Nuclear transport receptors depleted cytosol marginally reduces the mobility of

From the previous experiments, it was observed that RanQ69L and Impb (45-462) had a significant effect on the diffusional mobility of emerin to the NE. Since both of these factors contribute to NTR (nuclear transport receptor)-mediated NPC translocation (Ribbeck and Görlich, 2002b), the effect of NTRs on the mobility of emerin was determined. NTRs have hydrophobic properties that assist them to pass through the NPCs. These can be enriched by a hydrophobic interaction column, phenyl-sepharose (Ribbeck and Görlich, 2002b). To deplete NTRs, HeLa cytosol was subjected to binding to phenyl-sepharose. The efficiency of depletion was monitored by Western blotting for Importins b, 11, 13, 7 and Transportin 1 (Figure 28A). FRAP assays were performed using cytosol depleted of NTRs in semi-permeabilized cells. As shown in (Figure 28B and C), the depleted cytosol showed a reduced effect (20%) compared to cytosol with no depletion (30%), but the mobility was not as low as that observed in digitonin treated cells (less than 20%). Thus, NTRs depletion only has a marginal effect on the mobility of emerin to the NE.

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Figure 28. NTRs depleted cytosol has a reduced effect on the diffusion of emerin to the NE.

(A) Cytosol was depleted of nuclear transport factors (NTRs) by phenyl-sepharose. The efficacy of depletion was monitored by Western blotting using antibodies against Impb, Imp11, Imp 13, Imp7 and TNPO1. GAPDH was used as a loading control. (B) FRAP assays were performed on intact (- digitonin) and semi-permeabilized (+ digitonin) HeLa cells expressing mCherry-emerin using this depleted cytosol or untreated cytosol (+cytosol; +digitonin). Images were analyzed by a confocal microscope. The bleached areas are represented by circles. The scale bar corresponds to 10 µm. (C) The curve shows the normalized fluorescence intensities with error bars indicating the standard deviation from the mean of a total of 12 cells from two independent experiments per condition.

Cytosol supplementation affects the diffusional mobility of emerin in the ER

The differences in the mobility of emerin in the NE upon addition of cytosol and cytosolic factors prompted us to check for its mobility in the ER. FRAP assays were performed on cells that were transiently transfected with mCherry-emerin, permeabilized and incubated in the presence or absence of cytosol. As shown in Figure 29, in intact cells recovery of nearly 60% was observed, whereas in semi-permeabilized cells only 20%

recovery was obtained. This was in line with the NE targeting defect of emerin in semi-permeabilized cells. Next, cytosol was added to the semi-permeabilized cells to assess changes in mobility of emerin in the ER. Interestingly, the addition of cytosol slightly increased the recovery rate in semi-permeabilized cells to 30%. This suggests that the addition of soluble

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factors indeed affect the diffusional mobility of emerin in the ER. Additional experiments will have to be performed to monitor what factors in the cytosol are responsible for this effect.

Figure 29. Mobility of emerin in the ER measured by FRAP.

(A) HeLa cells transfected with mCherry-emerin were subjected to FRAP assays in the ER. The regions that were bleached are represented by circles. The recovery of emerin was monitored over time in intact (- digitonin) and semi-permeabilized cells (+ digitonin), which were treated in the presence or absence of cytosol (+ cytosol; + digitonin). Images were analyzed by a confocal microscope. The scale bar corresponds to 10 µm. (B) Quantification of normalized intensity at the ER under the conditions as mentioned in (A) with error bars indicating the standard deviation from the mean of a total of 12 cells per experiment per condition.

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5.3. Discussion

This chapter addresses the dynamics of several INM proteins with both single and multiple TMDs using FRAP assays. Under the experimental conditions used in this work, all the tested proteins showed a percentage recovery of 40 to 60% in intact cells (Figure 18). For emerin and Lap2b, similar percentages were observed, which were consistent with previous reports (Ostlund et al., 1999; Shimi et al., 2004; Wu et al., 2002). LBR had a lower percentage of recovery, possibly due to its binding to lamins and heterochromatin proteins (Ellenberg et al., 1997; Ostlund et al., 2006). Lower recovery rates were similarly observed for VAPB and PTP1B, suggesting that their mobility at the NE could also be affected by binding to proteins at the INM (James et al., 2019; Yip et al., 2012).

In contrast to the high percentage of recovery monitored in intact cells, a lower recovery was observed in permeabilized cells for all the proteins tested (Figure 19). The measured fluorescence recovery may not only result from the exchange of proteins within the NE but also from transport of proteins from the ER. The effect observed due to permeabilization could result from the loss of cytosolic factors. Alternatively, the reduction in recovery may also result from an effect that might alter the ER topology. The long-range diffusional mobility of proteins in the ER network, and the efficiency of targeting to the INM is reduced when ER-topology is altered (Pawar et al., 2017). Interestingly, the addition of cytosol partially increased the recovery of emerin but not for other proteins tested. This suggests that diffusion of emerin is dependent on the presence of cytosolic factors and/or on a proper ER-topology, whereas diffusion of other tested proteins might be dependent solely on proper maintenance of the ER network. However, further investigations have to be performed to monitor the effect of proper maintenance of ER-topology on the diffusion of the proteins tested in this work.

It has been previously reported that the mobility of emerin to the NE requires ATP (Zuleger et al., 2011) and that emerin also binds to cytoplasmic partners, which might affect its release from the ER (Lattanzi et al., 2000; Salpingidou et al., 2007; Zuleger et al., 2011).

The observation that the addition of cytosolic factors led to an increase in fluorescence recovery at the NE may reflect an increase in the efficiency of diffusion of emerin. It was also observed that the addition of RanQ69L, WGA and Impb (45-462) that block nuclear import, impaired the diffusion of emerin to the NE, further suggesting that the decrease in recovery observed in permeabilized cells was indeed due to a lower rate of diffusion of emerin from the ER to the NE. A cytosol dependence has also been observed for an INM protein SUN2 (Ungricht et al., 2015), further raising the question of whether additional molecular requirements add to the diffusion and retention model for efficient targeting of INM proteins.

Chapter 6:

Discussion

: Discussion

In eukaryotes, TA proteins with important roles in diverse processes can be found in different organelles like the mitochondria, peroxisome, ER, Golgi, plasma membrane, endosomes, lysosomes (Borgese et al., 2007) and also NE (Blenski and Kehlenbach, 2019;

Pfaff et al., 2016). Two different TA proteins that localize to the INM were studied in this work. The first is VAPB, which is known as an ER-resident protein, and the second is emerin, which is a well-characterized INM protein.

The entry site for several TA proteins that reside in the membranes of the secretory pathway is the ER (Behrens et al., 1996; Kutay et al., 1995; Linstedt et al., 1995). Even though VAPB is well studied as an ER protein, the biogenesis of the protein is not entirely understood. To characterize the biogenesis of VAPB, the possible role of the TRC40 pathway in the membrane insertion of VAPB was analyzed.

In addition to its already established ER localization, localization of VAPB to the INM was observed for the first time. Moreover, the interactome of VAPB at the INM was established by a new proximity-based approach Rapamycin and APEX-dependent identification of proteins by SILAC (RAPIDS) and the interactome at the INM and ER was validated by this approach.

Requirements for targeting of newly synthesized TA proteins from the ER to the INM have been studied only for a few proteins. In this work, diffusional mobility of several INM proteins were assessed in intact and semi-permeabilized cells and emerin was one of the proteins that was studied further in detail.