CELLCYCLEGROWTHSIZE · Cell Growth and Size Homeostasis in Proliferating Mammalian Cells

Cell Growth and Size Homeostasis in Proliferating Mammalian Cells

Comments: The cell cycle is a fundamental process, which underlies cell and organism development and reproduction. In essence, it is a continuous and unidirectional series of events that ultimately ensure DNA duplication and distribution into two daughter cells. Cell cycle events are often categorized into four phases known as Gap1 (G1), DNA synthesis (S), Gap2 (G2), and mitosis (M); however, each of these phases can be subdivided into discrete molecular milestones. Orderly progression through the cell cycle is achieved by several checkpoint mechanisms that, overall, secure genome stability over many generations. Indeed, an abnormally controlled cell cycle is a main cause of cancer. Another basic process is cell growth, i.e., increase in cell mass or size over time resulting from the biosynthesis of macromolecules, in particularly proteins. Proliferating cells grow continuously from birth to division, doubling their size during their life cycle. Although cells can progress through the cell cycle at different paces and grow at different rates, on the average G1 cells are smaller than cells in S-phase, which, in turn, are smaller than cells in G2 or mitosis. The life cycle of a proliferating cell ends by cell division, which effectively generates two new daughter cells that are approximately half the size of the mother cell. Cell cycle and cell growth are two distinct processes. In proliferating cells, these processes are coupled by a mechanism that is critical for maintaining one of the most basic features of the cell – its size. For this reason, cells proliferating under steady-state conditions maintain time-invariant distributions of both cell cycle phases and cell size. Cell cycle and cell growth are not always coupled; for instance, in early embryos, cells duplicate without growing, halving their size each cycle. The overall aim of this project was to study the cell cycle/cell growth/cell size interface in proliferating mammalian cells, with strong emphasize on developing methodologies to study this difficult question. We set up a fully functional lab suitable for studying suspension and adherent cells. We purchased an advanced Coulter Counter for cell volume measurements and built a replica of the mechanical cell synchronizer known as the “Baby-Machine” for cell cycle/cell growth studies. This machine is capable of synchronizing L1210 mouse pre-B cells at birth. In addition, we developed and optimized protocols for size-based separation for both adherent and unattached mammalian cells (Published; 2011). This information allowed us to study the cell cycle progression of large and small cell populations, relating G1/S transition to cell size at quiescent state. We also demonstrated that size-based separation by cytometry can be utilized for purifying cells at G1 phase (Published; 2013) and cytokinesis (Published; 2015) without using drugs or any other means of pre-synchronization. Relying on standard optical flow cytometry, these methodologies can be immediately integrated in research labs across the globe to facilitate cell cycle research. Five cell lines from both human and mouse origins (adherent: HeLa, BALB/c-3T3, MDCK; non-adherent: L1210, FL5.12), carrying one, and for the most part two cell cycle fluorescence markers, are now available in our lab. In combination with dry mass and buoyant mass measurements, we could study growth of single cells in relation to cell cycle milestones. This was a joint project with laboratories from MIT and Harvard, and a very fruitful one (Published; 2012 and 2013). Analysis of L1210 lymphoblasts revealed a decrease in growth rate variability at the G1-S phase transition, which suggests the presence of a growth rate threshold for maintaining size homeostasis. Interestingly, in L1210 growing in rich media growth rate rather than size seemed to form the threshold for passing the G1-S transition. However, in cells growing in limiting growth conditions, the length of the G1 phase was extended until the cells reached a signature size and, only then, traverse through the G1-S transition. Dry mass measurements revealed a size-dependent growth also in adherent cells, and surprising mass asymmetry in daughter cells. These measurements provided another proof for the strong need of a cell-autonomous mechanism that coordinates cell cycle with cell growth in a way that maintains a time-invariant cell size. Overall, we provided evidence for size feedback in populations and single mammalian cells relying on volume, mass, and light scattering metrics. To summarize, in line with the overall aims of this project we 1) optimized and developed new methodologies with potential use in cell growth and cell size research; 2) demonstrated a size-sensing mechanism in proliferating cell types; 3) demonstrated that such mechanisms are implemented at the G1-S transition; and 4) suggest that both size and growth rate are metrics cells can process and integrate into the cell cycle machinery. Size is a fundamental feature of all cells. Understanding how cell growth and cell cycle are coordinated in a way that maintain a constant cell size is important question in cell biology with relevance to normal and malignant tissue-homeostasis, organogenesis and development. I believe that our work, as presented in meetings and papers, will encourage others to investigate this fundamental, albeit neglected, field of research, and push for technological advancements that will facilitate this field of research. Amit Tzur, PhD. Mina and Everard Faculty of Life Sciences and the Bar-Ilan Institute of Nanotechnology and Advanced Materials. Building 206, B-440. Bar-Ilan University, Ramat Gan 52900, Israel TEL: +972-3-7384541 FAX: +972-3-7384058 Email: amit.tzur@biu.ac.il

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