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LIS1 and nuclear distribution gene E (NudE) are partner proteins in a conserved pathway regulating the function of dynein and microtubules. Here, we present data that cytoplasmic LEK1 (cytLEK1), a large protein containing a spectrin repeat and multiple leucine zippers, is a component of this pathway through its direct interaction with NudE, as determined by a yeast two-hybrid screen. We identified the binding domains in each molecule, and coimmunoprecipitation and colocalization studies confirmed the specificity of the interaction between cytLEK1 and NudE. Confocal deconvolution analysis revealed that cytLEK1 exhibits colocalization with endogenous NudE and with the known NudE binding partners, LIS1 and dynein. By localizing the NudE-binding domain of cytLEK1 to a small domain within the molecule, we were able to disrupt cytLEK1 function by using a dominant negative approach in addition to LEK1 knockdown and, thus, examine the role of the cytLEK1-NudE interaction in cells. Consistent with a defect in the LIS1 pathway, disruption of cytLEK1 function resulted in alteration of microtubule organization and cellular shape. The microtubule network of cells became tightly focused around the nucleus and resulted in a rounded cell shape. Additionally, cells exhibited a severe inability to repolymerize their microtubule networks after nocodazole challenge. Taken together, our studies revealed that cytLEK1 is essential for cellular functions regulated by the LIS1 pathway.cytoskeleton

SNAP-25 is a component of the SNARE complex that is involved in membrane docking and fusion. Using a yeast two-hybrid screen, we identify a novel interaction between SNAP-25 and cytoplasmic Lek1 (cytLEK1), a protein previously demonstrated to associate with the microtubule network. The binding domains within each protein were defined by yeast two-hybrid, coimmunoprecipitation, and colocalization studies. Confocal analyses reveal a high degree of colocalization between the proteins. In addition, the endogenous proteins can be isolated as a complex by immunoprecipitation. Further analyses demonstrate that cytLEK1 and SNAP-25 colocalize and coprecipitate with Rab11a, myosin Vb, VAMP2, and syntaxin 4, components of the plasma membrane recycling pathway. Overexpression of the SNAP-25-binding domain of cytLEK1, and depletion of endogenous Lek1 alters transferrin trafficking, consistent with a function in vesicle recycling. Taken together, our studies indicate that cytLEK1 is a link between recycling vesicles and the microtubule network through its association with SNAP-25. This interaction may play a key role in the regulation of the recycling endosome pathway.

Further analyses demonstrate that murine CENPF and syntaxin 4 colocalize with components of plasma membrane recycling: SNAP-25 and VAMP2. Depletion of endogenous CENPF disrupts GLUT4 trafficking whereas expression of a dominantnegative form of CENPF inhibits cell coupling. Taken together, these studies demonstrate that CENPF provides a direct link between proteins of the SNARE system and the microtubule network and indicate a diverse role for murine CENPF in vesicular transport. (Chen and Scheller, 2001;. Apposing SNARE proteins, which consist of vesicle-associated membrane proteins (VAMPs), plasma membrane-associated syntaxins, and cytoplasmic synaptosomal-associated proteins (SNAPs), form coiled-coil aggregates that are important in regulating membrane fusion events (Aikawa et al., 2006; Chen and Scheller, 2001;Martin et al., 1998;McMahon et al., 1993;Rowe et al., 1999). Plasma membrane trafficking of proteins between subcellular domains and translocation to the cell surface is mediated, in part, by SNARE proteins (Chen and Scheller, 2001;Jahn and Sudhof, 1999;Mallard et al., 2002;Sollner et al., 1993;Wilcke et al., 2000). The SNARE protein syntaxin 4 is an integral membrane protein that localizes to the plasma membrane and is essential in vesicular docking and fusion (Aikawa et al., 2006; Bajohrs et al., 2005; Band et al., 2002;Pooley et al., 2006). Specifically, syntaxins are critical in vesicular transport of GLUT4-containing vesicles in skeletal muscle, cardiomyocytes and adipose tissue after insulin stimulation (Bryant et al., 2002; Cain et al., 1992;Martin et al., 1996;Pessin et al., 1999). Determining the physical linkage of SNAREs to the microtubule network is essential for understanding the role of this cytoskeletal component in the myriad of trafficking events.In a recent study, we reported that murine CENPF physically associates with SNAP-25 (synaptosomal-associated protein 25), and together these proteins form a complex with Rab11a, myosin Vb, and VAMP2 in the recycling endosome pathway (Pooley et al., 2006). Furthermore, disruption of endogenous murine CENPF function by dominant-negative protein expression or protein knockdown severely retarded the recycling endosome network and transferrin trafficking (Chen and Scheller, 2001). Although this is the only report of CENPF regulating vesicular transport, the family has been shown to function with the cytoskeleton (Goodwin et al., 1999; Feng et al., 2006), and from this, we postulate that CENPF may have a extensive role in controlling SNARE-mediated vesicular transport by the microtubule network. Further data is crucial to establish the roles for CENPF in the diverse processes of vesicular transport.In the current study, using yeast two-hybrid (Y2H) and biochemical analyses, we demonstrate that syntaxin 4 and murine CENPF physically interact. These data are consistent with the hypothesis that murine CENPF is a critical component in the dynamic regulation of plasma membrane trafficking with the microtubule network through its interaction with SNARE ...

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Since the first description of a practical DNA amplification process many refinements have been described and automatic thermal cyclers have become standard laboratory equipment. PCR is now an essential tool for many biologists and the standard protocols are very simple and user friendly. The exponential amplification process provides nano gram quantities of

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