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      Phase transitions of multivalent proteins can promote clustering of membrane receptors

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          Clustering of proteins into micrometer-sized structures at membranes is observed in many signaling pathways. Most models of clustering are specific to particular systems, and relationships between physical properties of the clusters and their molecular components are not well understood. We report biochemical reconstitution on supported lipid bilayers of protein clusters containing the adhesion receptor Nephrin and its cytoplasmic partners, Nck and N-WASP. With Nephrin attached to the bilayer, multivalent interactions enable these proteins to polymerize on the membrane surface and undergo two-dimensional phase separation, producing micrometer-sized clusters. Dynamics and thermodynamics of the clusters are modulated by the valencies and affinities of the interacting species. In the presence of the Arp2/3 complex, the clusters assemble actin filaments, suggesting that clustering of regulatory factors could promote local actin assembly at membranes. Interactions between multivalent proteins could be a general mechanism for cytoplasmic adaptor proteins to organize membrane receptors into micrometer-scale signaling zones.

          DOI: http://dx.doi.org/10.7554/eLife.04123.001

          eLife digest

          The membrane that surrounds a cell is made up of a mixture of lipid molecules and proteins. Membrane proteins perform a wide range of roles, including transmitting signals into, and out of, cells and helping neighboring cells to stick together. To perform these tasks, these proteins commonly need to bind to other molecules—collectively known as ligands—that are found either inside or outside the cell.

          Membrane proteins are able to move around within the membrane, and in many systems, ligand binding causes the membrane proteins to cluster together. Although this clustering has been seen in many different systems, no general principles that describe how clustering occurs had been found.

          Now, Banjade and Rosen have constructed an artificial cell membrane to investigate the clustering of a membrane protein called Nephrin, which is essential for kidneys to function correctly. When it is activated, Nephrin interacts with protein ligands called Nck and N-WASP that are found inside cells and helps filaments of a protein called actin to form. These filaments perform a number of roles including enabling cells to adhere to each other and to move.

          In Banjade and Rosen's artificial system, when a critical concentration of ligands was exceeded, clusters of Nephrin, Nck and N-WASP suddenly formed. This suggests that the clusters form through a physical process known as ‘phase separation’. Banjade and Rosen found that this critical concentration depends on how strongly the proteins interact and the number of sites they possess to bind each other.

          Within the clusters, the three proteins formed large polymer chains. The clusters were mobile and, over time, small clusters coalesced into larger clusters. Even though the clusters persisted for hours, individual proteins did not stay in a given cluster for long and instead continuously exchanged back-and-forth between the cluster and its surroundings.

          When actin and another protein complex that interacts with N-WASP were added to the artificial membrane system, actin filaments began to form at the protein clusters. Banjade and Rosen suggest that such clusters act as ‘signaling zones’ that coordinate the construction of the actin filaments. Regions that are also found in many other signaling proteins mediate the interactions between Nephrin, Nck and N-WASP. Banjade and Rosen therefore suggest that phase separation and protein polymer formation could explain how many different types of membrane proteins form clusters.

          DOI: http://dx.doi.org/10.7554/eLife.04123.002

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          Most cited references 60

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          For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the analysis of scientific images. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.
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              Molecular mechanisms of invadopodium formation

              Invadopodia are actin-rich membrane protrusions with a matrix degradation activity formed by invasive cancer cells. We have studied the molecular mechanisms of invadopodium formation in metastatic carcinoma cells. Epidermal growth factor (EGF) receptor kinase inhibitors blocked invadopodium formation in the presence of serum, and EGF stimulation of serum-starved cells induced invadopodium formation. RNA interference and dominant-negative mutant expression analyses revealed that neural WASP (N-WASP), Arp2/3 complex, and their upstream regulators, Nck1, Cdc42, and WIP, are necessary for invadopodium formation. Time-lapse analysis revealed that invadopodia are formed de novo at the cell periphery and their lifetime varies from minutes to several hours. Invadopodia with short lifetimes are motile, whereas long-lived invadopodia tend to be stationary. Interestingly, suppression of cofilin expression by RNA interference inhibited the formation of long-lived invadopodia, resulting in formation of only short-lived invadopodia with less matrix degradation activity. These results indicate that EGF receptor signaling regulates invadopodium formation through the N-WASP–Arp2/3 pathway and cofilin is necessary for the stabilization and maturation of invadopodia.

                Author and article information

                Role: Reviewing editor
                eLife Sciences Publications, Ltd
                16 October 2014
                : 3
                [1 ]Department of Biophysics, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center , Dallas, United States
                Max Planck Institute of Molecular Cell Biology and Genetics , Germany
                Max Planck Institute of Molecular Cell Biology and Genetics , Germany
                Author notes
                Copyright © 2014, Banjade and Rosen

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                Funded by: Howard Hughes Medical Institute FundRef identification ID: http://dx.doi.org/10.13039/100000011
                Award ID: HHMI Collaborative Innovations Award (Work performed at MBL/HHMI Summer Institute)
                Award Recipient :
                Funded by: National Institute of General Medical Sciences FundRef identification ID: http://dx.doi.org/10.13039/100000057
                Award ID: R01-GM56322
                Award Recipient :
                Funded by: Welch Foundation FundRef identification ID: http://dx.doi.org/10.13039/100000928
                Award ID: I-1544
                Award Recipient :
                Funded by: Chilton Foundation
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Research Article
                Biophysics and Structural Biology
                Cell Biology
                Custom metadata
                A new and general mechanism describes the organization of membrane proteins and their cytoplasmic ligands into micrometer-scale clusters, based on polymerization and concomitant phase separation of multivalent proteins.


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