C-Terminal Motifs of the MTW1 Complex Cooperatively Stabilize Outer Kinetochore Assembly in Budding Yeast.

Kinetochores are macromolecular protein assemblies at centromeres that mediate accurate chromosome segregation during cell division. The outer kinetochore KNL1SPC105, MIS12MTW1, and NDC80NDC80 complexes assemble the KMN network, which harbors the sites of microtubule binding and spindle assembly checkpoint signaling. The buildup of the KMN network that transmits microtubule pulling forces to budding yeast point centromeres is poorly understood. Here, we identify 225 inter-protein crosslinks by mass spectrometry on KMN complexes isolated from Saccharomyces cerevisiae that delineate the KMN subunit connectivity for outer kinetochore assembly. C-Terminal motifs of Nsl1 and Mtw1 recruit the SPC105 complex through Kre28, and both motifs aid tethering of the NDC80 complex by the previously reported Dsn1 C terminus. We show that a hub of three C-terminal MTW1 subunit motifs mediates the cooperative stabilization of the KMN network, which is augmented by a direct NDC80-SPC105 association.

Increased expression of the coxsackie and adenovirus receptor downregulates αvß3 and αvß5 integrin expression and reduces cell adhesion and migration.

AIMS: Coxsackie and adenovirus receptor (CAR) is a tumor suppressor and a primary receptor for adenovirus type 5 (Ad5). Our study aims to examine the influence of forced expression of CAR in rhabdomyosarcoma cells (RD) on expression levels of integrins implicated in Ad5 entry, and the effect of CAR on cell-extracellular matrix adhesion and migration. MAIN METHODS: CAR expressing clones were established from RD cells by stable transfection. Flow cytometry was used to evaluate the expression of CAR and integrins. Adhesion was measured in plates previously coated with vitronectin or fibronectin. Boyden chambers were used to investigate migration. Transfection of cells with siRNA was used to achieve integrin silencing. Ad5-mediated transgene expression was measured by ß-gal staining. KEY FINDINGS: Increased expression of CAR in RD cells reduces the expression of αvß3 and αvß5 integrins. Cells overexpressing CAR exhibit significantly reduced adhesion to vitronectin and fibronectin, and reduced cell migration. Specifically silencing αvß3 integrin in RD cells reduced cell migration indicating that reduced migration could be the consequence of αvß3 integrin downregulation. This study also demonstrates the negative effect of reduced levels of αvß3 and αvß5 integrins on Ad5-mediated transgene expression with Ad5 retargeted to αv integrins. SIGNIFICANCE: The pharmacological upregulation of CAR aimed to increase Ad5-mediated transgene expression may actually downregulate αvß3 and αvß5 integrins and thus alter Ad5-mediated gene transfer. The mechanism of decreased cell migration, a prerequisite for metastasis and invasion, due to increased CAR expression may be explained by reduced αvß3 integrin expression.