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Open Access Publications from the University of California

Scholarly Works (2 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2024)

Stem cell differentiation is key to plant development, which utilizes asymmetric cell divisions (ACD) to maintain stem cells, promote growth, and encourage cell diversity. Previous work in Arabidopsis thaliana elucidated some pathways controlling stem cell dynamics during morphogenesis. In the leaf epidermis, pre-mitotic, oriented nuclear migrations in meristemoids, a transient stem cell in Arabidopsis thaliana, allow creation of daughter cells with differing sizes, fates, and placements. Pre-division, the nucleus uses microtubules to migrate away from a polarity crescent that controls division orientation. Post-division, it is known that the nucleus orients towards the polarity crescent using F-actin, but precise mechanisms, including relevant nucleators, are unknown. Additionally, while behavior of the nucleus during ACDs in meristemoids has been characterized, cytoskeletal regulators linking the nucleus to F-actin remain unknown. Time-lapse imaging of Arabidopsis seedlings treated with an F-actin Arp2/3 complex inhibitor (CK-666) and a control compound (CK-689), revealed that Arp2/3 complex-mediated F-actin controls nuclear migration post-division. Novel regulators of nuclear migration were found by phenotypic characterization of stomatal formation in loss-of-function mutants in protein components linking the cytoskeleton to the nuclear envelope. We found three candidate proteins (CRWN1, CRWN3, and SINE1) decreased stomatal density, while one (SUN1) increased stomatal density. This suggests ACD frequency is altered in these mutants and identifies new factors to probe in future studies. These candidate proteins, SUN1, CRWN1, CRWN3, and SINE1, may control nuclear migration in Arabidopsis thaliana, alongside Arp2/3 complex-mediated F-actin. Determining mechanisms behind nuclear migration gives insight into how organelle positioning controls ACDs and stomatal development.

    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2015)
    Centromeres are defined epigenetically in the majority of eukaryotes by the presence of chromatin containing the centromeric histone H3 variant CENP-A. Most species have a single gene encoding a centromeric histone variant whereas C. elegans has two: HCP-3 (also known as CeCENP-A) and CPAR-1. Prior RNAi replacement experiments showed that HCP-3 is the functionally dominant isoform, consistent with CPAR-1 not being detectable in embryos. GFP::CPAR-1 is loaded onto meiotic chromosomes in diakinesis and is enriched on bivalents until meiosis I. Here we show that GFP::CPAR-1 signal loss from chromosomes precisely coincides with homolog segregation during anaphase I. This loss of GFP::CPAR-1 signal reflects proteolytic cleavage between GFP and the histone fold of CPAR-1, as CPAR-1::GFP, in which GFP is fused to the C-terminus of CPAR-1, does not exhibit any loss of GFP signal. A focused candidate screen implicated separase, the protease that initiates anaphase by cleaving the kleisin subunit of cohesin, in this cleavage reaction. Examination of the N-terminal tail sequence of CPAR-1 revealed a putative separase cleavage site and mutation of the signature residues in this site eliminated the cleavage reaction, as visualized by retention of GFP::CPAR-1 signal on separating homologous chromosomes at the metaphase-anaphase transition of meiosis I. Neither cleaved nor uncleavable CPAR-1 were centromere-localized in mitosis and instead localized throughout chromatin, indicating that centromere activity has not been retained in CPAR-1. Although the functions of CPAR-1 and of its separase-dependent cleavage remain to be elucidated, this effort reveals a new substrate of separase and provides an in vivo biosensor to monitor separase activity at the onset of meiosis I anaphase.
      Cover page: Separase Cleaves the N-Tail of the CENP-A Related Protein CPAR-1 at the Meiosis I Metaphase-Anaphase Transition in C. elegansCreative Commons 'BY' version 4.0 license
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