ABSTRACT

A Physiological Unfolded Protein Response Impacts Progression Through the Cell CycleBy Soham Chowdhury

The cell cycle is a series of coordinated molecular steps that allow a progenitor cell to produce two daughter cells. During symmetrical cell division, a mother cell’s particular organelle makeup must also be inherited by the daughter cells. Multi-copy organelles such as mitochondria divide and are equally distributed between daughter cells. Single copy organelles such as the endoplasmic reticulum (ER) and the Golgi apparatus however must be first expanded, then fragmented, and finally partitioned to each daughter cell. Such organelle expansion requires the production of new membranes. Being that the ER is the site of endomembrane biosynthesis, we reasoned that ER expansion should precede and be necessary for mammalian cell division. The Unfolded Protein Response (UPR) is an evolutionarily conserved collection of signaling pathways that maintain ER health. Protein folding perturbations in the ER lumen as well disturbances of the ER membrane lipid bilayer trigger ER stress and activate the UPR. The UPR mitigates ER stress by increasing the biosynthetic and protein degradative capacities of the ER, including its physical expansion through endomembrane biogenesis. Here we show that as cells grow through interphase, their ER chaperone and foldase contents increase, as does the ER volume, indicating that ER expansion precedes cell division. Moreover, pharmacological inhibition of the UPR sensors IRE1 and ATF6 at steady-state delayed cell cycle progression. Furthermore, we found that the threshold for UPR activation is lower in the S/G2 stage of the cell cycle suggesting that ER expansion and increased ER protein-processing capacity is subsequent to genome duplication. Finally, our data indicate that IRE1 activity is dampened by the G2/M cell cycle checkpoint kinase PKMYT1 suggesting negative feedback control. Taken together, these findings suggest a physiological role for the UPR in coordinating the mammalian cell cycle

Throughout the cell cycle, genome duplication is coordinated with the multiplication and growth of organelles, which requires membrane biosynthesis at the endoplasmic reticulum (ER). By this reasoning, ER growth and increased ER function would be a pre-requisite for cell division. Because the unfolded protein response (UPR)—a fundamental homeostatic mechanism that maintains ER integrity—increases the size and protein-processing capacity of the ER, I reasoned that it may oversee ER physiology during the cell cycle. To investigate ER growth and activation of the UPR during the cell cycle, I optimized and characterized a well-described fluorescent reporter of cell cycle progression, known as the FAST-FUCCI system. This live-cell reporter enabled me to separate G1 and S/G2 cell populations by fluorescence activated cell sorting (FACS). My data show that mammalian cells increased in size and granularity during interphase. These hallmarks were correlated with an increase in ER-resident protein content, suggesting that the ER enlarges in preparation for cell division. Moreover, I found that inhibition of IRE1 via pharmacological agents delayed progression through the G1/S boundary. While investigating a plausible mechanism that could regulate UPR activity during the cell cycle, I identified PKMYT1, an ER- and Golgi apparatus-associated G2/M cell cycle checkpoint kinase, in a candidate-based approach. I found that IRE1 activity is suppressed by PKMYT1, suggesting that PKMYT1 exerts regulatory control over IRE1 prior to cell division. Preliminary data suggests that PKMYT1 and IRE1 do not physically interact, which suggests regulation via an unidentified intermediate or a transcriptomic regulation by downstream transcription factors, such as XBP1s. Taken together, my results provide evidence to suggest that mammalian cells engage a physiological UPR involving IRE1 signaling during cell cycle progression.