Surface pH changes suggest a role for H+/OH- channels in salinity response of Chara australis.

To understand salt stress, the full impact of salinity on plant cell physiology has to be resolved. Electrical measurements suggest that salinity inhibits the proton pump and opens putative H+/OH- channels all over the cell surface of salt sensitive Chara australis (Beilby and Al Khazaaly 2009; Al Khazaaly and Beilby 2012). The channels open transiently at first, causing a characteristic noise in membrane potential difference (PD), and after longer exposure remain open with a typical current-voltage (I/V) profile, both abolished by the addition of 1 mM ZnCl2, the main known blocker of animal H+ channels. The cells were imaged with confocal microscopy, using fluorescein isothiocyanate (FITC) coupled to dextran 70 to illuminate the pH changes outside the cell wall in artificial fresh water (AFW) and in saline medium. In the early saline exposure, we observed alkaline patches (bright fluorescent spots) appearing transiently in random spatial distribution. After longer exposure, some of the spots became fixed in space. Saline also abolished or diminished the pH banding pattern observed in the untreated control cells. ZnCl2 suppressed the alkaline spot formation in saline and the pH banding pattern in AFW. The osmotic component of the saline stress did not produce transient bright spots or affect banding. The displacement of H+ from the cell wall charges, the H+/OH- channel conductance/density, and self-organization are discussed. No homologies to animal H+ channels were found. Salinity activation of the H+/OH- channels might contribute to saline response in roots of land plants and leaves of aquatic angiosperms.

Is Wortmannin-Induced Reorganization of the trans-Golgi Network the Key to Explain Charasome Formation?

Wortmannin, a fungal metabolite and an inhibitor of phosphatidylinositol-3 (PI3) and phosphatidylinositol-4 (PI4) kinases, is widely used for the investigation and dissection of vacuolar trafficking routes and for the identification of proteins located at multivesicular bodies (MVBs). In this study, we applied wortmannin on internodal cells of the characean green alga Chara australis. Wortmannin was used at concentrations of 25 and 50 µM which, unlike in other cells, arrested neither constitutive, nor wounding-induced endocytosis via coated vesicles. Wortmannin caused the formation of "mixed compartments" consisting of MVBs and membranous tubules which were probably derived from the trans-Golgi network (TGN) and within these compartments MVBs fused into larger organelles. Most interestingly, wortmannin also caused pronounced changes in the morphology of the TGNs. After transient hypertrophy, the TGNs lost their coat and formed compact, three-dimensional meshworks of anastomosing tubules containing a central core. These meshworks had a size of up to 4 µm and a striking resemblance to charasomes, which are convoluted plasma membrane domains, and which serve to increase the area available for transporters. Our findings indicate that similar mechanisms are responsible for the formation of charasomes and the wortmannin-induced reorganization of the TGN. We hypothesize that both organelles grow because of a disturbance of clathrin-dependent membrane retrieval due to inhibition of PI3 and/or PI4 kinases. This leads to local inhibition of clathrin-mediated endocytosis during charasome formation in untreated cells and to inhibition of vesicle release from the TGN in wortmannin-treated cells, respectively. The morphological resemblance between charasomes and wortmannin-modified TGN compartments suggests that homologous proteins are involved in membrane curvature and organelle architecture.

Cholesterol modulates Orai1 channel function.

STIM1 (stromal interaction molecule 1) and Orai proteins are the essential components of Ca(2+) release-activated Ca(2+) (CRAC) channels. We focused on the role of cholesterol in the regulation of STIM1-mediated Orai1 currents. Chemically induced cholesterol depletion enhanced store-operated Ca(2+) entry (SOCE) and Orai1 currents. Furthermore, cholesterol depletion in mucosal-type mast cells augmented endogenous CRAC currents, which were associated with increased degranulation, a process that requires calcium influx. Single point mutations in the Orai1 amino terminus that would be expected to abolish cholesterol binding enhanced SOCE to a similar extent as did cholesterol depletion. The increase in Orai1 activity in cells expressing these cholesterol-binding-deficient mutants occurred without affecting the amount in the plasma membrane or the coupling of STIM1 to Orai1. We detected cholesterol binding to an Orai1 amino-terminal fragment in vitro and to full-length Orai1 in cells. Thus, our data showed that Orai1 senses the amount of cholesterol in the plasma membrane and that the interaction of Orai1 with cholesterol inhibits its activity, thereby limiting SOCE.