Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Mercadante, Dayna; Crowley, Elizabeth A; Manning, Amity L.

doi:10.3791/60255

Cited by 6 publications

(12 citation statements)

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“…To assess the temporal relationship between spindle dynamics and mitotic fate of cells following loss of cortical dynein activity, we performed timelapse imaging of cells stably expressing fluorescently-tagged tubulin (EGFP-tubulin) (Figure 3C) (Mercadante et al, 2019) and induced cells to have extra centrosomes through induction of PLK4 expression, as described above. Phase contrast and fluorescence images were captured every 2.5 minutes throughout mitosis and used to assess spindle structure, mitotic timing, and the number of progeny resulting from each division.…”

Section: Resultsmentioning

confidence: 99%

Cortical Dynein Drives Centrosome Clustering in Cells with Centrosome Amplification

Mercadante

Aaron

Olson

et al. 2022

Preprint

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During cell division, the microtubule nucleating and organizing organelle, known as the centrosome, is critical for the formation of the mitotic spindle. In cells with two centrosomes, each centrosome functions as an anchor point for microtubules, leading to the formation of a bipolar spindle and progression through a bipolar cell division. When extra centrosomes are present, multipolar spindles form and the parent cell may divide into more than two daughter cells. Cells that are born from multipolar divisions are not viable and hence clustering of extra centrosomes and progression to a bipolar division are critical determinants of viability in cells with extra centrosomes. We combine experimental approaches with computational modeling to define a role for cortical dynein in centrosome clustering. We show that centrosome clustering fails and multipolar spindles dominate when cortical dynein distribution or activity is experimentally perturbed. Our simulations further reveal that centrosome clustering is sensitive to the distribution of dynein on the cortex. Together, these results indicate that dynein's cortical localization alone is insufficient for effective centrosome clustering and instead, dynamic relocalization of dynein from one side of the cell to the other throughout mitosis promotes timely clustering and bipolar cell division in cells with extra centrosomes.

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Section: Resultsmentioning

confidence: 99%

Cortical Dynein Drives Centrosome Clustering in Cells with Centrosome Amplification

Mercadante

Aaron

Olson

et al. 2022

Preprint

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“…Live cell images were captured at 10 coordinates per condition at 5-minute intervals for 24hrs for each of 3 biological replicates. Mitotic timing was recorded from NEB until anaphase onset (Mercadante et al, 2019) for a minimum of 50 cells per replicate and significance between biological replicates (n=3) was determined using a students’ two-tailed t-test. All analyses were performed on unprocessed images.…”

Section: Methodsmentioning

confidence: 99%

Suv420 enrichment at the centromere limits Aurora B localization and function

Herlihy

Hahn

Crowley

et al. 2021

Preprint

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Centromere structure and function are defined by the epigenetic modification of histones at centromeric and pericentromeric chromatin. The constitutive heterochromatin found at pericentromeric regions is highly enriched for H3K9me3 and H4K20me3. While mis-expression of the methyltransferase enzymes, Suv39 and Suv420, that regulate these marks are common in disease, the consequences of such changes are not well understood. Our data show that increased centromere localization of Suv39 and Suv420 suppress centromere transcription and compromise localization of the mitotic kinase Aurora B: decreasing microtubule dynamics and compromising chromosome alignment and segregation. We find that inhibition of Suv420 methyltransferase activity partially restores Aurora B localization to centromeres and that restoration of the Aurora B-containing CPC to the centromere is sufficient to suppress mitotic errors that result when Suv420/H4K20me3 is enriched at centromeres. Consistent with a role for Suv39 and Suv420 in negatively regulating Aurora B, high expression of these enzymes corresponds with increased sensitivity to Aurora kinase inhibition in cancer cells suggesting that increased H3K9 and H4K20 methylation may be an underappreciated source of chromosome missegregation in cancer.

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“…RPE cells stably expressing α-tubulin-EGFP were seeded onto a 6-well plate. NIS elements HCA jobs software was used to enable multi-coordinate, multi-well imaging in a single z-stack (0.67 µm per pixel) [65]. Images were captured every 5 minutes for 16 hours.…”

Section: Live-cell Imagingmentioning

confidence: 99%

“…RPE cells stably expressing L304-EGFP-Tubulin (Addgene #64060, Watertown, MA) were generated by lentiviral transduction and placed under 10 g/mL Puromycin selection for 5-7 days. Expression of the tagged construct was confirmed by immunofluorescent imaging (76). RPE cells stably expressing GFP-centrin were previously described ( 77) and generously provided by Neil Ganem.…”

Section: Repulsive Forcementioning

confidence: 99%

“…Cells were captured with a Zyla sCMOS (Oxford Instruments, Belfast, UK) camera mounted on a Nikon Ti-E microscope (Nikon, Tokyo, Japan). A 60x Plan Apo oil immersion objective was used for fixed-cell imaging and live-cell imaging of RPE cells expressing GFP-centrin to visualize centrosomes (78), and a 20x CFI Plan Fluor objective was used for live-cell imaging of RPE cells expressing GFP-tubulin (76).…”

Section: Immunofluorescence Imagingmentioning

confidence: 99%

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Modeling reveals cortical dynein-dependent fluctuations in bipolar spindle length

Mercadante

Manning

Olson

2020

Preprint

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Proper formation and maintenance of the mitotic spindle is required for faithful cell division. While much work has been done to understand the roles of the key force components of the mitotic spindle, identifying the implications of force perturbations in the spindle remains a challenge. We developed a computational framework of the minimal force requirements for mitotic progression and used experimental approaches to further define and validate the model. Through simulations, we show that rather than achieving and maintaining a constant bipolar spindle length, oscillations in pole to pole distance occur that coincide with microtubule binding and force generation by cortical dynein. In the context of high kinesin-14 (HSET) activity, we reveal the requirement of high cortical dynein activity for bipolar spindle formation.

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Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Cited by 6 publications

References 0 publications

Cortical Dynein Drives Centrosome Clustering in Cells with Centrosome Amplification

Cortical Dynein Drives Centrosome Clustering in Cells with Centrosome Amplification

Suv420 enrichment at the centromere limits Aurora B localization and function

Modeling reveals cortical dynein-dependent fluctuations in bipolar spindle length

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