Linkage between the adherens junction (AJ) and the actin cytoskeleton is required for tissue development and homeostasis. In vivo findings indicated that the AJ proteins E-cadherin, β-catenin, and the filamentous (F)-actin binding protein αE-catenin form a minimal cadherin-catenin complex that binds directly to F-actin. Biochemical studies challenged this model since the purified cadherin-catenin complex does not bind F-actin in solution. Here we reconciled this difference. Using an optical trap-based assay, we showed that the minimal cadherin-catenin complex formed stable bonds with an actin filament under force. Bond dissociation kinetics can be explained by a catch bond model in which force shifts the bond from a weakly to a strongly bound state. These results may explain how the cadherin-catenin complex transduces mechanical forces at cell-cell junctions.

SUMMARY AAA+ unfoldases denature and translocate polypeptides into associated peptidases. We report direct observations of mechanical, force-induced protein unfolding by the ClpX unfoldase from E. coli, alone, and in complex with the ClpP peptidase. ClpX hydrolyzes ATP to generate mechanical force and translocate polypeptides through its central pore. Threading is interrupted by pauses that are found to be off the main translocation pathway. ClpX’s translocation velocity is force dependent, reaching a maximum of 80 aa/s near-zero force and vanishing at around 20 pN. ClpX takes 1, 2, or 3 nm steps, suggesting a fundamental step-size of 1 nm and a certain degree of intersubunit coordination. When ClpX encounters a folded protein, it either overcomes this mechanical barrier or slips on the polypeptide before making another unfolding attempt. Binding of ClpP decreases the slip probability and enhances the unfolding efficiency of ClpX. Under the action of ClpXP, GFP unravels cooperatively via a transient intermediate.

STIM1 is a transmembrane ER protein that serves as the primary ER calcium sensor for store-operated calcium entry (SOCE), an essential and widespread calcium signaling mechanism. Depletion of ER calcium causes STIM1 to relocate to ER-plasma membrane (PM) junctions where it binds and opens Orai1 channels. While many of the basic events of SOCE are now known, the underlying mechanisms remain unclear. Single-molecule imaging techniques can offer new and detailed mechanistic insights beyond what is possible using standard ensemble macroscopic approaches. We have tracked single particles of STIM1 and Orai1 proteins in cells to test a diffusion-trap model for their redistribution to ER-PM junctions. In cells with replete calcium stores, STIM1 moves by Brownian diffusion while Orai1 is slightly subdiffusive. After store depletion, STIM1 and Orai1 accumulate at junctions where they slowly diffuse at speeds consistent with formation of STIM-Orai complexes. Our data support the idea that only free STIM1 or Orai1 can escape the trap due to the physical constraints imposed by STIM-Orai binding across the junctional gap. STIM-Orai binding within junctions appears to be readily reversible, continuously generating free STIM1 and Orai1 which can exchange freely with extrajunctional pools. An important challenge is to understand the conformational changes that convert STIM1 to the active state that can bind and activate Orai1. To this end, we have applied single-molecule FRET techniques to examine spontaneous conformational changes in purified cytosolic fragments of STIM1 in vitro. Different regions of STIM1 display characteristic FRET signatures based on the number and amplitude of FRET states and the frequency of transitions among states. Continuing smFRET studies are aimed at tracking the sequence of conformational changes in STIM1 that couple the depletion of ER calcium to activation of calcium entry through Orai channels. 53-SympTuning the Taps: STIM1 and STIM2 Regulatory Mechanisms Cell homeostasis and IP3 dependent signaling rely on a precise regulation of basal and induced Ca 2þ concentrations; alterations of which can lead to defective signaling and abnormal growth. The two known isoforms STIM1 and STIM2 differentially sense the ER Ca 2þ content via their distinct luminal EF-hand Ca 2þ affinities. Upon dissociation of bound Ca 2þ , they oligomerize and translocate to plasma membrane near regions to trigger Ca 2þ entry by gating Orai channels. The exact contribution of STIM2 for immune cell Ca 2þ homeostasis is still controversial. We have identified a novel STIM2 splice variant, STIM2.1, which differs in a single exon inserted within the CRAC activating domain. Expression of STIM2.1 is ubiquitous but highest in naïve T-cells with expression relative to the conventional STIM2 changing upon activation or cell type. How and if this novel variant can resolve discrepancies regarding STIM2's physiological and pathophysiological role will be discussed. 54-SympGating Mechanisms of Store-Operated CRAC Channels Murali Prakriya. Ph...

Tissue morphogenesis requires the coordinated regulation of cellular behavior, which includes the orientation of cell division that defines the position of daughter cells in the tissue. Cell division orientation is instructed by biochemical and mechanical signals from the local tissue environment, but how those signals control mitotic spindle orientation is not fully understood. Here, we tested how mechanical tension across an epithelial monolayer is sensed to orient cell divisions. Tension across Madin-Darby canine kidney cell monolayers was increased by a low level of uniaxial stretch, which oriented cell divisions with the stretch axis irrespective of the orientation of the cell long axis. We demonstrate that stretch-induced division orientation required mechanotransduction through E-cadherin cell-cell adhesions. Increased tension on the E-cadherin complex promoted the junctional recruitment of the protein LGN, a core component of the spindle orientation machinery that binds the cytosolic tail of Ecadherin. Consequently, uniaxial stretch triggered a polarized cortical distribution of LGN. Selective disruption of trans engagement of E-cadherin in an otherwise cohesive cell monolayer, or loss of LGN expression, resulted in randomly oriented cell divisions in the presence of uniaxial stretch. Our findings indicate that E-cadherin plays a key role in sensing polarized tensile forces across the tissue and transducing this information to the spindle orientation machinery to align cell divisions.cell-cell adhesion | cell division orientation | mitotic spindle | mechanotransduction

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