The Drosophila Nesprin-1 homolog MSP300 is required for muscle autophagy and proteostasis.

Nesprin proteins, which are components of the linker of nucleoskeleton and cytoskeleton (LINC) complex, are located within the nuclear envelope and play prominent roles in nuclear architecture. For example, LINC complex proteins interact with both chromatin and the cytoskeleton. Here, we report that the Drosophila Nesprin MSP300 has an additional function in autophagy within larval body wall muscles. RNAi-mediated MSP300 knockdown in larval body wall muscles resulted in defects in the contractile apparatus, muscle degeneration and defective autophagy. In particular, MSP300 knockdown caused accumulation of cytoplasmic aggregates that contained poly-ubiquitylated cargo, as well as the autophagy receptor ref(2)P (the fly homolog of p62 or SQSTM) and Atg8a. Furthermore, MSP300 knockdown larvae expressing an mCherry-GFP-tagged Atg8a transgene exhibited aberrant persistence of the GFP signal within these aggregates, indicating failure of autophagosome maturation. These autophagy deficits were similar to those exhibited by loss of the endoplasmic reticulum (ER) fusion protein Atlastin (Atl), raising the possibility that Atl and MSP300 might function in the same pathway. In support of this possibility, we found that a GFP-tagged MSP300 protein trap exhibited extensive localization to the ER. Alteration of ER-directed MSP300 might abrogate important cytoskeletal contacts necessary for autophagosome completion.

Motor neuron activity enhances the proteomic stress caused by autophagy defects in the target muscle.

Several lines of evidence demonstrate that increased neuronal excitability can enhance proteomic stress. For example, epilepsy can enhance the proteomic stress caused by the expression of certain aggregation-prone proteins implicated in neurodegeneration. However, unanswered questions remain concerning the mechanisms by which increased neuronal excitability accomplishes this enhancement. Here we test whether increasing neuronal excitability at a particular identified glutamatergic synapse, the Drosophila larval neuromuscular junction, can enhance the proteomic stress caused by mutations in the ER fusion/GTPase gene atlastin (atl). It was previously shown that larval muscle from the atl2 null mutant is defective in autophagy and accumulates protein aggregates containing ubiquitin (poly-UB aggregates). To determine if increased neuronal excitability might enhance the increased proteomic stress caused by atl2, we activated the TrpA1-encoded excitability channel within neurons. We found that TrpA1 activation had no effect on poly-UB aggregate accumulation in wildtype muscle, but significantly increased poly-UB aggregate number in atl2 muscle. Previous work has shown that atl loss from either neuron or muscle increases muscle poly-UB aggregate number. We found that neuronal TrpA1 activation enhanced poly-UB aggregate number when atl was removed from muscle, but not from neuron. Neuronal TrpA1 activation enhanced other phenotypes conferred by muscle atl loss, such as decreased pupal size and decreased viability. Taken together, these results indicate that the proteomic stress caused by muscle atl loss is enhanced by increasing neuronal excitability.

Atl (atlastin) regulates mTor signaling and autophagy in Drosophila muscle through alteration of the lysosomal network.

ABBREVIATIONS: atl atlastin; ALR autophagic lysosome reformation; ER endoplasmic reticulum; GFP green fluorescent protein; HSP hereditary spastic paraplegia; Lamp1 lysosomal associated membrane protein 1 PolyUB polyubiquitin; RFP red fluorescent protein; spin spinster; mTor mechanistic Target of rapamycin; VCP valosin containing protein.

The Drosophila melanogaster attP40 docking site and derivatives are insertion mutations of msp-300.

The ɸC31 integrase system is widely used in Drosophila melanogaster to allow transgene targeting to specific loci. Over the years, flies bearing any of more than 100 attP docking sites have been constructed. One popular docking site, termed attP40, is located close to the Nesprin-1 orthologue msp-300 and lies upstream of certain msp-300 isoforms and within the first intron of others. Here we show that attP40 causes larval muscle nuclear clustering, which is a phenotype also conferred by msp-300 mutations. We also show that flies bearing insertions within attP40 can exhibit decreased msp-300 transcript levels in third instar larvae. Finally, chromosomes carrying certain "transgenic RNAi project" (TRiP) insertions into attP40 can confer pupal or adult inviability or infertility, or dominant nuclear clustering effects in certain genetic backgrounds. These phenotypes do not require transcription from the insertions within attP40. These results demonstrate that attP40 and insertion derivatives act as msp-300 insertional mutations. These findings should be considered when interpreting data from attP40-bearing flies.

Roles for RNA export factor, Nxt1, in ensuring muscle integrity and normal RNA expression in Drosophila.

The mRNA export pathway is responsible for the transport of mRNAs from the nucleus to the cytoplasm, and thus is essential for protein production and normal cellular functions. A partial loss of function allele of the mRNA export factor Nxt1 in Drosophila shows reduced viability and sterility. A previous study has shown that the male fertility defect is due to a defect in transcription and RNA stability, indicating the potential for this pathway to be implicated in processes beyond the known mRNA transport function. Here we investigate the reduced viability of Nxt1 partial loss of function mutants, and describe a defect in growth and maintenance of the larval muscles, leading to muscle degeneration. RNA-seq revealed reduced expression of a set of mRNAs, particularly from genes with long introns in Nxt1 mutant carcass. We detected differential expression of circRNA, and significantly fewer distinct circRNAs expressed in the mutants. Despite the widespread defects in gene expression, muscle degeneration was rescued by increased expression of the costamere component tn (abba) in muscles. This is the first report of a role for the RNA export pathway gene Nxt1 in the maintenance of muscle integrity. Our data also links the mRNA export pathway to a specific role in the expression of mRNA and circRNA from common precursor genes, in vivo.