MiniCORVET is a Vps8-containing early endosomal tether in Drosophila
- Eötvös Loránd University, Hungary
- Institute of Genetics, Biological Research Centre, Hungarian Academy of Sciences, Hungary
- Hungarian Academy of Sciences, Hungary
Figures
Figure 1 with 2 supplements
Vps8 is highly expressed in nephrocytes and hemocytes.
(A–C) These panels show various tissues of larvae expressing a Vps8-HA reporter under the control of genomic vps8 promoter sequences. (A) Low magnification image of the upper gastrointestinal tract. …
Figure 1—figure supplement 1
Vps8 is highly expressed in larval nephrocytes and hemocytes.
(A) In the map of the cg10144/vps8 locus, red arrows indicate the primers used to amplify genomic vps8 sequences that were used to generate the vps8-HA transgene (shown enlarged). This construct …
Figure 1—figure supplement 2
Vps8 expression in various adult and pupal tissues.
(A–H) These panels show various adult tissues expressing the Vps8-HA reporter. (I) This panel shows a 30 hr pupal wing from an animal expressing the Vps8-HA reporter. The reporter is strongly …
Figure 2
Vps8 localizes to early endosomes in nephrocytes.
(A–C) These panels illustrate the general structure of garland nephrocytes. (A) Ultrastructure of a wild type garland nephrocyte. (B–C) Fluorescent images of garland cells expressing the late …
Figure 3 with 1 supplement
Generation of a vps8 null mutant.
(A) Map of the vps8/cg10144 locus. CRISPR/Cas9-induced deletions in vps81 mutants are indicated with red lines. The sequence of the region containing the first microdeletion and the resulting early …
Figure 3—figure supplement 1
Loss of Vps8 results in ectopic immune activation.
(A) This chart shows the results of qPCR analyses from animals of the indicated genotypes, using primers that are not sensitive to the presence of frameshift mutations in vps8 mutants. The mRNA …
Figure 4 with 1 supplement
Loss of Vps8 impairs endocytic trafficking in garland nephrocytes and hemocytes.
(A–C) Garland cells take up silver nitrate from the hemolymph and store it in vesicles, which are visible as brown dots. Compared to control or rescue animals, garland nephrocytes from vps8 mutants …
Figure 4—figure supplement 1
Loss of Vps8 impairs endocytic trafficking in garland nephrocytes and hemocytes.
(A,B) Garland nephrocytes from homozygous vps81 mutants contain practically no detectable silver inclusions (similar to vps81/Df animals shown in Figure 4B), and this effect can be rescued by the …
Figure 5 with 2 supplements
The biogenesis of late endosomes and lysosomes is impaired in vps8 mutant garland nephrocytes.
(A–C) Late endosomes are fragmented in garland nephrocytes lacking Vps8, unlike early endosomes. In wild type control or rescued cells, a layer of Rab7-positive endosomes (green) is found under the …
Figure 5—figure supplement 1
The biogenesis of late endosomes is impaired in homozygous vps8 mutant garland nephrocytes.
(A) Rab7 positive late endosomes are fragmented in homozygous vps81 mutant garland nephrocytes, similar to vps81/Df cells shown in Figure 5B. (B) Late endosome defects of vps81 homozygotes are …
Figure 5—figure supplement 2
The endosomal compartment of larval wing disc cells is normal in vps8 mutant larvae.
(A,B) Larval wing discs stained with cortical actin phalloidin and extracellular domain-specific anti-Notch. Wing disc structure and the pattern of endosomal Notch are similar in control (A) and …
Figure 6 with 1 supplement
Vps8 forms a miniCORVET complex with the class C Vps proteins Dor/Vps18, Car/Vps33A and Vps16A.
(A) Endogenous Car, Dor and Vps16A proteins coprecipitate with Vps8-HA. (B) Vps8 interacts with Dor in yeast two-hybrid experiments. Yeast colony growth on synthetic medium lacking Ade indicates a …
Figure 6—figure supplement 1
Vps8 coprecipitates the class C Vps proteins Dor/Vps18, Car/Vps33A and Vps16A from larval lysates.
Endogenous Car, Dor and Vps16A proteins coprecipitate with Vps8-HA from larval lysates, similar to adult samples shown in Figure 6A.
Figure 7 with 1 supplement
MiniCORVET promotes endosomal fusion upstream of HOPS.
Garland nephrocytes lacking the miniCORVET subunits Dor/Vps18 (B) or Vps16A (C) have fragmented Rab7-positive late endosomes compared to controls (A), whilst the early endosomal Rbsn-5 signal …
Figure 7—figure supplement 1
Additional miniCORVET and HOPS mutant nephrocyte data.
(A–C) Garland cells stained with anti-Rbsn-5 and Rab7. Knockdown of car (A) leads to fragmentation of late endosomes. In contrast, RNAi silencing of vps39 (B) or vps11 (C) results in the enlargement …
Figure 8 with 1 supplement
Loss of miniCORVET leads to fragmentation of endosomes, unlike HOPS defects.
Vps81 mutant (B) garland nephrocytes lack normal sized α-vacuoles and only small vesicles can be found, unlike in control (A,D) or rescued (C) cells. In contrast, nephrocytes lacking either the HOPS …
Figure 8—figure supplement 1
Additional miniCORVET and HOPS mutant nephrocyte data.
Low magnification ultrastructural images of garland nephrocytes. Large α-vacuoles containing a single dense core that are clearly visible in control cells (A) are absent from cells lacking …
Figure 9 with 2 supplements
Vps8 localization to early endosomes depends on Rab5 and miniCORVET subunits, but not the HOPS complex.
(A–F) Images from garland nephrocytes expressing Vps8-HA in different genetic backgrounds (as indicated) were stained with anti-HA (magenta) and anti-Rab5 (green). Plot profiles of the framed areas …
Figure 9—figure supplement 1
The early endosomal localization of Vps8 is independent of Rab7 and requires Car/Vps33A and Vps16A.
All images show garland nephrocytes expressing Vps8-HA in different genetic backgrounds, stained with anti-HA (magenta) and anti-Rab5 or GFP (green). Panels (A’–D’) show the plot profiles of the …
Figure 9—figure supplement 2
Validation of knockdown efficiencies for RNAi lines used in this study.
(A) Western blots from larvae ubiquitously expressing car, vps16a or dor RNAi (driven by tub-Gal4). In all three cases, the corresponding protein product practically disappears from the blots in …
Figure 10
Rbsn-5 binds to Dor/Vps18 and is also required for early endosomal fusions.
(A) Rbsn-5 directly binds to Dor, based on prominent growth of yeast colonies on synthetic medium lacking Ade in yeast two hybrid experiments. (B) Rab7-positive late endosomes are fragmented in …
Figure 11
A cartoon illustrating garland nephrocytes in wild type, miniCORVET and/or HOPS loss-of-function animals.
In control cells, fusion of early endosomes leads to formation of large electron lucent late endosomes (α-vacuoles) that contain a single core. These vacuoles mature into degradative endolysosomes …
Additional files
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Supplementary file 1
Nucleotide sequence of the wild type vps8/CG10144 gene (from the translational start codon to the stop codon) and the deletions present in the vps81 allele.
- https://doi.org/10.7554/eLife.14226.024
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Supplementary file 2
Detailed Vps8-HA proteomic data.
The table contains all peptides/proteins identified in the precipitate from Vps8-HA animals, which were absent in controls. Acc#: UNIPROT accession number. Num Unique: number of unique peptides identified for the listed protein.% Cov: The percentage indicates the sequence coverage by the identified peptides. MW: molecular weight in Daltons. Both% Cov and MW were calculated from the genomic sequence listed in the database that may differ from the mature protein. DROME: Drosophila melanogaster.
- https://doi.org/10.7554/eLife.14226.025
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Supplementary file 3
Genotype of animals used in this study.
- https://doi.org/10.7554/eLife.14226.026
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Supplementary file 4
Additional table showing statistical tests, N values and p-values.
- https://doi.org/10.7554/eLife.14226.027
