CLAN formation in spreading cells is DEX sensitive, occurs independent of the matrix substrate, and is not regulated by either PKC or PI–3K signaling pathways. The vertices of CLANs contain specific protein complexes (vertisomes) that are known to regulate actin filament formation and hence may regulate CLAN formation.
Keywords: trabecular meshwork • cytoskeleton • extracellular matrix