Biology subjects

Krizaj, D.

Publications and source records attributed to Krizaj, D..

3 recordsLinked to original sources

bioRxiv2020

Membrane cholesterol regulates TRPV4 function, cytoskeletal expression and the cellular response to tension

Despite the association of cholesterol with debilitating pressure-related diseases, its role in mechanotransduction is not well understood. We investigated the relationship between mechanical strain, free membrane cholesterol, actin cytoskeleton, and activation of stretch- activated TRPV4 (transient receptor potential vanilloid isoform 4) channel in human trabecular meshwork (TM) cells. Physiological levels of cyclic stretch resulted in time- dependent decreases in membrane cholesterol/phosphatidylcholine ratio and upregulation of stress fibers. Depletion of free membrane cholesterol with m-{beta}-cyclodextrin (M{beta}CD) augmented TRPV4 activation by the agonist GSK1016790A, swelling and strain, with the effects reversed by cholesterol supplementation. M{beta}CD increased membrane expression of TRPV4, caveolin-1 and flotillin. Caveolin-1 antibody partially precipitated a truncated [~]75 kDa variant whereas the majority of TRPV4 did not colocalize or interact with caveolae or lipid rafts, indicating that TRPV4 is mainly localized outside of cholesterol-enrichedmembrane domains. M{beta}CD induced currents in TRPV4-expressing Xenopus laevis oocytes. Thus, while the membrane C/P ratio reflects the biomechanical milieu, trabecular transduction of mechanical information is modulated by the membrane cholesterol content. Diet, cholesterol metabolism and mechanical stress might modulate the conventional outflow pathway and intraocular pressure in glaucoma and diabetes.

cell biology↗

bioRxiv2020

Mechanoreciprocity requires interactions between TRPV4 channels, the Rho pathway and actin cytoskeleton

Intraocular pressure (IOP) is dynamically regulated by the trabecular meshwork (TM), a mechanosensitive tissue that protects the eye from injury through dynamic regulation of aqueous humor outflow from the anterior chamber of the eye. IOP-dependent increases in TM stiffness and contractility drive open angle glaucoma but the mechanotransduction mechanisms that regulate these processes remain poorly understood. We used fluorescence imaging and biochemical analyses to investigate cytoskeletal and focal adhesion remodeling in human TM cells stimulated with cyclic strain. The cells showed enhanced F-actin polymerization, increased number and size of focal adhesions, and activation of the Rho-associated protein kinase (ROCK). Stretch-induced activation of the small GTPase RhoA, and tyrosine phosphorylations of focal adhesion proteins paxillin, focal adhesion kinase (FAK), vinculin and zyxin were time-dependently inhibited by HC-067047, an antagonist of transient receptor potential vanilloid 4 (TRPV4) channels, and the ROCK inhibitor Y-27632. TRPV4 and ROCK activation were required for zyxin translocation and increase in the number/size of focal adhesions in stretched cells. Y-27632 blocked actin polymerization without affecting calcium influx induced by membrane stretch and the TRPV4 agonist GSK1016790A. These results reveal that mechanical tuning of TM cells requires parallel activation of TRPV4, integrins and ROCK, with chronic stress leading to sustained remodeling of the cytoskeleton and focal complexes.

cell biology↗

bioRxiv2020

Mechanotransduction and dynamic outflow regulation in trabecular meshwork requires Piezo1 channels

Mechanosensitivity of the trabecular meshwork (TM) is a key determinant of intraocular pressure (IOP) yet our understanding of the molecular mechanisms that subserve it remains in its infancy. Here, we show that mechanosensitive Piezo1 channels modulate the TM pressure response via calcium signaling and dynamics of the conventional outflow pathway. Pressure steps evoked fast, inactivating cation currents and calcium signals that were inhibited by Ruthenium Red, GsMTx4 and Piezo1 shRNA. Piezo1 expression was confirmed by transcript and protein analysis, and by visualizing Yoda1-mediated currents and [Ca2+]i elevations in primary human TM cells. Piezo1 activation was obligatory for transduction of physiological shear stress and was coupled to reorganization of F-actin cytoskeleton and focal adhesions. The importance of Piezo1 channels as pressure sensors was shown by the GsMTx4 -dependence of the pressure-evoked current and conventional outflow function. We also demonstrate that Piezo1 collaborates with the stretch-activated TRPV4 channel, which mediated slow, delayed currents to pressure steps. Collectively, these results suggest that TM mechanosensitivity utilizes kinetically, regulatory and functionally distinct pressure transducers to inform the cells about force-sensing contexts. Piezo1-dependent control of shear flow sensing, calcium homeostasis, cytoskeletal dynamics and pressure-dependent outflow suggests a novel potential therapeutic target for treating glaucoma. Significance StatementTrabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humor drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humor outflow are understood at the molecular level. We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure. Its activation resulted in intracellular signals that altered organization of the cytoskeleton and cell-extracellular matrix contacts, and modulated the trabecular component of aqueous outflow whereas another channel, TRPV4, mediated a delayed mechanoresponse. These findings provide a new mechanistic framework for trabecular mechanotransduction and its role in the regulation of fast fluctuations in ocular pressure, as well as chronic remodeling of TM architecture that epitomizes glaucoma.

physiology↗