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Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes
Published on: December 20, 2014
Golgi-Associated Protein JAKMIP2 Is Linked to the Centrosome and Performs Microtubule-Related Functions
Evgeniia Ulas1,2, Ilya Brodsky1, Petros Padaryan1,3
1A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia.
None:
Microtubules are present in all eukaryotes, and their spatial organization in the cells depends on the function of microtubule-organizing centers (MTOCs). Various organelles may act in this capacity, including the centrosome, Golgi, nuclear envelope, endosomes, and others. The molecular mechanisms that facilitate microtubule nucleation and/or anchoring at MTOCs are diverse. Many proteins can participate in these processes while localized to different MTOCs-either simultaneously on several or alternately on each of them. Here we studied the Golgi-associated protein JAKMIP2 in various cells using the methods of fluorescent multichannel confocal microscopy with subsequent image analysis via our own algorithm, transfection with a genetic construct encoding a fused protein of interest, and microtubule recovery monitoring during nocodazole washout. We demonstrated for the first time that JAKMIP2 is present on centrosomes in various cells. We also found that its abundance at this location is dependent on the cell cycle stage. Furthermore, we showed that an excess of JAKMIP2 specifically impairs centrosome function as a MTOC. Finally, our data indicates that exogenous JAKMIP2 slows down centrosomal microtubule nucleation and may also affect their anchoring. Our findings make a new contribution to the existing knowledge of the molecular mechanisms of the centrosome's function as a MTOC.
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