Isolation and partial characterization of Drosophila myoblasts from primary cultures of embryonic cells.

Bernstein, Sanford I.; Fyrberg, Eric; Donady, J. James

doi:10.1083/jcb.78.3.856

Cited by 22 publications

(20 citation statements)

References 18 publications

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“…As earlier studies were largely based on muscle-specific morphological features, such as multiple nuclei in primary myotubes, as well as on myofibril structures observed using light and/or electron microscopy (Bernstein et al, 1978;Echalier, 1997), we set out to confirm and extend previous analyses by following myogenesis in primary culture using muscle-specific molecular markers. We developed conditions for RNAi by culturing cells in the presence of doublestranded RNAs (dsRNAs), and used it to identify genes involved in muscle maintenance and integrity.…”

Section: Introductionmentioning

confidence: 72%

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RNA interference screening inDrosophilaprimary cells for genes involved in muscle assembly and maintenance

Bai

Binari

et al. 2008

Development

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To facilitate the genetic analysis of muscle assembly and maintenance, we have developed a method for efficient RNA interference (RNAi) in Drosophila primary cells using double-stranded RNAs (dsRNAs). First, using molecular markers, we confirm and extend the observation that myogenesis in primary cultures derived from Drosophila embryonic cells follows the same developmental course as that seen in vivo. Second, we apply this approach to analyze 28 Drosophila homologs of human muscle disease genes and find that 19 of them, when disrupted, lead to abnormal muscle phenotypes in primary culture. Third, from an RNAi screen of 1140 genes chosen at random, we identify 49 involved in late muscle differentiation. We validate our approach with the in vivo analyses of three genes. We find that Fermitin 1 and Fermitin 2, which are involved in integrin-containing adhesion structures, act in a partially redundant manner to maintain muscle integrity. In addition, we characterize CG2165, which encodes a plasma membrane Ca 2+ -ATPase, and show that it plays an important role in maintaining muscle integrity. Finally, we discuss how Drosophila primary cells can be manipulated to develop cell-based assays to model human diseases for RNAi and small-molecule screens.

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Section: Introductionmentioning

confidence: 72%

“…Embryonic primary cell cultures were established as described previously (Bernstein et al, 1978). Briefly, eggs were collected on molasses plates streaked with killed yeast paste for 2 hours and incubated for an additional 4 hours at 25°C.…”

Section: Embryonic Primary Cell Culturesmentioning

confidence: 99%

RNA interference screening inDrosophilaprimary cells for genes involved in muscle assembly and maintenance

Bai

Binari

et al. 2008

Development

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“…These studies were carried out in isolated primary myoblasts for clear visualization of cell-cell contacts. Cells were isolated as described previously (Bai et al, 2008;Bai et al, 2009;Bernstein et al, 1978) and behave similarly in our hands (see Fig. S2 in the supplementary material).…”

Section: Asymmetric Localization Of Mbc Active Rac1 and Actin Foci Imentioning

confidence: 99%

“…Drosophila primary myoblast cultures were prepared as described previously (Bai et al, 2009;Bernstein et al, 1978) with the following modifications. Embryos were sterilized for 10 minutes at 25°C in 50% bleach, washed and disrupted at 0.01-0.02 g embryos/ml.…”

Section: Preparation Of Primary Cellsmentioning

confidence: 99%

Asymmetric Mbc, active Rac1 and F-actin foci in the fusion-competent myoblasts during myoblast fusion inDrosophila

et al. 2011

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Myoblast fusion is an intricate process that is initiated by cell recognition and adhesion, and culminates in cell membrane breakdown and formation of multinucleate syncytia. In the Drosophila embryo, this process occurs asymmetrically between founder cells that pattern the musculature and fusion-competent myoblasts (FCMs) that account for the bulk of the myoblasts. The present studies clarify and amplify current models of myoblast fusion in several important ways. We demonstrate that the non-conventional guanine nucleotide exchange factor (GEF) Mbc plays a fundamental role in the FCMs, where it functions to activate Rac1, but is not required in the founder cells for fusion. Mbc, active Rac1 and F-actin foci are highly enriched in the FCMs, where they localize to the Sns:Kirre junction. Furthermore, Mbc is crucial for the integrity of the F-actin foci and the FCM cytoskeleton, presumably via its activation of Rac1 in these cells. Finally, the local asymmetric distribution of these proteins at adhesion sites is reminiscent of invasive podosomes and, consistent with this model, they are enriched at sites of membrane deformation, where the FCM protrudes into the founder cell/myotube. These data are consistent with models promoting actin polymerization as the driving force for myoblast fusion.

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“…23 4 55e. 6'$7 (6). Cultured cells were lysed directly in RNA extraction buffer, and RNA was isolated by phenolchloroform extraction (5).…”

mentioning

confidence: 99%

Alternative RNA splicing generates transcripts encoding a thorax-specific isoform of Drosophila melanogaster myosin heavy chain.

Bernstein¹,

Hansen²,

Becker³

et al. 1986

Mol. Cell. Biol.

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Genomic and cDNA sequencing studies show that transcripts from the muscle myosin heavy-chain (MHC) gene of Drosophila melanogaster are alternatively spliced, producing RNAs that encode at least two MHC isoforms with different C termini. Transcripts encoding an MHC isoform with 27 unique C-terminal amino acids accumulate during both larval and adult muscle differentiation. Transcripts for the second isoform encode one unique C-terminal amino acid and accumulate almost exclusively in pupal and adult thoracic segments, the location of the indirect flight muscles. The 3' splice acceptor site preceding the thorax-specific exon is unusually purine rich and thus may serve as a thorax-specific splicing signal. We suggest that the alternative C termini of these two MHC isoforms control myofilament assembly and may play a role in generating the distinctive myofilament organizations of flight muscle and other muscle types.

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Isolation and partial characterization of Drosophila myoblasts from primary cultures of embryonic cells.

Abstract: We describe a method for preparing highly enriched cultures of Drosophila

Cited by 22 publications

References 18 publications

RNA interference screening inDrosophilaprimary cells for genes involved in muscle assembly and maintenance

RNA interference screening inDrosophilaprimary cells for genes involved in muscle assembly and maintenance

Asymmetric Mbc, active Rac1 and F-actin foci in the fusion-competent myoblasts during myoblast fusion inDrosophila

Alternative RNA splicing generates transcripts encoding a thorax-specific isoform of Drosophila melanogaster myosin heavy chain.

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