Live Imaging of &lt;em&gt;Drosophila&lt;/em&gt; Larval Neuroblasts

Lerit, Dorothy A.; Plevock, Karen M.; Rusan, Nasser M.

doi:10.3791/51756

Cited by 31 publications

(32 citation statements)

References 38 publications

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“…A 22 × 22-mm number 1.5 coverslip (VWR) was placed on top of the discs, as described before (Lerit et al 2014). For mu2 focus tracking, the medium was supplemented with 400 μM trimethoprim.…”

Section: Imagingmentioning

confidence: 99%

A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

et al. 2016

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Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.

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Section: Imagingmentioning

confidence: 99%

A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

et al. 2016

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“…Live-cell imaging of developing embryos and dissected larval brains was performed as previously described [59][60][61] . Images were captured on a Zeiss Lightsheet Z1 microscope using a 20X (N.A.…”

Section: Live-cell Imaging and Data Analysismentioning

confidence: 99%

In vivostudy of gene expression with an enhanced dual-color fluorescent transcriptional timer

He¹,

Binari

Huang

et al. 2019

Preprint

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Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns.

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“…Whole testes were dissected from 3 rd instar larvae in Drosophila Schneider's medium (Life Technologies) containing Antibiotic-Antomycotic (Life Technologies), and were mounted in the same medium for imaging on a 50 mm gas-permeable lumox dish (Sarstedt). The medium was surrounded by Halocarbon 700 oil (Sigma) to support a glass coverslip (22x22 mm, #1.5, Fisher) that was placed on top of the medium (Lerit et al, 2014). For colchicine treatments, whole testes were placed in a glass-bottom dish (MatTek) filled with Schneider's medium.…”

Section: Live Spermatocyte Imagingmentioning

confidence: 99%

A novel, dynein-independent mechanism focuses the endoplasmic reticulum around spindle poles in dividingDrosophilaspermatocytes

Karabasheva

Smyth

2019

Preprint

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In dividing animal cells the endoplasmic reticulum (ER) concentrates around the poles of the spindle apparatus by associating with astral microtubules (MTs), and this association is essential for proper ER partitioning to progeny cells. The mechanisms that associate the ER with astral MTs are unknown. Because astral MT minus-ends are anchored by centrosomes at spindle poles, we tested the hypothesis that the MT minus-end motor dynein mediates ER concentration around spindle poles. Live in vivo imaging of Drosophila spermatocytes undergoing the first meiotic division revealed that dynein is required for ER concentration around centrosomes during interphase. In marked contrast, however, dynein suppression had no effect on ER association with astral MTs and concentration around spindle poles in early M-phase. Importantly though, there was a sudden onset of ER-astral MT association in Dhc64C RNAi cells, revealing activation of an M-phase specific mechanism. ER redistribution to spindle poles also did not require non-claret disjunctional (ncd), the other known Drosophila MT minus-end motor, nor Klp61F, a MT plus-end motor that generates spindle poleward forces. Collectively, our results suggest that a novel, M-phase specific mechanism of ER-MT association that is independent of MT minus-end motors is required for proper ER partitioning in dividing cells.

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Live Imaging of <em>Drosophila</em> Larval Neuroblasts

Cited by 31 publications

References 38 publications

A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

In vivostudy of gene expression with an enhanced dual-color fluorescent transcriptional timer

A novel, dynein-independent mechanism focuses the endoplasmic reticulum around spindle poles in dividingDrosophilaspermatocytes

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