Cdc42, a small GTPase of the Rho family, stands out as playing a central role in establishing cell polarity in all eukaryotic cells, irrespective of the biological context.
Cdc42 during budding yeast polarization
Budding yeast can polarize in response to two main stimuli: they undergo polarized growth, which leads to budding, and they respond to pheromone gradient during mating by forming a shmoo. These stimuli both lead to Cdc42 recruitment and activation at the site of polarized growth through distinct signalling cascades. Far1p associates with the Cdc42p-GEF Cdc24p and plays a key role in Cdc42p activation. During budding, Far1p degradation allows Cdc24p exit form the nucleus and, during mating, it binds Gß and thereby recruits Cdc24p to the site of polarization. The active, GTP-bound form of Cdc42p regulates multiple direct or indirect effectors, which control several cell functions. The co-ordinate polarization of septins, actin and microtubule structures, and of membrane trafficking, allows a polarized growth that leads during budding to the formation of a bud and during mating to the formation of a shmoo.
Cdc42 and polarity establishment in multicellular organisms
General mechanisms of cell polarization appear to be conserved throughout evolution. However, the complexity encountered in multicellular organisms has led to an even more diverse range of Cdc42 regulators and downstream effectors.
In multicellular organisms, cell polarity is determined primarily by external stimuli. Contact receptors such as integrins and cadherins, as well as receptors for soluble ligands such as chemokines, allow individual cells to sense their environment and organize polarity accordingly. This is controlled by Cdc42 and, as in yeast, the localized recruitment and activation of Cdc42 is likely to be a key event leading to cell polarization. In multicellular organisms, cells respond external signals that promote cell polarization and are transduced by different families of receptors that trigger signalling pathways that eventually recruit and activate a Cdc42-GEF.
Multiple Cdc42 targets mediate polarization
Cdc42 regulates the actin cytoskeleton
One of the best-characterized targets of Cdc42 and Rac is the p65PAK family of serine/threonine kinases that associates with F-actin in membrane ruffles and lamellipodia at the leading edge of polarized migrating cells, to cell-cell contacts in epithelial cells, and in phagocytic cups in neutrophils and macrophages. It apparently plays an important role in actin rearrangements by regulating LIM kinase, which in turn phosphorylates and inactivates the actin-severing protein cofilin.
In its GTP-bound form, Cdc42 binds to and activates WASp, the product of the gene mutated in Wiskott-Aldrich syndrome. WASp in turn recruits and activates the Arp2/3 complex leading to actin polymerization and filopodia formation that is closely coupled to polarity establishment. In migrating cells might help cells sense the local environment and transmit this information to the polarization machinery. One other interesting activity of Cdc42 is its ability to activate Rac, providing a mechanism to couple the polarization and protrusion machineries during directed cell migration.
Cdc42 regulates the microtubule cytoskeleton
Cell polarization is often characterized by microtubule reorganization. The microtubule cytoskeleton is required for polarization of the Caenorhabditis elegans embryo, in which the positioning of the asymmetric spindle is dependent on microtubule-mediated forces. These are regulated by the PAR proteins (PAR-1 to PAR-6), which are asymmetrically localized. This PAR complex is highly conserved throughout eukaryotes. In its active form, Cdc42 interacts with a semi-CRIB motif and the adjacent PDZ domain of PAR-6 inducing a conformational change in PAR-6 that activates the aPKC. Glycogen synthase kinase 3ß (GSK3ß) activity is spatially inhibited by PKC-induced phosphorylation and this leads to the association of the adenomatous polyposis coli protein (APC) with microtubule plus-ends This is required for centrosome reorientation in migrating cells, perhaps regulating microtubule dynamics or microtubule plus-end capture, or both, at the leading edge. The Par6-aPKC complex also interacts with the tumour suppressor Lethal giant larvae (Lgl), with aPKC phosphorylating it. Lgl localizes to the leading edge of migrating cells as well and a non-phosphorylatable Lgl mutant inhibits aspects of polarization.
Cdc42 can also contribute to the polarization of the microtubule system by promoting microtubule capture at the plasma membrane that might allow the generation of forces that reorient the microtubule cytoskeleton. Cdc42 binds to and recruits IQGAP1, which interacts with the microtubule-plus-end-associated protein Clip-170. Interfering with IQGAP1 binding to Cdc42 or Clip-170 alters cell polarization and microtubule organization.
Cdc42 and the formation of intercellular junctions
In mammalian cells, the Par6-aPKC complex contributes to the formation of tight junctions in a process that initiates epithelial polarization. It is believed that Par3 is recruited to sites of cell-cell contact by junctional adhesion molecule (JAM), a trans-membrane molecule found in tight junctions . Feedback loops probably control this localization, because inhibition of aPKC disrupts Par3 localization. Lgl and Par6/aPKC are then recruited, whereupon Lgl becomes phosphorylated and relocalizes to the baso-lateral membrane.
Cdc42 regulates membrane traffic
In polarized cells, membrane traffic is oriented towards one particular membrane of the cell. In migrating cells, for instance, most of the exocytic machinery faces the front edge. In particular, the Golgi is localized in front of the nucleus in the direction of migration. This localization is dependent upon the integrity of the microtubule cytoskeleton. In migrating astrocytes, the Cdc42-Par6-aPKC pathway controls the orientation of the Golgi, probably indirectly by modifying microtubule organization. Moreover, active Cdc42 binds directly to the coatomer complex and regulates vesicular trafficking between the endoplasmic reticulum and Golgi. Cdc42 also controls the exit of baso-lateral proteins from the trans-Golgi network and facilitates the targeting of exocytic vesicles. Cdc42 can activate the small GTPase, RalA, which interacts directly with Sec5, a component of the exocyst.
