Figure 3. Cochlin mechanosensing and proposed model of potential associated TM changes.

(A) Western analysis of purified recombinant cochlin (*) using anti-cochlin antibody before after sheer stress, revealing shear stress-induced mulitmers. Cochlin was subjected to fluid shear of 3 µl/min for 150 cycles. (B) Comparison of proteolytic digestion (in percentage) of native (left side) and multimerized (right side) cochlin by trypsin (open bars) and proteinase K (filled bars) showing slower digestion of multimerized cochlin. Digestion of multimerized and native cochlin (10 µg) was performed using 0.01 µg/10 µg Trypsin or 0.05 µg/10 µg Proteinase K. After incubation at room temperature, the samples were boiled using 1 mM DTT at 100°C for 1 minute and analyzed on SDS-PAGE. Densitometric analyses data from three independent experiments (mean± standard deviation) has been presented. (C) A representative Western analysis of native and multimerized cochlin subjected to subcatalytic amounts of trypsin and proteinase K digestion for 10 minutes at room temperature. Purified recombinant cochlin (*), native monomeric cochlin and multimeric cochlin (initial amount 1 µg) has been depicted as Mo and Mu respectively. Digestion was performed by 0. 1 µg/10 µg Trypsin or 0.5 µg/10 µg Proteinase K. Digestion was stopped by heating at 100°C, separated on a 10% reducing SDS-PAGE and probed with chicken polyclonal antibody against cochlin. Proposed model of cochlin mechanosensing and associated global change in TM. (D) Illustration depicting that the cochlin constitutively secreted by the normal TM cells is degraded by the proteases (dashed lines denoting degraded proteins). (E) Cochlin in the presence of fluctuating shear stress (or elevated divalent cations), forms multimers which are resistant to proteolysis. Multimerized cochlin may potentially interact with the transmembrane proteins TREK-1 or slc44a2 either directly, functionally, or indirectly leading to events that may result in cytoskeletal reorganization. (F) Confocal microscopy image showing the sieve like structure of TM (arrow). A representative area of TM with rectangular arrangement of cells is highlighted by a yellow box. SC = Schlemm's canal; TM = trabecular meshwork. The magnified view of rectangular arrangement of cells highlighted by a yellow box also has been shown. (G) The line diagram depicts the filter like structure of the TM. A magnified view mimicking one such rectangle as in (F) with an additional cell in the middle of the rectangle has been shown. Notice the change in orientation of the middle cell opens up interstitial space for additional fluid flow indicated by dashed arrows. (H) When incremental space opening is insufficient, larger orientation changes by cells in the middle and those forming rectangle are necessary to increase fluid flow. Newly opened space is indicated by arrow head. The dark lines and dark dashed lines are to indicate orientation changes. (I) Cartoon illustrating concerted orientation changes in several individual cells (dashed lines) leading to a global change in the structure of the sieve like TM. (J) Confocal microscopy image of cochlin transfected TM cells (left; i) expressing cochlin (pink) and tiny aggregates of cochlin (arrows) secreted into the media. These small cochlin aggregates coalesce (arrow) to form larger deposits as shown in human glaucomatous TM probed for cochlin (green; right; ii). Scale bar = 25 µm (left) and 50 µm (right). (K) Confocal microscopy image of trilayer of cochlin transfected TM cells on a PVDF membrane exposed to continuous fluctuation in fluid shear stress in an Ussing type chamber showing the development of cochlin deposits (red; arrows).