Developing Methods to Study Dynamic Biological Processes in Single Cells With Microscopy

Abstract

Many aspects of intracellular dynamics are difficult to infer from bulk experiments: cell-cycle specific responses are hidden by averaging over an asynchronous population, spatial aspects are invisible, and many of the perturbations that cells respond to vary greatly between cells due to spontaneous, stochastic events. Some of these challenges can be addressed by using quantitative time-lapse microscopy. Great strides have been made in terms of imaging modalities with higher resolution, and fluorescent probes with improved properties. However, the methods for handling cells have lagged behind. Many assays are low-throughput, keep cells under poorly controlled conditions, are confounded by artefacts (e.g. due to the point-spread function of light when cells are close to each other), struggle to quantify components present in low numbers, and are often limited in terms of imaging individual cells in changing populations. In this thesis, we developed several methods to deal with these technological challenges, and applied these new tools to various biological processes. First we present a detailed and reproducible protocol for a platform termed MACS–microfluidics assisted cell screening. MACS provides a single device that can be used with a wide-range of cell types from E. coli to human red blood cells. With MACS, cells are subject to mechanical pressing from a PDMS valve that can exert controlled pressure on cells. By employing high pressure, MACS can squeeze some of the water from E. coli cells, reducing the cytoplasmic diffusion rate and allowing individual fluorescent proteins to be visualized and counted as punctate spots. It can also be used to force E. coli cells to uptake normally impermeable dyes or retrieve specific cells of interest. Second we introduce a platform based on a modified version of the mother machine that allows us to track lineages of cells as they enter and exit stationary phase multiple times. We show that with proper design, this platform quantitatively reproduces the properties of the changing batch culture. As a proof of principle, we track cells expressing an rpos transcriptional reporter in this device through entry and exit from stationary phase. We show that cells entering stationary phase exhibit a mixed ‘timer-adder’ mode of cell size regulation, producing widely distributed stationary phase cell lengths. Upon exit, the cells instead turn into nearly perfect ‘sizers’, quickly returning to the mean length in exponential growth. The individual cells also varied greatly in when they turned off division as they entered stationary phase, with some cells dividing 2-3 more times than others. We found that, as long as cells do not spend too much time in stationary phase, this pattern was perfectly compensated by the opposite effect during exit from stationary phase, where cells that divided more times during entry divided fewer times during exit. Finally, we applied these tools to study an archetypal, noisy biological system–plasmid replication control–in single cells. We describe a technique, based on the DNA binding protein MalI, that allows plasmids to be visualized as fluorescent spots that, unlike many conventional DNA binding proteins, does not appear to interfere with native plasmid function. Comparing the full distribution of copy numbers we obtain with this method to results obtained using a highly tested replication arrest assay (unpublished), we found a strong agreement for all statistical properties extracted. Strikingly, given the constraints on noise control for self-replicators like plasmids, both assays show that the copy number distribution of R1 is strongly sub-Poisson, particularly at the left tail of the distribution. Specifically, essentially all cells nearing division length had a copy number of four or higher despite being present in just seven copies on average in those cells. Furthermore, with the MalI counting assay, plasmid localization was analyzed and found to be distributed throughout the cell, with signs of being weakly excluded from the nuclear region. This contrasts with previous studies that observed strong foci only at the poles of the cells; however, these studies relied on methods which we have observed to create localization artefacts.