Drosophila Larval NMJ Dissection

Brent, Jonathan Robert; Werner, Kristen M.; McCabe, Brian D.

doi:10.3791/1107

Cited by 102 publications

(95 citation statements)

References 5 publications

(4 reference statements)

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“…Adult brains and ventral nerve cords were dissected using established techniques (44). Larval brains were dissected and stained as previously described (45,46). They were labeled with mouse anti-flag (1:500) (Sigma-Aldrich) and rat anti-ELAV (1:500) (DSHB).…”

Section: Methodsmentioning

confidence: 99%

The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span

et al. 2011

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The fatal adult motor neuron disease amyotrophic lateral sclerosis (ALS) shares some clinical and pathological overlap with frontotemporal dementia (FTD), an early-onset neurodegenerative disorder. The RNA/DNAbinding proteins fused in sarcoma (FUS; also known as TLS) and TAR DNA binding protein-43 (TDP-43) have recently been shown to be genetically and pathologically associated with familial forms of ALS and FTD. It is currently unknown whether perturbation of these proteins results in disease through mechanisms that are independent of normal protein function or via the pathophysiological disruption of molecular processes in which they are both critical. Here, we report that Drosophila mutants in which the homolog of FUS is disrupted exhibit decreased adult viability, diminished locomotor speed, and reduced life span compared with controls. These phenotypes were fully rescued by wild-type human FUS, but not ALS-associated mutant FUS proteins. A mutant of the Drosophila homolog of TDP-43 had similar, but more severe, deficits. Through cross-rescue analysis, we demonstrated that FUS acted together with and downstream of TDP-43 in a common genetic pathway in neurons. Furthermore, we found that these proteins associated with each other in an RNA-dependent complex. Our results establish that FUS and TDP-43 function together in vivo and suggest that molecular pathways requiring the combined activities of both of these proteins may be disrupted in ALS and FTD. IntroductionAmyotrophic lateral sclerosis (ALS) is a fatal adult neurodegenerative disorder characterized by progressive motor system dysfunction and loss of cortical and spinal motor neurons. Though predominantly a sporadic disease, 5%-10% of ALS cases are linked to heritable mutations in a diverse range of proteins (1). Recently, mutations in 2 related proteins, 43 kDa transactive response TAR DNA-binding protein-43 (TDP-43) and fused in sarcoma (FUS; also known as TLS), have been associated with both familial ALS (fALS) and frontotemporal dementia (FTD), the second most common early-onset dementia, which shares some clinical and pathological overlap with ALS. TDP-43 was initially identified as the primary component of abnormal protein aggregates found in patients with both sporadic ALS and FTD (2, 3). Subsequent to its purification from aggregates, mutations in TDP-43 were found in approximately 4% of fALS cases. TDP-43 is an evolutionarily conserved protein with 2 RNA recognition motifs (RRMs) and a C-terminal glycinerich region where the majority of ALS-and FTD-associated mutations occur. TDP-43 can shuttle between the nucleus and cytoplasm and has been linked to both DNA transcription and RNA splicing in addition to many other cellular processes (4, 5). Following the identification of TDP-43, mutations in FUS were also linked to ALS and FTD cases (6, 7). Both dominant and recessive inherited mutations of FUS in fALS have been described (6), and mutations of FUS in fALS may be more common than TDP-43 mutations (8). fALS mutant FUS proteins can aberrantly loc...

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Section: Methodsmentioning

confidence: 99%

The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span

et al. 2011

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“…In all cases, embryos were devitellinized by vortexing in a 1:1 methanol:heptane solution. Larvae were dissected in ice-cold HL3.1 as previously described (Brent et al, 2009) and fixed with 10% formalin (Sigma, HT501128-4L). Embryos and larvae were mounted in ProLong Gold (Invitrogen) for fluorescent stainings.…”

Section: Immunohistochemistrymentioning

confidence: 99%

Muscle length and myonuclear position are independently regulated by distinct Dynein pathways

2012

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SUMMARYVarious muscle diseases present with aberrant muscle cell morphologies characterized by smaller myofibers with mispositioned nuclei. The mechanisms that normally control these processes, whether they are linked, and their contribution to muscle weakness in disease, are not known. We examined the role of Dynein and Dynein-interacting proteins during Drosophila muscle development and found that several factors, including Dynein heavy chain, Dynein light chain and Partner of inscuteable, contribute to the regulation of both muscle length and myonuclear positioning. However, Lis1 contributes only to Dynein-dependent muscle length determination, whereas CLIP-190 and Glued contribute only to Dynein-dependent myonuclear positioning. Mechanistically, microtubule density at muscle poles is decreased in CLIP-190 mutants, suggesting that microtubule-cortex interactions facilitate myonuclear positioning. In Lis1 mutants, Dynein hyperaccumulates at the muscle poles with a sharper localization pattern, suggesting that retrograde trafficking contributes to muscle length. Both Lis1 and CLIP-190 act downstream of Dynein accumulation at the cortex, suggesting that they specify Dynein function within a single location. Finally, defects in muscle length or myonuclear positioning correlate with impaired muscle function in vivo, suggesting that both processes are essential for muscle function.

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“…For most experiments, fixation used 4% paraformaldehyde. For examination of larval muscle attachment sites, fillet preparations of wandering third instar larvae were prepared in PBS, as described previously (Brent et al, 2009). For Johnston's organ staining, adult heads with the proboscis removed were fixed in 4% PFA for 90 minutes at 4°C.…”

Section: Immunostaining and Microscopymentioning

confidence: 99%

Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila

et al. 2013

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SUMMARYCbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. Here, we demonstrate that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. We investigate the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function.

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Drosophila Larval NMJ Dissection

Cited by 102 publications

References 5 publications

The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span

The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span

Muscle length and myonuclear position are independently regulated by distinct Dynein pathways

Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila

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