Fig 1.
Vps13c03628 encodes for a truncated Vps13 protein.
(A) Schematic representation of the Vps13 gene and the genomic localization, RNA and protein is depicted. The epitopes of the polyclonal Vps13 antibodies (Vps13 NT and Vps13 #62) are indicated.(B) Relative levels of Vps13 mRNA in control and Vps13 mutant flies were determined by Q-PCR. Mean and SEM (n = 2) are plotted. (C) Western blot analysis of Vps13 protein in control and Vps13 mutant fly heads using the Vps13 #62 antibody. β-Actin was used as a loading control. (D) Western blot analysis of the level of Vps13 protein in control and Vps13 mutant fly head extracts analyzed with the Vps13 NT antibody. α-tubulin was used as a loading control. (E) Lysates of the heads of control flies and whole control flies were analyzed for Vps13 levels. α-tubulin was used as a loading control. (F) Lysates of the heads of control flies, Vps13 mutant flies and three excision lines were analyzed for Vps13 levels. Human VPS13A was detected in samples of Hek293 cells. Drosophila samples and human samples were run on the same gel, separated by a lane containing the molecular weight standards, after transfer of the membrane, the marker lane was split to detect human and Drosophila VPS13 separately using species specific antibodies. The marker lane was used to align the blots after antibody detection. α-tubulin was used as a loading control.
Fig 2.
Vps13 co-fractionates with Rab7 and Rab5.
(A) Western blot analysis of control fly head samples fractionated into a cytosolic and membrane fraction from postnuclear supernatant (PNS). EGFR was used as a membrane marker and GAPDH as a cytosolic marker. (B) Membrane fractions from control fly heads treated with 1 M KCl, Na2CO3 pH 11 or 6 M urea were centrifuged to separate the soluble and insoluble (membrane containing) fractions. The level of Vps13 was determined in these fractions. Markers for peripheral membrane proteins (GM130), integral membrane proteins (EGFR) and the cytosolic proteins (GAPDH) were used. The “Vps13 lysate” lane contains a lysate derived from Vps13 homozygous mutant fly heads, as expected no Vps13 is detected, demonstrating the specificity of the antibody against Vps13. (C) Membranes from control fly heads were fractionated on a sucrose gradient. Western blot analysis was performed to analyze the distribution of Vps13 in relation to markers associated with membranes of various organelles: Rab7 (late endosomes), Rab5 (early endosomes), GM130 (golgi), Lamp1 (lysosomes) and ATP5A (mitochondria). (D) Immunoisolation of membranes from fraction 14 of the sucrose gradient using Vps13 NT, Rab7 and Rab5 antibodies. (E) Quantification of the sucrose gradient fractionation of Fig 2C.
Fig 3.
Vps13 mutant flies show a decreased life span, age dependent impairment of locomotor function and neurodegeneration.
(A) Life span analysis of isogenic control and Vps13 mutant flies. (B) The fraction of dead flies of total flies used,observed within the indicated time intervals. (C) Life span curve of Vps13 mutant flies and three excision lines. (D) Climbing behavior was analyzed by determining the percentage of isogenic control and Vps13 mutant flies (4 and 17 days old) able to climb 5 cm against gravity within 15 seconds. Mean and SEM are plotted (n = 5). For statistical analysis a two-tailed students T-test was used. P<0.001 is ***. (E) Fly heads (20 day old) of control and homozygous Vps13 mutant flies were fixed, dehydrated and embedded in epon. Sections, visualizing a cross section of the complete brain, were stained with toluidine blue. The scale bar indicates 50 μm.(F) Higher magnification images of the boxed area’s in Fig E. The central complex is denoted with a dotted line. The scale bar indicates 25 μm.
Fig 4.
Impaired Vps13 function leads to defects in protein homeostasis.
(A) Percentage of isogenic control and Vps13 mutant flies that eclosed at increasing temperatures. (B) Percentage of homozygous Vps13 mutant flies and excision line flies that eclosed at 29°C. (C) Percentage of flies of various genotypes that eclosed at 29°C. Two independent deficiency lines (lacking a genomic area containing the Vps13 gene) were crossed with Vps13/ CyO heterozygous flies. Eclosion rate of the following genotypes was analyzed: Vps13/+, Df #7535/+, Vps13/Df #7535, Df #7534/+ and Vps13/Df #7534. (D) Percentage of Vps13 flies that eclosed at 22°C on food with increasing concentrations of L-canavanine. (E) Western blot analysis of lysates of 1 day old control and Vps13 mutant fly heads. Ubiquitylated proteins, K48 ubiquitylated proteins and K63 ubiquitylated proteins were detected. All quantifications show the mean and SEM of at least three independent experiments per condition. For statistical analysis a two-tailed students T-test was used in combination with a Welch’s correction if necessary. P<0.05 is *, P<0.01 is ** and P<0.001 is ***.
Fig 5.
Central nervous system of larval and adult Vps13 mutants contain protein aggregates.
(A) Ventral nerve cords of control, Vps13 mutant, Vps13/Df #7534 and Vps13/Df #7535 third instar larvae were stained for ubiquitylated proteins and DAPI. The presence of DAPI indicates areas where nuclei of neuronal cell bodies or glial cells are located. DAPI negative regions represent areas mainly containing axonal and synaptic structures [33,35]. The areas in the grey boxes are shown below as higher magnification images. The scale bar indicates 50 μm. (B) Quantification of the number of ubiquitylated protein puncta in the ventral nerve cord. (C) Staining of 1 day old adult control brains using DAPI. The grey box denotes the area in the brain where the two antennal lobes are located. The presence of DAPI indicates areas where nuclei of neuronal or glial cell bodies are located. The center area which is negative for DAPI contains axons and synaptic structures [34]. The scale bar indicates 50 μm. (D) Quantification of the number of puncta of ubiquitylated proteins in the antennal lobes derived from 1 day old isogenic controls, Vps13 mutants and excision line 3. (E) Staining of brains derived from 1 day old controls, Vps13 mutants and excision line 3 flies for ubiquitylated proteins, Ref(2)p and DAPI. The scale bar indicates 20 μm Arrows indicate colocalization of Ref(2)P and Ubiquitin positive foci. All quantifications show the mean and SEM of at least three independent experiments per condition. For statistical analysis a two-tailed students T-test was used in combination with a Welch’s correction if necessary. P<0.05 is *, P<0.01 is ** and P<0.001 is ***.
Fig 6.
Overexpression of HsVps13A rescues phenotypes of Vps13 mutants.
(A) Samples from fly heads of Actin-GAL4 / + (as a control) and Actin-GAL4 / UAS-HsVps13A (HsVps13A expressing) flies were separated into a membrane and cytosol fraction and analyzed by Western blot for HsVps13A levels. EGFR and GAPDH used as controls for membrane and cytosolic proteins, respectively. (B) Eclosion rate of Vps13 mutant flies a Actin-GAL4/+ (control) or Actin-GAL4/UAS-HsVp13A (HsVps13A expressing) background at 25°C. (C) Ubiquitylated proteins from samples of 1 day old fly head extracts of Vps13/CyO; Actin-GAL4/+ (as a control), Vps13/ Vps13; Actin-GAL4/+ (representing homozygous mutants) and Vps13/ Vps13; Actin-GAL4/UAS-HsVps13A (representing homozygous mutants expressing human VPS13A). (D) Representative picture of ubiquitylated protein staining of the third instar larval ventral nerve cord of Vps13/CyO; Actin-GAL4/+ (as a control), Vps13/ Vps13; Actin-GAL4/+ and Vps13/ Vps13; Actin-GAL4/UAS-HsVps13A. Arrows indicate accumulations of ubiquitylated positive structures. The scale bar indicates 50 μm and 12,5 μm in the enlargement. (E) Quantification of the number of puncta in third instar larval ventral nerve cord of the experiment presented in Fig 6D. (F) Life span curve of Vps13/ Vps13; Actin-GAL4/+ and Vps13/ Vps13; Actin-GAL4/UAS-HsVps13A. All quantifications show the mean and SEM of at least three independent experiments per condition. For statistical analysis a two-tailed students T-test was used in combination with a Welch’s correction if necessary. P<0.05 is *, P<0.01 is ** and P<0.001 is ***.