Integrin trafficking at a glance.
Ontology highlight
ABSTRACT:
SUBMITTER: Bridgewater RE
PROVIDER: S-EPMC3462077 | biostudies-literature | 2012 Aug
REPOSITORIES: biostudies-literature
Publications
Integrin trafficking at a glance.
Bridgewater Rebecca E RE Norman Jim C JC Caswell Patrick T PT
Journal of cell science 20120801 Pt 16
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Project description:The integrin αvβ6 is expressed at low levels in most normal healthy tissue but is very often upregulated in a disease context including cancer and fibrosis. Integrins use endocytosis and trafficking as a means of regulating their surface expression and thus their functions, however little is known of how this process is regulated in the context of αvβ6. As αvβ6 is a major target for the development of therapeutics in cancer and fibrosis, understanding these dynamics is critical in the development of αvβ6-targeted therapies. Following development of a flow cytometry-based assay to measure ligand (A20FMDV2 or LAP)-bound αvβ6 endocytosis, an siRNA screen was performed to identify which genes were responsible for internalising αvβ6. These data identified 15 genes (DNM2, CBLB, DNM3, CBL, EEA1, CLTC, ARFGAP3, CAV1, CYTH2, CAV3, CAV2, IQSEC1, AP2M1, TSG101) which significantly decreased endocytosis, predominantly within dynamin-dependent pathways. Inhibition of these dynamin-dependent pathways significantly reduced αvβ6-dependent migration (αvβ6-specific migration was 547 ± 128 under control conditions, reduced to 225 ± 73 with clathrin inhibition, and 280 ± 51 with caveolin inhibition). Colocalization studies of αvβ6 with endosome markers revealed that up to 6 h post-internalisation of ligand, αvβ6 remains in Rab11-positive endosomes in a perinuclear location, with no evidence of αvβ6 degradation up to 48 h post exposure to A20FMDV2. Additionally, 60% of ligand-bound αvβ6 was recycled back to the surface by 6 h. With studies ongoing using conjugated A20FMDV2 to therapeutically target αvβ6 in cancer and fibrosis, these data have important implications. Binding of A20FMDV2 seemingly removes much of the αvβ6 from the cell membrane, and upon its recycling, a large fraction appears to still be in the ligand-bound state. While these results are observed with A20FMDV2, these data will be of value in the design of αvβ6-specific therapeutics and potentially the types of therapeutic load.
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Project description:Membrane traffic is an important regulator of cell migration through the endocytosis and recycling of cell surface receptors such as integrin heterodimers. Intracellular nanovesicles (INVs) are transport vesicles that are involved in multiple membrane trafficking steps, including the recycling pathway. The only known marker for INVs is tumor protein D54 (TPD54/TPD52L2), a member of the TPD52-like protein family. Overexpression of TPD52-like family proteins in cancer has been linked to poor prognosis and an aggressive metastatic phenotype, which suggests cell migration may be altered under these conditions. Here, we show that TPD54 directly binds membrane and associates with INVs via a conserved positively charged motif in its C terminus. We describe how other TPD52-like proteins are also associated with INVs, and we document the Rab GTPase complement of all INVs. Depletion of TPD52-like proteins inhibits cell migration and invasion, while their overexpression boosts motility. We show that inhibition of migration is likely due to altered recycling of α5β1 integrins in INVs.
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Project description:Recycling endosomes maintain plasma membrane homeostasis and are important for cell polarity, migration, and cytokinesis. Yet, the molecular machineries that drive endocytic recycling remain largely unclear. The CORVET complex is a multi-subunit tether required for fusion between early endosomes. Here we show that the CORVET-specific subunits Vps3 and Vps8 also regulate vesicular transport from early to recycling endosomes. Vps3 and Vps8 localise to Rab4-positive recycling vesicles and co-localise with the CHEVI complex on Rab11-positive recycling endosomes. Depletion of Vps3 or Vps8 does not affect transferrin recycling, but delays the delivery of internalised integrins to recycling endosomes and their subsequent return to the plasma membrane. Consequently, Vps3/8 depletion results in defects in integrin-dependent cell adhesion and spreading, focal adhesion formation, and cell migration. These data reveal a role for Vps3 and Vps8 in a specialised recycling pathway important for integrin trafficking.
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Project description:ObjectiveCoagulation initiation by tissue factor (TF) is regulated by cellular inhibitors, cell surface availability of procoagulant phosphatidylserine, and thiol-disulfide exchange. How these mechanisms contribute to keeping TF in a noncoagulant state and to generating prothrombotic TF remain incompletely understood.Approach and resultsHere, we study the activation of TF in primary macrophages by a combination of pharmacological, genetic, and biochemical approaches. We demonstrate that primed macrophages effectively control TF cell surface activity by receptor internalization. After cell injury, ATP signals through the purinergic receptor P2rx7 induce release of TF+ microvesicles. TF cell surface availability for release onto microvesicles is regulated by the GTPase arf6 associated with integrin α4β1. Furthermore, microvesicles proteome analysis identifies activation of Gαi2 as a participating factor in the release of microvesicles with prothrombotic activity in flowing blood. ATP not only prevents TF and phosphatidylserine internalization but also induces TF conversion to a conformation with high affinity for its ligand, coagulation factor VII. Although inhibition of dynamin-dependent internalization also exposes outer membrane procoagulant phosphatidylserine, the resulting TF+ microvesicles distinctly lack protein disulfide isomerase and high affinity TF and fail to produce fibrin strands typical for microvesicles generated by thrombo-inflammatory P2rx7 activation.ConclusionsThese data show that procoagulant phospholipid exposure is not sufficient and that TF affinity maturation is required to generate prothrombotic microvesicles from a variety of cell types. These findings are significant for understanding TF-initiated thrombosis and should be considered in designing functional microvesicles-based diagnostic approaches.
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Project description:Melanosomes are melanin-containing organelles that provide pigmentation and protection from solar UV radiation to the skin. In melanocytes, melanosomes mature and traffic to dendritic tips, where they are transferred to adjacent epidermal keratinocytes through pathways that involve microtubule networks and the actin cytoskeleton. However, the role of scaffold proteins in these processes is poorly understood. Integrin-linked kinase (ILK) is a scaffold protein that regulates microtubule stability and F-actin dynamics. Here we show that ILK is necessary for normal trafficking of melanosomes along microtubule tracks. In the absence of ILK, immature melanosomes are not retained in perinuclear regions, and mature melanosome trafficking along microtubule tracks is impaired. These deficits can be attenuated by microtubule stabilization. Microtubules are also necessary for the formation of dendrites in melanocytes, and Ilk inactivation reduces melanocyte dendricity. Activation of glycogen synthase kinase-3 (GSK-3) interferes with microtubule assembly. Significantly, inhibition of GSK-3 activity or exogenous expression of the GSK-3 substrate collapsin response mediator protein 2 (CRMP2) in ILK-deficient melanocytes restored dendricity. ILK is also required for normal melanin transfer, and GSK-3 inhibition in melanocytes partially restored melanin transfer to neighboring keratinocytes. Thus, our work shows that ILK is a central modulator of melanosome movements in primary epidermal melanocytes and identifies ILK and GSK-3 as important modulators of melanin transfer to keratinocytes, a key process for epidermal UV photoprotection.
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Project description:The laminin-binding integrins, α3β1 and α6β1, are needed for tumor metastasis and their surface expression is regulated by endocytic recycling. β1 integrins share the Rab11 recycling machinery, but the trafficking of α3β1 and α6β1 are distinct by an unknown mechanism. Using a mouse PDX tumor model containing human metastatic prostate cancer, Rab11 family interacting protein 5 (Rab11-FIP5) was identified as a lead candidate for α6β1 trafficking. Rab11-FIP5 and its membrane-binding domain were required for α6β1 recycling, without affecting the other laminin-binding integrin (i.e., α3β1) or unrelated membrane receptors like CD44, transferrin receptor, or E-cadherin. Depletion of Rab11-FIP5 resulted in the intracellular accumulation of α6β1 in the Rab11 recycling compartment, loss of cell migration on laminin, and an unexpected loss of α6β1 recycling in cell-cell locations. Taken together, these data demonstrate that α6β1 is distinct from α3β1 via Rab11-FIP5 recycling and recycles in an unexpected cell-cell location.Implications: Rab11-FIP5-dependent α6β1 integrin recycling may be selectively targeted to limit migration of prostate cancer cells into laminin-rich tissues. Mol Cancer Res; 16(8); 1319-31. ©2018 AACR.
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