Modulation of nucleocytoplasmic trafficking by retention in cytoplasm or nucleus

Abstract

Nuclear protein transport processes have largely been studied using in vitro semi-intact cell systems where high concentrations of nuclear localizing substrates are used, and cytoplasmic components such as the microtubule (MT) network, are either absent or damaged. Here we use the fluorescence recovery after photobleaching (FRAP) technique to analyze the nucleocytoplasmic flux of distinct fluorescently tagged proteins over time in living cultured cells. FRAP was performed in different parts of the cell to analyze the kinetics of nucleocytoplasmic trafficking and intranuclear/cytoplasmic mobility of the tumor suppressor Rb protein and a SV40 large tumor antigen (T-ag) derivative containing the nuclear localization sequence (NLS), both fused to green fluorescent protein (GFP). The results indicate that proteins carrying the T-ag NLS are highly mobile in the nucleus and cytoplasm. Rb, in contrast, is largely immobile in both cellular compartments, with similar nuclear import and export kinetics. Rb nuclear export was CRM-1-mediated, with its reduced mobility in the cytoplasm in part due to association with MTs. Overall our results show that nuclear and cytoplasm retention modulates the rates of nuclear protein import and export in intact cells.

70-kDa Heat Shock Cognate Protein hsc70 Mediates Calmodulin-dependent Nuclear Import of the Sex-determining Factor SRY

“…Image analysis was performed using NIH ImageJ software as described above. Data were fitted to a sigmoidal curve and the Fn/c max and initial rates (from the first linear portion of the recovery curve) were determined using Sigmaplot software, as described previously (17)(18)(19).…”

“…Image analysis was performed using ImageJ as described above. Results were expressed as the fractional recovery of Fn/c (Fn/c of respective time points divided by prebleach value), and data were fitted exponentially according to the formula y ϭ a(1Ϫe Ϫbx ) as described (17)(18)(19)(20) to determine the maximal recovery and halftime (t1 ⁄ 2 ). The initial rate was determined using results for the Fn/c between 0 and 100 s post-bleaching.…”

“…Taken together, these results indicate that hsc70 is required for CaM-dependent nuclear import of SRY in intact cells. The role of hsc70 in enhancing SRY nuclear import in living cells was assessed using the established FRAP technique (18). Briefly, an area corresponding to 50% of the nucleus of cells transfected to express full length GFP-SRY fusion protein in the absence or presence of hsc70 siRNA was photobleached, and the return of nuclear fluorescence from the translocation of unbleached, cytoplasmic fluorescent protein into the nucleus monitored by CLSM for up to 280 s (see Fig.…”

Roles of nuclear trafficking in infection by cytoplasmic negative-strand RNA viruses: paramyxoviruses and beyond

“…RABV P3) proteins Moseley et al, 2007bMoseley et al, , 2009Roth et al, 2007). Analogous regulable interactions of proteins with intranuclear structures such as cellular genomic DNA and nuclear bodies can provide another means to control protein localization within the nuclear compartment (Bernardi et al, 2004;Roth et al, 2009).…”

Mechanism of Microtubule-facilitated “Fast Track” Nuclear Import

“…Image analysis was performed as above to determine the Fn and Fc values at all time intervals during the photobleaching experiment. Results were then expressed as fractional recovery of Fn/c (Fn/c of respective time points divided by the corresponding prebleach value), and data were fitted exponentially according to the formula y ϭ a(1 Ϫ e Ϫbx ) as described previously (9,16,22,(27)(28)(29)(30)(31) to determine the maximal recovery and half-time (t1 ⁄ 2 ). Results for Fc between 20 and 120 s were used to determine the rate of cytoplasmic decay (31).…”