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Ian Grierson, Andrew D. Heath, Natalie Pollock, Laura M. Currie, Yalin Zheng; Comparison Between Polygonal Actin Arrangements (paas) And Cross-linked Actin Networks (clans) In Cultured Trabecular Meshwork (tm) Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3255.
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PAAs are temporary polygonal, hub and spoke arrangements of actin seen in the first few hours of tissue culture while the cells are settling but then are lost. CLANs are also hub and spoke arrangements of actin that are slowly induced in confluent cultures by steroids, TGFbeta and others and persist. We wished to determine whether or not CLANs and PAAs are one in the same thing so that rapidly forming PAAs can legitimately be used as a model for the slower forming CLANs that we know to form in vivo and are associated with glaucoma in a way that is poorly understood.
We stain actin in TM cells both in vitro and in situ using phalloid-FITC and image cells either with conventional immunofluorescence or by confocal microscopy. We have a large collection of PAAs and CLANs images and from these over 50 images were selected and handed over in a masked fashion to our reading team who used Image J Software (NIH) to measure CLAN and PAA territories, height, circularity, incidence of hubs per unit area, hub and spoke dimensions and other parameters.
TM cell CLANs had similar dimensional characteristics whether they were from normal or glaucoma donors. CLANS in cultured cells and those from ex vivo tissue were also indistinguishable except the latter were marginally smaller. PAAs were over twice the size of CLANs. They were circular (0.81) whereas CLANs were not (0.49). PAAs had a dome shape and were often located over the nucleus (height 3-8um) while CLANs were flat plate-like structures. In addition the circular hubs (over 0.9 for both) were twice the area in CLANs than PAAs and the spokes were significantly longer in CLANs than PAAs (all tests significant at P<0.05 or better Mann Whitney U Test).
We were interested to find that CLANs from different sources were fundamentally similar. PAAs may serve as a rapidly forming model to study some CLAN characteristics but they are by no means the same thing. It does seem that PAAs and CLANs have only superficial similarities and although these structures have the same design blueprint they are dimensionally dissimilar. We intend to make mathematical and structural models using our current data of PAAs and CLANs to explore their contribution to TM cell rigidity.
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