The Minimal Cadherin-Catenin Complex Binds to Actin Filaments under Force

Abstract

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...

Adherens Junctions Revisualized: Organizing Cadherins as Nanoassemblies

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“…First, cadherins associate physically with F-actin. This is mediated by a-catenin (Buckley et al, 2014;Pokutta et al, 2002;Rimm et al, 1995), a ubiquitous component of the cadherin molecular complex, and also by a diverse array of other actin-binding proteins whose association with cadherins may be context dependent (reviewed in . Second, the cadherin-associated actin cytoskeleton is dynamic, undergoing turnover and assembly both during junction assembly and at steady state (Kovacs et al, 2011;Yamada et al, 2005).…”

Actin Rings of Power

“…Actin within the actin-cadherin ring at the contact edge is thought to be predominantly organized in non-branched bundles oriented parallel to the cell membrane (Buckley et al, 2014). This organization appears to be dependent on the activity of a-catenin, which has been shown to bind not only to actin but also to other actin-associated factors, such as actinin, Ajuba, paxillin, EPLIN, and ZO-1 (Abe and Takeichi, 2008;Itoh et al, 1997;Maiden, and Hardin, 2011;Nola et al, 2011).…”

A Perspective on the Role of Myosins as Mechanosensors

“…Mechanical signals from the internal and external environments are sensed and integrated at these sites, leading to changes in cellular signaling. Many proteins found at these sites have been identified as mechanosensitive, including the b-catenin-aE-cadherin complex in adherens junctions that increases its actin affinity in the presence of force (45). In focal adhesions, stretching of the integrinbinding protein talin causes the exposure of cryptic binding sites for both vinculin and integrins, leading to a strengthening of the adhesions (46,47).…”