Thermally resistant nuclease in Serratia marcescens hinders PCR reactions and degrades PCR products.

Polymerase chain reaction (PCR) is an important tool for exogenous gene acquisition and recombinants identification. There exist two problems when using Serratia marcescens as a template for PCR amplification: amplified PCR products are rapidly degraded, and the results of PCR amplification are unstable. The aim of the present work was to elucidate the reasons for this. By mixing PCR products amplified from Escherichia coli DH5α with S. marcescens supernatant or pellet, we found that the DNA-degrading substance in S. marcescens is thermally resistant and present both intracellularly and extracellularly. We then determined that it is protein, and most likely S. marcescens nuclease, that degrades PCR products since the addition of SDS and EDTA can effectively inhibit or block the degradation of PCR products. By knocking out the S. marcescens nuclease encoding gene, nucA, we confirmed that the nuclease is responsible for the degradation of PCR products and the instability of PCR amplification. This work is the first to show that the S. marcescens nuclease is temporarily and partially inhibited by high temperatures during PCR and recovers rapidly at room temperature after PCR.

TRACES: a Freely Accessible, Semi-automated Pipeline for Detection, Tracking, and Quantification of Fluorescently Labeled Cellular Structures.

Subcellular structures exhibit diverse behaviors in different cellular processes, including changes in morphology, abundance, and relative spatial distribution. Faithfully tracking and quantifying these changes are essential to understand their functions. However, most freely accessible methods lack integrated features for tracking multiple objects in different spectral channels simultaneously. To overcome these limitations, we have developed TRACES (Tracking of Active Cellular Structures), a customizable and open-source pipeline capable of detecting, tracking, and quantifying fluorescently labeled cellular structures in up to three spectral channels simultaneously at single-cell level. Here, we detail step-by-step instructions for performing the TRACES pipeline, including image acquisition and segmentation, object identification and tracking, and data quantification and visualization. We believe that TRACES will be a valuable tool for cell biologists, enabling them to track and measure the spatiotemporal dynamics of subcellular structures in a robust and semi-automated manner.

Condensation of pericentrin proteins in human cells illuminates phase separation in centrosome assembly.

At the onset of mitosis, centrosomes expand the pericentriolar material (PCM) to maximize their microtubule-organizing activity. This step, termed centrosome maturation, ensures proper spindle organization and faithful chromosome segregation. However, as the centrosome expands, how PCM proteins are recruited and held together without membrane enclosure remains elusive. We found that endogenously expressed pericentrin (PCNT), a conserved PCM scaffold protein, condenses into dynamic granules during late G2/early mitosis before incorporating into mitotic centrosomes. Furthermore, the N-terminal portion of PCNT, enriched with conserved coiled-coils (CCs) and low-complexity regions (LCRs), phase separates into dynamic condensates that selectively recruit PCM proteins and nucleate microtubules in cells. We propose that CCs and LCRs, two prevalent sequence features in the centrosomal proteome, are preserved under evolutionary pressure in part to mediate liquid-liquid phase separation, a process that bestows upon the centrosome distinct properties critical for its assembly and functions.

Evidence for anaphase pulling forces during C. elegans meiosis.

Anaphase chromosome movement is thought to be mediated by pulling forces generated by end-on attachment of microtubules to the outer face of kinetochores. However, it has been suggested that during C. elegans female meiosis, anaphase is mediated by a kinetochore-independent pushing mechanism with microtubules only attached to the inner face of segregating chromosomes. We found that the kinetochore proteins KNL-1 and KNL-3 are required for preanaphase chromosome stretching, suggesting a role in pulling forces. In the absence of KNL-1,3, pairs of homologous chromosomes did not separate and did not move toward a spindle pole. Instead, each homolog pair moved together with the same spindle pole during anaphase B spindle elongation. Two masses of chromatin thus ended up at opposite spindle poles, giving the appearance of successful anaphase.

Three-dimensional Reconstruction and Quantification of Proteins and mRNAs at the Single-cell Level in Cultured Cells.

Gene expression is often regulated by the abundance, localization, and translation of mRNAs in both space and time. Being able to visualize mRNAs and protein products in single cells is critical to understand this regulatory process. The development of single-molecule RNA fluorescence in situ hybridization (smFISH) allows the detection of individual RNA molecules at the single-molecule and single-cell levels. When combined with immunofluorescence (IF), both mRNAs and proteins in individual cells can be analyzed simultaneously. However, a precise and streamlined quantification method for the smFISH and IF combined dataset is scarce, as existing workflows mostly focus on quantifying the smFISH data alone. Here we detail a method for performing sequential IF and smFISH in cultured cells (as described in Sepulveda et al., 2018 ) and the subsequent statistical analysis of the smFISH and IF data via three-dimensional (3D) reconstruction in a semi-automatic image processing workflow. Although our method is based on analyzing centrosomally enriched mRNAs and proteins, the workflow can be readily adapted for performing and analyzing smFISH and IF data in other biological contexts.

Co-translational protein targeting facilitates centrosomal recruitment of PCNT during centrosome maturation in vertebrates.

As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here, we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment of PCNT mRNA, its translation near centrosomes, and requirement of intact polysomes for PCNT mRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism.