2. You are examining the behavior of two centrosomal proteins Centrin and Cep192

Question

2. You are examining the behavior of two centrosomal proteins Centrin and Cep192 by FRAP analysis. Both proteins are expressed as GFP-tagged forms and localize to the centrosome (the centrosome is indicated by red the arrow, the plasma membrane is indicated by the dashed line). p.b. is before bleaching, the subsequent images show time points during recovery from photobleaching (time is indicated in minutes and the time course shows the fluorescent signal at the centrosome over time, always focusing on the centrosome). Which of the following conclusions can you draw from this experiment? The legend to this figure as described in the research paper by Zhu et al., 2008. Current Biology 18, 136-141) FRA analysis Hela cel expressing GFP4 ged mu ne Centrin-2 o muri e Cep182. I a es ere taken be re p.b.) and immediately a tot bleaching 0) and every 2 min a e ards. I sets s o 6-fold na ni ations of the cen roso na e ion. The scale bar e resents 10 n (1 point) Centrin-GFP O Both, Centrin and Cep192 are stably associated with the centrosome and do not exchange with cytosolic subunits. O Centrin-GFP displays a more dynamic behavior than Cep192 o Cep192-GFP is more dynamic than Centrin Cep192-GFP The dynamic behavior of Cep192 and centrin is similar O The dynamic behavior of Cep192 depends on the cell cycle stage

Explanation / Answer

Cep192 is a human centrosomal protein that plays an important role in centriole duplication and centrosomal maturation. Centrin is a stable centriolar protein is always associated with the centriole.

In fluorescence recovery after photobleaching (FRAP), there is irreversible bleaching of fluorescent probes with high intensity light. Post bleaching, recovery of fluorescence due to movement of surrounding intact probes into the bleached spot is monitored. Hence, first the specific cells are labeled with a fluorescent probe. The cell is images, then photobleached and the recovery of fluorescence over time is imaged. In the experiment, the 0 hr time will be the image before photobleaching. The expression of both centrin-GFP and Cep192-GFP can be seen. After photobleaching, the expression of fluorescently tagged Centrin-GFP is not recovered, indicating no movement of the intact fluorescent probes. However, after photobleaching, the expression of Cep192 GFP can be recovered. At 16, 18 and 20 minutes, Cep192 is possibly seen at the two ends of mitotic spindle.

This study indicates that centrin is stably associated with the centriolar region. Hence, it does not move to cause recovery of the fluorescent probe. On the other hand, Cep-GFP is very mobile, being associated with different regions of the centriole. Hence, it is in dynamic equilibrium with its cytoplasmic pool. As the stage of the cell cycle is not monitored, it cant be said that the dynamic behavior of cep192 depends on the cell cycle stage.

Option is : Cep192-GFP is more dynamic than centrin.

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