Integrin Cytoplasmic Tail Research Articles

The α1 integrin cytoplasmic tail interacts with phosphoinositides and interferes with Akt activation

Integrin α1β1 is an adhesion receptor that binds to collagen and laminin. It regulates cell adhesion, cytoskeletal organization, and migration. The cytoplasmic tail of the α1 subunit consists of 15 amino acids and contains six positively charged lysine residues. In this study, we present evidence that the α1 integrin cytoplasmic tail (α1CT) directly associates with phosphoinositides, preferentially with phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). Since the association was disrupted by calcium, magnesium and phosphate ions, this interaction appears to be in ionic nature. Here, the peptide-lipid interaction was driven by the conserved KIGFFKR motif. The exchange of both two potential phospholipid-binding lysines for glycines in the KIGFFKR motif increased α1β1 integrin-specific adhesion and F-actin cytoskeleton formation compared to cells expressing the unmodified α1 subunit, whereas only mutation of the second lysine at position 1171 increased levels of constitutively active α1β1 integrins on the cell surface. In addition, enhanced focal adhesion formation and increased phosphorylation of focal adhesion kinase, but decreased phosphorylation of AKT was observed in these cells. We conclude that the KIGFFKR motif, and in particular lysine1171 is involved in the dynamic regulation of α1β1 integrin activity and that the interaction of α1CT with phosphoinositides may contribute to this process.

Surface-induced phase separation of reconstituted nascent integrin clusters on lipid membranes

Integrin adhesion complexes are essential membrane-associated cellular compartments for metazoan life. The formation of initial integrin adhesion complexes is a dynamic process involving focal adhesion proteins assembled at the integrin cytoplasmic tails and the inner leaflet of the plasma membrane. The weak multivalent protein interactions within the complex and with the plasma membrane suggest that liquid-liquid phase separation could play a role in the nascent adhesion assembly. Here, we report that solid-supported lipid membranes supplemented with phosphoinositides induce the phase separation of minimal integrin adhesion condensates composed of integrin β1 tails, kindlin, talin, paxillin, and FAK at physiological ionic strengths and protein concentrations. We show that the presence of phosphoinositides is key to enriching kindlin and talin on the lipid membrane, which is necessary to further induce the phase separation of paxillin and FAK at the membrane. Our data demonstrate that lipid membrane surfaces set the local solvent conditions for steering the membrane-localized phase separation even in a regime where no condensate formation of proteins occurs in bulk solution.

Epithelial morphogenesis in the Drosophila egg chamber requires Parvin and ILK.

Integrins are the major family of transmembrane proteins that mediate cell-matrix adhesion and have a critical role in epithelial morphogenesis. Integrin function largely depends on the indirect connection of the integrin cytoplasmic tail to the actin cytoskeleton through an intracellular protein network, the integrin adhesome. What is currently unknown is the role of individual integrin adhesome components in epithelia dynamic reorganization. Drosophila egg chamber consists of the oocyte encircled by a monolayer of somatic follicle epithelial cells that undergo specific cell shape changes. Egg chamber morphogenesis depends on a developmental array of cell-cell and cell-matrix signalling events. Recent elegant work on the role of integrins in the Drosophila egg chamber has indicated their essential role in the early stages of oogenesis when the pre-follicle cells assemble into the follicle epithelium. Here, we have focused on the functional requirement of two key integrin adhesome components, Parvin and Integrin-Linked Kinase (ILK). Both proteins are expressed in the developing ovary from pupae to the adult stage and display enriched expression in terminal filament and stalk cells, while their genetic removal from early germaria results in severe disruption of the subsequent oogenesis, leading to female sterility. Combining genetic mosaic analysis of available null alleles for both Parvin and Ilk with conditional rescue utilizing the UAS/Gal4 system, we found that Parvin and ILK are required in pre-follicle cells for germline cyst encapsulation and stalk cell morphogenesis. Collectively, we have uncovered novel developmental functions for both Parvin and ILK, which closely synergize with integrins in epithelia.

Tension Enhances the Binding Affinity of β1 Integrin by Clamping Talin Tightly: An Insight from Steered Molecular Dynamics Simulations.

Integrin activation is a predominant step for cell-cell and cell-ECM interactions. Talin and Kindlin are mechanosensitive adaptor proteins that bind to the integrin cytoplasmic tail and mediate integrin activation, cytoskeleton rearrangement, and focal adhesion assembly. However, knowledge about how Talin and Kindlin synergistically assist integrin activation remains unclear. Here, we performed so-called "ramp-clamp" SMD simulations, which modeled the mechanosignaling from Kindlin, to investigate the effect of tension on the interaction of the β1 integrin cytoplasmic tail with the Talin-F3 domain. The present results showed that mild but not excessive stretching enhanced the binding of integrin with Talin. This mechanical regulation on integrin affinity to Talin referred to an event cascade, in which under stretching, the integrin cytoplasmic tail adopted allostery in response to the mechanical stimulus, remodeling of integrin in favor of Talin-association ensued, and finally, a stable, close-knit complex was formed. In the cascade, the torsion angle transition of integrin was the cue for the stable interaction of the complex under tensile force. The present work suggested a model for Talin and Kindlin to synergistically activate integrin. It should help understand integrin activation and its mechanochemical regulation mechanism, integrin-related innate cellular immune responses, cell adhesion, cell-cell interaction, and integrin-related drug development.

Regulation of Dynamic Cell Adhesion by Integrin-Integrin Crosstalk

Most cells express several integrins. The integrins are able to respond to various cellular functions and needs by modifying their own activation state, but in addition by their ability to regulate each other by activation or inhibition. This crosstalk or transdominant regulation is strictly controlled. The mechanisms resulting in integrin crosstalk are incompletely understood, but they often involve intracellular signalling routes also used by other cell surface receptors. Several studies show that the integrin cytoplasmic tails bind to a number of cytoskeletal and adaptor molecules in a regulated manner. Recent work has shown that phosphorylations of integrins and key intracellular molecules are of pivotal importance in integrin-cytoplasmic interactions, and these in turn affect integrin activity and crosstalk. The integrin β-chains play a central role in regulating crosstalk. In addition to Integrin-integrin crosstalk, crosstalk may also occur between integrins and related receptors, including other adhesion receptors, growth factor and SARS-CoV-2 receptors.

MiRNA-200c-3p targets talin-1 to regulate integrin-mediated cell adhesion

The ability of integrins on the cell surface to mediate cell adhesion to the extracellular matrix ligands is regulated by intracellular signaling cascades. During this signaling process, the talin (TLN) recruited to integrin cytoplasmic tails plays the critical role of the major adaptor protein to trigger integrin activation. Thus, intracellular levels of TLN are thought to determine integrin-mediated cellular functions. However, the epigenetic regulation of TLN expression and consequent modulation of integrin activation remain to be elucidated. Bioinformatics analysis led us to consider miR-200c-3p as a TLN1-targeting miRNA. To test this, we have generated miR-200c-3p-overexpressing and miR-200c-3p-underexpressing cell lines, including HEK293T, HCT116, and LNCaP cells. Overexpression of miR-200c-3p resulted in a remarkable decrease in the expression of TLN1, which was associated with the suppression of integrin-mediated cell adhesion to fibronectin. In contrast, the reduction in endogenous miR-200c-3p levels led to increased expression of TLN1 and enhanced cell adhesion to fibronectin and focal adhesion plaques formation. Moreover, miR-200c-3p was found to target TLN1 by binding to its 3′-untranslated region (UTR). Taken together, our data indicate that miR-200c-3p contributes to the regulation of integrin activation and cell adhesion via the targeting of TLN1.

Phosphorylation of Kindlins and the Control of Integrin Function.

Integrins serve as conduits for the transmission of information between cells and their extracellular environment. Signaling across integrins is bidirectional, transducing both inside-out and outside-signaling. Integrin activation, a transition from a low affinity/avidity state to a high affinity/avidity state for cognate ligands, is an outcome of inside-signaling. Such activation is particularly important for the recognition of soluble ligands by blood cells but also influences cell-cell and cell-matrix interactions. Integrin activation depends on a complex series of interactions, which both accelerate and inhibit their interconversion from the low to the high affinity/avidity state. There are three components regarded as being most proximately involved in integrin activation: the integrin cytoplasmic tails, talins and kindlins. The participation of each of these molecules in integrin activation is highly regulated by post-translation modifications. The importance of targeted phosphorylation of integrin cytoplasmic tails and talins in integrin activation is well-established, but much less is known about the role of post-translational modification of kindlins. The kindlins, a three-member family of 4.1-ezrin-radixin-moesin (FERM)-domain proteins in mammals, bind directly to the cytoplasmic tails of integrin beta subunits. This commentary provides a synopsis of the emerging evidence for the role of kindlin phosphorylation in integrin regulation.

The F1 loop of the talin head domain acts as a gatekeeper in integrin activation and clustering.

Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the β integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn2+ Here we show that kindlin-1 can replace Mn2+ to mediate β3 integrin clustering induced by the talin head, but not that induced by the F2-F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for β3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of α- and β3-integrins, in order to activate the β3 integrin heterodimer, further detailing the mechanism by which the talin-kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the β3 integrin tail required the newly identified compact FERM fold of the talin head, which positions the F1 loop next to the inner membrane clasp of the talin-bound integrin heterodimer.This article has an associated First Person interview with the first author of the paper.

Word games and molecular recognition – The creative control of endothelial cell function

In this paper we postulate that figures of language and structural features, which are characteristic of poetic writings and word games, have direct correlates in the cell/cell and cell/matrix recognition processes. We shall discuss the particular example of endothelial cells engagement into angiogenesis (the making of new blood vessels in adult vertebrates)—an activity that requires intense extracellular matrix (ECM) remodeling and Cell Adhesion Molecules (CAMs) rearrangement—to argue that angiogenesis controls are organized into semiotic dimensions. CAMs attach cells to the ECM; they are mainly composed of integrins (transmembrane receptors) that bind selectively to different ECM components. After ECM binding, the cytoplasmic tails of integrins within the cell will begin to interact with a wide range of recruited factors that, in turn, regulate integrin clustering in the cell membrane. These recruited factors also activate signaling pathways that link activated integrins to later microfilament system remodeling during cell migration. Ultimately CAMs work as functional protein networks—as the adaptor molecules in an organic code, the Adhesion Code—controlling cell migration/proliferation transition through the continual rearrangement of both ECM adhesion and Actin polymerization. Additionally, orchestrating the transition from clonal growth to differentiation, the interaction between CAMs and ECM components will trigger specific Signal Transduction pathways. We shall examine some attempts to conceptualize “somatic cell function” in the recent specialized literature, which introduces the notion of hierarchic organization into levels i.e. molecular, sub-cellular and cellular and describes an informational flow of increasing complexity versus decreasing number of entities through the levels. Beyond the syntactic level—the specific recognition of discrete ECM motifs by integrin heterodimers—we shall examine the semantic and pragmatic levels at which the composition and architecture of multimolecular complexes will dictate cell recognition events and cell fate decisions. These higher-level codes will be compared to the dynamics of the word games of Lewis Carroll, e.g. “doublets” and “magic squares.” In such creative games words are subjected to synthetic transformations that have to conform to semantic rules, but are ultimately constrained by meaning, as concrete pragmatics. This gives us a model for analyzing angiogenesis control as a word game.

Kindlin‐3 recruitment to the plasma membrane in neutrophils precedes high affinity integrin activation

Integrin‐mediated adhesion of circulating leukocytes to the blood vessel wall starts by arrest from rolling. Activation of integrins by inside‐out signaling involves conformational changes from an inactive bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin‐3 is critically required for leukocyte adhesion. Mutations of kindlin‐3 cause leukocyte adhesion deficiency type III (LAD‐III) syndrome, characterized by severe bleeding (a platelet adhesion defect) and recurring infections (a neutrophil adhesion defect). The clinical significance of kindlin‐3 has been extensively studied; however, the molecular mechanism by which kindlin‐3 regulates the β2 integrin activation is unknown. Kindlin‐3 can bind the β2 integrin cytoplasmic tail at a site distinct from the talin‐1 binding site. It remains unclear whether kindlin‐3 affects individual integrin conformation and/or integrin clustering. Here we monitored the spatiotemporal dynamics of kindlin‐3 and β2 integrin conformation changes during neutrophil rolling, arrest and spreading under flow (6 dyn/cm2) using high‐resolution quantitative dynamic footprinting (qDF) microscopy in combination with an integrin conformation‐specific antibody reporting assay and kindlin‐3 fusion reporters. We find that kindlin‐3 is recruited to the plasma membrane during neutrophil adhesion prior to induction of H+ β2 integrin conformation, indicating that kindlin‐3 is required for H+ integrin conformation. Indeed, deletion of kindlin‐3 in neutrophil‐like HL‐60 cells completely abolishes high affinity β2 integrin. Together, our data suggest that kindlin‐3 is permissive for the high affinity β2 integrin conformation.Support or Funding InformationThis work was supported by funding from the National Institutes of Health, USA (NIH, P01‐HL078784).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LCP1 preferentially binds clasped αMβ2 integrin and attenuates leukocyte adhesion under flow.

Integrins are α/β heterodimers that interconvert between inactive and active states. In the active state the α/β cytoplasmic domains recruit integrin-activating proteins and separate the transmembrane and cytoplasmic (TMcyto) domains (unclasped TMcyto). Conversely, in the inactive state the α/β TMcyto domains bind integrin-inactivating proteins, resulting in the association of the TMcyto domains (clasped TMcyto). Here, we report the isolation of integrin cytoplasmic tail interactors using either lipid bicelle-incorporated integrin TMcyto domains (α5, αM, αIIb, β1, β2 and β3 integrin TMcyto) or a clasped, lipid bicelle-incorporated αMβ2 TMcyto. Among the proteins found to preferentially bind clasped rather than the isolated αM and β2 subunits was L-plastin (LCP1, also known as plastin-2), which binds to and maintains the inactive state of αMβ2 integrin in vivo and thereby regulates leukocyte adhesion to integrin ligands under flow. Our findings offer a global view on cytoplasmic proteins interacting with different integrins and provide evidence for the existence of conformation-specific integrin interactors.

Interaction Analyses of 14-3-3ζ, Dok1, and Phosphorylated Integrin β Cytoplasmic Tails Reveal a Bi-molecular Switch in Integrin Regulation

Integrins are hetero-dimeric (α and β subunits) type I transmembrane proteins that facilitate cell adhesion and migration. The cytoplasmic tails (CTs) of integrins interact with a plethora of intra-cellular proteins that are required for integrin bidirectional signaling. In particular, the β CTs of integrins are known to recruit a variety of cytosolic proteins that often have overlapping recognition sites. However, the chronological sequence of β CTs/cytosolic proteins interactions remains to be fully characterized. Previous studies have shown that the scaffold protein 14-3-3ζ binds to phosphorylated β CTs in activated integrins, whereas interactions of Dok-1 with phosphorylated β CTs maintained integrins in the resting state. In this study, we examined the binding interactions between 14-3-3ζ, Dok1, and phosphorylated integrin β2 and β3 CTs. We show that the scaffold protein 14-3-3ζ interacts with the phosphotyrosine binding (PTB) domain of Dok1 even in the absence of the phosphorylated integrin β CTs. The interactions were mapped onto the β-sheet region of the PTB domain of Dok1. Furthermore, we provide evidence that the 14-3-3ζ/Dok1 binary complex is able to bind to their cognate phosphorylated sequence motifs in the integrin β CTs. We demonstrate that Thr phosphorylated pTTT β2 CT or pTST β3 CT can bind to 14-3-3ζ that is in complex with the Dok1 PTB domain, whereas Ser phosphorylated β2 CT or Tyr phosphorylated β3 CT interacted with Dok1 in 14-3-3ζ/Dok1 complex. Based on these data, we propose that 14-3-3ζ/Dok1 complex could serve as a molecular switch providing novel molecular insights into the regulating integrin activation.

ProLIF - quantitative integrin protein-protein interactions and synergistic membrane effects on proteoliposomes.

Integrin transmembrane receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. Diverse methods have been used to investigate interactions between integrins and intracellular proteins, and predominantly include peptide-based pulldowns and biochemical immuno-isolations from detergent-solubilised cell lysates. However, quantitative methods to probe integrin-protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here, we describe 'protein-liposome interactions by flow cytometry' (denoted ProLIF), a technique to reconstitute recombinant integrin transmembrane domains (TMDs) and cytoplasmic tail (CT) fragments in liposomes as individual subunits or as αβ heterodimers and, via flow cytometry, allow rapid and quantitative measurement of protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. Moreover, the effect of membrane composition, such as PI(4,5)P2 incorporation, on protein recruitment to the integrin CTs can be analysed. ProLIF requires no specific instrumentation and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.This article has associated First Person interviews with the first authors of the paper (see doi: 10.1242/jcs.223644 and doi: 10.1242/jcs.223719).

Peptides derived from the integrin \u03b2 cytoplasmic tails inhibit angiogenesis

BackgroundIntegrins are essential regulators of angiogenesis. However, the antiangiogenic potential of peptides derived from the integrin cytoplasmic tails (CT) remains mostly undetermined.MethodsHere we designed a panel of membrane-penetrating peptides (termed as mβCTPs), each comprising a C-terminal NxxY motif from one of the conserved integrin β CTs, and evaluated their antiangiogenic ability using both in vitro and in vivo approaches.ResultsWe found that mβ3CTP, mβ5CTP and mβ6CTP, derived respectively from the integrin β3, β5 and β6 CTs, but not others, exhibit antiangiogenic ability. Interestingly, we observed that the integrin β3, β5 and β6 CTs but not others are able to interact with β3-endonexin. In addition, the antiangiogenic core in mβ3CTP is identical to a previously identified β3-endonexin binding region in the integrin β3 CT, indicating that the antiangiogenic mβCTPs may function via their binding to β3-endonexin. Consistently, knockdown of endogenous β3-endonexin in HUVECs significantly suppresses tube formation, suggesting that β3-endonexin is proangiogenic. However, neither treatment with the antiangiogenic mβCTPs nor knockdown of endogenous β3-endonexin affects integrin-mediated HUVEC adhesion and migration, indicating that their antiangiogenic effect may not rely on directly regulating integrin activity. Importantly, both treatment with the antiangiogenic mβCTPs and knockdown of endogenous β3-endonexin in HUVECs inhibit VEGF expression and cell proliferation, thereby providing mechanistic explanations for the functional consequences.ConclusionOur results suggest that the antiangiogenic mβCTPs can interact with β3-endonexin in vascular endothelial cells and suppress its function in regulating VEGF expression and cell proliferation, thus disclosing a unique pathway that may be useful for developing novel antiangiogenic strategies.

AMPK negatively regulates tensin-dependent integrin activity.

Tight regulation of integrin activity is paramount for dynamic cellular functions such as cell matrix adhesion and mechanotransduction. Integrin activation is achieved through intracellular interactions at the integrin cytoplasmic tails and through integrin-ligand binding. In this study, we identify the metabolic sensor AMP-activated protein kinase (AMPK) as a β1-integrin inhibitor in fibroblasts. Loss of AMPK promotes β1-integrin activity, the formation of centrally located active β1-integrin- and tensin-rich mature fibrillar adhesions, and cell spreading. Moreover, in the absence of AMPK, cells generate more mechanical stress and increase fibronectin fibrillogenesis. Mechanistically, we show that AMPK negatively regulates the expression of the integrin-binding proteins tensin1 and tensin3. Transient expression of tensins increases β1-integrin activity, whereas tensin silencing reduces integrin activity in fibroblasts lacking AMPK. Accordingly, tensin silencing in AMPK-depleted fibroblasts impedes enhanced cell spreading, traction stress, and fibronectin fiber formation. Collectively, we show that the loss of AMPK up-regulates tensins, which bind β1-integrins, supporting their activity and promoting fibrillar adhesion formation and integrin-dependent processes.

Integrin and cadherin clusters: A robust way to organize adhesions for cell mechanics.

Recent studies at the nanometer scale have revealed that relatively uniform clusters of adhesion proteins (50-100 nm) constitute the modular units of cell adhesion sites in both cell-matrix and cell-cell adhesions. Super resolution microscopy and membrane protein diffusion studies both suggest that even large focal adhesions are formed of 100 nm clusters that are loosely aggregated. Clusters of 20-50 adhesion molecules (integrins or cadherins) can support large forces through avidity binding interactions but can also be disassembled or endocytosed rapidly. Assembly of the clusters of integrins is force-independent and involves gathering integrins at ligand binding sites where they are stabilized by cytoplasmic adhesion proteins that crosslink the integrin cytoplasmic tails plus connect the clusters to the cell cytoskeleton. Cooperative-signaling events can occur in a single cluster without cascading to other clusters. Thus, the clusters appear to be very important elements in many cellular processes and can be considered as a critical functional module.

Conformational equilibrium of talin is regulated by anionic lipids

A critical step in the activation of integrin receptors is the binding of talin to the cytoplasmic domain of the β subunits. This interaction leads to separation of the integrin α and β transmembrane domains and significant conformational changes in the extracellular domains, resulting in a dramatic increase in integrin's affinity for ligands. It has long been shown that the membrane bilayer also plays a critical role in the talin–integrin interaction. Anionic lipids are required for proper interaction, yet the specificity for specific anionic headgroups is not clear. In this report, we document talin–membrane interactions with bilayers of controlled composition using Nanodiscs and a FRET based binding and structural assay. We confirm that recruitment of the talin head domain to the membrane surface is governed by charge in the absence of other adapter proteins. In addition, measurement of the donor–acceptor distance is consistent with the hypothesis that anionic lipids promote a conformational change in the talin head domain allowing interaction of the F3 domain with the phospholipid bilayer. The magnitude of the F3 domain movement is altered by the identity of the phospholipid headgroup with phosphatidylinositides promoting the largest change. Our results suggest that phoshpatidylinositol-4,5-bisphosphate plays key a role in converting talin head domain to a conformation optimized for interactions with the bilayer and subsequently integrin cytoplasmic tails.

The Membrane Lipid Composition Regulates The Activity of Talin

Integrins plays a central role in the dynamics of cell‐cell and cell to extra‐cellular matrix interactions and signaling. A key step in the activation of integrin receptors is the binding of the cytoplasmic domain of integrin subunits by talin. This interaction leads to separation of the integrin transmembrane domains and significant conformational changes in the extracellular domains, resulting in a dramatic increase in integrin's affinity for ligands. Talin is a 470 kDa adapter protein that consists of a N‐terminal head domain (THD) and a C‐terminal rod domain. THD is homologous to to the domain found in band 4.1/ezrin/radixin/moesin family of proteins (FERM domain) with uncanonical linear arrangement of F0–F3 domains. A second mechanism in the regulation of integrin activation is directly related to the fact that full‐length talin adopts an auto‐inhibited conformation. In the cytosol, the rod domain binds THD, preventing talin interactions with the membrane surface and the integrin cytoplasmic domain. The membrane bilayer also plays a critical role in the talin ‐integrin interaction, where anionic lipids are required for proper interaction, yet a detailed picture of the interplay between talin and the membrane has remained elusive. We describe a new fluorescence based assay for probing talin – membrane interactions with Nanodisc bilayers of controlled composition. We show that recruitment of THD to the membrane surface is governed by the absolute charge. Measurement of the donor‐acceptor distances reveals that anionic lipids promote a conformational change in the THD favoring a direct interaction of the F3 domain with the phospholipid bilayer. The magnitude of this conformational change is regulated by the identity of the phospholipid headgroup with phosphatidylinositides (e.g. PI4P and PIP2) promoting the largest conformational motions. In addition, the binding of THD to bilayers containing PIP2 is insensitive to the presence of the inhibitory segments of the talin rod domain, while the association of THD to other tested anionic lipid bilayers are strongly inhibited by purified talin rod domains. Furthermore, we have mapped the activation to the interaction of PIP2 with the F2–F3 domain of the THD. Thus, we show that PIP2 plays a central role in the converting talin to a conformation optimized for interactions with integrin cytoplasmic tails and regulation of the auto‐inhibited form of talin, which are essential for integrin inside‐out activation.Support or Funding InformationSupported by NIH grant GM101048.

Changes in single-molecule integrin dynamics linked to local cellular behavior.

Recent advances in light microscopy permit visualization of the behavior of individual molecules within dense macromolecular ensembles in live cells. It is now conceptually possible to relate the dynamic organization of molecular machinery to cellular function. However, inherent heterogeneities, as well as disparities between spatial and temporal scales, pose substantial challenges in deriving such a relationship. New approaches are required to link discrete single-molecule behavior with continuous cellular-level processes. Here we combined intercalated molecular and cellular imaging with a computational framework to detect reproducible transient changes in the behavior of individual molecules that are linked to cellular behaviors. Applying our approach to integrin transmembrane receptors revealed a spatial density gradient underlying characteristic molecular density increases and mobility decreases, indicating the subsequent onset of local protrusive activity. Integrin mutants further revealed that these density and mobility transients are separable and depend on different binding domains within the integrin cytoplasmic tail. Our approach provides a generalizable paradigm for dissecting dynamic spatiotemporal molecular behaviors linked to local cellular events.

Kindlin-3 interacts with the ribosome and regulates c-Myc expression required for proliferation of chronic myeloid leukemia cells

Kindlins are FERM-containing cytoplasmic proteins that regulate integrin-mediated cell-cell and cell-extracellular matrix (ECM) attachments. Kindlin-3 is expressed in hematopoietic cells, platelets, and endothelial cells. Studies have shown that kindlin-3 stabilizes cell adhesion mediated by ß1, ß2, and ß3 integrins. Apart from integrin cytoplasmic tails, kindlins are known to interact with other cytoplasmic proteins. Here we demonstrate that kindlin-3 can associate with ribosome via the receptor for activated-C kinase 1 (RACK1) scaffold protein based on immunoprecipitation, ribosome binding, and proximity ligation assays. We show that kindlin-3 regulates c-Myc protein expression in the human chronic myeloid leukemia cell line K562. Cell proliferation was reduced following siRNA reduction of kindlin-3 expression and a significant reduction in tumor mass was observed in xenograft experiments. Mechanistically, kindlin-3 is involved in integrin α5ß1-Akt-mTOR-p70S6K signaling; however, its regulation of c-Myc protein expression could be independent of this signaling axis.