Goldstein Lab at UNC Chapel Hill
Research Projects
How are mitotic spindles positioned by cell-cell signaling?
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A cell's division orientation determines the microenvironment of the resulting daughter cells, including their cell contacts and proximity to mitogenic cues. Proper regulation of cell division orientation is critical both during development when cells divide frequently and tissues are patterned, and in established tissues to maintain the proper number and organization of cells. We set out to better understand how Wnt-dependent mitotic spindle positioning occurs in the early C. elegans embryo, by identifying some of the key proteins that become localized by cell-cell signaling. Using Cas9-triggered homologous recombination, we inserted fluorescent protein sequences into the endogenous loci of 23 candidate genes, and assessed each protein's localization in the early C. elegans embryo (Figure). We found that Wnt signaling positions the mitotic spindle without inducing the asymmetric localization of known spindle orienting proteins.
We conclude that the canonical model of oriented cell division, in which a few known proteins are asymmetrically localized at the cell cortex in response to an extrinsic cue, cannot be the sole mechanism by which signaling pathways direct cell divisions. Our study suggests other possibilities, including roles for two asymmetrically localized proteins, Dishevelled and APC, in regulating mitotic spindle positioning. Heppert et al. Genetics 2018 |
Localization of fluorescently tagged proteins in 4-cell stage C. elegans embryos.
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What fluorescent proteins should the field use for in vivo imaging?
C. elegans embryos labeled with a membrane associated protein fused to each of the seven fluorescent proteins compared in this study.
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As cell biologists, we use fluorescent proteins everyday to label proteins and study their dynamics in living cells. Recent advances in genome editing techniques such as CRISPR have expanded these possibilities even further. However, we constantly found ourselves asking which fluorescent protein to use for a given experiment. To address this problem, I performed a systematic comparison of bright, commonly used, green and red fluorescent proteins in vivo, in an animal model system. Head-to-head comparisons of fluorescent proteins revealed that their performance in vivo, did not always match predictions based on brightness and photostability measurements made in vitro. These results underscore the importance of testing fluorescent proteins in vivo in various systems, and suggest that factors beyond the intrinsic brightness of fluorescent proteins may govern their in vivo performance.
Heppert et. al. Molecular Biology of the Cell 2016. |
