Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network.

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of α-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster, and then examining the microtubule networks in the neuromuscular junction and muscle cells, we accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

α-Tubulin acetylation at lysine 40 regulates dendritic arborization and larval locomotion by promoting microtubule stability in Drosophila.

Posttranslational modification of tubulin increases the dynamic complexity and functional diversity of microtubules. Acetylation of α-tubulin at Lys-40 is a highly conserved posttranslational modification that has been shown to improve the flexibility and resilience of microtubules. Here we studied the in vivo functions of α-tubulin acetylation by knocking-out Atat, the Drosophila α-tubulin acetyltransferase, and by mutating Lys-40 to Arg in α1-tubulin. We found a reduction in the dendritic arborization of larval class I dendritic arborization (da) neurons in both mutants. The dendritic developmental defects in atat mutants could be reversed by enhancing the stability of microtubules either through knocking down the microtubule severing protein Katanin 60 or through overexpressing tubulin-specific chaperone E, suggesting that α-tubulin deacetylation impairsed dendritic morphology by decreasing the stability of microtubules. Using time-lapse recordings, we found that atat and α1-tubulinK40R mutations dramatically increased the number of dendritic protrusions that were likely to be immature dendritic precursors. Finally, we showed that both Atat and α-tubulin acetylation were required in class I da neurons to control larval locomotion. These findings add novel insight into the current knowledge of the role of α-tubulin acetylation in regulating neuronal development and functions.

High miR-3648 expression and low APC2 expression are associated with shorter survival and tumor progression in NSCLC.

BACKGROUND: Emerging studies have demonstrated that microRNAs (miRNAs) play crucial roles in the carcinogenesis of many developing human tumors. However, the clinical significance and biological function of microRNA-3648 (miR-3648) in non-small cell lung cancer (NSCLC) have been largely undefined. METHODS: The expression of miR-3648 and the mRNA of adenomatous polyposis coli 2 (APC2) in NSCLC tissues and cell lines were analyzed using quantitative real-time RT-PCR. The prognostic value of miR-3648 and APC2 was examined using the Kaplan-Meier method and Cox regression analyses. Experiments using NSCLC cells were conducted to explore the influences of miR-3648 on tumor cell proliferation, migration and invasion. RESULT: Increased expression of miR-3648 was observed in NSCLC tissues and cell lines compared with the corresponding controls (all P<0.05). miR-3648 expression was associated with the differentiation, lymph node metastasis and TNM stage (all P<0.05) of NSCLC patients, and high expression of miR-3648 was associated with poor overall survival rate. NSCLC cell proliferation, migration and invasion were significantly enhanced by miR-3648 overexpression. The further luciferase reporter assay and expression results showed that the decreased APC2 might also be a prognostic biomarker, and served as a target of miR-3648 in NSCLC. CONCLUSION: The findings from the present study indicate that the overexpression of miR-3648 serves as a useful biomarker for the prediction of prognosis in NSCLC, and promotes tumor cell proliferation, migration and invasion. APC2, as another prognosis-related molecule, may be a target of miR-3648 in NSCLC.