Karen P. Wedaman

Movement of motor and cargo along cilia

Intraflagellar transport (IFT) is important in the formation and maintenance of many cilia, such as the motile cilia that drive the swimming of cells and embryos, the nodal cilia that generate left-right asymmetry in vertebrate embryos, and the sensory cilia that detect sensory stimuli in some animals. The heterotrimeric kinesin-II motor protein drives the anterograde transport of macromolecular complexes, called rafts, along microtubule tracks from the base of the cilium to its distal tip, whereas cytoplasmic dynein moves the rafts back in the retrograde direction. We have used fluorescence microscopy to visualize for the first time the intracellular transport of a motor and its cargo in vivo. We observed the anterograde movement of green fluorescent protein (GFP)-labelled kinesin-II motors and IFT rafts within sensory cilia on chemosensory neurons in living Caenorhabditis elegans.

Regulation of Ceramide Biosynthesis by TOR Complex 2

Ceramides and sphingoid long-chain bases (LCBs) are precursors to more complex sphingolipids and play distinct signaling roles crucial for cell growth and survival. Conserved reactions within the sphingolipid biosynthetic pathway are responsible for the formation of these intermediates. Components of target of rapamycin complex 2 (TORC2) have been implicated in the biosynthesis of sphingolipids in S. cerevisiae; however, the precise step regulated by this complex remains unknown. Here we demonstrate that yeast cells deficient in TORC2 activity are impaired for de novo ceramide biosynthesis both in vivo and in vitro. We find that TORC2 regulates this step in part by activating the AGC kinase Ypk2 and that this step is antagonized by the Ca2+/calmodulin-dependent phosphatase calcineurin. Because Ypk2 is activated independently by LCBs, the direct precursors to ceramides, our data suggest a model wherein TORC2 signaling is coupled with LCB levels to control Ypk2 activity and, ultimately, regulate ceramide formation.

Yeast TOR signaling: a mechanism for metabolic regulation.

Understanding how cell growth is regulated in response to environmental signals remains a challenging biological problem. Recent studies indicate the TOR (target of rapamycin) kinase acts within an intracellular regulatory network used by eukaryotic cells to regulate their growth according to nutrient availability. This network affects all aspects of gene expression, including transcription, translation, and protein stability, making TOR an excellent candidate as a global regulator of cellular activity. Here we review our recent studies of two specific transcriptional outputs controlled by TOR in the budding yeast, S. cerevisiae: (1) positive regulation of genes involved in ribosome biogenesis, and (2) negative regulation of genes required for de novo biosynthesis of glutamate and glutamine. These studies have raised the important issue as to how diverse nutritional cues can pass through a common signaling pathway and yet ultimately generate distinct transcriptional responses.

8 Tor-signaling and Tor-interacting proteins in yeast

The Tor (target of rapamycin) signaling pathway is an important mechanism used by eukaryotic cells to regulate their growth in response to nutrient-related environmental cues. Recent studies have revealed that the two Tor kinases in S. cerevisiae, Tor1p and Tor2p, regulate gene expression at several levels, including transcription, translation, intracellular protein trafficking, as well as protein stability. How the activity of each kinase is controlled remains to be elucidated, however, as does the nature of potential upstream regulatory signals. Here we review recent efforts to address these issues by focusing on two areas related to Tor signaling: (1) transcriptional control of genes required for the de novo biosynthesis of glutamate and glutamine and (2) characterization of interacting partners of Tor1p and Tor2p. These studies have converged in unanticipated ways to yield new insights into how these kinases may function both to receive as well as transmit nutritional information in yeast.