ABSTRACT
The human ubiquitin proteasome system, composed of over 700 ubiquitin ligases (E3s) and deubiquitinases (DUBs), has been difficult to characterize systematically and phenotypically. We performed chemical-genetic CRISPR-Cas9 screens to identify E3s/DUBs whose loss renders cells sensitive or resistant to 41 compounds targeting a broad range of biological processes, including cell cycle progression, genome stability, metabolism, and vesicular transport. Genes and compounds clustered functionally, with inhibitors of related pathways interacting similarly with E3s/DUBs. Some genes, such as FBXW7, showed interactions with many of the compounds. Others, such as RNF25 and FBXO42, showed interactions primarily with a single compound (methyl methanesulfonate for RNF25) or a set of related compounds (the mitotic cluster for FBXO42). Mutation of several E3s with sensitivity to mitotic inhibitors led to increased aberrant mitoses, suggesting a role for these genes in cell cycle regulation. Our comprehensive CRISPR-Cas9 screen uncovered 466 gene-compound interactions covering 25% of the interrogated E3s/DUBs.
Subject(s)
CRISPR-Cas Systems , Mitosis , Signal Transduction , Ubiquitin-Protein Ligases , Ubiquitin , Cell Line , Humans , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Fertilization occurs during female meiosis in most animals, which raises the question of what prevents the sperm DNA from interacting with the meiotic spindle. In this study, we find that Caenorhabditis elegans sperm DNA stays in a fixed position at the opposite end of the embryo from the meiotic spindle while yolk granules are transported throughout the embryo by kinesin-1. In the absence of F-actin, the sperm DNA, centrioles, and organelles were transported as a unit with the yolk granules, resulting in sperm DNA within 2 µm of the meiotic spindle. F-actin imaging revealed a cytoplasmic meshwork that might restrict transport in a size-dependent manner. However, increasing yolk granule size did not slow their velocity, and the F-actin moved with the yolk granules. Instead, sperm contents connect to the cortical F-actin to prevent interaction with the meiotic spindle.
Subject(s)
Actins/metabolism , Caenorhabditis elegans/metabolism , DNA/metabolism , Meiosis , Oocytes/metabolism , Sperm-Ovum Interactions , Spermatozoa/metabolism , Spindle Apparatus/metabolism , Actins/genetics , Animals , Animals, Genetically Modified , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA/genetics , Genotype , Kinesins/genetics , Kinesins/metabolism , Male , Microscopy, Fluorescence , Microscopy, Video , Phenotype , Profilins/genetics , Profilins/metabolism , RNA Interference , Sperm Motility , Time Factors , Time-Lapse ImagingABSTRACT
Objective: To describe the characteristics of craniocervical posture of children aged between 6 and 11 years and its relationship to their sagittal skeletal classification. Material and Methods: This descriptive cross-sectional study involved 107 children (55 girls - 52 boys), aged between 6 and 11 years. The sample included no previous orthodontically/orthopedic treated and systemically healthy children. After proper calibration, lateral skull radiographs, taken for diagnosis purpose for maxillary orthopedic treatment, were obtained by the same operator in natural head position. A radiographic analysis was made using a NEMOTEC software: 13 variables were registered: age, gender, ANB angle (to classify sagittal skeletal relationships) and 10 variables related to craniocervical posture: cervical lordosis, hyoid triangle, craniocervical angle, intervertebral spaces: C0-C1, C1-C2 and distances NSL-Ver, NLVer, ML-Ver, OPT-Hor, CVT-Hor. To evaluate the reliability of measures, 15 randomly selected radiographs were re-measured by the same investigator two weeks after the initial analysis. Results: Intra-class correlation coefficients were in a range of 0.945-0.996. Lordosis, CCA, C1-C2, OPT-Hor y CVT-Hor, values were higher in male than in female children (p<0.05). No statistically significant differences were found among groups of sagittal skeletal relationships, but class III children had a tendency to higher craniocervical flexion; 66.3% of the studied group presented rectified lordotic curvature and class II subjects presented increased values of NSL-Ver, NL-Ver and MLVer. Class I children had the lowest values for OPT-Hor and CVT-Hor. Conclusion: All craniocervical postural variables were higher in boys than in girls. No differences were found in this study between cervical postural variables with different malocclusion.
