Ensconsin is a conserved microtubule-associated protein (MAP) that interacts dynamically with microtubules, but its cellular function has remained elusive. We show that Drosophila ensconsin is required for all known kinesin-1-dependent processes in the polarized oocyte without detectable effects on microtubules. ensconsin is also required in neurons. Using a single molecule assay for kinesin-1 motility in Drosophila ovary extract, we show that recruitment to microtubules and subsequent motility is severely impaired without ensconsin. Ensconsin protein is enriched at the oocyte anterior and apically in polarized epithelial cells, although required for localization of posterior determinants. Par-1 is required for ensconsin localization and directly phosphorylates it at conserved sites. Our results reveal an unexpected function of a MAP, promoting productive recruitment of a specific motor to microtubules, and an additional level of kinesin regulation. Furthermore, spatial control of motor recruitment can provide additional regulatory control in Par-1 and microtubule-dependent cell polarity.

How a given Ras prreotein coordinates multiple signaling inputs and outputs is a fundamental issue of signaling specificity. Schizosaccharomyces pombe contains one Ras, Ras1, that has two distinct outputs. Ras1 activates Scd1, a presumptive guanine nucleotide exchange factor (GEF) for Cdc42, to control morphogenesis and chromosome segregation, and Byr2, a component of a mitogen-activated protein kinase cascade, to control mating. So far there is only one established Ras1 GEF, Ste6. Paradoxically, ste6 null (ste6⌬) mutants are sterile but normal in cell morphology. This suggests that Ste6 specifically activates the Ras1-Byr2 pathway and that there is another GEF capable of activating the Scd1 pathway. We thereby characterized a potential GEF, Efc25. Genetic data place Efc25 upstream of the Ras1-Scd1, but not the Ras1-Byr2, pathway. Like ras1⌬ and scd1⌬, efc25⌬ is synthetically lethal with a deletion in tea1, a critical element for cell polarity control. Using truncated proteins, we showed that the C-terminal GEF domain of Efc25 is essential for function and regulated by the N terminus. We conclude that Efc25 acts as a Ras1 GEF specific for the Scd1 pathway. While ste6 expression is induced during mating, efc25 expression is constitutive. Moreover, Efc25 overexpression renders cells hyperelongated and sterile; the latter can be rescued by activated Ras1. This suggests that Efc25 can recruit Ras1 to selectively activate Scd1 at the expense of Byr2. Reciprocally, Ste6 overexpression can block Scd1 activation. We propose that external signals can partly segregate two Ras1 pathways by modulating GEF expression and that GEFs can influence how Ras is coupled to specific effectors.Ras G proteins act as molecular switches for signal transduction pathways that are important for cell proliferation, differentiation, cell death, and organization of the cytoskeleton (reviewed in reference 29). In humans, there are three RAS genes (H-, K-, and N-RAS) which encode four Ras proteins with more than 90% identity in amino acid sequence. The biochemical properties of these Ras proteins are very similar and straightforward. Ras can bind either GTP or GDP. In the resting state of the cell, Ras is primarily GDP bound and inactive. In response to signals, Ras switches to the active GTP-bound state, a process catalyzed by guanine nucleotide exchange factors (GEFs). Activated Ras stimulates effector proteins to turn on downstream pathways. How a given Ras protein functions in the cell, however, is anything but straightforward. By one count, there are at least three Ras effectors (Raf, phosphatidylinositol 3-kinase, and Ral GDS; reviewed in reference 29) and three families of GEFs containing at least five members (Sos1, Sos2, GRF1/Cdc25Mm, GRF2, and GRP [2]). Under in vitro conditions, most known Ras effectors and GEFs can frequently interact with more than one Ras protein, but how they actually match up with one another in the cell is poorly understood.We use the fission yeast Schizosaccharomyces pombe as a genetic model organism to study ...

In fission yeast, Scd1͞Ral1 is a putative guanine nucleotide exchange factor for Cdc42sp and also acts as a Ras1 effector necessary for the regulation of cytoskeleton organization. In this study, we have characterized a protein, Moe1, that binds directly to Scd1. A moe1 null (⌬) mutant exhibits numerous phenotypes indicative of abnormal microtubule functioning, including an abnormality in the spindle. moe1⌬ mutants are resistant to microtubule destabilizing agents; moreover, moe1⌬ rescued the growth defects of tubulin mutants containing unstable microtubules. These results suggest that Moe1 induces instability in microtubules. Biochemical and subcellular localization studies suggest that Moe1 and Scd1 colocalize in the nucleus. Furthermore, loss of function in Scd1 or Ras1 also induced abnormality in the spindle and is synthetically lethal with moe1⌬ producing cells that lack a detectable spindle. These data demonstrate that Moe1 is a component of the Ras1 pathway necessary for proper spindle formation in the nucleus. Human and nematode Moe1 both can substitute for yeast Moe1, indicating that the function of Moe1 in spindle formation has been conserved substantially during evolution.

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