Endocytic Trafficking of Integrins in Cell Migration Nikki R. Paul, Guillaume Jacquemet, Patrick T. Caswell  Current Biology  Volume 25, Issue 22, Pages R1092-R1105 (November 2015) DOI: 10.1016/j.cub.2015.09.049 Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 1 Clathrin-dependent endocytosis of integrins and adhesion turnover in migrating cells. (A) Dab2-dependent clathrin-dependent endocytosis of inactive β1 integrins from the dorsal surface allows re-delivery to the ventral plasma membrane. (B) The R-Ras–Rin2 complex promotes clathrin-dependent endocytosis of β1 integrins by recruiting Rab5-GTP to adhesions. The Rab5–Rin2 complex subsequently acts to localise R-Ras-GTP to endosomes to activate Rac via Tiam1. (C) Microtubule-dependent adhesion turnover is mediated by the kinesin KIF15, which promotes recruitment of Dab2 to the ventral plasma membrane. Dynamin is recruited by FAK and is required for vesicle scission. (D) Eps8 binds the NPxY/NxxY motif of β integrins via its PTB domain. Eps8 also binds RN-tre, which inhibits Rab5-mediated internalisation of β1 (but not β3) integrins. (E) Numb undergoes cycles of Par-3–aPKC-mediated phosphorylation/dephosphorylation, which regulates endocytosis of β1 and β3 integrins at the leading edge of the cell. Current Biology 2015 25, R1092-R1105DOI: (10.1016/j.cub.2015.09.049) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 2 Clathrin-independent endocytosis of integrins and adhesion turnover in migrating cells. (A) Stimulation of the PDGF receptor (PDGFR) induces circular dorsal ruffles on the dorsal surface of the cell and promotes internalisation of integrins via macropinocytosis. (B) Clathrin-independent carriers (CLICs) that are distributed towards the leading edge of the cell permit β1-integrin internalisation. They require extracellular galectin-3 (Gal-3) and intracellular GRAF1, which forms a complex with phosphorylated FAK. (C) In the trailing uropod of leukocytes, endocytosis of LFA-1 (αLβ2 integrin) is dependent upon caveolin and is regulated by GPCR signalling. (D) α5β1 integrin is internalised from sub-nuclear fibrillar adhesions via a clathrin- and dynamin-independent mechanism that requires Arf4 and Scar/WAVE. Ligand-bound integrins are delivered to lysosomes to activate mTOR complex 1 (mTORC1). (E) Syndecans are adhesion receptors that bind ECM ligands. Syndecan-4 binding to fibronectin (FN) activates PKCα and RhoG, and this promotes caveolar endocytosis of β1 integrins at the leading edge. Current Biology 2015 25, R1092-R1105DOI: (10.1016/j.cub.2015.09.049) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 3 Directional trafficking of integrins. (A) In neutrophils, integrins are internalised at the rear of the migrating cell and recycled to the leading edge via the perinuclear recycling compartment (PNRC), in a process that is dependent upon calcium signalling. (B) In rear-steering epithelial cells, the kinesin KIF1C drives the movement of integrin-containing vesicles from the perinuclear region along microtubules to the tip of the trailing cell tail. This allows maturation of adhesions at the rear of the cell, providing directionality towards guidance cues and promotes persistent migration. (C) In cancer cells migrating in 3D ECM, α5β1 integrins are internalised at the leading edge and sorted into Rab25-positive endosomes; inactive integrins return to the plasma membrane at the cell front, whereas active integrins are trafficked to chloride intracellular channel 3 (CLIC3)-positive lysosomes. Subsequent recycling of active integrins to the cell rear and an increase in Src activity drive forward movement. Current Biology 2015 25, R1092-R1105DOI: (10.1016/j.cub.2015.09.049) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 4 Integrin recycling in cancer cell invasion. (A) In 3D ECM with a low fibronectin content, trafficking of αvβ3 integrin promotes invasion, presumably through redistribution of this integrin to the leading edge resulting in high Rac activity and lamellipodial-like protrusions. However, αvβ3 (and αvβ3 recycling), or expression of miRNAs through Dicer, suppress the association of α5β1 with RCP. This results in low levels of invasion in the presence of the α5β1 ligand fibronectin. (B) In 3D ECM with a high fibronectin content, when αvβ3 integrin is inhibited with small cyclic peptides or soluble ligands such as osteopontin, or when mutant p53 inhibits p63 and Dicer, α5β1 integrin is recruited by RCP and rapidly recycled to the plasma membrane. This is coordinated by diacylglycerol kinase (DGK)-α which produces phosphatidic acid (PA) from diacylglycerol (DAG) to recruit RCP-containing vesicles to the pseudopodial tip. RCP and α5β1 integrin recruit RTKs, such as EGFR, and promote their trafficking and localised signalling. This drives PKB/Akt-mediated phosphorylation of RacGAP1, which is recruited to the scaffold IQGAP and inhibits Rac. This in turn allows localised RhoA activity, which drives the formation of actin spikes and bundles through ROCK-mediated activation of FHOD3. Current Biology 2015 25, R1092-R1105DOI: (10.1016/j.cub.2015.09.049) Copyright © 2015 Elsevier Ltd Terms and Conditions