Abstract
Although Schyzosaccharomyces pombe is one of the principal model organisms for studying the cell cycle, surprisingly few methods have characterized S. pombe growth on the single cell level, and no methods exist capable of analyzing thousands of cells and tens of thousands of cell division events. We developed an automated microfluidic platform permitting S. pombe to be grown on-chip for several days under defined and changeable conditions. We developed an image processing pipeline to extract and quantitate several physiological parameters including cell length, time to division, and elongation rate without requiring synchronization of the culture. Over a period of 50 hours our platform analyzed over 100000 cell division events and reconstructed single cell lineages up to 10 generations in length. We characterized cell lengths and division times in a temperature shift experiment in which cells were initially grown at 30°C and transitioned to 25°C. Although cell length was identical at both temperatures at steady-state, we observed transient changes in cell length if the temperature shift took place during a critical phase of the cell cycle. We further show that cells born with normal length do divide over a wide range of cell lengths and that cell length appears to be controlled in the second generation, were large newly born cells have a tendency to divide more rapidly and thus at a normalized cell size. The platform is thus applicable to measure fine-details in cell cycle dynamics, should be a useful tool to decipher the molecular mechanism underlying size homeostasis, and will be generally applicable to study processes on the single cell level that require large numbers of precision measurements and single cell lineages.
Highlights
Cells have been historically studied with population-level measurements, on the assumption that an individual cell’s phenotype is well-described by the population average
Microfluidic Device and Image Processing We designed a microfluidic device consisting of 120 microchemostat growth chambers based on a design previously applied to large-scale single cell studies in S. cerevisiae (Fig. 1) [25]
A temperature sensitive process that regulates cell-division length appears to be active during this part of the cell-cycle and possibly remains active till division. Those cells that divide at abnormally large cell-size, give rise to abnormally large daughter cells (Fig. 5B), but cell division length is normal in the second generation. These results show that cell division is robust at steady-state, but is susceptible to temperature shifts if the shift occurs during a critical phase of the cell cycle and that these abnormal cell-division lengths are corrected in the generation
Summary
Cells have been historically studied with population-level measurements, on the assumption that an individual cell’s phenotype is well-described by the population average. Recent studies have shown that considerable variations in mRNA levels, protein levels, doubling time, and size exist between isogenic cells [1,2,3,4,5]. Clonal cultures exhibit large variation on the single cell level. This variation raises questions related to how robustness can be achieved, information is processed and transmitted over several cell-cycles. Tools to measure single cell variability are becoming available [6,7], but generally lack both throughput and precision. Several approaches exist for measuring growth of microbial cells [8]. Different strains or clones are pooled and grown in liquid culture, with each strain or clone carrying a unique marker. The relative abundance of each strain/clone can be determined using generation sequencing [9], microarray analysis [10,11], FACS [12], or plating [13], from which the relative fitness for each strain/clone can be determined
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