Journal: Frontiers in Cell and Developmental Biology

Article Title: tsCRISPR based identification of Rab proteins required for the recycling of Drosophila TRPL ion channel

doi: 10.3389/fcell.2024.1444953

Figure Lengend Snippet: Recycling of TRPL to the rhabdomere depends on RabX2. (A) Localization of TRPL in eye cross-sections of Ey - uS-Cas9/+ control flies and RabX2 CRISPR mutants in d, d-l, and d-l-d light conditions. Flies were dark-adapted for 72 h after eclosure, then exposed to orange light for 16 h and were subsequently returned to darkness for another 2 h. Sections were probed with α-Cnx99A and α-TRPL antibodies. Rhabdomeres were stained with phalloidin and nuclei with DAPI. Images at the bottom show magnifications of the indicated areas above. Scale bar: 10 μm. (B) Quantification of TRPL content in rhabdomeres. Signals in R1-R6 rhabdomeres were normalized to R7 signals and values obtained after the initial dark adaptation were set to 100%. Statistically significant differences analyzed by a two-way ANOVA calculation with Bonferroni correction are indicated (**** p < 0.0001, *** p < 0.001) Error bars: SEM (n = 3–7). (C) Colocalization of TRPL with the ER marker Cnx99A after 16 h of orange light illumination. Colocalization was assessed using Pearson correlation. The TRPL channel was chosen for the selection of the areas to be quantified. Statistically significant differences as analyzed by an unpaired t-test (ns not significant) Error bars: SEM (n = 5–6).

Article Snippet: To verify efficient gene disruption for RabX2 , we employed T7 endonuclease (NEB, # M0689L) digestion of hybridized PCR amplified genomic regions covering the sgRNA target sites of the RabX2 gene (ALLinTM Mega HiFi Mastermix (highQu, Cat#HLM0201), forward primer: CGA CTT GAC GAT GAG CCA CTT; reverse primer: CAC ATG GCG CCG TAT CTC CTT).

Techniques: Control, CRISPR, Staining, Marker, Selection

Journal: Frontiers in Cell and Developmental Biology

Article Title: tsCRISPR based identification of Rab proteins required for the recycling of Drosophila TRPL ion channel

doi: 10.3389/fcell.2024.1444953

Figure Lengend Snippet: In RabX2 mutants TRPL is released from the ER after 15 min dark adaptation but does not reach the rhabdomere after 90 min. (A) Localization of TRPL in cross-sections of Ey-uS-Cas9/+ control and RabX2 CRISPR mutant ommatidia. After 16 h of orange light exposure, flies were dark-adapted for 15 min, 45 min, and 90 min. Sections were probed with α-Cnx99A and with α-TRPL antibodies. Rhabdomeres were stained with phalloidin and nuclei with DAPI. Images at the bottom show magnifications of the indicated areas above. Scale bar: 10 μm. (B) Colocalization of TRPL with the ER marker Cnx99A after 16 h of orange light illumination and 15 min of dark adaptation. Colocalization was assessed using Pearson correlation. The TRPL channel was chosen for the selection of the areas to be quantified. Statistically significant differences as analyzed by a two-way ANOVA calculation with Bonferroni correction are indicated (ns not significant) Error bars: SEM (n = 4–5).

Article Snippet: To verify efficient gene disruption for RabX2 , we employed T7 endonuclease (NEB, # M0689L) digestion of hybridized PCR amplified genomic regions covering the sgRNA target sites of the RabX2 gene (ALLinTM Mega HiFi Mastermix (highQu, Cat#HLM0201), forward primer: CGA CTT GAC GAT GAG CCA CTT; reverse primer: CAC ATG GCG CCG TAT CTC CTT).

Techniques: Control, CRISPR, Mutagenesis, Staining, Marker, Selection

Journal: Frontiers in Cell and Developmental Biology

Article Title: tsCRISPR based identification of Rab proteins required for the recycling of Drosophila TRPL ion channel

doi: 10.3389/fcell.2024.1444953

Figure Lengend Snippet: Knock-out of RabX2 leads to TRPL accumulation at the trans-Golgi. (A) Localization of TRPL in eye cross and longitudinal -sections of Ey-uS-Cas9/+ control flies and RabX2 knock-out flies. After 3 days of dark adaption and 16 h of orange light exposure, flies were dark-adapted for 15 min, 45 min, and 90 min. Sections were probed with α-Golgin245 (labelling trans-Golgi) and α-TRPL antibodies. Rhabdomeres were stained with phalloidin and nuclei with DAPI. Images at the bottom show magnifications of the indicated areas above. Scale bar: 10 μm. (B) Colocalization of TRPL with Golgin245 after 45 min or 90 min of dark adaptation. Colocalization was assessed using Pearson correlation. The TRPL channel was chosen for the selection of the areas to be quantified. Statistically significant differences analyzed by a two-way ANOVA calculation with Bonferroni correction are indicated (**** p < 0.0001, ns not significant) Error bars: SEM (n = 3–5) (C) Localization of RabX2-YFP in eye longitudinal sections of Rh1 > Gal4/UAS-RabX2-YFP flies. Sections were probed with α-Golgin245 (labelling trans-Golgi) antibody. Rhabdomeres were stained with phalloidin and nuclei with DAPI. Images at the bottom show magnifications of the indicated areas above. Scale bar: 10 μm. (D) Quantification for colocalization of RabX2-YFP with Golgin245. The Golgin245 channel was chosen for the selection of the areas to be quantified. Colocalization was assessed using Pearson correlation. Error bars: SEM (n = 5).

Article Snippet: To verify efficient gene disruption for RabX2 , we employed T7 endonuclease (NEB, # M0689L) digestion of hybridized PCR amplified genomic regions covering the sgRNA target sites of the RabX2 gene (ALLinTM Mega HiFi Mastermix (highQu, Cat#HLM0201), forward primer: CGA CTT GAC GAT GAG CCA CTT; reverse primer: CAC ATG GCG CCG TAT CTC CTT).

Techniques: Knock-Out, Control, Staining, Selection

Journal: Frontiers in Cell and Developmental Biology

Article Title: tsCRISPR based identification of Rab proteins required for the recycling of Drosophila TRPL ion channel

doi: 10.3389/fcell.2024.1444953

Figure Lengend Snippet: Rab3 and RabX2 are important components of TRPL recycling. In the dark, TRPL is localized to the rhabdomeric membrane and translocates to the endosomal network within 2 h after light adaptation. From there, it is transported to the ER within 12 h, where it is temporarily stored. Upon renewed dark adaptation, TRPL is transported from the ER via the trans-Golgi network back to the rhabdomeric membrane within 90 min. Anterograde transport due to de novo synthesis of TRPL occurs via the cis- and trans-Golgi networks (green arrows). A small fraction of internalized TRPL is degraded via the lysosomal degradation pathway (red arrows). Rab3 was identified at the late endosome and is essential for TRPL transport to the ER as well as for proper recycling (burgundy). In contrast, knock-out of RabX2 resulted in accumulation of TRPL at the trans-Golgi (magenta). Marker proteins for the different membrane enclosed compartments are indicated by dots (turquoise).

Article Snippet: To verify efficient gene disruption for RabX2 , we employed T7 endonuclease (NEB, # M0689L) digestion of hybridized PCR amplified genomic regions covering the sgRNA target sites of the RabX2 gene (ALLinTM Mega HiFi Mastermix (highQu, Cat#HLM0201), forward primer: CGA CTT GAC GAT GAG CCA CTT; reverse primer: CAC ATG GCG CCG TAT CTC CTT).

Techniques: Membrane, Knock-Out, Marker

Journal: Molecular Cell

Article Title: RNF14-dependent atypical ubiquitylation promotes translation-coupled resolution of RNA-protein crosslinks

doi: 10.1016/j.molcel.2023.10.012

Figure Lengend Snippet:

Article Snippet: Q5 Master Mix , New England BioLabs , Cat# M0494L.

Techniques: Virus, Recombinant, Protease Inhibitor, Mutagenesis, Plasmid Preparation, Mass Spectrometry, Western Blot, Software