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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2018)

Maintenance of the mammalian intestine throughout life relies on rapidly dividing stem cells located at the base of invaginating crypts in both the small intestine and the colon. These crypts constitute a protective niche to provide signals conducive to stem cell proliferation and maintenance, while preventing exposure to harmful toxins that may be present in the gut lumen. During homeostasis, gut stem cells respond to signals from the niche to either self-renew or differentiate to generate specialized epithelial cells. Intestinal and colonic stem cells are both specifically marked by the Wnt target gene Lgr5. While much has been learned about intestinal stem cell biology through the use of Lgr5-labeled transgenic mouse models, relatively little is known about how stem cell behavior differs between small intestine and colon, particularly in human. Colon cancer is far more common than small intestinal cancers and is thought to arise from malignant transformation of gut stem cells during aging. Yet how the stem cells of small intestine and colon differ molecularly under homeostatic conditions and during aging remains unclear. Three-dimensional organoids, which permit long-term self-renewal of mammalian gut stem cells in vitro, provide a convenient and accessible system for investigating regional differences in stem cell behavior. Additionally, the epigenetic clock – a measure of age based on age-dependent DNA methylation changes – provides a quantitative tool for analyzing aging of different tissues and cell types.

Here, we show that colon stem cells grown in culture proliferate slower than small intestinal stem cells, have decreased stem cell function, and have a greater tendency to differentiate. In addition to stem cell behavioral differences in culture, we find that human small intestine and colon exhibit different aging profiles as measured by the epigenetic clock. We demonstrate that small intestine crypt cells have decreased epigenetic clock aging rates compared to colon crypts and to surrounding small intestine mucosal cells, and these regional differences in epigenetic aging appear to be maintained in vitro.

    Cover page: Molecular differences between human small intestine and colon stem and progenitor cells
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2020)

    Background & aims

    The epithelia of the intestine and colon turn over rapidly and are maintained by adult stem cells at the base of crypts. Although the small intestine and colon have distinct, well-characterized physiological functions, it remains unclear if there are fundamental regional differences in stem cell behavior or region-dependent degenerative changes during aging. Mesenchyme-free organoids provide useful tools for investigating intestinal stem cell biology in vitro and have started to be used for investigating age-related changes in stem cell function. However, it is unknown whether organoids maintain hallmarks of age in the absence of an aging niche. We tested whether stem cell-enriched organoids preserved the DNA methylation-based aging profiles associated with the tissues and crypts from which they were derived.

    Methods

    To address this, we used standard human methylation arrays and the human epigenetic clock as a biomarker of age to analyze in vitro-derived, 3-dimensional, stem cell-enriched intestinal organoids.

    Results

    We found that human stem cell-enriched organoids maintained segmental differences in methylation patterns and that age, as measured by the epigenetic clock, also was maintained in vitro. Surprisingly, we found that stem cell-enriched organoids derived from the small intestine showed striking epigenetic age reduction relative to organoids derived from colon.

    Conclusions

    Our data validate the use of organoids as a model for studying human intestinal aging and introduce methods that can be used when modeling aging or age-onset diseases in vitro.
      Cover page: DNA Methylation Analysis Validates Organoids as a Viable Model for Studying Human Intestinal Aging
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2017)
      Ageing results in loss of tissue homeostasis across taxa. In the intestine of Drosophila melanogaster, ageing is correlated with an increase in intestinal stem cell (ISC) proliferation, a block in terminal differentiation of progenitor cells, activation of inflammatory pathways, and increased intestinal permeability. However, causal relationships between these phenotypes remain unclear. Here, we demonstrate that ageing results in altered localization and expression of septate junction proteins in the posterior midgut, which is quite pronounced in differentiated enterocytes (ECs) at tricellular junctions (TCJs). Acute loss of the TCJ protein Gliotactin (Gli) in ECs results in increased ISC proliferation and a block in differentiation in intestines from young flies, demonstrating that compromised TCJ function is sufficient to alter ISC behaviour in a non-autonomous manner. Blocking the Jun N-terminal kinase signalling pathway is sufficient to suppress changes in ISC behaviour, but has no effect on loss of intestinal barrier function, as a consequence of Gli depletion. Our work demonstrates a pivotal link between TCJs, stem cell behaviour, and intestinal homeostasis and provides insights into causes of age-onset and gastrointestinal diseases.
        Cover page: Tricellular junctions regulate intestinal stem cell behaviour to maintain homeostasis
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2013)
        The RAF serine/threonine kinases regulate cell growth through the MAPK pathway, and are targeted by small-molecule RAF inhibitors (RAFis) in human cancer. It is now apparent that protein multimers play an important role in RAF activation and tumor response to RAFis. However, the exact stoichiometry and cellular location of these multimers remain unclear because of the lack of technologies to visualize them. In the present work, we demonstrate that photoactivated localization microscopy (PALM), in combination with quantitative spatial analysis, provides sufficient resolution to directly visualize protein multimers in cells. Quantitative PALM imaging showed that CRAF exists predominantly as cytoplasmic monomers under resting conditions but forms dimers as well as trimers and tetramers at the cell membrane in the presence of active RAS. In contrast, N-terminal truncated CRAF (CatC) lacking autoinhibitory domains forms constitutive dimers and occasional tetramers in the cytoplasm, whereas a CatC mutant with a disrupted CRAF-CRAF dimer interface does not. Finally, artificially forcing CRAF to the membrane by fusion to a RAS CAAX motif induces multimer formation but activates RAF/MAPK only if the dimer interface is intact. Together, these quantitative results directly confirm the existence of RAF dimers and potentially higher-order multimers and their involvement in cell signaling, and showed that RAF multimer formation can result from multiple mechanisms and is a critical but not sufficient step for RAF activation.
          Cover page: Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2015)
          Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRas(G12D), a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRas(G12D) is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.
            Cover page: Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway
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