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  • formestane Subcellular domains where Rac is active show limi

    2022-05-13

    Subcellular domains where Rac is active show limited Rho activity and vice versa (Fig. 4). This distinctive patterning in Rho activity is achieved via the downstream Rac effector Par6, an important part of the so-called Par polarity complex. In primary epithelium, the cytoskeleton and associated Rho GTPases provide the initial polarity cue by directing the localization of the Par6/aPKC complex to the apical side of the cell and other polarity complexes to the lateral (e.g. Bazooka) and basolatareal membranes (e.g. Dlg/Scrib/Lgl complex) (Chen & Zhang, 2013). The binding of Cdc42 to Par6 activates the associated aPKC (Horikoshi et al., 2009), which subsequently activates the Rho degrading ubiquitin ligase Smurf1, thereby reducing Rho availability at the leading edge (Wang et al., 2003). Alternatively, in response to integrin-engagement, Rac inactivates Rho via activating p190RhoGAP (Bustos et al., 2008, Nimnual et al., 2003, Wildenberg et al., 2006). In addition, the Rac-activated kinase PAK1 phosphorylates, and thereby inactivates, a set of RhoA GEFs, such as PDZ-RhoGEF, GEF-H1, p115RhoGEF and NET1 (Alberts et al., 2005, Barac et al., 2004, Callow et al., 2005, Meiri et al., 2014, Nalbant et al., 2009, Rosenfeldt et al., 2006, Zenke et al., 2004). However, while the role of RhoA at the trailing edge is well conserved in 3D cellular environents (Yamazaki, Kurisu, & Takenawa, 2009), it was suggested that the formation of protrusions at the leading edge of motile formestane requires both Rac1 and RhoA activity in an oscillatory manner (Kim et al., 2015). In fact, the use of FRET-based biosensors revealed that Rho is also active at the leading edge (Pertz, Hodgson, Klemke, & Hahn, 2006), where it is activated immediately in front of Rac and Cdc42 in lammelipodia (Machacek et al., 2009). The recent development of optogenetic tools to alter subcellular distribution of RhoA to control cell and tissue mechanics will undoubtedly further strengthen our knowledge on subcellular domain of Rho GTPases during cell migration (Valon, Marin-Llaurado, Wyatt, Charras, & Trepat, 2017). Active RhoA also limits Rac activation via ROCK through inhibition of the recruitment of β-Pix to focal adhesions, thereby locally limiting Rac activation (Kuo et al., 2011, Sanz-Moreno et al., 2008). Interestingly, although RhoB has opposite effects compared to RhoA and RhoC on cancer progression, RhoB also affect Rac activity negatively. Loss of RhoB, which is often observed during lung cancer progression, results in the activation of Rac1 and EMT (Bousquet et al., 2015). However, the mechanism by which RhoB affects Rac1 activty is a multistep process and not a direct inhibitory effect of the one on the other. Here, downregulation of RhoB first results in the activation of Akt1 which further activates and the Rac GEF Trio, thus increasing active Rac1. Thus, through mutually excluding cross-talk between RhoA and Rac, opposing functions in the regulation of focal adhesions, membrane protrusions and actomyosin contractility, are confined to subcellular compartments where their functions are required for effective cell migration.
    Rho proteins in collective migration Although it was generally assumed that cancer cells migrate as single cells, it has now become clear that cancer cells can migrate both collectively and individually and that cells dynamically and reversibly change their migratory behavior. At a cell biological level, most malignant cell types can invade as cohesive multicellular units in which cells migrate with intact cell-cell contacts and that loosening of cell-cell contacts is sufficient to permit this (Friedl & Gilmour, 2009). Mesenchymal migration of carcinoma cells is activated by extracellular factors that induce EMT. Within the tumor, where EMT inducing factors are less present, carcinoma cells migrate in such multicellular clusters, following tracks through the ECM created by cancer associated fibroblasts. By studying collective migration in a co-culture system of carcinoma cells and stromal fibroblasts, movement patterns of leading fibroblast were found to be driven by RhoA-dependent actomyosin contractility in concert with MMP activity, which creates tracks through the ECM. Interestingly, the trailing carcinoma cells use Cdc42-dependent actomyosin contractility, highlighting a new aspect of cooperation between Rho and Cdc42 (Gaggioli et al., 2007). While the leader cell of collective migration is often a cancer associated fibroblast, it could also be a cancer cell on the edge of an epithelial sheet. On the edge of a tumor, cells can undergo EMT, becoming leader cells that form membrane protrusions in the direction of cell movement, dependent on Rac and Cdc42 activity, and degrade ECM by secretion of proteases, similar to the mesenchymal movement described above. Cells that are further into a migrating sheet or strand maintain intact cell-cell junctions and do not undergo EMT. A correct front-rear polarity is important for collective cell migration and is maintained by upstream regulators of Rac such as the Scribble and Par polarity complexes. The Par complex consists of Par6, which upon Cdc42 binding recruits Par3 and aPKC. This complex is recruited to the leading edge where it regulates the activity of Rac via the Rac GEFs Tiam1 and Tiam2 (Chen and Macara, 2005, Nishimura et al., 2005, Wang et al., 2012). Importantly, while Rho/ROCK dependent actomyosin contractility is required for collective cell migration (Friedl et al., 2014, Ridley, 2011, Zegers and Friedl, 2014), contractility needs to be limited to not disrupt cell-cell junctions. In addition to activating Rac, the Par complex also locally inhibits Rho near cell-cell junctions through regulating the localization of Rnd3, an atypical Rho GTPase devoid of GTP hydrolytic activity (Hidalgo-Carcedo et al., 2011), while it inhibits Rho at membrane protrusions through regulating Smurf1 (Wang et al., 2003). In pancreatic cancer cells, lower expression of Par3 correlates with a more invasive phenotype and lower patient survival. Thus, the loss of Par3 in pancreatic cancer might be the result of aberrant Rho activation and actomyosin contractility.