A RIAM/lamellipodin-talin-integrin complex forms the tip of sticky fingers that guide cell migration.

The leading edge of migrating cells contains rapidly translocating activated integrins associated with growing actin filaments that form 'sticky fingers' to sense extracellular matrix and guide cell migration. Here we utilized indirect bimolecular fluorescence complementation to visualize a molecular complex containing a Mig-10/RIAM/lamellipodin (MRL) protein (Rap1-GTP-interacting adaptor molecule (RIAM) or lamellipodin), talin and activated integrins in living cells. This complex localizes at the tips of growing actin filaments in lamellipodial and filopodial protrusions, thus corresponding to the tips of the 'sticky fingers.' Formation of the complex requires talin to form a bridge between the MRL protein and the integrins. Moreover, disruption of the MRL protein-integrin-talin (MIT) complex markedly impairs cell protrusion. These data reveal the molecular basis of the formation of 'sticky fingers' at the leading edge of migrating cells and show that an MIT complex drives these protrusions

Myosin IIA/IIB restrict adhesive and protrusive signaling to generate front–back polarity in migrating cells

Myosin IIA and IIB synergistically generate front–back polarity through their effects on actomyosin bundling formation and stability, and adhesion maturation, which are mediated by localized Rac GEF depletion

Integrin-associated complexes form hierarchically with variable stoichiometry in nascent adhesions.

BackgroundA complex network of putative molecular interactions underlies the architecture and function of cell-matrix adhesions. Most of these interactions are implicated from coimmunoprecipitation studies using expressed components, but few have been demonstrated or characterized functionally in living cells.ResultsWe introduce fluorescence fluctuation methods to determine, at high spatial and temporal resolution, "when" and "where" molecular complexes form and their stoichiometry in nascent adhesions (NAs). We focus on integrin-associated molecules implicated in integrin activation and in the integrin-actin linkage in NAs and show that these molecules form integrin-containing complexes hierarchically within the adhesion itself. Integrin and kindlin reside in a molecular complex as soon as adhesions are visible; talin, although also present early, associates with the integrin-kindlin complex only after NAs have formed and in response to myosin II activity. Furthermore, talin and vinculin association precedes the formation of the integrin-talin complex. Finally, α-actinin enters NAs periodically and in clusters that transiently associate with integrins. The absolute number and stoichiometry of these molecules varies among the molecules studied and changes as adhesions mature.ConclusionsThese observations suggest a working model for NA assembly whereby transient α-actinin-integrin complexes help nucleate NAs within the lamellipodium. Subsequently, integrin complexes containing kindlin, but not talin, emerge. Once NAs have formed, myosin II activity promotes talin association with the integrin-kindlin complex in a stoichiometry consistent with each talin molecule linking two integrin-kindlin complexes

Integrin-associated complexes form hierarchically with variable stoichiometry in nascent adhesions.

BackgroundA complex network of putative molecular interactions underlies the architecture and function of cell-matrix adhesions. Most of these interactions are implicated from coimmunoprecipitation studies using expressed components, but few have been demonstrated or characterized functionally in living cells.ResultsWe introduce fluorescence fluctuation methods to determine, at high spatial and temporal resolution, "when" and "where" molecular complexes form and their stoichiometry in nascent adhesions (NAs). We focus on integrin-associated molecules implicated in integrin activation and in the integrin-actin linkage in NAs and show that these molecules form integrin-containing complexes hierarchically within the adhesion itself. Integrin and kindlin reside in a molecular complex as soon as adhesions are visible; talin, although also present early, associates with the integrin-kindlin complex only after NAs have formed and in response to myosin II activity. Furthermore, talin and vinculin association precedes the formation of the integrin-talin complex. Finally, α-actinin enters NAs periodically and in clusters that transiently associate with integrins. The absolute number and stoichiometry of these molecules varies among the molecules studied and changes as adhesions mature.ConclusionsThese observations suggest a working model for NA assembly whereby transient α-actinin-integrin complexes help nucleate NAs within the lamellipodium. Subsequently, integrin complexes containing kindlin, but not talin, emerge. Once NAs have formed, myosin II activity promotes talin association with the integrin-kindlin complex in a stoichiometry consistent with each talin molecule linking two integrin-kindlin complexes

CD81 regulates cell migration through its association with Rac GTPase

CD81 is a member of the tetraspanin family that has been described to have a key role in cell migration of tumor and immune cells. To unravel the mechanisms of CD81-regulated cell migration, we performed proteomic analyses that revealed an interaction of the tetraspanin C-terminal domain with the small GTPase Rac. Direct interaction was confirmed biochemically. Moreover, microscopy cross-correlation analysis demonstrated the in situ integration of both molecules into the same molecular complex. Pull-down experiments revealed that CD81-Rac interaction was direct and independent of Rac activation status. Knockdown of CD81 resulted in enhanced protrusion rate, altered focal adhesion formation, and decreased cell migration, correlating with increased active Rac. Reexpression of wild-type CD81, but not its truncated form lacking the C-terminal cytoplasmic domain, rescued these effects. The phenotype of CD81 knockdown cells was mimicked by treatment with a soluble peptide with the C-terminal sequence of the tetraspanin. Our data show that the interaction of Rac with the C-terminal cytoplasmic domain of CD81 is a novel regulatory mechanism of the GTPase activity turnover. Furthermore, they provide a novel mechanism for tetraspanin-dependent regulation of cell motility and open new avenues for tetraspanin-targeted reagents by the use of cell-permeable peptides. © 2013 Jo et al.PI080794 and PI11/01645 from the Instituto de Salud Carlos III; SAF2011-25834 and ERC AdG-2011; BIO2009-07990 from the Ministerio de Educación y Ciencia; CAM BIO/0194/2006 from Comunidad de Madrid, and RECAVA RD06/0014 from the Fondo de Investigaciones Sanitarias (Ministerio de Sanidad y Consumo, Instituto Salud Carlos III); National Institutes of Health grant GM23244 and the Cell Migration Consortium (U54 GM064346)Peer Reviewe

Detection and Correction of Blinking Bias in Image Correlation Transport Measurements of Quantum Dot Tagged Macromolecules

Semiconductor nanocrystals or quantum dots (QDs) are becoming widely used as fluorescent labels for biological applications. Here we demonstrate that fluorescence fluctuation analysis of their diffusional mobility using temporal image correlation spectroscopy is highly susceptible to systematic errors caused by fluorescence blinking of the nanoparticles. Temporal correlation analysis of fluorescence microscopy image time series of streptavidin-functionalized (CdSe)ZnS QDs freely diffusing in two dimensions shows that the correlation functions are fit well to a commonly used diffusion decay model, but the transport coefficients can have significant systematic errors in the measurements due to blinking. Image correlation measurements of the diffusing QD samples measured at different laser excitation powers and analysis of computer simulated image time series verified that the effect we observe is caused by fluorescence intermittency. We show that reciprocal space image correlation analysis can be used for mobility measurements in the presence of blinking emission because it separates the contributions of fluctuations due to photophysics from those due to transport. We also demonstrate application of the image correlation methods for measurement of the diffusion coefficient of glycosyl phosphatidylinositol-anchored proteins tagged with QDs as imaged on living fibroblasts

A RIAM/lamellipodin-talin-integrin complex forms the tip of sticky fingers that guide cell migration.

The leading edge of migrating cells contains rapidly translocating activated integrins associated with growing actin filaments that form 'sticky fingers' to sense extracellular matrix and guide cell migration. Here we utilized indirect bimolecular fluorescence complementation to visualize a molecular complex containing a Mig-10/RIAM/lamellipodin (MRL) protein (Rap1-GTP-interacting adaptor molecule (RIAM) or lamellipodin), talin and activated integrins in living cells. This complex localizes at the tips of growing actin filaments in lamellipodial and filopodial protrusions, thus corresponding to the tips of the 'sticky fingers.' Formation of the complex requires talin to form a bridge between the MRL protein and the integrins. Moreover, disruption of the MRL protein-integrin-talin (MIT) complex markedly impairs cell protrusion. These data reveal the molecular basis of the formation of 'sticky fingers' at the leading edge of migrating cells and show that an MIT complex drives these protrusions

Membrane Lateral Diffusion and Capture of CFTR within Transient Confinement Zones

The cystic fibrosis transmembrane conductance regulator (CFTR) channel interacts with scaffolding and other proteins that are expected to restrict its lateral movement, yet previous studies have reported predominantly free diffusion. We examined the lateral mobility of CFTR channels on live baby hamster kidney cells using three complementary methods. Channels bearing an extracellular biotinylation target sequence were labeled with streptavidin conjugated with fluorescent dyes (Alexa Fluor 488 or 568) or quantum dots (qDot605). Fluorescence recovery after photobleaching and image correlation spectroscopy of the dye-labeled channels revealed a significant immobile population (∼50%), which was confirmed by direct single particle tracking (SPT) of qDot605-labeled CFTR. Adding 10 histidine residues at the C-terminus of CFTR to mask the postsynaptic density 95, Discs large, ZO-1 (PDZ) binding motif abolished its association with EBP50/NHERF1, reduced the immobile fraction, and increased mobility. Other interactions that are not normally detected on this timescale became apparent when binding of PDZ domain proteins was disrupted. SPT revealed that CFTR(His-10) channels diffuse randomly, become immobilized for periods lasting up to 1 min, and in some instances are recaptured at the same location. The impact of transient confinement on the measured diffusion using the three fluorescence techniques were assessed using computer simulations of the biological experiments. Finally, the impact of endosomal CFTR on mobility measurements was assessed by fluorescence correlation spectroscopy. These results reveal unexpected features of CFTR dynamics which may influence its ion channel activity