Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity

Dewey, Evan B.; Johnston, Christopher A.

doi:10.1091/mbc.e17-04-0219

Cited by 10 publications

(18 citation statements)

References 92 publications

(130 reference statements)

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“…In contrast to these results, we recently demonstrated that loss of the spectraplakin gene, Short stop (Shot), induces apoptosis in Drosophila wing discs in the absence of JNK activation 15 . Shot is a large actin-microtubule (MT) crosslinking protein that organizes polarized MTs arrays and participates in axon growth, neuromuscular junction maintenance, and cell membrane dynamics 16 .…”

Section: Introductionmentioning

confidence: 68%

“…We next sought to delineate the molecular underpinning for the apoptotic response following shot RNAi expression. We previously showed that shot RNAi treatment in cultured S2 cells leads to an elevated incidence of lagging and bridged chromosomes during anaphase 15 . Errors in chromosome segregation are associated with DNA damage and chromosome instability in other cell systems 4 , 19 , and we thus wondered if shot loss might induce these effects in vivo.…”

Section: Resultsmentioning

confidence: 99%

“…Shot localizes to mitotic spindle poles and directly binds Actin-related protein 1 (Arp1), the filamentous component of the Dynein-activating Dynactin complex. In addition to triggering apoptosis in vivo, Shot loss leads to SAC-dependent mitotic delay and DNA segregation errors in cell culture 15 . Many of these mitotic defects are shared with SAS-4 knockdown, yet their apoptotic responses have opposite JNK dependencies.…”

Section: Introductionmentioning

confidence: 99%

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Loss of the spectraplakin gene Short stop induces a DNA damage response in Drosophila epithelia

Dewey

Parra

Johnston

2020

Sci Rep

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Epithelia are an eminent tissue type and a common driver of tumorigenesis, requiring continual precision in cell division to maintain tissue structure and genome integrity. Mitotic defects often trigger apoptosis, impairing cell viability as a tradeoff for tumor suppression. Identifying conditions that lead to cell death and understanding the mechanisms behind this response are therefore of considerable importance. Here we investigated how epithelia of the Drosophila wing disc respond to loss of Short stop (Shot), a cytoskeletal crosslinking spectraplakin protein that we previously found to control mitotic spindle assembly and chromosome dynamics. In contrast to other known spindle-regulating genes, Shot knockdown induces apoptosis in the absence of Jun kinase (JNK) activation, but instead leads to elevated levels of active p38 kinase. Shot loss leads to double-strand break (DSB) DNA damage, and the apoptotic response is exacerbated by concomitant loss of p53. DSB accumulation is increased by suppression of the spindle assembly checkpoint, suggesting this effect results from chromosome damage during error-prone mitoses. Consistent with DSB induction, we found that the DNA damage and stress response genes, Growth arrest and DNA damage (GADD45) and Apoptosis signal-regulating kinase 1 (Ask1), are transcriptionally upregulated as part of the shot-induced apoptotic response. Finally, co-depletion of Shot and GADD45 induced significantly higher rates of chromosome segregation errors in cultured cells and suppressed shot-induced mitotic arrest. Our results demonstrate that epithelia are capable of mounting molecularly distinct responses to loss of different spindle-associated genes and underscore the importance of proper cytoskeletal organization in tissue homeostasis.

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Section: Introductionmentioning

confidence: 68%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Loss of the spectraplakin gene Short stop induces a DNA damage response in Drosophila epithelia

Dewey

Parra

Johnston

2020

Sci Rep

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“…Here we provide a detailed protocol for live imaging of mitotic S2 cells using methods we recently described 10 . Our method utilizes stably transfected cells with fluorescent markers for microtubules and centromeres whose expression are under control of a copper-inducible promotor (metallothionein; pMT).…”

Section: Introductionmentioning

confidence: 99%

“…Figure 1C. This video is adapted and republished with permission from 10 (https:// www.molbiolcell.org/info-for-authors). Please click here to download this video.…”

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confidence: 99%

Use of <em>Drosophila</em> S2 Cells for Live Imaging of Cell Division

Dewey

Parra

Johnston

2019

JoVE

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Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed-and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:α-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as 'CID' gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

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Melatonin as an oncostatic agent: Review of the modulation of tumor microenvironment and overcoming multidrug resistance

et al. 2022

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Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity

Cited by 10 publications

References 92 publications

Loss of the spectraplakin gene Short stop induces a DNA damage response in Drosophila epithelia

Loss of the spectraplakin gene Short stop induces a DNA damage response in Drosophila epithelia

Use of <em>Drosophila</em> S2 Cells for Live Imaging of Cell Division

Melatonin as an oncostatic agent: Review of the modulation of tumor microenvironment and overcoming multidrug resistance

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