CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25.

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.

Immunity in heifers 12 months after vaccination with a multivalent vaccine containing a United States Leptospira borgpetersenii serovar Hardjo isolate.

OBJECTIVE: To evaluate immunity induced by a multivalent vaccine containing a US Leptospira borgpetersenii serovar Hardjo type hardjo bovis (LHB) isolate in heifers challenged 12 months after vaccination. DESIGN: Prospective vaccine challenge study. ANIMALS: 36 one-month old Holstein heifers. PROCEDURES: 18 heifers were vaccinated at 4 and 8 weeks of age with an inactivated vaccine containing Leptospira fractions. Additionally, 18 heifers were vaccinated at the same age with the same vaccine without any Leptospira fractions. All heifers were challenged with a US-origin LHB 12 months following booster vaccination. Urine samples were collected weekly for 8 weeks after challenge, and serum was collected at -1, 28, and 56 days after challenge for serologic testing. At 8 weeks after challenge, all heifers were necropsied, and kidney and reproductive system samples were collected for bacteriologic culture. RESULTS: 4 of 18 vaccinates had positive results of bacteriologic culture of urine samples, but only at 1 time point. All control heifers had positive results of bacteriologic culture of urine samples for at least 5 time points. Vaccinates had negative results of bacteriologic culture of kidney and reproductive system samples following necropsy, whereas all control heifers had positive results of bacteriologic culture of kidney samples and 5 of 18 had positive results of bacteriologic culture of reproductive system samples. CONCLUSIONS AND CLINICAL RELEVANCE: The vaccine administered to calves at 1 month of age prevented leptospire colonization of kidney and reproductive system tissue and significantly reduced urine shedding following challenge 12 months after vaccination. This vaccine provides an opportunity to protect calves at an early age from becoming infected and ultimately from becoming an LHB reservoir.

Cardiac-specific deletion of the microtubule-binding protein CENP-F causes dilated cardiomyopathy.

CENP-F is a large multifunctional protein with demonstrated regulatory roles in cell proliferation, vesicular transport and cell shape through its association with the microtubule (MT) network. Until now, analysis of CENP-F has been limited to in vitro analysis. Here, using a Cre-loxP system, we report the in vivo disruption of CENP-F gene function in murine cardiomyocytes, a cell type displaying high levels of CENP-F expression. Loss of CENP-F function in developing myocytes leads to decreased cell division, blunting of trabeculation and an initially smaller, thin-walled heart. Still, embryos are born at predicted mendelian ratios on an outbred background. After birth, hearts lacking CENP-F display disruption of their intercalated discs and loss of MT integrity particularly at the costamere; these two structures are essential for cell coupling/electrical conduction and force transduction in the heart. Inhibition of myocyte proliferation and cell coupling as well as loss of MT maintenance is consistent with previous reports of generalized CENP-F function in isolated cells. One hundred percent of these animals develop progressive dilated cardiomyopathy with heart block and scarring, and there is a 20% mortality rate. Importantly, although it has long been postulated that the MT cytoskeleton plays a role in the development of heart disease, this study is the first to reveal a direct genetic link between disruption of this network and cardiomyopathy. Finally, this study has broad implications for development and disease because CENP-F loss of function affects a diverse array of cell-type-specific activities in other organs.

Murine CENPF interacts with syntaxin 4 in the regulation of vesicular transport.

Syntaxin 4 is a component of the SNARE complex that regulates membrane docking and fusion. Using a yeast two-hybrid screen, we identify a novel interaction between syntaxin 4 and cytoplasmic murine CENPF, a protein previously demonstrated to associate with the microtubule network and SNAP-25. The binding domain for syntaxin 4 in CENPF was defined by yeast two-hybrid assay and co-immunoprecipitation. Confocal analyses in cell culture reveal a high degree of colocalization between endogenously expressed proteins in interphase cells. Additionally, the endogenous SNARE proteins can be isolated as a complex with CENPF in immunoprecipitation experiments. 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 dominant-negative 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.

Cytoplasmic LEK1 is a regulator of microtubule function through its interaction with the LIS1 pathway.

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.