Nucleoid and cytoplasmic localization of small RNAs in Escherichia coli.

Bacterial small RNAs (sRNAs) regulate protein production by binding to mRNAs and altering their translation and degradation. sRNAs are smaller than most mRNAs but larger than many proteins. Therefore it is uncertain whether sRNAs can enter the nucleoid to target nascent mRNAs. Here, we investigate the intracellular localization of sRNAs transcribed from plasmids in Escherichia coli using RNA fluorescent in-situ hybridization. We found that sRNAs (GlmZ, OxyS, RyhB and SgrS) have equal preference for the nucleoid and cytoplasm, and no preferential localization at the cell membrane. We show using the gfp mRNA (encoding green fluorescent protein) that non-sRNAs can be engineered to have different proportions of nucleoid and cytoplasmic localization by altering their length and/or translation. The same localization as sRNAs was achieved by decreasing gfp mRNA length and translation, which suggests that sRNAs and other RNAs may enter the densely packed DNA of the nucleoid if they are sufficiently small. We also found that the Hfq protein, which binds sRNAs, minimally affects sRNA localization. Important implications of our findings for engineering synthetic circuits are: (i) sRNAs can potentially bind nascent mRNAs in the nucleoid, and (ii) localization patterns and distribution volumes of sRNAs can differ from some larger RNAs.

Recruitment of Polo-like kinase couples synapsis to meiotic progression via inactivation of CHK-2.

Meiotic chromosome segregation relies on synapsis and crossover (CO) recombination between homologous chromosomes. These processes require multiple steps that are coordinated by the meiotic cell cycle and monitored by surveillance mechanisms. In diverse species, failures in chromosome synapsis can trigger a cell cycle delay and/or lead to apoptosis. How this key step in 'homolog engagement' is sensed and transduced by meiotic cells is unknown. Here we report that in C. elegans, recruitment of the Polo-like kinase PLK-2 to the synaptonemal complex triggers phosphorylation and inactivation of CHK-2, an early meiotic kinase required for pairing, synapsis, and double-strand break (DSB) induction. Inactivation of CHK-2 terminates DSB formation and enables CO designation and cell cycle progression. These findings illuminate how meiotic cells ensure CO formation and accurate chromosome segregation.

Most animals, plants, and fungi reproduce sexually, meaning that the genetic information from two parents combines during fertilization to produce offspring. This parental genetic information is carried within the reproductive cells in the form of chromosomes. Reproductive cells in the ovaries or testes first multiply through normal cell division, but then go through a unique type of cell division called meiosis. During meiosis, pairs of chromosomes ­ the two copies inherited from each parent ­ must find each other and physically line up from one end to the other. As they align side-by-side with their partners, chromosomes also go through a mixing process called recombination, during which regions of one chromosome cross over to the paired chromosome to exchange information. Scientists are still working to understand how this process of chromosome alignment and crossing-over is controlled. If chromosomes fail to line up or cross over during meiosis, eggs or sperm can end up with too many or too few chromosomes. If these faulty reproductive cells combine during fertilization this can lead to birth defects and developmental problems. To minimize this problem, reproductive cells have a quality control mechanism during meiosis called "crossover assurance", which limits how often mistakes occur. Zhang et al. have investigated how cells can tell if their chromosomes have accomplished this as they undergo meiosis. They looked at egg cells of the roundworm C. elegans, whose meiotic processes are similar to those in humans. In C. elegans, a protein called CHK-2 regulates many of the early events during meiosis. During successful meiosis, CHK-2 is active for only a short amount of time. But if there are problems during recombination, CHK-2 stays active for longer and prevents the cell division from proceeding. Zhang et al. uncovered another protein that affects for how long CHK-2 stays switched on. When chromosomes align with their partners, a protein called PLK-2 sticks to other proteins at the interface between the aligned chromosomes. A combination of microscopy and test tube experiments showed that when PLK-2 is bound to this specific location, it can turn off CHK-2. However, if the chromosome alignment fails, PLK-2 is not activated to switch off CHK-2. Therefore, CHK-2 is only switched off when the chromosomes are properly aligned and move on to the next step in crossing-over, which then allows meiosis to proceed. Thus, PLK-2 and CHK-2 work together to detect errors and to slow down meiosis if necessary. Further experiments in mammalian reproductive cells will reveal how similar the crossover assurance mechanism is in different organisms. In the future, improved understanding of quality control during meiosis may eventually lead to improvements in assisted reproduction.

EzColocalization: An ImageJ plugin for visualizing and measuring colocalization in cells and organisms.

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.

Systematic and general method for quantifying localization in microscopy images.

Quantifying the localization of molecules with respect to other molecules, cell structures and intracellular regions is essential to understanding their regulation and actions. However, measuring localization from microscopy images is often difficult with existing metrics. Here, we evaluate a metric for quantifying localization termed the threshold overlap score (TOS), and show it is simple to calculate, easy to interpret, able to be used to systematically characterize localization patterns, and generally applicable. TOS is calculated by: (i) measuring the overlap of pixels that are above the intensity thresholds for two signals; (ii) determining whether the overlap is more, less, or the same as expected by chance, i.e. colocalization, anti-colocalization, or non-colocalization; and (iii) rescaling to allow comparison at different thresholds. The above is repeated at multiple threshold combinations to generate a TOS matrix to systematically characterize the relationship between localization and signal intensities. TOS matrices were used to identify and distinguish localization patterns of different proteins in various simulations, cell types and organisms with greater specificity and sensitivity than common metrics. For all the above reasons, TOS is an excellent first line metric, particularly for cells with mixed localization patterns.