Proximity-Dependent Biotinylation (BioID) of Integrin Interaction Partners.

Integrins are heterodimeric adhesion receptors that maintain cell-extracellular matrix (ECM) interactions in diverse tissue microenvironments. They mediate cell adhesion and signaling through the assembly of large cytoplasmic multiprotein complexes that focally connect with the cytoskeleton. Integrin adhesion complexes (IAC) are specialized by the type of integrin-ECM contact and are sensitive to mechanical forces. Thus, they encrypt context-dependent information about the microenvironment in their composition. Signals mediated through IACs modulate many aspects of cell behavior, which allows cells to adapt to their surroundings. To gain insights into their function, IACs have been isolated from cultured cells and explored by proteomics. IACs are insoluble by nature and held together by transient/weak interactions, which makes it challenging to isolate intact IACs. Usually all IACs coupled to a specified ECM, which may employ different integrins, are isolated. Here we describe an alternative method based on proximity-dependent biotin identification (BioID), where specific integrin interaction partners are labeled in live cells and isolated without the need to isolate intact IACs.

The membrane-distal regions of integrin α cytoplasmic domains contribute differently to integrin inside-out activation.

Functioning as signal receivers and transmitters, the integrin α/ß cytoplasmic tails (CT) are pivotal in integrin activation and signaling. 18 α integrin subunits share a conserved membrane-proximal region but have a highly diverse membrane-distal (MD) region at their CTs. Recent studies demonstrated that the presence of α CTMD region is essential for talin-induced integrin inside-out activation. However, it remains unknown whether the non-conserved α CTMD regions differently regulate the inside-out activation of integrin. Using αIIbß3, αLß2, and α5ß1 as model integrins and by replacing their α CTMD regions with those of α subunits that pair with ß3, ß2, and ß1 subunits, we analyzed the function of CTMD regions of 17 α subunits in talin-mediated integrin activation. We found that the α CTMD regions play two roles on integrin, which are activation-supportive and activation-regulatory. The regulatory but not the supportive function depends on the sequence identity of α CTMD region. A membrane-proximal tyrosine residue present in the CTMD regions of a subset of α integrins was identified to negatively regulate integrin inside-out activation. Our study provides a useful resource for investigating the function of α integrin CTMD regions.

Integrin alpha chains exhibit distinct temporal and spatial localization patterns in epithelial cells of the Drosophila ovary.

Integrins are heterodimeric transmembrane receptors that modulate cell adhesion, migration, and signaling. Multiple integrin chains contribute to development and morphogenesis of a given tissue. Here, we analyze the expression of Drosophila integrin alpha chains in the ovarian follicular epithelium, a model for tissue morphogenesis and cell migration. We find expression throughout development of the beta chain, betaPS. Alpha chains, however, exhibit both spatial and temporal expression differences. alphaPS1 and alphaPS2 integrins are detected during early and mid-oogenesis on apical, lateral, and basal membranes with the betaPS chain, whereas alphaPS3-family integrins (alphaPS3, alphaPS4, alphaPS5) are expressed in anterior cells late in oogenesis. Surprisingly, we find that alphaPS3-family integrins are dispensable for dorsal appendage morphogenesis but play a role in the final length of the egg, suggesting redundant functions of integrins in a simple tissue. We also demonstrate roles for alphaPS3betaPS integrin in border cell migration and in stretch cells.

Zebrafish integrin-linked kinase is required in skeletal muscles for strengthening the integrin-ECM adhesion complex.

Mechanical instability of skeletal muscle cells is the major cause of congenital muscular dystrophy. Here we show that the zebrafish lost-contact mutant, that lacks a functional integrin-linked kinase (ilk) gene, suffers from mechanical instability of skeletal muscle fibres. With genetic and morpholino knock-down experiments we demonstrate that: 1) laminin, itgalpha7, Ilk and beta-parvin are all critical for mechanical stability in skeletal muscles. 2) Ilk acts redundantly with the dystrophin/dystroglycan adhesion complex in maintaining mechanical stability of skeletal muscles. 3) Ilk protein is recruited to the myotendinous junctions, which requires the ECM component laminin and the presence of itgalpha7 in the sarcolemma. 4) Ilk, unexpectedly, is dispensable for formation of the adhesion complex. Ilk, however, is required for strengthening the adhesion of the muscle fibre with the ECM and this activity requires the presence of a functional kinase domain in Ilk. 5) We identified a novel interaction between Ilk and the mechanical stretch sensor protein MLP. Thus, Ilk is an essential intracellular component downstream of laminin and itgalpha7, providing strengthening of skeletal muscle fibre adhesion with the ECM and therefore qualified as a novel candidate gene for congenital muscular dystrophy.

Developmental and regional differences in the consolidation of long-term potentiation.

The alpha5beta1 integrin is present in high concentrations in the apical dendrites of pyramidal neurons in adult rats but is virtually absent in the basal dendrites. Moreover, alpha5beta1 does not appear in apical dendritic branches until the third post-natal week. Given that integrins contribute to the consolidation of synaptic plasticity, these results raise the possibility of developmental and regional differences in the stability of long-term potentiation (LTP). The present study tested this point using a LTP reversal paradigm in field CA1 of hippocampal slices. In accord with earlier reports, low-frequency afferent stimulation (5 Hz) introduced 30 s after theta burst stimulation (TBS) completely reversed LTP but was ineffective 30 min and 60 min later in slices from adult rats. The same low-frequency trains caused a partial reversal of LTP when applied 30 and 60 min post-TBS in slices from 21-day-old rats and a complete reversal at all time points in slices from 10-day-old rats. LTP in the basal dendrites of adult rats did not fully consolidate; i.e. potentiation was partially reversed by low-frequency stimulation even after delays of 30 or 60 min. Moreover, spaced (10 min) applications of 5- Hz pulses beginning at 30 min post-TBS completely erased LTP. The reversal effect in both apical and basal dendrites was blocked by N-methyl-D-aspartic acid receptor antagonists but an integrin antagonist had differential effects across the two dendritic domains. These results constitute evidence that the stability of LTP increases with age in the apical dendrites but remains incomplete even in adulthood in the basal dendrites. The possibilities that the developmental and regional variations in LTP consolidation are correlated with integrin expression and linked to different types of memory processing are discussed.