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The formation of cortical actin arrays in human trabecular meshwork cells in response to cytoskeletal disruption.

ABSTRACT: The cytoskeleton of human trabecular meshwork (HTM) cells is known to be altered in glaucoma and has been hypothesized to reduce outflow facility through contracting the HTM tissue. Latrunculin B (Lat-B) and Rho-associated protein kinase (ROCK) inhibitors disrupt the actin cytoskeleton and are in clinical trials as glaucoma therapeutics. We have previously reported a transient increase in HTM cell stiffness peaking at 90 min after Lat-B treatment with a return to pretreatment values after 270 min. We hypothesize that changes in actin morphology correlate with alterations in cell stiffness induced by Lat-B but this is not a general consequence of other cytoskeletal disrupting agents such as Rho kinase inhibitors. We treated HTM cells with 2 µM Lat-B or 100 µM Y-27632 and allowed the cells to recover for 30-270 min. While examining actin morphology in Lat-B treated cells, we observed striking cortical actin arrays (CAAs). The percentage of CAA positive cells (CPCs) was time dependent and exceeded 30% at 90 min and decreased after 270 min. Y-27632 treated cells exhibited few CAAs and no changes in cell stiffness. Together, these data suggest that the increase in cell stiffness after Lat-B treatment is correlated with CAAs.

SUBMITTER: Murphy KC

PROVIDER: S-EPMC4178168 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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The formation of cortical actin arrays in human trabecular meshwork cells in response to cytoskeletal disruption.

Murphy Kaitlin C KC Morgan Joshua T JT Wood Joshua A JA Sadeli Adeline A Murphy Christopher J CJ Russell Paul P

Experimental cell research 20140630 1

The cytoskeleton of human trabecular meshwork (HTM) cells is known to be altered in glaucoma and has been hypothesized to reduce outflow facility through contracting the HTM tissue. Latrunculin B (Lat-B) and Rho-associated protein kinase (ROCK) inhibitors disrupt the actin cytoskeleton and are in clinical trials as glaucoma therapeutics. We have previously reported a transient increase in HTM cell stiffness peaking at 90 min after Lat-B treatment with a return to pretreatment values after 270 mi ...[more]

PMID: 24992043

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Project description:Changes in the actin cytoskeleton, especially the formation of cross-linked actin networks (CLANs) are thought to contribute to the increased intraocular pressure observed in primary open-angle and steroid-induced glaucoma. To better understand the effects of glucocorticoids, we employed a shotgun method to analyze global changes in the cytoskeleton and integrin signaling pathways following dexamethasone (DEX) treatment of human trabecular meshwork (HTM) cells. RNA and cell lysates were obtained from HTM cells incubated with or without DEX. Changes in protein expression were determined by mass spectrometry (MS) following differential centrifugation of cell lysates to enrich for low-abundance cytoskeletal and signaling proteins, proteolytic digestion, and a titanium dioxide column to enrich for phosphopeptides. Results were validated by Western blots. Changes in RNA levels were determined with gene arrays and RT-PCR. Overall, MS identified 318 cytoskeleton associated proteins. Five of these proteins (PDLIM1, FGFR1OP, leiomodin-1, ZO-2 and LRP16A) were only detected in DEX-treated cells by MS. However, only PDLIM1 showed a statistically significant increase at the RNA level. Other proteins with differences at both the RNA and protein levels included ?3 integrin, caveolin-1, Borg2, raftlin1, PI-3 kinase regulatory subunit ?, transgelin, and filamin B. By immunofluorescence microscopy filamin B and PDLIM1 showed enhanced expression in human trabecular meshwork cells, but only PDLIM1 demonstrated significant localization within CLANs. Finally, MS showed that some of the cytoskeleton proteins (Borg2, leiomodin-1, LRP16A, raftlin1 and CKAP4) contained phosphorylated residues. This study suggests that DEX affects the expression of cytoskeleton proteins at the transcriptional and translational level and shows that a combined genomic and proteomic approach can be used for rapid analysis of proteins in the TM. It also shows that DEX altered the expression of components (PDLIM1 and ?3 integrins) involved in CLAN formation and provides new findings into the effects of glucocorticoids on the cytoskeleton.

Dataset Information

The formation of cortical actin arrays in human trabecular meshwork cells in response to cytoskeletal disruption.

Publications

The formation of cortical actin arrays in human trabecular meshwork cells in response to cytoskeletal disruption.

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