The Cdc42 GTPase – a central organizer of cell polarization
Small GTPases of the Rho family play essential roles in eukaryotic cell polarization. These signaling molecules act as switches, being active in GTP-bound and inactive in GDP-bound forms. In yeast, the Rho-family GTPase Cdc42 is a key regulator. Cdc42 is active at cell poles where is promotes polarized cell growth. How the cell activates Cdc42 specifically at cell poles, how the size of the Cdc42-active zone is defined, and how Cdc42 then promotes polarized growth are some of the questions we are aiming to address.
We have developed a functionally-tagged Cdc42 allele, in which a fluorescent protein, mCherry or sfGFP, is inserted in a non-conserved internal site. This tool permits to investigate the localization and dynamic nature of Cdc42. The image on the left shows how Cdc42 (top) is distributed on most cellular membranes, whereas its active form (middle) is present exclusively at the poles of the cell, where the cell is growing. In its active form, Cdc42 promotes the assembly of the actin cytoskeleton and the exocytosis of growth vesicles.
Organization of actin cables for polarized cell growth
In yeast cells, three main actin structures have been described during the mitotic growth cycle: the cytokinetic actin ring, necessary for cell division, actin patches, which represent sites of endocytosis, and actin cables, long bundles of largely parallel linear actin filaments. Actin cables are assembled by For3, a member of the formin family of actin nucleators. These cables serve as tracks for myosin V-dependent transport of cargoes towards sites of polarized cell growth. Cargoes include membrane material and cell wall remodeling components essential for polarized cell growth. We have previously shown that actin cables undergo retrograde actin flow, with actin polymerization taking place at cell poles, where For3 is localized, and pushing the cable into the cell interior. For3 undergoes similar flow, detaching from cell poles and traveling with the cable into the cell interior. In addition, we have shown that type V myosins also contribute to the organization of actin cables.
While wildtype fission yeast cells transport vesicles towards cell tips along actin cables, we recently showed that this transport can be re-routed along microtubules to achieve very similar cell shape. To this aim, we engineered a chimeric motor protein between a kinesin motor domain and the cargo-binding region of the major type V myosin Myo52. This chimeric motor transports myosin cargos along microtubules and restores viability and elongated shape to cells lacking actin cables. Thus, cells are plastic enough to permit this re-wiring. This also demonstrates that vesicular transport to cell poles is sufficient for polarized cell growth, whatever route is used. The video-abstract below describes this study.
Role of the exocyst for polarized cell growth
Myosin V transport and actin cables, while important for polarized cell growth, are not essential for it. Indeed, fission yeast cells also use a second Cdc42-dependent polarization strategy. Secretion of cell wall-remodelling components at cell poles requires the exocyst complex, an eight-subunit complex that facilitates fusion of vesicles with the plasma membrane. Localization of this complex to cell poles is independent of the cytoskeleton. Disruption of actin cables or of the exocyst does not block polarized growth, but double disruption prevents polarized growth. For polar growth at cell poles, fission yeast thus rely both on the actin cables that transport vesicle cargoes and on the exocyst complex that promotes fusion of these vesicles with the plasma membrane.