Xu and F. show that knockdown of Rab18 reduces the size of focal adhesions (FAs) and influences their dynamics. Furthermore, we found that Rab18, by directly interacting with the endoplasmic reticulum (ER)-resident protein kinectin-1, controls the anterograde kinesin-1Cdependent transport of the ER required for the maturation of nascent FAs and protrusion orientation toward a chemoattractant. Altogether, our data support a model in which Rab18 regulates kinectin-1 transport toward the cell surface to form ERCFA contacts, thus promoting FA growth and cell migration during chemotaxis. Introduction Rab proteins constitute the largest family within the Ras superfamily of LRP8 antibody small GTPases. The first Rabs were identified in yeast in the 1980s (Gallwitz et al., 1983; Schmitt et al., 1986), and today >60 members have been revealed in humans (Zhen and Stenmark, 2015). Rab proteins are master regulators of intracellular membrane traffic, and by localizing to different membrane compartments, they control the specificity of vesicular transport and ensure that the cargoes are transported to their correct destinations within the cell (Wandinger-Ness and Zerial, 2014; Zhen and Stenmark, 2015). Rabs function as molecular switches that alternate between an active GTP-bound state and an inactive GDP-bound state. Upon membrane recruitment, Rab proteins in their GTP-bound state can bind a variety of different effector molecules, including sorting adaptors, tethering factors, fusion regulators, kinases, phosphatases, and motor proteins (Gillingham et al., 2014). More recently, Rab proteins have been shown to take part in other cellular processes. These small GTPases can indeed regulate the mitotic spindle and abscission during cell division (Gibie?a and Prekeris, 2018; Kouranti et al., 2006), apical lumen formation and polarization of epithelial cells (Bryant et al., 2010), nutrient sensing, and signaling (Thomas et al., 2014). Interestingly, an increasing amount of evidence shows that Rab proteins are implicated in the processes of cell migration and invasion (Borg et al., 2014; Linford et al., 2012; Palamidessi et al., 2008; Vestre et al., 2019). This function is often connected to their role in mediating intracellular transport, but can also be associated with their ability to regulate cytoskeleton dynamics (Borg et al., 2014; Kjos et al., 2018; Lanzetti et al., 2004; Linford et al., 2012; Palamidessi et al., 2008). Indeed, Rab proteins can influence cytoskeleton dynamics, for example through cross-talk with Rho GTPases (Borg et al., 2014; Bravo-Cordero et al., 2016; Chevallier et al., 2009; Jian et al., 2016; Margiotta et al., 2017; Palamidessi et al., 2008; Vestre et al., 2019). Rab18 is one of the most highly conserved Rab GTPases (Kl?pper et al., 2012). It localizes to the ER and lipid droplets (LDs), and it has been described to regulate LD growth and maturation by establishing contacts between LDs and the ER (Li et al., 2019; Martin et al., 2005; Ozeki et Imirestat al., 2005; Xu et al., 2018). Depletion of Rab18 causes defects in LD morphology but also in ER tubule integrity (Carpanini et al., 2014; Gerondopoulos et al., 2014; Jayson et al., 2018). Furthermore, Rab18 is Imirestat also reported to regulate Imirestat ER trafficking (Dejgaard et al., 2008), as well as secretory granule transport (Vazquez-Martinez et al., 2007). Loss-of-function mutations in gene causing altered ER morphology have been identified in Warburg micro syndrome, a human neurological and developmental disorder in which Rab18 has a critical role as a regulator of neuronal migration and morphogenesis (Bem et al., 2011; Gerondopoulos et al., 2014; Wu et al., 2016). However, how the loss-of-function of a protein regulating ER trafficking and morphology results in defects of cell migration is poorly understood, which emphasizes the importance of further investigating the contribution of Rab18 to this process. We therefore elucidate the role of Rab18 in cell migration and the underlying mechanism. In particular, we investigate whether this function is connected to kinectin-1 (KNT1) function. KNT1 is an integral transmembrane protein that connects the ER to the microtubule motor kinesin-1 (Ong et al., 2000). Evidence indicates Imirestat that KNT1-kinesin interaction mediates the anterograde transport from the ER to aid FA development and maturation during cell migration, but what regulates the KNT1-kinesinCmediated transportation from the ER towards the leading edge continues to be unresolved (Ng et al., 2016; Zhang et al., 2010). Right here, we see that Rab18 interacts with KNT1. Furthermore, we present that this connections must mediate Imirestat the anterograde ER transportation to market ERCFA contacts and therefore, FA maturation. Certainly, impairment of Rab18 recruitment to KNT1 reproduces the previously defined phenotype attained upon KNT1 depletion with cells exhibiting flaws in FA development. Furthermore, Rab18 knockdown impacts protrusion orientation toward a chemoattractant, recommending that the connections.