Integrin Trafficking Regulated by Rab21 Is Necessary for Cytokinesis

Abstract

Objective-Coagulation 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 Results-Here, 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 Gα i2 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. Conclusions-These 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. Visual Overview-An online visual overview is available for this article. The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/ Highlights • Tissue factor (TF) cell surface availability is controlled by integrin α4β1-and arf6-regulated trafficking. • Microvesicles generated by pharmacological interruption of TF-integrin internalization differ in protein composition and function from mi-crovesicles released by P2rx7 cell injury signaling. • Maturation of TF to a high affinity state is a key determinant for the prothrombotic activity of TF + microvesicles in blood.

Cytokinesis and cancer

“…Importantly, the RAB21 gene was found mutated in ovarian carcinoma cell lines and in a prostate cancer tumour sample. Cells containing these mutations accumulate multinucleated profiles, in line with the notion that Rab21 controls cytokinesis [79]. KLHDC8B gene was recently characterized and it was found to be disrupted by translocations found in a family in which multiple individuals developed Classical Hodgkin lymphoma.…”

A chemical biology screen reveals a role for Rab21-mediated control of actomyosin contractility in fibroblast-driven cancer invasion

“…These data led us to propose that Rab21 delivers integrin a5 to the plasma membrane where it locally signals to the contractile machinery. Interestingly, a connection between Rab21, integrins and actomyosin regulation has been reported during cytokinesis (Pellinen et al, , 2008. This is probably through the activation of Rho-ROCK function as ligand-bound integrin a5 can signal to RhoGEFs and thereby increase Rho activity (Ren et al, 1999;Dubash et al, 2007;Huveneers et al, 2008).…”

Liprin-α1, ERC1 and LL5 identify a polarized, dynamic compartment implicated in cell migration

“…There is growing evidence that internalized integrins can be rapidly recycled at the front of migrating cells: localized endocytic and exocytic traffic of integrins can spatially restrict receptor distribution to promote the turnover of adhesions and signaling molecules at the cell front and to support protrusion . It is known that b1 integrin can be internalized (Pellinen et al, 2008;Shi and Sottile, 2008) in both inactive and ligand-bound active conformations (Ng et al, 1999;Valdembri et al, 2009;Dozynkiewicz et al, 2012), and that the two pools might be recycled through distinct routes (Arjonen et al, 2012). The negative effect of the lack of liprin-a1, ERC1a or LL5 on the specific internalization of active b1 integrin supports the hypothesis that the dynamic structures identified in this study are directly or indirectly involved in the internalization of integrins, thus contributing to the turnover of focal adhesions at the front of migrating cells.…”