Methods to analyze novel liaisons between endosomes and centrosomes

Hung, Hui-Fang; Hehnly, Heidi; Doxsey, Stephen

doi:10.1016/bs.mcb.2015.03.024

Cited by 4 publications

(6 citation statements)

References 30 publications

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“…We confirmed the increase in phosphorylation seen with the FRET-PACT biosensor by isolating mitotic centrosomes ( Hung et al. , 2015 ) from control and Gravin-depleted cells.…”

Section: Resultssupporting

confidence: 59%

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Gravin regulates centrosome function through PLK1

Colicino

Garrastegui

Freshour

et al. 2018

MBoC

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We propose to understand how the mitotic kinase PLK1 drives chromosome segregation errors, with a specific focus on Gravin, a PLK1 scaffold. In both three-dimensional primary prostate cancer cell cultures that are prone to Gravin depletion and Gravin short hairpin RNA (shRNA)–treated cells, an increase in cells containing micronuclei was noted in comparison with controls. To examine whether the loss of Gravin affected PLK1 distribution and activity, we utilized photokinetics and a PLK1 activity biosensor. Gravin depletion resulted in an increased PLK1 mobile fraction, causing the redistribution of active PLK1, which leads to increased defocusing and phosphorylation of the mitotic centrosome protein CEP215 at serine-613. Gravin depletion further led to defects in microtubule renucleation from mitotic centrosomes, decreased kinetochore-fiber integrity, increased incidence of chromosome misalignment, and subsequent formation of micronuclei following mitosis completion. Murine Gravin rescued chromosome misalignment and micronuclei formation, but a mutant Gravin that cannot bind PLK1 did not. These findings suggest that disruption of a Gravin–PLK1 interface leads to inappropriate PLK1 activity contributing to chromosome segregation errors, formation of micronuclei, and subsequent DNA damage.

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“…We confirmed the increase in phosphorylation seen with the FRET-PACT biosensor by isolating mitotic centrosomes ( Hung et al. , 2015 ) from control and Gravin-depleted cells.…”

Section: Resultssupporting

confidence: 59%

“…HEK 293ad cells were treated with 100 nM nocodazole for 18 h. A mitotic shake-off was then performed, and centrosome isolation was carried out as described in Hung et al. (2015) .…”

Section: Methodsmentioning

confidence: 99%

Gravin regulates centrosome function through PLK1

Colicino

Garrastegui

Freshour

et al. 2018

MBoC

Self Cite

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Section: Accessibility Transparency Data Sharing and Precisionmentioning

confidence: 99%

Achieving Reproducibility and Closed-Loop Automation in Biological Experimentation with an IoT-Enabled Lab of the Future

Miles¹,

Lee²

2018

SLAS Technology

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A robotic cloud laboratory driven by a state-of-the-art unified laboratory operating system integrates automated hardware, humans, and sensors. This lab of the future system enables researchers to transparently and collaboratively create, optimize, and organize biological experiments to achieve more reproducible results, perform around-the-clock experimentation, and more efficiently navigate the vast parameter space of biology.

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“…6d). We provide evidence that Piezo proteins exhibit pericentrosomal localization, likely through an association with endosomes that dynamically localize to the centrosome 52, 53, 56, 71 and that both Piezo hyperactivation and inhibition can induce supernumerary centrosomes. This intriguing result is potentially consistent with the observation of similar arthrogryposis phenotypes in humans carrying either PIEZO2 gain- and loss-of-function mutations 17–21 .…”

Section: Discussionmentioning

confidence: 81%

“…9b). Sorting nexins, including Snx5, are responsible for trafficking endosomes to specific intracellular destinations 51–56 . They often bind to C-termini of transmembrane endosome cargo proteins that protrude from endosome membranes into the cytoplasm 57 .…”

Section: Resultsmentioning

confidence: 99%

Piezo mechanosensory channels regulate centrosome integrity

David

Martinez

et al. 2022

Preprint

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Piezo1 and 2 are evolutionarily conserved mechanosensory cation channels known to function on the cell surface by responding to external pressure and transducing a mechanically activated Ca2+ current. Here we show that both Piezo1 and 2 also exhibit concentrated intracellular localization at centrosomes. Both Piezo1 and 2 loss-of-function and Piezo1 activation by the small molecule Yoda1 produced supernumerary centrosomes due to inappropriate centriole disengagement. Using a centrosome-localized GCaMP Ca2+-sensitive reporter, we show that perturbations in Piezo modulate Ca2+ local concentration at centrosomes. We designed a photoactivable Yoda1 analog (caged-Yoda1) and revealed that its photoactivation specifically at centrosomes leads to rapid premature centriole disengagement within minutes. We identified the sorting nexin Snx5, which is involved in endocytic uptake and trafficking, as a protein-interactor with the conserved Piezo C-terminal domain, and Snx5 also co-localizes with Piezo1 and 2 at centrosomes. Moreover, inhibition of Polo-like-kinase 1 (PLK1) abolishes Yoda1-induced centriole disengagement. Collectively, these data suggest that Piezo1 and 2 in pericentrosomal endosomes control centrosome integrity, likely by maintaining local Ca2+ within a defined range through mechanotransduction of cell intrinsic forces from microtubules.

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Methods to analyze novel liaisons between endosomes and centrosomes

Cited by 4 publications

References 30 publications

Gravin regulates centrosome function through PLK1

Gravin regulates centrosome function through PLK1

Achieving Reproducibility and Closed-Loop Automation in Biological Experimentation with an IoT-Enabled Lab of the Future

Piezo mechanosensory channels regulate centrosome integrity

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