Coordination between cytoskeletal organization, cell contraction and extracellular matrix development, is depended on LOX for aneurysm prevention.

Distinct, seemingly independent, cellular pathways affecting intracellular machineries or extracellular matrix (ECM) deposition and organization, have been implicated in aneurysm formation. One of the key genes associated with the pathology in both humans and mice is Lysyl oxidase (LOX), a secreted ECM-modifying enzyme, highly expressed in medial vascular smooth muscle cells. To dissect the mechanisms leading to aneurysm development, we conditionally deleted Lox in smooth muscle cells. We find that cytoskeletal organization is lost following Lox deletion. Cell culture assays and in vivo analyses demonstrate a cell-autonomous role for LOX affecting myosin light chain phosphorylation and cytoskeletal assembly resulting in irregular smooth muscle contraction. These results not only highlight new intracellular roles for LOX, but notably they link between multiple processes leading to aneurysm formation suggesting LOX coordinates ECM development, cytoskeletal organization and cell contraction required for media development and function.

The SOAR of STIM1 interacts with plasma membrane lipids to form ER-PM contact sites.

Depletion of Ca2+ from the endoplasmic reticulum (ER) causes the ER Ca2+ sensor STIM1 to form membrane contact sites (MCSs) with the plasma membrane (PM). At the ER-PM MCS, STIM1 binds to Orai channels to induce cellular Ca2+ entry. The prevailing view of this sequential process is that STIM1 interacts with the PM and with Orai1 using two separate modules: a C-terminal polybasic domain (PBD) for the interaction with PM phosphoinositides and the STIM-Orai activation region (SOAR) for the interaction with Orai channels. Here, using electron and fluorescence microscopy and protein-lipid interaction assays, we show that oligomerization of the SOAR promotes direct interaction with PM phosphoinositides to trap STIM1 at ER-PM MCSs. The interaction depends on a cluster of conserved lysine residues within the SOAR and is co-regulated by the STIM1 coil-coiled 1 and inactivation domains. Collectively, our findings uncover a molecular mechanism for formation and regulation of ER-PM MCSs by STIM1.

Bidirectional regulation of calcium release-activated calcium (CRAC) channel by SARAF.

Store-operated calcium entry (SOCE) through the Ca2+ release-activated Ca2+ (CRAC) channel is a central mechanism by which cells generate Ca2+ signals and mediate Ca2+-dependent gene expression. The molecular basis for CRAC channel regulation by the SOCE-associated regulatory factor (SARAF) remained insufficiently understood. Here we found that following ER Ca2+ depletion, SARAF facilitates a conformational change in the ER Ca2+ sensor STIM1 that relieves an activation constraint enforced by the STIM1 inactivation domain (ID; aa 475-483) and promotes initial activation of STIM1, its translocation to ER-plasma membrane junctions, and coupling to Orai1 channels. Following intracellular Ca2+ rise, cooperation between SARAF and the STIM1 ID controls CRAC channel slow Ca2+-dependent inactivation. We further show that in T lymphocytes, SARAF is required for proper T cell receptor evoked transcription. Taking all these data together, we uncover a dual regulatory role for SARAF during both activation and inactivation of CRAC channels and show that SARAF fine-tunes intracellular Ca2+ responses and downstream gene expression in cells.