The Use of Infrared Fluorescent Dyes in Immunofluorescence Microscopy

Abstract

AbstractCentrosomes are microtubule organizing centers that regulate assembly of the mitotic spindle apparatus (1). Excess centrosomes such as those observed in a variety of human tumors generate aberrant mitotic spindles, and the duplication of centrosomes must be tightly controlled (2). The Mps1 protein kinase localizes to centrosomes and is required for centrosome duplication (3–5). However, Mps1 has many cellular localizations including kinetochores (3, 6–8) and nuclear pores (7), and has additional functions related to the mitotic spindle assembly checkpoint (9, 10). Recent evidence suggests that centro-some duplication specifically requires the centrosomal Mps1 pool, and that the accumulation of Mps1 at centrosomes is regulated by mutually exclusive Cdk2 phosphorylation and proteasome-mediated degradation (5). However, different means of inhibiting Cdk2 activity have different consequences for the centrosomal and whole-cell pools of Mps1 (5). Both inhibition of Cdk2 with the small molecule roscovitine and the depletion of Cdk2 activity with cyclin A-specific siRNAs leads to the loss of Mps1 from centrosomes. However, while Mps1 can be almost completely depleted from cells by roscovitine (3, 5), whole-cell Mps1 levels are virtually unchanged by cyclin A-specific siRNAs (5). In contrast, while overexpression of cyclin A only modestly increases in whole-cell Mps1 levels, it causes a 2.5-fold increase in centrosomal Mps1 levels that is sufficient to cause the over production of centrosomes during S-phase arrest (5).KeywordsMitotic SpindleCentrosome DuplicationInhibit Cdk2 ActivityDark Humidify ChamberRound Glass CoverslipThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.