A functionally neutral single chain antibody to measure beta‐1 integrin uptake and recycling

Integrin‐mediated cell adhesion and signaling are critical for many physiological processes. The dynamic turnover of integrins and their associated adhesion complexes through endocytic and recycling pathways has emerged as an important mechanism for controlling cell migration and invasion in cancer. Thus, the regulation of integrin trafficking and how this may be altered by disease‐specific molecular mechanisms has generated considerable interest. However, current tools available to study integrin trafficking may cause artifacts and/or do not provide adequate kinetic information. Here, we report the generation of a functionally neutral and monovalent single chain antibody to quantitatively and qualitatively measure β1 integrin trafficking in cells. Our novel probe can be used in a variety of assays and allows for the biochemical characterization of rapid recycling of endogenous integrins. We also demonstrate its potential utility in live cell imaging, providing proof of principle to guide future integrin probe design.     

Mechanisms of integrin activation and trafficking

Integrin adhesion receptors are essential for the normal function of most multicellular organisms, and defective integrin activation or integrin signaling is associated with an array of pathological conditions. Integrins are regulated by conformational changes, clustering, and trafficking, and regulatory mechanisms differ strongly between individual integrins and between cell types. Whereas integrins in circulating blood cells are activated by an inside-out-induced conformational change that favors high-affinity ligand binding, β1-integrins in adherent cells can be activated by force or clustering. In addition, endocytosis and recycling play an important role in the regulation of integrin turnover and integrin redistribution in adherent cells, especially during dynamic processes such as cell migration and invasion. Integrin trafficking is strongly regulated by their cytoplasmic tails, and the mechanisms are now being identified.     

Competitive binding of Rab21 and p120RasGAP to integrins regulates receptor traffic and migration

Integrin trafficking from and to the plasma membrane controls many aspects of cell behavior including cell motility, invasion, and cytokinesis. Recruitment of integrin cargo to the endocytic machinery is regulated by the small GTPase Rab21, but the detailed molecular mechanisms underlying integrin cargo recruitment are yet unknown. Here we identify an important role for p120RasGAP (RASA1) in the recycling of endocytosed α/β1-integrin heterodimers to the plasma membrane. Silencing of p120RasGAP attenuated integrin recycling and augmented cell motility. Mechanistically, p120RasGAP interacted with the cytoplasmic domain of integrin α-subunits via its GAP domain and competed with Rab21 for binding to endocytosed integrins. This in turn facilitated exit of the integrin from Rab21- and EEA1-positive endosomes to drive recycling. Our results assign an unexpected role for p120RasGAP in the regulation of integrin traffic in cancer cells and reveal a new concept of competitive binding of Rab GTPases and GAP proteins to receptors as a regulatory mechanism in trafficking.     

The β3‐integrin endothelial adhesome regulates microtubule‐dependent cell migration

Integrin β3 is seen as a key anti‐angiogenic target for cancer treatment due to its expression on neovasculature, but the role it plays in the process is complex; whether it is pro‐ or anti‐angiogenic depends on the context in which it is expressed. To understand precisely β3's role in regulating integrin adhesion complexes in endothelial cells, we characterised, by mass spectrometry, the β3‐dependent adhesome. We show that depletion of β3‐integrin in this cell type leads to changes in microtubule behaviour that control cell migration. β3‐integrin regulates microtubule stability in endothelial cells through Rcc2/Anxa2‐driven control of active Rac1 localisation. Our findings reveal that angiogenic processes, both in vitro and in vivo, are more sensitive to microtubule targeting agents when β3‐integrin levels are reduced.     

SHARPIN is an endogenous inhibitor of β1-integrin activation

Regulated activation of integrins is critical for cell adhesion, motility and tissue homeostasis. Talin and kindlins activate β1-integrins, but the counteracting inhibiting mechanisms are poorly defined. We identified SHARPIN as an important inactivator of β1-integrins in an RNAi screen. SHARPIN inhibited β1-integrin functions in human cancer cells and primary leukocytes. Fibroblasts, leukocytes and keratinocytes from SHARPIN-deficient mice exhibited increased β1-integrin activity, which was fully rescued by re-expression of SHARPIN. We found that SHARPIN directly binds to a conserved cytoplasmic region of integrin α-subunits and inhibits recruitment of talin and kindlin to the integrin. Therefore, SHARPIN inhibits the critical switching of β1-integrins from inactive to active conformations.     

Numb controls integrin endocytosis for directional cell migration with aPKC and PAR-3

Migrating cells extend protrusions to establish new adhesion sites at their leading edges. One of the driving forces for cell migration is the directional trafficking of cell-adhesion molecules such as integrins. Here, we show that the endocytic adaptor protein Numb is an important component of the machinery for directional integrin trafficking in migrating cells. Numb binds to integrin-betas and localizes to clathrin-coated structures (CCSs) at the substratum-facing surface of the leading edge. Numb inhibition by RNAi impairs both integrin endocytosis and cell migration toward integrin substrates. Numb is regulated by phosphorylation since the protein is released from CCSs and no longer binds integrins when phosphorylated by atypical protein kinase C (aPKC). Because Numb interacts with the aPKC binding partner PAR-3, we propose a model in which polarized Numb phosphorylation contributes to cell migration by directing integrin endocytosis to the leading edge.     

SNX27 mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling receptors

Endocytic sorting of signalling receptors between recycling and degradative pathways is a key cellular process controlling the surface complement of receptors and, accordingly, the cell's ability to respond to specific extracellular stimuli. The β2 adrenergic receptor (β2AR) is a prototypical seven-transmembrane signalling receptor that recycles rapidly and efficiently to the plasma membrane after ligand-induced endocytosis. β2AR recycling is dependent on the receptor's carboxy-terminal PDZ ligand and Rab4. This active sorting process is required for functional resensitization of β2AR-mediated signalling. Here we show that sequence-directed sorting occurs at the level of entry into retromer tubules and that retromer tubules are associated with Rab4. Furthermore, we show that sorting nexin 27 (SNX27) serves as an essential adaptor protein linking β2ARs to the retromer tubule. SNX27 does not seem to directly interact with the retromer core complex, but does interact with the retromer-associated Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex. The present results identify a role for retromer in endocytic trafficking of signalling receptors, in regulating a receptor-linked signalling pathway, and in mediating direct endosome-to-plasma membrane traffic.     

Quantitative proteomics identifies a Dab2/integrin module regulating cell migration

Clathrin-associated endocytic adapters recruit cargoes to coated pits as a first step in endocytosis. We developed an unbiased quantitative proteomics approach to identify and quantify glycoprotein cargoes for an endocytic adapter, Dab2. Surface levels of integrins β1, α1, α2, and α3 but not α5 or αv chains were specifically increased on Dab2-deficient HeLa cells. Dab2 colocalizes with integrin β1 in coated pits that are dispersed over the cell surface, suggesting that it regulates bulk endocytosis of inactive integrins. Depletion of Dab2 inhibits cell migration and polarized movement of integrin β1 and vinculin to the leading edge. By manipulating intracellular and surface integrin β1 levels, we show that migration speed correlates with the intracellular integrin pool but not the surface level. Together, these results suggest that Dab2 internalizes integrins freely diffusing on the cell surface and that Dab2 regulates migration, perhaps by maintaining an internal pool of integrins that can be recycled to create new adhesions at the leading edge.     

Binding of autotaxin to integrins localizes lysophosphatidic acid production to platelets and mammalian cells

Autotaxin (ATX) is a secreted lysophospholipase D that generates the bioactive lipid mediator lysophosphatidic acid (LPA). We and others have reported that ATX binds to integrins, but the function of ATX-integrin interactions is unknown. The recently reported crystal structure of ATX suggests a role for the solvent-exposed surface of the N-terminal tandem somatomedin B-like domains in binding to platelet integrin αIIbβ(3). The opposite face of the somatomedin B-like domain interacts with the catalytic phosphodiesterase (PDE) domain to form a hydrophobic channel through which lysophospholipid substrates enter and leave the active site. Based on this structure, we hypothesize that integrin-bound ATX can access cell surface substrates and deliver LPA to cell surface receptors. To test this hypothesis, we investigated the integrin selectivity and signaling pathways that promote ATX binding to platelets. We report that both platelet β1 and β3 integrins interact in an activation-dependent manner with ATX via the SMB2 domain. ATX increases thrombin-stimulated LPA production by washed platelets ~10-fold. When incubated under conditions to promote integrin activation, ATX generates LPA from CHO cells primed with bee venom phospholipase A(2), and ATX-mediated LPA production is enhanced more than 2-fold by CHO cell overexpression of integrin β(3). The effects of ATX on platelet and cell-associated LPA production, but not hydrolysis of small molecule or detergent-solubilized substrates, are attenuated by point mutations in the SMB2 that impair integrin binding. Integrin binding therefore localizes ATX activity to the cell surface, providing a mechanism to generate LPA in the vicinity of its receptors.     

BFA‐induced compartments from the Golgi apparatus and trans‐Golgi network/early endosome are distinct in plant cells

Brefeldin A (BFA) is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. At low concentrations (5–10 μg ml^?1), BFA caused both the Golgi apparatus and trans‐Golgi network (TGN), an early endosome (EE) equivalent in plant cells, to form visible aggregates in transgenic tobacco BY‐2 cells. Here we show that these BFA‐induced aggregates from the Golgi apparatus and TGN are morphologically and functionally distinct in plant cells. Confocal immunofluorescent and immunogold electron microscope (EM) studies demonstrated that BFA‐induced Golgi‐ and TGN‐derived aggregates are physically distinct from each other. In addition, the internalized endosomal marker FM4‐64 co‐localized with the TGN‐derived aggregates but not with the Golgi aggregates. In the presence of the endocytosis inhibitor tyrphostin A23, which acts in a dose‐ and time‐dependent manner, SCAMP1 (secretory carrier membrane protein 1) and FM4‐64 are mostly excluded from the SYP61‐positive BFA‐induced TGN aggregates, indicating that homotypic fusion of the TGN rather than de novo endocytic trafficking is important for the formation of TGN/EE‐derived BFA‐induced aggregates. As the TGN also serves as an EE, continuously receiving materials from the plasma membrane, our data support the notion that the secretory Golgi organelle is distinct from the endocytic TGN/EE in terms of its response to BFA treatment in plant cells. Thus, the Golgi and TGN are probably functionally distinct organelles in plants.     

Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.     

Stimulation-dependent recycling of integrin beta1 regulated by ARF6 and Rab11

In comparison to the internalization pathways of endocytosis, the recycling pathways are less understood. Even less defined is the process of regulated recycling, as few examples exist and their underlying mechanisms remain to be clarified. In this study, we examine the endocytic recycling of integrin β1, a process that has been suggested to play an important role during cell motility by mediating the redistribution of integrins to the migrating front. External stimulation regulates the endocytic itinerary of β1, mainly at an internal compartment that is likely to be a subset of the recycling endosomes. This stimulation-dependent recycling is regulated by ARF6 and Rab11, and also requires the actin cytoskeleton in an ARF6-dependent manner. Consistent with these observations being relevant for cell motility, mutant forms of ARF6 that affect either actin rearrangement or recycling inhibit the motility of a breast cancer cell line.     

Adenovirus endocytosis

Pathogen entry into cells occurs by direct penetration of the plasma membrane, clathrin-mediated endocytosis, caveolar endocytosis, pinocytosis or macropinocytosis. For a particular agent, the infectious pathways are typically restricted, reflecting a tight relationship with the host. Here, we survey the uptake process of human adenovirus (Ad) type 2 and 5 and integrate it into the cell biology of endocytosis. Ad2 and Ad5 naturally infect respiratory epithelial cells. They bind to a primary receptor, the coxsackie virus B Ad receptor (CAR). The CAR-docked particles activate integrin coreceptors and this triggers a variety of cell responses, including endocytosis. Ad2/Ad5 endocytosis is clathrin-mediated and involves the large GTPase dynamin and the adaptor protein 2. A second endocytic process is induced simultaneously with viral uptake, macropinocytosis. Together, these pathways are associated with viral infection. Macropinocytosis requires integrins, F-actin, protein kinase C and small G-proteins of the Rho family, but not dynamin. Macropinocytosis per se is not required for viral uptake into epithelial cells, but it appears to be a productive entry pathway of Ad artificially targeted to the high-affinity Fcgamma receptor CD64 of hematopoietic cells lacking CAR. In epithelial and hematopoietic cells, the macropinosomal contents are released to the cytosol. This requires viral signalling from the surface and coincides with particle escape from endosomes and infection. It emerges that incoming Ad2 and Ad5 distinctly modulate the endocytic trafficking and disrupt selective cellular compartments. These features can be exploited for effective artificial targeting of Ad vectors to cell types of interest.     

Shear stress stimulates integrin β1 trafficking and increases directional migration of cancer cells via promoting deacetylation of microtubules

In egress routes of malignancy, cancer cells are constantly subjected to shear stress imposed by blood/lymph flow. Increasing evidence points toward the regulatory roles of shear stress in tumor cell adhesion and motility. Although it is known that integrin endocytic trafficking governs focal adhesion (FA) turnover and cell migration, the effect and biological consequences of low shear stress (LSS) on integrin trafficking remain unclear. Here, we identified the critical role of integrin β1 trafficking and caveolin-1 (Cav-1) mediated endocytosis in LSS-induced cell directional migration. LSS altered the distribution of integrin β1 in MDA-MB-231 cells and significantly promoted its internalization and recycling, which in turn facilitated FA turnover and directional cell migration. Furthermore, LSS induced cytoskeleton remodeling, which was required for internalization of integrin β1. LSS down-regulated the acetylation level of microtubules (MTs) via activating ROCK/HDAC6 pathway, resulting in elevation of MTs dynamics, Cav-1 motility, and Cav-1-dependent integrin β1 recycling. We also showed that high HDAC6 expression was a ROCK-dependent prognostic factor, which was correlated with poor outcomes in breast cancer patients. Taken together, these results defined a novel mechanism by which LSS enhanced integrin β1 trafficking via actin cytoskeleton remodeling and ROCK/HDAC6 mediated deacetylation of MTs, thereby promoting FAs turnover and directional cell migration.     

Adenovirus endocytosis

Pathogen entry into cells occurs by direct penetration of the plasma membrane, clathrin-mediated endocytosis, caveolar endocytosis, pinocytosis or macropinocytosis. For a particular agent, the infectious pathways are typically restricted, reflecting a tight relationship with the host. Here, we survey the uptake process of human adenovirus (Ad) type 2 and 5 and integrate it into the cell biology of endocytosis. Ad2 and Ad5 naturally infect respiratory epithelial cells. They bind to a primary receptor, the coxsackie virus B Ad receptor (CAR). The CAR-docked particles activate integrin coreceptors and this triggers a variety of cell responses, including endocytosis. Ad2/Ad5 endocytosis is clathrin-mediated and involves the large GTPase dynamin and the adaptor protein 2. A second endocytic process is induced simultaneously with viral uptake, macropinocytosis. Together, these pathways are associated with viral infection. Macropinocytosis requires integrins, F-actin, protein kinase C and small G-proteins of the Rho family, but not dynamin. Macropinocytosis per se is not required for viral uptake into epithelial cells, but it appears to be a productive entry pathway of Ad artificially targeted to the high-affinity Fcgamma receptor CD64 of hematopoietic cells lacking CAR. In epithelial and hematopoietic cells, the macropinosomal contents are released to the cytosol. This requires viral signalling from the surface and coincides with particle escape from endosomes and infection. It emerges that incoming Ad2 and Ad5 distinctly modulate the endocytic trafficking and disrupt selective cellular compartments. These features can be exploited for effective artificial targeting of Ad vectors to cell types of interest.     

Selective alterations in biosynthetic and endocytic protein traffic in Madin-Darby canine kidney epithelial cells expressing mutants of the small GTPase Rac1

Madin-Darby canine kidney (MDCK) cells expressing constitutively active Rac1 (Rac1V12) accumulate a large central aggregate of membranes beneath the apical membrane that contains filamentous actin, Rac1V12, rab11, and the resident apical membrane protein GP-135. To examine the roles of Rac1 in membrane traffic and the formation of this aggregate, we analyzed endocytic and biosynthetic trafficking pathways in MDCK cells expressing Rac1V12 and dominant inactive Rac1 (Rac1N17). Rac1V12 expression decreased the rates of apical and basolateral endocytosis, whereas Rac1N17 expression increased those rates from both membrane domains. Basolateral-to-apical transcytosis of immunoglobulin A (IgA) (a ligand for the polymeric immunoglobulin receptor [pIgR]), apical recycling of pIgR-IgA, and accumulation of newly synthesized GP-135 at the apical plasma membrane were all decreased in cells expressing Rac1V12. These effects of Rac1V12 on trafficking pathways to the apical membrane were the result of the delivery and trapping of these proteins in the central aggregate. In contrast to abnormalities in apical trafficking events, basolateral recycling of transferrin, degradation of EGF internalized from the basolateral membrane, and delivery of newly synthesized pIgR from the Golgi to the basolateral membrane were all relatively unaffected by Rac1V12 expression. Rac1N17 expression had little or no effect on these postendocytic or biosynthetic trafficking pathways. These results show that in polarized MDCK cells activated Rac1 may regulate the rate of endocytosis from both membrane domains and that expression of dominant active Rac1V12 specifically alters postendocytic and biosynthetic membrane traffic directed to the apical, but not the basolateral, membrane.     

EphA2 Signaling Following Endocytosis: Role of Tiam1

Eph receptors and their membrane‐bound ligands, the ephrins, represent a complex subfamily of receptor tyrosine kinases (RTKs). Eph/ephrin binding can lead to various and opposite cellular behaviors such as adhesion versus repulsion, or cell migration versus cell‐adhesion. Recently, Eph endocytosis has been identified as one of the critical steps responsible for such diversity. Eph receptors, as many RTKs, are rapidly endocytosed following ligand‐mediated activation and traffic through endocytic compartments prior to degradation. However, it is becoming obvious that endocytosis controls signaling in many different manners. Here we showed that activated EphA2 are degraded in the lysosomes and that about 35% of internalized receptors are recycled back to the plasma membrane. Our study is also the first to demonstrate that EphA2 retains the capacity to signal in endosomes. In particular, activated EphA2 interacted with the Rho family GEF Tiam1 in endosomes. This association led to Tiam1 activation, which in turn increased Rac1 activity and facilitated Eph/ephrin endocytosis. Disrupting Tiam1 function with RNA interference impaired both ephrinA1‐dependent Rac1 activation and ephrinA1‐induced EphA2 endocytosis. In summary, our findings shed new light on the regulation of EphA2 endocytosis, intracellular trafficking and signal termination and establish Tiam1 as an important modulator of EphA2 signaling .     

Integrins regulate centrosome integrity and astrocyte polarization following a wound

In response to a wound, astrocytes in culture extend microtubule‐rich processes and polarize, orienting their centrosomes and Golgi apparatus woundside. β1 Integrin null astrocytes fail to extend processes toward the wound, and are disoriented, and often migrate away orthogonal, to the wound. The centrosome is unusually fragmented in β1 integrin null astrocytes. Expression of a β1 integrin cDNA in the null background yields cells with intact centrosomes that polarize and extend processes normally. Fragmented centrosomes rapidly assemble following integrin ligation and cell attachment. However, several experiments indicated that cell adhesion is not necessary. For example, astrocytes in suspension expressing a chimeric β1 subunit that can be activated by an antibody assemble centrosomes suggesting that β1 activation is sufficient to cause centrosome assembly in the absence of cell adhesion. siRNA knockdown of PCM1, a major centrosomal protein, inhibits cell polarization, consistent with the notion that centrosomes are necessary for polarity and that integrins regulate polarity via centrosome integrity. Screening inhibitors of molecules downstream of integrins indicate that neither FAK nor ILK is involved in regulation of centrosome integrity. In contrast, blebbistatin, a specific inhibitor of non‐muscle myosin II (NMII), mimics the response of β1 integrin null astrocytes by disrupting centrosome integrity and cell polarization. Blebbistatin also inhibits integrin‐mediated centrosome assembly in astrocytes attaching to fibronectin, consistent with the hypothesis that NMII functions downstream of integrins in regulating centrosome integrity. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 73: 333–353, 2013.