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Ian Grierson, Steven O'Reilly, Natalie M. Pollock, Laura M. Currie; Cross-linked Actin Networks (clans) In Cultured Trabecular Meshwork (tm) Cells Have An Epitheliod Shape And Are Immobile. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4654.
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© ARVO (1962-2015); The Authors (2016-present)
Cytoskeletal CLANs are induced in cultured TM cells by various agents but rarely in all cells despite days of exposure. The present study was conducted to determine whether CLAN formation was associated with a particular cell appearance or activity.
Primary bovine TM cells (BTMC) were grown in media with 10% serum but maintained pre-confluent in 1% serum with minimum cell division and exposed to three CLAN inducing agents dexamethasone, TGFβ2 and decorin for 7 days. The cells were examined by phase microscopy, subjected to time-lapse microscopy, fixed and then stained with phalloidin Alexa-fluor 488 (Invitrogen) prior to immunoflourescent and phase microscopy with image analysis (Image-J).
Prior to confluence, BTMC adopt 3 distinct shapes spindle, epitheliod and kite of which spindle predominate (averages for each 65, 20 and 15%) however after exposure to our CLAN inducers more BTMC are epitheliod (40, 40 and 20%). Masked immunofluorescent and phase fields were taken using x40 microscope lenses and from these images (200 per experiment) cells with 2 different actin patterns were selected - those which clearly had a CLAN arrangement and those without CLANs but prominent stress fibers. For each cell we then switched to the phase image, drew its outline and categorised it as being epitheliod, spindle or kite. Image analysis showed that, irrespective of stimulant, cells with CLANs were more circular than those with stress fibers only (P<0.05 or better). Categorisation showed that CLAN containing cells were predominantly, but not exclusively, epitheliod (70.4% with DEX, 77.2% with TGFβ2, 79.2% with decorin). Stress fibers only cells were predominantly spindle (70.7% with DEX, 74.7% with TGFβ2, 82.1% with decorin). Subsequent time lapse experiments showed that BTMC without CLANs were highly mobile cells whereas those with CLANs were immobile irrespective of shape (391 cells examined).
Our findings show that CLANs are associated with BTMC that are immobile and epitheliod and in phenotypically heterogeneous primary cell populations why not all cells form these structures .
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