A growing number of diseases are attributed to dysfunction of cilia and cilia-associated proteins (ciliopathies). In epithelial cells, cilia-like microtubule (MT)-based membrane protrusions are rapidly formed in response to protein toxins (e.g. C. difficile toxin CDT), which ADP-ribosylate actin and cause partial depolymerization of the actin cytoskeleton. The toxin-inducible protrusions are highly dynamic structures that contain cilia-like microtubules, which promote ante- and retrograde vesicle transport and recruit signaling molecules strikingly similar to normal cilia. In this project, we will utilize these toxin-induced MT-based protrusions as a readily accessible model system to obtain novel insight into the formation, structure, dynamics and function (vesicle transport and signaling) of primary cilia. One major goal is the elucidation of the functions of GTP-binding proteins of the septin family. Septins have been implicated as diffusion barrier at the ciliary transition zone, but their precise function and regulation has remained elusive. Using the toxin-induced MT model, we will study the roles of septins in the initiation of MT-based protrusions, and determine the cytosolic and membrane-associated binding partners of septins. The toxin-induced MT model will allow us to further clarify the regulation of septins by small GTPases, especially Cdc42 and its effectors Borgs. We will test the hypothesis that functional membrane barriers are formed by septins and actin, determining the content and compartmentalization of toxin-induced MT-based protrusions by fluorescence microscopy, mass spectrometry and molecular biological methods. The role of small GTPases, kinesins and dynein in vesicle traffic in MT-based protrusions will be elucidated. We will also determine whether the toxin-induced MT-protrusions can form signaling platforms similar to cilia. Exploiting the toxin-induced MT protrusions as a less complex and rapidly inducible model to study cilia, the proposal will obtain novel insight into cilia physiology, promoting the understanding of cilia-related diseases.