Abstract
Fluid flow through large interstitial spaces is sensed at the cellular level, and mechanistic responses to flow changes enables expansion or contraction of the cells modulating the surrounding area and brings about changes in fluid flow. In the anterior eye chamber, aqueous humor, a clear fluid, flows through trabecular meshwork (TM), a filter like region. Cochlin, a secreted protein in the extracellular matrix, was identified in the TM of glaucomatous patients but not controls by mass spectrometry. Cochlin undergoes shear induced multimerization and plays a role in mechanosensing of fluid shear. Cytoskeletal changes in response to mechanosensing in the ECM by cochlin will necessitate transduction of mechanosensing. TREK-1, a stretch activated outward rectifying potassium channel protein known to act as mechanotransducer was found to be expressed in TM. Cochlin expression results in co-expression of TREK-1 and filopodia formation. Prolonged cochlin expression results in expression and subsequent secretion of annexin A2, a protein known to play a role in cytoskeletal remodeling. Cochlin interacts with TREK-1 and annexin A2. Cochlin-TREK-1 interaction has functional consequences and results in changes in cell shape and motility. Annexin A2 expression and secretion follows cochlin-TREK-1 syn-expression and correlates with cell elongation. Thus cytoskeleton changes in response to fluid shear sensed by cochlin are further mediated by TREK-1 and annexin A2.
Highlights
A number of late onset and progressive diseases for example, glaucoma and idiopathic intracranial hypertension are associated with fluid flow abnormalities
Two distinct components are envisaged for regulation of fluid flow: a mechanosensor residing at the extracellular matrix (ECM) and transmembrane mechanotransducers residing at the cell surface
We present evidence that cochlin-TREK-1 and cochlin-annexin A2 interaction is commensurate with changes in trabecular meshwork (TM) cell shape and motility which changes the filter like structure of TM affecting aqueous outflow
Summary
A number of late onset and progressive diseases for example, glaucoma and idiopathic intracranial hypertension are associated with fluid flow abnormalities. Fixed cells were initially blocked in 16PBS+0.2% BSA for 1 h and incubated with the respective primary antibodies for cochlin, TREK1, actin, annexin A2, alpha-tectorin, green fluorescence protein (GFP) and diaphonous related formin-1 in the dilution factor of 1:200 in 16 Media for excreted protein analysis was collected at 24 h post transfection and subjected to ELISA probing for cochlin (hCochlin#3, Aves Labs Inc.) and annexin A2 (Santa Cruz Biotechnology Inc).
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