skip to main content

Explore Research Products in the NSF-PAR

Title: The human origin recognition complex is essential for pre-RC assembly, mitosis, and maintenance of nuclear structure
The origin recognition complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre-RC complexes at origins of DNA replication. Here, using tiling-sgRNA CRISPR screens, we report that each subunit of ORC and CDC6 is essential in human cells. Using an auxin-inducible degradation system, we created stable cell lines capable of ablating ORC2 rapidly, revealing multiple cell division cycle phenotypes. The primary defects in the absence of ORC2 were cells encountering difficulty in initiating DNA replication or progressing through the cell division cycle due to reduced MCM2-7 loading onto chromatin in G1 phase. The nuclei of ORC2-deficient cells were also large, with decompacted heterochromatin. Some ORC2-deficient cells that completed DNA replication entered into, but never exited mitosis. ORC1 knockout cells also demonstrated extremely slow cell proliferation and abnormal cell and nuclear morphology. Thus, ORC proteins and CDC6 are indispensable for normal cellular proliferation and contribute to nuclear organization.  more » « less
Award ID(s):
1350041
NSF-PAR ID:
10276652
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ; ;
Date Published:
Journal Name:
eLife
Volume:
10
ISSN:
2050-084X
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ( , mBio)
    Boyle, Jon P. (Ed.)
    ABSTRACT Mitogen-activated protein kinases (MAPKs) are a conserved family of protein kinases that regulate signal transduction, proliferation, and development throughout eukaryotes. The apicomplexan parasite Toxoplasma gondii expresses three MAPKs. Two of these, extracellular signal-regulated kinase 7 (ERK7) and MAPKL1, have been implicated in the regulation of conoid biogenesis and centrosome duplication, respectively. The third kinase, MAPK2, is specific to and conserved throughout the Alveolata, although its function is unknown. We used the auxin-inducible degron system to determine phenotypes associated with MAPK2 loss of function in Toxoplasma . We observed that parasites lacking MAPK2 failed to duplicate their centrosomes and therefore did not initiate daughter cell budding, which ultimately led to parasite death. MAPK2-deficient parasites initiated but did not complete DNA replication and arrested prior to mitosis. Surprisingly, the parasites continued to grow and replicate their Golgi apparatus, mitochondria, and apicoplasts. We found that the failure in centrosome duplication is distinct from the phenotype caused by the depletion of MAPKL1. As we did not observe MAPK2 localization at the centrosome at any point in the cell cycle, our data suggest that MAPK2 regulates a process at a distal site that is required for the completion of centrosome duplication and the initiation of parasite mitosis. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that can cause severe and fatal disease in immunocompromised patients and the developing fetus. Rapid parasite replication is critical for establishing a productive infection. Here, we demonstrate that a Toxoplasma protein kinase called MAPK2 is conserved throughout the Alveolata and essential for parasite replication. We found that parasites lacking MAPK2 protein were defective in the initiation of daughter cell budding and were rendered inviable. Specifically, T. gondii MAPK2 (TgMAPK2) appears to be required for centrosome replication at the basal end of the nucleus, and its loss causes arrest early in parasite division. MAPK2 is unique to the Alveolata and not found in metazoa and likely is a critical component of an essential parasite-specific signaling network. 
    more » « less
  2. ; ; ; ; ; ; ( , G3 Genes|Genomes|Genetics)
    Abstract

    In the green alga Chlamydomonas reinhardtii, regulation of the cell cycle in response to external cues is critical for survival in a changing environment. The loss of the nuclear COMPROMISED HYDROLYSIS OF TRIACYLGLYCEROLS 7 (CHT7) protein affects the expression of many genes especially in response to nitrogen availability. Cells lacking CHT7 exhibit abnormal cell morphology following nitrogen deprivation and fail to resume normal cell division after N resupply. To investigate the function of CHT7 in the regulation of cell cycle-related pathways, cells were synchronized, and RNA-seq analysis was performed during various stages of the cell cycle. In the cht7 mutant following nitrogen deprivation, the cells were not dividing, but a subset of cell cycle genes involved in DNA replication and mitosis were found to be derepressed, suggesting that the CHT7 protein plays a role in cell cycle regulation that is opposite to that of the mitotic cyclin-dependent kinases. Furthermore, genes for cell wall synthesis and remodeling were found to be abnormally induced in nondividing cht7 cells; this misregulation may deplete cellular resources and thus contribute to cell death following nitrogen deprivation. Lastly, 43 minimally characterized kinases were found to be highly misregulated in cht7. Further analysis suggested that some of these CHT7-regulated kinases may be related to the MAP3K and Aurora-like kinases, while others are unique. Together, these results suggest a role of CHT7 in transcriptional regulation of the cell cycle and reveal several pathways and genes whose expression appears to be subject to a CHT7-mediated regulatory network.

     
    more » « less
  3. ; ; ; ( , Molecular Plant-Microbe Interactions®)
    Tar spot is a devasting corn disease caused by the obligate fungal pathogen Phyllachora maydis. Since its initial identification in the United States in 2015, P. maydis has become an increasing threat to corn production. Despite this, P. maydis has remained largely understudied at the molecular level due to difficulties surrounding its obligate lifestyle. Here, we generated a significantly improved P. maydis nuclear and mitochondrial genome using a combination of long- and short-read technologies and also provide the first transcriptomic analysis of primary tar spot lesions. Our results show that P. maydis is deficient in inorganic nitrogen utilization, is likely heterothallic, and encodes for significantly more protein coding genes, including secreted enzymes and effectors, than previous determined. Furthermore, our expression analysis suggests that following primary tar spot lesion formation, P. maydis might reroute carbon flux away from DNA replication and cell division pathways and towards pathways previously implicated in having significant roles in pathogenicity, such as autophagy and secretion. Together, our results identified several highly expressed unique secreted factors that likely contribute to host recognition and subsequent infection, greatly increasing our knowledge of the biological capacity of P. maydis, which have much broader implications for mitigating tar spot of corn. 
    more » « less
  4. ; ; ; ; ( , Proceedings of the National Academy of Sciences)
    How cells regulate their cell cycles is a central question for cell biology. Models of cell size homeostasis have been proposed for bacteria, archaea, yeast, plant, and mammalian cells. New experiments bring forth high volumes of data suitable for testing existing models of cell size regulation and proposing new mechanisms. In this paper, we use conditional independence tests in conjunction with data of cell size at key cell cycle events (birth, initiation of DNA replication, and constriction) in the model bacterium Escherichia coli to select between the competing cell cycle models. We find that in all growth conditions that we study, the division event is controlled by the onset of constriction at midcell. In slow growth, we corroborate a model where replication-related processes control the onset of constriction at midcell. In faster growth, we find that the onset of constriction is affected by additional cues beyond DNA replication. Finally, we also find evidence for the presence of additional cues triggering initiations of DNA replication apart from the conventional notion where the mother cells solely determine the initiation event in the daughter cells via an adder per origin model. The use of conditional independence tests is a different approach in the context of understanding cell cycle regulation and it can be used in future studies to further explore the causal links between cell events. 
    more » « less
  5. ; ; ; ; ; ; ( , Development)
    ABSTRACT Regulation of cell cycle progression is essential for cell proliferation during regeneration following injury. After appendage amputation, the axolotl (Ambystoma mexicanum) regenerates missing structures through an accumulation of proliferating cells known as the blastema. To study cell division during blastema growth, we generated a transgenic line of axolotls that ubiquitously expresses a bicistronic version of the fluorescent ubiquitination-based cell-cycle indicator (FUCCI). We demonstrate near-ubiquitous FUCCI expression in developing and adult tissues, and validate these expression patterns with DNA synthesis and mitosis phase markers. We demonstrate the utility of FUCCI for live and whole-mount imaging, showing the predominantly local contribution of cells during limb and tail regeneration. We also show that spinal cord amputation results in increased proliferation at least 5 mm from the site of injury. Finally, we use multimodal staining to provide cell type information for cycling cells by combining fluorescence in situ hybridization, EdU click-chemistry and immunohistochemistry on a single FUCCI tissue section. This new line of animals will be useful for studying cell cycle dynamics using in situ endpoint assays and in vivo imaging in developing and regenerating animals. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email