Biology subjects

Cahoon, C. K.

Publications and source records attributed to Cahoon, C. K..

6 recordsLinked to original sources

bioRxiv2022

Sexual dimorphic regulation of recombination by the synaptonemal complex

In sexually reproducing organisms, germ cells faithfully transmit the genome to the next generation by forming haploid gametes, such as eggs and sperm. Although most meiotic proteins are conserved between eggs and sperm, many aspects of meiosis are sexually dimorphic. The mechanisms regulating recombination display sex-specific differences in multiple organisms such that the same proteins in each sex are being utilized in different ways to produce sexually dimorphic outcomes. The synaptonemal complex (SC), a large ladder-like structure that forms between homologous chromosomes, is essential for regulating meiotic chromosome organization and promoting recombination. To assess whether sex-specific differences in the SC underpin sexually dimorphic aspects of meiosis, we examined two Caenorhabditis elegans SC central region proteins, SYP-2 and SYP-3, in oogenesis and spermatogenesis and uncovered sex-specific roles for the SYPs in regulating meiotic recombination. We find that SC composition is regulated by sex-specific mechanisms throughout meiotic prophase I. During pachytene, both oocytes and spermatocytes differentially regulate the stability of SYP-2 and SYP-3 within an assembled SC, with increased SYP-2 dynamics in spermatocytes and increased SYP-3 dynamics in oocytes. Further, we uncover that the relative amount of SYP-2 and SYP-3 within the SC is independently regulated in both a sex-specific and a recombination-dependent manner. Specifically, we find that SYP-2 regulates the early steps of recombination in both sexes, while SYP-3 controls the timing and positioning of crossover recombination events across the genomic landscape in only oocytes. Taken together, we demonstrate dosage-dependent regulation of individual SC components with sex-specific functions in recombination. These sexual dimorphic features of the SC provide insights into how spermatogenesis and oogenesis adapted similar chromosome structures to differentially regulate and execute recombination.

developmental biology↗

bioRxiv2021

Conditional immobilization for live imaging C. elegans using auxin-dependent protein depletion

The visualization of biological processes using fluorescent proteins and dyes in living organisms has enabled numerous scientific discoveries. The nematode Caenorhabditis elegans is a widely used model organism for live imaging studies since the transparent nature of the worm enables imaging of nearly all tissues within a whole, intact animal. While current techniques are optimized to enable the immobilization of hermaphrodite worms for live imaging, many of these approaches fail to successfully restrain the smaller male worms. To enable live imaging of worms of both sexes, we developed a new genetic, conditional immobilization tool that uses the auxin inducible degron (AID) system to immobilize both hermaphrodites and male worms for live imaging. Based on chromosome location, mutant phenotype, and predicted germline consequence, we identified and AID-tagged three candidate genes (unc-18, unc-104, and unc-52). Strains with these AID-tagged genes were placed on auxin and tested for mobility and germline defects. Among the candidate genes, auxin-mediated depletion of UNC-18 caused significant immobilization of both hermaphrodite and male worms that was also partially reversible upon removal from auxin. Notably, we found that male worms require a higher concentration of auxin for a similar amount of immobilization as hermaphrodites, thereby suggesting a potential sex-specific difference in auxin absorption and/or processing. In both males and hermaphrodites, depletion of UNC-18 did not largely alter fertility, germline progression, nor meiotic recombination. Finally, we demonstrate that this new genetic tool can successfully immobilize both sexes enabling live imaging studies of sexually dimorphic features in C. elegans. ARTICLE SUMMARYC. elegans is a powerful model system for visualizing biological processes in live cells. In addition to the challenge of suppressing the worm movement for live imaging, most immobilization techniques only work with hermaphrodites. Here, we describe a new genetic immobilization tool that conditionally immobilizes both worm sexes for live imaging studies. Additionally, we demonstrate that this tool can be used for live imaging the C. elegans germline without causing large defects to germline progression or fertility in either sex.

cell biology↗

bioRxiv2021

Homologous locus pairing is a transient, diffusion-mediated process in meiotic prophase

The pairing of homologous chromosomes (homologs) in meiosis is essential for distributing the correct numbers of chromosomes into haploid gametes. In budding yeast, pairing depends on the formation of 150-200 Spo11-mediated double-strand breaks (DSBs) that are distributed among 16 homolog pairs, but it is not known if all, or only a subset of these DSBs, contribute to the close juxtaposition of homologs. Having established a system to measure the position of fluorescently tagged chromosomal loci in 3D space over time, we analyzed locus trajectories to determine how frequently, and how long, loci spend colocalized or apart. Continuous imaging revealed highly heterogeneous cell-to-cell behavior of foci, with the majority of cells exhibiting a "mixed" phenotype where foci move into and out of proximity, even at late stages of prophase, suggesting that the axial structures of the synaptonemal complex may be more dynamic than anticipated. The observed plateaus of the mean-squared change in distance (MSCD) between foci informed the development of a biophysical model of two diffusing polymers that captures the loss of centromere linkages as cells enter meiosis, nuclear confinement, and the formation of Spo11-dependent linkages. The predicted number of linkages per chromosome in our theoretical model closely approximates the small number (~2-4) of estimated synapsis-initiation sites, suggesting that excess DSBs have negligible effects on the overall juxtaposition of homologs. These insights into the dynamic in-terchromosomal behavior displayed during homolog pairing demonstrate the power of combining time-resolved in vivo analysis with modeling at the granular level. Significance StatementEssential for sexual reproduction, meiosis is a specialized cell division required for the production of haploid gametes. Critical to this process is the pairing, recombination, and segregation of homologous chromosomes (homologs). While pairing and recombination are linked, it is not known how many linkages are sufficient to hold homologs in proximity. Here, we reveal that random diffusion and the placement of a small number of linkages are sufficient to establish the apparent "pairing" of homologs. We also show that colocalization between any two loci is more dynamic than anticipated. Our study is the first to provide observations of live interchromosomal dynamics during meiosis and illustrates the power of combining single-cell measurements with theoretical polymer modeling.

genetics↗

bioRxiv2020

Automated and customizable quantitative image analysis of whole C. elegans germlines

Arranged in a spatial-temporal gradient for germ cell development, the adult germline of Caenorhabditis elegans is an excellent system for understanding the generation, differentiation, function, and maintenance of germ cells. Imaging whole C. elegans germlines along the distal-proximal axis enables powerful cytological analyses of germ cell nuclei as they progress from the pre-meiotic tip through all the stages of meiotic prophase I. To enable high-throughput image analysis of whole C. elegans gonads, we developed a custom algorithm and pipelines to function with image processing software that enables: 1) quantification of cytological features at single nucleus resolution from immunofluorescence images; and, 2) assessment of these individual nuclei based on their position within the germline. We demonstrate the capability of our quantitative image analysis approach by analyzing multiple cytological features of meiotic nuclei in whole C. elegans germlines. First, we quantify double strand DNA breaks (DSBs) per nucleus by analyzing DNA-associated foci of the recombinase RAD-51 at the single-nucleus resolution in the context of whole germline progression. Second, we quantify the DSBs that are licensed for crossover repair by analyzing foci of MSH-5 and COSA-1 when they associate with the synaptonemal complex during meiotic prophase progression. Finally, we quantify P-granule composition across the whole germline by analyzing the colocalization of PGL-1 and ZNFX-1 foci. Our image analysis pipeline is an adaptable and useful method for researchers spanning multiple fields utilizing the C. elegans germline as a model system.

cell biology↗

bioRxiv2020

Excess crossovers impede faithful meiotic chromosome segregation in C. elegans

During meiosis, at least one crossover must form between each pair of homologous chromosomes to ensure their proper partitioning. However, most organisms limit the number of crossovers by a phenomenon called crossover interference; why this occurs is not well understood. Here we investigate the functional consequences of extra crossovers in Caenorhabditis elegans. Using a fusion chromosome that exhibits a high frequency of supernumerary crossovers, we find that essential chromosomal structures are mispatterned, subjecting chromosomes to improper spindle forces and leading to congression and segregation defects. Moreover, we uncover mechanisms that counteract these errors; anaphase I chromosome bridges were often able to resolve in a LEM-3 nuclease dependent manner, and tethers between homologs that persisted were frequently resolved during Meiosis II by a second mechanism. This study thus provides evidence that excess crossovers impact chromosome patterning and segregation, and also sheds light on how these errors are corrected.

cell biology↗

bioRxiv2019

Synaptonemal complex proteins direct and constrain the localization of crossover-promoting proteins during Caenorhabditis elegans meiosis

Crossovers (COs) between homologous chromosomes are critical for meiotic chromosome segregation and form in the context of the synaptonemal complex (SC), a meiosis-specific structure that assembles between aligned homologs. During Caenorhabditis elegans meiosis, central region components of the SC (SYP proteins) are essential to repair double-strand DNA breaks (DSBs) as COs, but the roles of these SYP proteins in promoting CO formation are poorly understood. Here, we investigate the relationships between the SYP proteins and conserved CO-promoting factors by examining the immunolocalization of these factors in meiotic mutants where SYP proteins are absent, reduced, or mis-localized. Although COs do not form in syp null mutants, CO-promoting proteins COSA-1, MSH-5, and ZHP-3 nevertheless become co-localized at a variable number of DSB-dependent sites during late prophase, reflecting an inherent affinity of these factors for DSB repair sites. In contrast, in mutants where SYP proteins are present but form aggregates or display abnormal synapsis, CO-promoting proteins consistently track with SYP-1 localization. Moreover, CO-promoting proteins usually localize to a single site per SYP-1 structure, even in SYP aggregates or in mutants where SC forms between sister-chromatids, suggesting that CO regulation occurs within these structures. Further, we find that sister chromatids in the meiotic cohesin mutant rec-8 require both CO-promoting proteins and the SC to remain connected. Taken together, our findings support a model in which SYP proteins promote CO formation by directing and constraining the localization of CO-promoting factors to ensure that CO maturation occurs only between properly aligned homologous chromosomes.\n\nArticle SummaryErrors during meiosis are the leading cause of birth defects and miscarriages in humans. Thus, the coordinated control of meiosis events is critical for the faithful inheritance of the genome each generation. The synaptonemal complex (SC) is a meiosis-specific structure that assembles between homologs chromosomes and is critical for the establishment and regulation of crossovers, which ensure the accurate segregation of the homologous chromosomes at meiosis I. Here we show that the SC proteins function to regulate crossovers by directing and constraining the localization of proteins involved in promoting the formation of crossovers.

genetics↗