Live and fixed imaging of translation sites at single mRNA resolution in the Drosophila embryo

Vinter, Daisy J.; Hoppe, Caroline; Ashe, Hilary L.

doi:10.1016/j.xpro.2021.100812

Cited by 5 publications

(5 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The protocol was adapted from previous publications [49, 50]. Late stage 16 embryos of the desired genotypes were collected at 22°C and dechorionated by incubating in a solution of 50% bleach and 50% water for 1.5 minutes, followed by extensive rinsing in water to stop the reaction.…”

Section: Methodsmentioning

confidence: 99%

Whole organism and tissue specific analysis of pexophagy inDrosophila

Barone,

Marcello,

Urbé

et al. 2023

Preprint

View full text Add to dashboard Cite

Peroxisomes are essential organelles involved in critical metabolic processes in animals such as fatty acid oxidation, ether phospholipid production and reactive oxygen species detoxification. We have generated transgenicDrosophila melanogastermodels expressing fluorescent reporters for the selective autophagy of peroxisomes, a process known as pexophagy. Using light sheet microscopy, we have been able to obtain a global overview of pexophagy levels, across the entire organism at different stages of development. Tissue specific control of pexophagy is exemplified by areas of peroxisome abundance but minimal pexophagy observed in clusters of oenocytes, which are the major site of long chain fatty acid synthesis. They are surrounded by epithelial cells where pexophagy is much more evident. Enhancement of pexophagy was achieved by feeding flies with the iron chelator deferiprone, in line with past results using mammalian cells. Specific drivers were used to visualise pexophagy in neurons, in which we tested the role of two proteins proposed to regulate pexophagy. Firstly, depletion of CG8814, theDrosophilahomologue of the yeast protein Atg37, had no noticeable impact on pexophagy. In contrast, specific depletion in the larval central nervous system of Hsc70-5, theDrosophilahomologue of the chaperone HSPA9/Mortalin, led to a substantial elevation in pexophagy.

show abstract

Section: Methodsmentioning

confidence: 99%

Whole organism and tissue specific analysis of pexophagy inDrosophila

Barone,

Marcello,

Urbé

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…By far the most popular systems for real-time transcription detection are the MS2 [28,29] and PP7 [30][31][32][33] systems, whereby MS2 coat protein (MCP) and PP7 coat protein (PCP), fused to fluorophores, bind MS2 and PP7 RNA stem loops, respectively. These have been used to generate a broad array of transgenic animals and computational tools to track gene expression timing in live imaging experiments [19,[34][35][36][37][38][39]. In addition to these popular mRNA visualization options, multiple other aptamer-coat protein pairs are currently available for transgenic studies including BglG The two-part aptamer-binding eIF4a [47] provides an intriguing twist on the system, providing a reliable, straight-forward method to label RNA without ubiquitous background fluorescence by fusing the 2 parts to split fluorophores [48,49] (Fig 1E).…”

Section: Advanced Tools For Visualizing Subcellular Functions In Vivomentioning

confidence: 99%

Opticool: Cutting-edge transgenic optical tools

Fenelon,

Krause,

Koromila

2024

PLoS Genet

View full text Add to dashboard Cite

Only a few short decades have passed since the sequencing of GFP, yet the modern repertoire of transgenically encoded optical tools implies an exponential proliferation of ever improving constructions to interrogate the subcellular environment. A myriad of tags for labeling proteins, RNA, or DNA have arisen in the last few decades, facilitating unprecedented visualization of subcellular components and processes. Development of a broad array of modern genetically encoded sensors allows real-time, in vivo detection of molecule levels, pH, forces, enzyme activity, and other subcellular and extracellular phenomena in ever expanding contexts. Optogenetic, genetically encoded optically controlled manipulation systems have gained traction in the biological research community and facilitate single-cell, real-time modulation of protein function in vivo in ever broadening, novel applications. While this field continues to explosively expand, references are needed to assist scientists seeking to use and improve these transgenic devices in new and exciting ways to interrogate development and disease. In this review, we endeavor to highlight the state and trajectory of the field of in vivo transgenic optical tools.

show abstract

“…When co-expressed in the same cell, the coat protein can bind to the stem loop with high affinity and thus decorate the mRNA in living cells [ 126 ]. This technique (predominantly using the MS2 bacteriophage) has been used to great success in Drosophila [ 127 ] and has even been optimised for single-molecule resolution at low laser power [ 128 , 129 , 130 ].…”

Section: In Vivo Exploration Of P Body Biogenesismentioning

confidence: 99%

Section: In Vivo Exploration Of P Body Biogenesismentioning

confidence: 99%

“…Once translated the epitope is recognised by a constitutively expressed cytoplasmic binding partner conjugated to a fluorophore. Multiple copies of the epitope amplify the fluorescent signal, allowing for the visualisation of single molecules of the nascent transcript in real time [129,130,[135][136][137][138][139][140][141]. Live [126] Yes [128] Yes [128] Low [126] Yes [142] Yes [128] Yes [127,129,130] smFISH RNA localisation [131] Fixed [131] Yes [131] Yes [131] High [131] No [131] Yes [54,[101][102][103][104][105][106] Yes [54,[101][102][103][104][105][106] Multiplexed smFISH mRNA decay [132] Fixed [132] Yes [132] Yes [132] High [132] No [132] Yes [132] Yes …”

Section: Focus On: Techniques For the Study Of P Bodies In Drosophilamentioning

confidence: 99%

See 1 more Smart Citation

Relating the Biogenesis and Function of P Bodies in Drosophila to Human Disease

Wilby,

Weil

2023

Genes

View full text Add to dashboard Cite

Drosophila has been a premier model organism for over a century and many discoveries in flies have furthered our understanding of human disease. Flies have been successfully applied to many aspects of health-based research spanning from behavioural addiction, to dysplasia, to RNA dysregulation and protein misfolding. Recently, Drosophila tissues have been used to study biomolecular condensates and their role in multicellular systems. Identified in a wide range of plant and animal species, biomolecular condensates are dynamic, non-membrane-bound sub-compartments that have been observed and characterised in the cytoplasm and nuclei of many cell types. Condensate biology has exciting research prospects because of their diverse roles within cells, links to disease, and potential for therapeutics. In this review, we will discuss processing bodies (P bodies), a conserved biomolecular condensate, with a particular interest in how Drosophila can be applied to advance our understanding of condensate biogenesis and their role in disease.

show abstract

Live and fixed imaging of translation sites at single mRNA resolution in the Drosophila embryo

Cited by 5 publications

References 30 publications

Whole organism and tissue specific analysis of pexophagy inDrosophila

Whole organism and tissue specific analysis of pexophagy inDrosophila

Opticool: Cutting-edge transgenic optical tools

Relating the Biogenesis and Function of P Bodies in Drosophila to Human Disease

Contact Info

Product

Resources

About