We report a motiliy in the flagella of the green alga Chlamydomonas that is unrelated to dynein-based flgelar beating. This motility, referred to as intraflageUar transport, was observed as the rapid bidirectional movement of granule-like particles along the length of the ftgella. IntraflageUar transport could be experimentally separated from other, previously reported, nonbeat fgellar motilities. EM of flageUla showed groups of nonvesicular, lollipop-shaped structures positioned between the outer doublet microtubules and the flagellar membrane. Movement of these complexes along the length of the flagella may be responsible for intraflagellar transport. While reexamining these three motilities with videoenhanced differential interference-contrast (DIC) microscopy, we were surprised to find a fourth, previously unobserved, nonbeat motility within the flagella of Chlamydomonas. In prior light microscopic studies, the flagella of Chlamydomonas have always been observed as relatively featureless high-contrast rods. Therefore, the visualization of granule-like particles moving bidirectionally along the length ofthe flagella, apparently between the microtubular axoneme and flagellar membrane, was striking. In addition, improved fixation methods for EM have allowed for the routine observation, in thin section, of complexes between the flagellar membrane and the axonemal microtubules. The movement of these complexes may account for this motility, referred to as intraflagellar transport (IFT). for video-enhanced DIC microscopy because their flagella are =50%o longer than those of C. reinhardtii. The paralyzed flagellar dynein triple-mutant ida2 ida4 oda6 of C. reinhardtii was from R. Kamiya (Nagoya University). Logarithmicphase, synchronously dividing cultures were grown in minimal medium (MI) (8) on a 12 hr:12 hr light/dark cycle with continuous aeration. MATERIALS AND METHODSFlagellar Regeneration and Resorption. Flagellar regeneration was induced by pH-shock (9). After deflagellation, the cells were washed with fresh MI medium. Flagellar resorption was induced by placing the cells in a low-Ca2+, high-Na+ medium (10) or also, with C. reinhardtii, by adding 3-isobutyl-l-methylxanthine to MI medium at a final concentration of 0.5 mM (11).Video Microscopy. For all experiments, cultures were diluted 1:5 with double-distilled water and placed between two acid-washed no. 1 coverslips (Corning) supported with 1-mm plastic shims affixed with Vaseline. When required, polystyrene beads with a 0.3-,&m diameter (Polysciences), washed four times with double-distilled water, were mixed with the cells to a final dilution of 1:500. Dilution of the culture medium enhanced the attachment of beads to the flagellar membrane. All experiments were done at room temperature using a Zeiss Axiovert microscope. The optics and method ofdigital data acquisition have been detailed (12), except that the light source used in this study was passed through a fiber optic scrambler to obtain full and even illumination of the condenser (1.4 N.A.) ...

Abstract. The Chlamydomonas FLAIO gene was shown to encode a flagellar kinesin-like protein (Walther, Z., M. Vashishtha, and J. L. Hall. 1994. J. Cell Biol. 126:175-188). By using a temperature-sensitive allele of FLAIO, we have determined that the FLA10 protein is necessary for both the bidirectional movement of polystyrene beads on the flagellar membrane and intraflagellar transport (IFT), the bidirectional movement of granule-like particles beneath the flagellar membrane (Kozminski, K. G., K. A. Johnson, P.Forscher, and J. L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. (USA). 90:5519-5523). In addition, we have correlated the presence and position of the IFT particles visualized by light microscopy with that of the electron dense complexes (rafts) observed beneath the flagellar membrane by electron microscopy. A role for FLA10 in submembranous or flagellar surface motility is also strongly supported by the immunolocalization of FLA10 to the region between the axonemal outer doublet microtubules and the flagellar membrane.

Polarized delivery and incorporation of proteins and lipids to specific domains of the plasma membrane is fundamental to a wide range of biological processes such as neuronal synaptogenesis and epithelial cell polarization. The exocyst complex is specifically localized to sites of active exocytosis and plays essential roles in secretory vesicle targeting and docking at the plasma membrane. Sec3p, a component of the exocyst, is thought to be a spatial landmark for polarized exocytosis. In a search for proteins that regulate the localization of the exocyst in the budding yeast Saccharomyces cerevisiae, we found that certain cdc42 mutants affect the polarized localization of the exocyst proteins. In addition, we found that these mutant cells have a randomized protein secretion pattern on the cell surface. Biochemical experiments indicated that Sec3p directly interacts with Cdc42 in its GTP-bound form. Genetic studies demonstrated synthetically lethal interactions between cdc42 and several exocyst mutants. These results have revealed a role for Cdc42 in exocytosis. We propose that Cdc42 coordinates the vesicle docking machinery and the actin cytoskeleton for polarized secretion.

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein–protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express ∼90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein–protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

Oxysterol-binding protein (OSBP)-related protein Kes1/Osh4p is implicated in nonvesicular sterol transfer between membranes in Saccharomyces cerevisiae. However, we found that Osh4p associated with exocytic vesicles that move from the mother cell into the bud, where Osh4p facilitated vesicle docking by the exocyst tethering complex at sites of polarized growth on the plasma membrane. Osh4p formed complexes with the small GTPases Cdc42p, Rho1p and Sec4p, and the exocyst complex subunit Sec6p, which was also required for Osh4p association with vesicles. Although Osh4p directly affected polarized exocytosis, its role in sterol trafficking was less clear. Contrary to what is predicted for a sterol-transfer protein, inhibition of sterol binding by the Osh4p Y97F mutation did not cause its inactivation. Rather, OSH4 Y97F is a gain-of-function mutation that causes dominant lethality. We propose that in response to sterol binding and release Osh4p promotes efficient exocytosis through the co-ordinate regulation of Sac1p, a phosphoinositide 4-phosphate (PI4P) phosphatase, and the exocyst complex. These results support a model in which Osh4p acts as a sterol-dependent regulator of polarized vesicle transport, as opposed to being a sterol-transfer protein.