Regulation of proteostasis during cell division

Abstract

Misfolding and aggregation of proteins not only disrupt their functions but also perturb the functions of other proteins by co-aggregation. These processes challenge protein homeostasis (proteostasis) and subsequently lead to cellular aging and neurodegenerative disorders. To maintain proteostasis, cells utilize several protein quality control (PQC) systems including the regulation of protein synthesis, folding, degradation, and the spatial sequestration of misfolded protein aggregates. How cells employ PQC systems is extensively studied, however, less is known about how cells regulate their proteostasis during division.

To investigate how PQC systems maintain proteostasis in dividing cells, firefly luciferase with two destabilizing mutations (FlucDM), a live-cell proteostasis reporter, was fused to enhanced green fluorescent protein (eGFP) and expressed in mammalian cells undergoing symmetric division. Upon heat shock, FlucDM-eGFP formed protein aggregates in mitotic cells, indicating that the FlucDM system effectively reports proteostasis during cell division. To further explore how mitotic cells regulate proteostasis, small-molecule inhibitors were utilized to disrupt key elements of the proteostasis network and live-cell imaging was used to monitor the aggregation of FlucDM-eGFP. The results demonstrated that the 26S proteasome and heat shock protein 70 (Hsp70) played critical roles in eliminating misfolded proteins during mitosis. Moreover, both the attenuation of protein translation and the induction of endoplasmic reticulum (ER) stress relieved proteostasis stress in mitotic cells. Interestingly, the inhibitor of the 26S proteasome alone induced protein aggregates in interphase cells but not in mitotic cells, suggesting that mitotic cells possess a stronger ability to relieve proteostasis stress caused by proteasome inhibition.

Unexpectedly, in various mammalian cell lines, ER-targeted FlucDM-eGFP (ERFlucDM- eGFP) assembled into protein aggregates in the nucleus. Interestingly, these aggregates were encapsulated by the ER membrane, similar to the structural feature of the nucleoplasmic reticulum (NR). Similar protein aggregates also formed in cells overexpressing other ER-targeted proteins such as ER-targeted eGFP. However, either perturbation of ER homeostasis by small molecules or the overexpression of human pathological misfolding-prone proteins in the ER did not induce similar aggregates. Surprisingly, ER-FlucDM-eGFP aggregates no longer formed upon the knockdown of binding immunoglobulin protein (BiP), an ER-resident member of the Hsp70 chaperone family. Furthermore, immunofluorescence staining showed that BiP colocalized with these aggregates. This suggested that despite its expected role in disaggregating misfolded protein aggregates, BiP may facilitate the aggregation of misfolding-prone proteins. Moreover, aggregates in mitotic cells exhibited faster fluorescence recovery kinetics after photobleaching than those in interphase cells. Live-cell imaging demonstrated that these aggregates were cleared only during cell division. Proteomic analysis revealed that ER-FlucDM-eGFP overexpression upregulated multiple proteins related to the unfolded protein response (UPR) and several proteins associated with cell cycle regulation. Using small-molecule inhibitors, it was found that aggregate clearance required the inactivation of cyclin-dependent kinase 1 (Cdk1) and the activity of Aurora kinase A. Moreover, the inhibition of the Hsp70 family chaperones, especially BiP, not only abolished aggregate clearance but also altered the ER membrane structure in dividing cells, suggesting mitotic ER remodeling facilitates aggregate clearance. The following perturbations on ER remodeling by both the disassembly of microtubules and the knockdown of the ER membrane fusion protein atlastins inhibited aggregate clearance. Furthermore, overexpression of the ER luminal spacer cytoskeleton-linking membrane protein 63 (CLIMP63) facilitated the clearance of aggregates. Notably, the total protein abundance of ER-FlucDM-eGFP remained mostly unchanged before and after cell division, suggesting that aggregate clearance primarily results from ER remodeling rather than protein degradation.

In conclusion, this study reveals several novel mechanisms regulating proteostasis in mammalian dividing cells. Results from Chapter 3 and Chapter 5 indicate that misfolding-prone proteins are less likely to aggregate during cell division. Results from Chapter 4 and Chapter 5 demonstrate that interphase cells confine misfolded proteins from the ER to the nucleus and assemble them into aggregates. These aggregates are cleared during cell division, a process dependent on ER remodeling.