Long-term<i>in vivo</i>imaging of<i>Drosophila</i>larvae

Kakanj, Parisa; Eming, Sabine A.; Partridge, Linda; Leptin, Maria

doi:10.1101/744383

Cited by 5 publications

(8 citation statements)

References 118 publications

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“…C9100‐50; 1,000 × 1,000 pixels) controlled by Volocity v.6.3.57, with 488‐nm and 561‐nm channels. z‐stacks were taken with a step size of 0.28 μm with 60×/1.2 and 0.24 μm with 40×/1.3 (45–95 z ‐stacks, covering a 10–30‐μm depth) both for live and fixed specimens (Kakanj et al , 2020). All figures and videos show merged z ‐stacks.…”

Section: Methodsmentioning

confidence: 99%

Autophagy–mediated plasma membrane removal promotes the formation of epithelial syncytia

et al. 2022

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Epithelial wound healing in Drosophila involves the formation of multinucleate cells surrounding the wound. We show that autophagy, a cellular degradation process often deployed in stress responses, is required for the formation of a multinucleated syncytium during wound healing, and that autophagosomes that appear near the wound edge acquire plasma membrane markers. In addition, uncontrolled autophagy in the unwounded epidermis leads to the degradation of endo‐membranes and the lateral plasma membrane, while apical and basal membranes and epithelial barrier function remain intact. Proper functioning of TORC1 is needed to prevent destruction of the larval epidermis by autophagy, in a process that depends on phagophore initiation and expansion but does not require autophagosomes fusion with lysosomes. Autophagy induction can also affect other sub‐cellular membranes, as shown by its suppression of experimentally induced laminopathy‐like nuclear defects. Our findings reveal a function for TORC1‐mediated regulation of autophagy in maintaining membrane integrity and homeostasis in the epidermis and during wound healing.

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Section: Methodsmentioning

confidence: 99%

Autophagy–mediated plasma membrane removal promotes the formation of epithelial syncytia

et al. 2022

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“…Whole larval images were acquired on either a Zeiss SteREO Discovery.V12 (Zeiss Achromat S 0.63x FWD 107mm objective) and Zeiss AxioCam ICc 5 camera (images in Fig 1 ), or on a Leica MZ10 F stereoscope (Leica Plan APO 1.0x objective #10450028) and Zeiss AxioCam MRc r2.1 camera (images in Fig 2 ). To immobilize larvae for imaging, wandering L3 larvae were anesthetized using di-ethyl ether for 3.5 minutes as described in (Kakanj et al, 2020). Larval and adult tissues were prepared for imaging by dissecting in 1x PBS, fixing in 1x PBS, 3.7% paraformaldehyde, 0.3% Triton-X for 30 minutes, and staining with Hoechst 33342 (1:1,500, Life Technologies, Carlsbad, CA, #c10339).…”

Section: Methodsmentioning

confidence: 99%

Distinct responses to rare codons in select Drosophila tissues

Allen

Stewart

Rogers

et al. 2022

Preprint

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Codon usage bias has long been appreciated to influence protein production. Yet, relatively few studies have analyzed the impacts of codon usage on tissue-specific mRNA and protein expression. Here, we use codon-modified reporters to perform an organism-wide screen in Drosophila melanogaster for distinct tissue responses to codon usage bias. These reporters reveal a cliff-like decline of protein expression near the limit of rare codon usage in endogenously expressed Drosophila genes. Near the edge of this limit, however, we find the testis and brain are uniquely capable of expressing rare codon-enriched reporters. We define a new metric of tissue-specific codon usage, the tissue-apparent Codon Adaptation Index, to reveal a conserved enrichment for rare codon usage in the endogenously expressed genes of both Drosophila and human testis. We further demonstrate a role for rare codons in restricting protein expression of an evolutionarily young gene, RpL10Aa, to the Drosophila testis. Rare codon-mediated restriction of this testis-specific protein is critical for female fertility. Our work highlights distinct responses to rarely used codons in select tissues, revealing a critical role for codon bias in tissue biology.

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“…Next, we investigated whether ubi-Gal4 MagHigh could be used to activate gene expression in 3 rd instar larvae. By adapting a protocol for imaging anesthetized larvae (Kakanj et al, 2020), we found that multiphoton illumination turned on His2B:RFP expression in a ROI of the wing disc pouch (Figures S3B and S3C). Furthermore, by specifically photoactivating the presumptive notum region of one of the two wing imaginal discs, we recovered strong His2B:RFP expression in the corresponding pupal hemi-notum, illustrating the possibility of gene activation on one side of the animal (Figures 3C and 3D).…”

Section: Shinegal4 Is Effective In Multiple Tissues and At Different Life Cycle Stagesmentioning

confidence: 99%

“…After 3 washes in PBS, embryos were placed on a MatTek dish (MatTek Life Sciences) and covered with PBS for live imaging. Larva For two photon activation experiments, we adapted the method described in (Kakanj et al, 2020) to anesthetize and mount living larvae for tissues imaging in order to perform small-scale experiments and increase viability at pupal stage. Briefly, using a red light in a dark room, 7 to 8 third instar larvae were collected with a soft brush, and placed in small homemade plastic cages allowing vapor exchange (see Figure 4 (Kakanj et al, 2020)).…”

Section: Ll Open Accessmentioning

confidence: 99%