Investigating The Spatiotemporal Distribution Patterns of PCID2 between The Nucleus And The Centrosome

Abstract

PCID2 is a subunit of the TREX‐2 mRNA export complex that is also involved in the process of CRM1‐mediated nuclear protein export. Nuclear export is driven by RanGTP, which triggers a structural modification of CRM1 resulting in increased affinity for the leucine‐rich NES. Subsequent GTP hydrolysis following translocation across the NPC causes the CRM1‐cargo complex to dissociate. Karyopherins can also serve functional roles at non‐nuclear locations in the cell. For example, both CRM1 and Ran are found at the centrosome in addition to the nucleus. Specific factors facilitate mitosis by restricting the centrosome to duplicate once during the cell cycle through alternating nuclear and centrosomal localization. Specifically, CRM1 has an integral role in localizing nucleophosmin to the centrosomes to facilitate this “licensing” process. Leptomycin B inhibition of CRM1 causes dissociation of nucleophosmin from the centrosomes. In doing so it leads to centrosome reduplication within the same cell cycle. In addition to its nuclear function, PCID2 is also localized to the centrosome in a subset of cells. The function of PCID2 at the centrosome is not yet elucidated, but we demonstrate here that PCID2 is in fact present in CRM1 complexes. This evidence gives us reason to believe that PCID2 may serve a role at the centrosome that is supplementary to that of CRM1 and Ran. We hypothesize that PCID2 is localized to the centrosome as a part of the CRM1‐RanGTP complex and may translocate from the nucleus to the centrosome. To investigate the relationship between PCID2 in the nucleus and at the centrosome, we performed immunofluorescence of PCID2 in HeLa cells treated with Leptomycin B, a CRM1‐dependent nuclear export inhibitor. Preliminary results shows that LMB treatment causes a decrease in nuclear intensity of CRM1 and a potential decrease in PCID2, while causing an increase in the incidence of cells exhibiting punctate staining of PCID2. Because we know that LMB treatment leads to unregulated duplication of centrosomes, these findings suggest a correlation between increased PCID2 punctate staining and multiple centrosome duplications. This is consistent with our previous work showing that PCID2 is most commonly localized to the centrosome in G2 when duplication is occurring. These findings could also imply that PCID2 has a role in the localization of centrosomal components or mitotic proteins to the centrosome from the nucleus. Future experiments will investigate whether increased PCID2 punctate staining is indicative of a role in excess centrosome duplication or whether LMB is instead altering the CRM1/PCID2 localization pattern, causing centrosomal accumulation of PCID2.