Journal: Journal of Histochemistry and Cytochemistry

Article Title: Immunocytochemical Labeling of Rhabdomeric Proteins in Drosophila Photoreceptor Cells Is Compromised by a Light-dependent Technical Artifact

doi: 10.1369/0022155419859870

Figure Lengend Snippet: Structure and arrangement of rhabdomeres from Drosophila photoreceptor cells. (A) Transmission electron micrograph of a cross section through photoreceptor cells R1-7 from wild type flies as indicated. (B) Light microscopic fluorescence image of an ommatidial cross section from wild type flies stained with Alexa Fluor 546 conjugated phalloidin (red) and DAPI (4’,6-diamidino-2-phenylindole, blue) to visualize the rhabdomeric actin cytoskeleton and nuclei of corresponding photoreceptor cells, respectively. (C) Schematics of fluorescence-tagged phototransduction proteins, rhodopsin Rh1 and ion channels TRP (transient receptor potential) or TRPL (TRP-like) that are expressed in photoreceptor cells R1-6 and were used in this study. INAD, inactivation no afterpotential D. Scale bar represents 2 µm.

Article Snippet: The following primary antibodies were used: rabbit α-TRPL, mouse α-TRP, and mouse α-Rh1 (MAb83F6 and 4C5, Developmental Studies Hybridoma Bank, Iowa City, IA).

Techniques: Transmission Assay, Fluorescence, Staining

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Immunocytochemical Labeling of Rhabdomeric Proteins in Drosophila Photoreceptor Cells Is Compromised by a Light-dependent Technical Artifact

doi: 10.1369/0022155419859870

Figure Lengend Snippet: Antibody staining artifact of rhabdomeric proteins is emphasized in illuminated photoreceptor cells. Immunocytochemical analyses of ommatidial cryosections were performed in dark adapted animals and upon 5 min of orange light exposition to initiate phototransduction in flies transgenically expressing either (A) TRPL::eGFP, (B) TRP::eGFP, or (C) Rh1::eGFP heterozygously in R1-6 photoreceptor cells. Signal patterns of respective antibody staining and fluorescence tags matched perfectly in case of dark adaptation or inhibition of the phototransduction cascade ( norpA P24 ) under illumination, but differed drastically when phototransduction was activated. In the latter case, antibodies detected only proteins at the rhabdomeric base whereas eGFP-tagged variants were evidently still present throughout the entire rhabdomeres. Scale bar represents 5 µm.

Article Snippet: The following primary antibodies were used: rabbit α-TRPL, mouse α-TRP, and mouse α-Rh1 (MAb83F6 and 4C5, Developmental Studies Hybridoma Bank, Iowa City, IA).

Techniques: Staining, Expressing, Fluorescence, Inhibition

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Immunocytochemical Labeling of Rhabdomeric Proteins in Drosophila Photoreceptor Cells Is Compromised by a Light-dependent Technical Artifact

doi: 10.1369/0022155419859870

Figure Lengend Snippet: Light-induced staining patterns of TRPL fusion proteins after vitamin A deprivation or labeled with alternative chromophores. (A,C) Ommatidial cryosections were prepared from 1-3 day old TRPL::SNAP or TRPL::eGFP expressing flies after 30 min of illumination with orange light. Sections were stained with α-TRPL or by self-labeling of TRPL::SNAP with the fluorescent substrates 505-Star (green) or TMR-Star (magenta). (B) Immunoblot analysis of endogenous Rh1 expression in TRPL::SNAP and TRPL::eGFP flies with and without vitamin A deprivation. Proteins from 4 Drosophila heads probed with antibody α-Rh1 which detects rhodopsin at ca. 30 kDa. Tubulin was used as loading control. Scale bar in A and C represents 2.5 µm.

Article Snippet: The following primary antibodies were used: rabbit α-TRPL, mouse α-TRP, and mouse α-Rh1 (MAb83F6 and 4C5, Developmental Studies Hybridoma Bank, Iowa City, IA).

Techniques: Staining, Labeling, Expressing, Western Blot, Control

Journal: Journal of Cell Science

Article Title: CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

doi: 10.1242/jcs.178764

Figure Lengend Snippet: CULD is a new photoreceptor-enriched CUB/LDLa-domain family protein. (A) Domain organization of CULD. Indicated are the CUB domain(s), LDLa domain and predicted transmembrane motif (TM) of CULD, Drosophila NETO, mouse NETO1 and human NETO1. The domains were predicated by InterPro ( http://www.ebi.ac.uk/InterProScan/ ). (B) The alignment of the CUB domain and LDLa domain among Drosophila NETO, mouse NETO1, and human NETO1. The top two rows are the alignment of CUB domain, and the alignment of LDLa domains is on the bottom. Yellow shows residues conserved across all proteins, blue shows residues conserved across some of the proteins and green shows similar residues. (C) CULD is a transmembrane protein. The cytoplasmic (c) and membranal (m) fractions of the ninaE-culd-rfp head extracts were separated and western blots are probed with anti-tubulin, anti-Rh1 and anti-RFP antibodies. Extracts from four heads were loaded for each lane. (D) Expression of culd is enriched in photoreceptor cells. Heads from Pculd-rfp flies at 70% pupal development express RFP (red) driven by the culd promoter are shown. Eye tissue was stained with anti-PDH (green) and anti-RFP (red) antibodies. The photoreceptor cells are indicated by arrows.

Article Snippet: The blots were probed with primary antibodies against tubulin (mouse, 1:2000, Developmental Studies Hybridoma Bank), Rh1 (mouse, 1:2000 dilution, Developmental Studies Hybridoma Bank), GFP (rabbit, 1:2000, Torrey Pines Biolabs, Houston, TX), RFP (rabbit, 1:2000, Biovision, Milpitas, CA), TRPL (rabbit, 1:1000, Fisher, Waltham, MA), TRP (rabbit, 1:2000; ), INAD (rat, 1:2000; ), PDH (rabbit, 1:2000; ) and NORPA (rabbit, 1:2000; ).

Techniques: Western Blot, Expressing, Staining

Journal: Journal of Cell Science

Article Title: CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

doi: 10.1242/jcs.178764

Figure Lengend Snippet: Mutations in culd increase the protein levels of TRPL and Rh1 and decrease light sensitivity. (A) Organization of the culd locus and two culd alleles. The culd 1 allele consists of a PiggyBac insertion in the third intron of culd . The culd 2 allele lacks most of the second and third exons, a deletion induced by gene targeting. (B) Both culd 1 and culd 2 mutations disrupt culd transcription. gpdh served as a loading control. The genotypes are as follows: wild-type (wt), w 1118 ; excised , a precise excision of culd 1 ; rescue , ninaE-culd-rfp, culd 1 . (C) Western blotting revealed that culd loss-of-function increased TRPL levels and the amount of Rh1 aggregations. Protein extracts from half a head were loaded for each lane. Four-day-old flies raised under 12-h-light–12-h-dark cycles were used. (D) ERG recordings from 4-day-old wt ( cn bw ) and culd 1 ( cn bw ; culd 1 ) flies. Flies were dark-adapted for 2 min before exposed to five pulses of white light of increasing intensities. The maximal light intensity is 300 Lux (10 −1 ). (E) Quantification of the ERG amplitudes of wt and culd 1 flies in the intensity of 0.3 Lux (10 −4 in D). Error bars represent s.d. ( n =7). *** P <0.001 (unpaired t -test).

Article Snippet: The blots were probed with primary antibodies against tubulin (mouse, 1:2000, Developmental Studies Hybridoma Bank), Rh1 (mouse, 1:2000 dilution, Developmental Studies Hybridoma Bank), GFP (rabbit, 1:2000, Torrey Pines Biolabs, Houston, TX), RFP (rabbit, 1:2000, Biovision, Milpitas, CA), TRPL (rabbit, 1:1000, Fisher, Waltham, MA), TRP (rabbit, 1:2000; ), INAD (rat, 1:2000; ), PDH (rabbit, 1:2000; ) and NORPA (rabbit, 1:2000; ).

Techniques: Control, Western Blot

Journal: Journal of Cell Science

Article Title: CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

doi: 10.1242/jcs.178764

Figure Lengend Snippet: Rh1 and TRPL accumulate within culd 1 photoreceptors. (A,B) The left row shows a schematic diagrams of cross-sectional (A) and longitudinal views (B) of photoreceptor cells from a single ommatidium. The rhabdomere (R) is indicated. The right rows show the whole-mount staining of Rh1 in cross-sectional (A) and longitudinal (B) views. Eyes from ninaE-rh1-gfp flies were dissected and stained for Rh1 (red). GFP fluorescence of Rh1–GFP was directly observed (green). (C–E) Rh1 and TRPL accumulated in large vesicles within the cytoplasm of culd 1 photoreceptor cells. Compound eyes from (C) wild-type (wt) ( ninaE-trpl-gfp ), (D) culd 1 ( cn bw; ninaE-trpl-gfp culd 1 ) and (E) rescue ( cn bw; ninaE-culd-rfp culd 1 ) flies were dissected and immunostained for Rh1. The ninaE-trpl-gfp transgene was present in all genotypes and GFP signals were directly observed. Two-day-old flies with white eyes were placed in the dark for 12 h before being exposed to 2000 Lux white light for 2.5 h. Scale bars: 10 µm.

Article Snippet: The blots were probed with primary antibodies against tubulin (mouse, 1:2000, Developmental Studies Hybridoma Bank), Rh1 (mouse, 1:2000 dilution, Developmental Studies Hybridoma Bank), GFP (rabbit, 1:2000, Torrey Pines Biolabs, Houston, TX), RFP (rabbit, 1:2000, Biovision, Milpitas, CA), TRPL (rabbit, 1:1000, Fisher, Waltham, MA), TRP (rabbit, 1:2000; ), INAD (rat, 1:2000; ), PDH (rabbit, 1:2000; ) and NORPA (rabbit, 1:2000; ).

Techniques: Staining, Fluorescence

Journal: Journal of Cell Science

Article Title: CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

doi: 10.1242/jcs.178764

Figure Lengend Snippet: Mutation of arr1 blocked the intracellular accumulation of Rh1 in culd 1 . (A,B) Rh1 colocalized with Arr1 in culd 1 flies. Eyes from cn bw; ninaE-arr1-bfp (wild-type, wt) and cn bw; ninaE-arr1-bfp, culd 1 ( culd 1 ) flies were dissected and stained for Rh1 (red) and TRP (blue). BFP fluorescence of Arr1–BFP was directly observed (green). (C) Retinas of wt ( w 1118 ), culd 1 ( cn bw; culd 1 ), arr1 1 ( arr1 1 cn bw ), and arr1 1 ; culd 1 ( arr1 1 cn bw; culd 1 ) flies were dissected and immunostained for Rh1 (red) and TRP (green). Two-day-old flies with white eyes raised under 12-h-light–12-h-dark cycles were used and placed in the dark for 12 h, then exposed to 2000 Lux white light for 7 h. Scale bars: 10 µm.

Article Snippet: The blots were probed with primary antibodies against tubulin (mouse, 1:2000, Developmental Studies Hybridoma Bank), Rh1 (mouse, 1:2000 dilution, Developmental Studies Hybridoma Bank), GFP (rabbit, 1:2000, Torrey Pines Biolabs, Houston, TX), RFP (rabbit, 1:2000, Biovision, Milpitas, CA), TRPL (rabbit, 1:1000, Fisher, Waltham, MA), TRP (rabbit, 1:2000; ), INAD (rat, 1:2000; ), PDH (rabbit, 1:2000; ) and NORPA (rabbit, 1:2000; ).

Techniques: Mutagenesis, Staining, Fluorescence

Journal: Journal of Cell Science

Article Title: CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

doi: 10.1242/jcs.178764

Figure Lengend Snippet: CULD locates in TRPL- or Rh1-positive endocytic vesicles in photoreceptor cells. (A) Cross-sectional images of retina dissected from ninaE-culd-HA ( cn bw; ninaE-culd-HA ) flies were stained with anti-HA antibody for detecting CULD (green) and with phalloidin for labeling the rhabdomere (red). (B) Longitudinal images of retina dissected from cn bw; ninaE- culd-rfp/+ flies were labeled with anti-Rh1 antibody for detecting Rh1 (green) and with anti-RFP for detecting CULD (red). (C) Retinas of cn bw; ninaE-culd-rfp/ninaE-trpl-gfp flies were stained with anti-RFP (red for CULD) and anti-GFP (green for TRPL) antibodies. (D) Retinas of cn bw; ninaE-culd-HA/ninaE-rab5-rfp flies were labeled with anti- HA (blue for CULD) and anti-RFP (red for RFP) antibodies. Two-day-old flies with white eyes were placed in the dark for 12 h before exposed to 2000 Lux white light for 2.5 h. Scale bars: 10 µm. (E) Quantification of the colocalization between CULD and the endocytic Rh1, TRPL and Rab5. Number of vesicles positive for CULD and/or Rh1, TRPL or Rab5 were counted in confocal sections as described in Materials and Methods, and divided by the total number of CULD-positive vesicles. Images of retinas from three different flies and at least five 20 µm×20 µm areas of each retina were quantified. Error bars represent s.d.

Article Snippet: The blots were probed with primary antibodies against tubulin (mouse, 1:2000, Developmental Studies Hybridoma Bank), Rh1 (mouse, 1:2000 dilution, Developmental Studies Hybridoma Bank), GFP (rabbit, 1:2000, Torrey Pines Biolabs, Houston, TX), RFP (rabbit, 1:2000, Biovision, Milpitas, CA), TRPL (rabbit, 1:1000, Fisher, Waltham, MA), TRP (rabbit, 1:2000; ), INAD (rat, 1:2000; ), PDH (rabbit, 1:2000; ) and NORPA (rabbit, 1:2000; ).

Techniques: Staining, Labeling

Journal: Journal of Cell Science

Article Title: CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

doi: 10.1242/jcs.178764

Figure Lengend Snippet: Defective light-dependent turnover of Rh1 and TRPL in culd 1 flies. (A,B) Colocalization between (A) Rab5–RFP (red) and (B) Rab7–RFP with TRPL–GFP (green) in ommatidia. Three-day-old flies with ninaE-trpl-gfp and ninaE-rab5-rfp or ninaE-rab7-rfp transgenes in either wild-type or culd 1 background were placed in the dark for 12 h, before exposed to orange light for 2 h. Scale bar: 10 µm. (C) Quantification of the percentage of TRPL and Rab5 double-positive vesicles among TRPL-positive vesicles (TRPL+Rab5+/TRPL+) and the percentage of TRPL and Rab7 double-positive vesicles among TRPL-positive vesicles (TRPL+Rab7+/TRPL+) are shown for the experiments in A and B. At least nine ommatidia were quantified for each sample as described in Materials and Methods. Error bars indicate s.d. *** P <0.001; ns, not significant (unpaired t -test). (D) Light-induced Rh1 degradation was blocked in culd 1 flies. Western blots of heads were from ninaE-rh1-gfp; cn bw; culd 1 /TM6B (wt) and ninaE-rh1-gfp; cn bw; culd 1 ( culd 1 ) flies exposed to blue light for the indicated periods of time. INAD served as a loading control, and protein extracts from half of a fly head were loaded for each line. (E) Western blot analysis of heads from ninaE-trpl-gfp (wt) or cn bw; ninaE-trpl-gfp, culd 1 ( culd 1 ) flies that were exposed to orange light for indicated periods of time. Protein extracts of one head were loaded for each line. Two-day-old wt or culd 1 flies with white eyes were used.

Article Snippet: The blots were probed with primary antibodies against tubulin (mouse, 1:2000, Developmental Studies Hybridoma Bank), Rh1 (mouse, 1:2000 dilution, Developmental Studies Hybridoma Bank), GFP (rabbit, 1:2000, Torrey Pines Biolabs, Houston, TX), RFP (rabbit, 1:2000, Biovision, Milpitas, CA), TRPL (rabbit, 1:1000, Fisher, Waltham, MA), TRP (rabbit, 1:2000; ), INAD (rat, 1:2000; ), PDH (rabbit, 1:2000; ) and NORPA (rabbit, 1:2000; ).

Techniques: Western Blot, Control