UC Berkeley

Chapter 1: Insight into the function and regulation of biological molecules can often be obtained by determining which cell structures and other molecules they localize with (i.e. colocalization). Here we describe an open source plugin for ImageJ called EzColocalization to visualize and measure colocalization in microscopy images. EzColocalization is designed to be easy to use and customize for researchers with minimal experience in quantitative microscopy and computer programming. Features of EzColocalization include: (i) tools to select individual cells and organisms from images; (ii) filters to select specific types of cells and organisms based on physical parameters and signal intensity; (iii) heat maps and scatterplots to visualize the localization patterns of reporters; (iv) multiple metrics to measure colocalization for two or three reporters; (v) metric matrices to systematically measure colocalization at multiple combinations of signal intensity thresholds; and (vi) data tables that provide detailed information on each cell in a sample. These features make EzColocalization well-suited for experiments with low reporter signal, complex patterns of localization, and heterogeneous populations of cells and organisms.

Chapter 2: In meiosis, programmed double-stranded breaks in DNA are induced to enable recombination between chromosomes. A subset of these breaks are eventually processed to become crossovers between homologous chromosomes, wherein sections of chromosomes are exchanged. The majority are repaired as non-crossovers. However, the mechanism by which cells regulate the number and assortment of crossovers amongst an excess of double-stranded breaks remains unclear. Prior work in our lab has suggested proteins may move along chromosomes through the SC, and that this movement may determine crossover regulation. Here, we characterize the movement of numerous chromosomal proteins in live C. elegans using fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). We see a wide variation in protein mobility, and specifically find that synaptonemal complex proteins are dynamic, and that four RING domain containing proteins that regulate crossovers, the ZHPs, are all dynamic in contrast to other crossover factors. We also tested the dosage dependence of these ZHPs. Using mutants and knockdowns we increase and decrease the effective concentrations of these ZHPs, demonstrating their concentration sensitivity in C. elegans. Together, our results are consistent with a model in which crossovers are regulated by a system of dynamically interacting ZHPs and other factors that establish a pattern of crossovers based on their interactions and concentrations.

Chapter 3: Meiosis is a specialized cell division which incorporates recombination between homologous chromosomes and a decrease in the ploidy of daughter cells. In meiosis, an excess of programmed double-stranded breaks are formed in DNA. Amongst these sites, most will be repaired as non-crossovers and a subset will be repaired as crossovers, with at least one crossover formed per pair of homologous chromosomes. Prior to formation of an established crossover, numerous crossover intermediates form at potential crossover sites with reduced amounts of crossover proteins. While many of the essential and nonessential crossover proteins have been characterized, the enzymatic activities which catalyze the formation of crossovers and crossover intermediates are unknown. Work in multiple orthologous systems has demonstrated the importance of the C. elegans ortholog of CDK-2 in forming crossovers. Additionally, the crossover essential protein COSA-1 in C. elegans is a cyclin-like protein. Based on these findings, CDK-2 was investigated as a crossover-dependent protein in C. elegans. Here, we show that CDK-2 localizes to crossovers, crossover formation depends on its continued activity, and localization of crossover proteins to both crossover intermediates and mature crossovers depends on CDK-2 presence. Together, our characterization implicates CDK-2 as a likely partner of COSA-1 in designating, reinforcing and forming crossovers in C. elegans.