1. Volume 28, Issue 2, Pages 287-295.e6 (January 2018)
Vps13D Encodes a Ubiquitin-Binding Protein that Is Required for the Regulation of Mitochondrial Size and Clearance 
Allyson L. Anding, Chunxin Wang, Tsun-Kai Chang, Danielle A. Sliter, Christine M. Powers, Kay Hofmann, Richard J. Youle, Eric H. Baehrecke 
Current Biology 
Volume 28, Issue 2, Pages e6 (January 2018)
DOI: /j.cub
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2. Current Biology 2018 28, 287-295.e6DOI: (10.1016/j.cub.2017.11.064)
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3. Figure 1
Vps13D Functions in Programmed Cell Size Reduction and Atg8 Puncta Formation in the Drosophila Intestine
(A) Control and Vps13D knockdown (green) cells in the Drosophila midgut stained with DAPI (blue).
(B) Quantitation of control wild-type and Vps13D knockdown cell size from at least 76 cells in at least nine intestines of either control or knockdown cell clones. Error bars, mean ± SEM; statistical significance: Student’s t test.
(C) Schematic of the Vps13 family. Drosophila Vps13D is the only Vps13 family member with a UBA domain. Atg8-interacting motifs analyzed in the manuscript are marked with arrowheads.
(D–D′′) Clonal knockdown of Vps13D (green cells) impairs mCherry-Atg8a puncta formation (red).
(E) Quantitation of mCherry-Atg8a puncta in control and Vps13D knockdown cell clones from at least 28 clones in four intestines. Error bars, mean ± SEM; statistical significance: Student’s t test.
(F–F′′) MiMIC insertion Vps13D mutant cell clones phenocopy Vps13D knockdown failure in cell size reduction and mCherry-Atg8a autophagy reporter puncta formation (red). The mutant clone (−/−; top cell) and an example of a heterozygous control cell (+/−; bottom cell) are outlined in white.
(G) Quantitation of Vps13DMI11101 mutant and heterozygous control (green) cell size from clones from three intestines. Error bars, mean ± SEM; statistical significance: Student’s t test.
(H–J′) Atg1 (H and H′), Atg8a (I and I′), and Vps13D (J and J′) knockdown (green) cells in the Drosophila midgut stained with DAPI (blue) and ubiquitin (fk2) antibody (red). Results are representative of at least three intestines per genotype.
(K–K′′) Intestines expressing LAMP-GFP (green) were dissected 2 hr after puparium formation and stained with DAPI (blue) and Vps13D antibody (red). Results are representative of at least three intestines.
Scale bars in all images represent 50 μm. See also Figures S1 and S2 and Table S1.
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4. Figure 2
Vps13D Function Is Required for Mitochondrial Clearance and Size Control in the Drosophila Intestine
(A and B) Mito-GFP in control gut (A) and Vps13D knockdown midguts (B) 2 hr after puparium formation. Results are representative of at least three biological replicates. Scale bars represent 50 μm.
(C–C′′) MiMIC insertion Vps13D mutant midgut cells (lacking GFP) possess persistent mitochondrial ATP5A protein compared to neighboring control cells (GFP-positive), indicating a defect in the clearance of mitochondria. Scale bars represent 50 μm.
(D–F) Knockdown of Vps13D (E and F) results in enlarged midgut mitochondria compared to mitochondria from control w1118 (D) animal midguts. Results are representative of at least three biological replicates.
See also Figure S3.
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5. Figure 3
Vps13D Ubiquitin Binding Is Required for Mitochondrial Size in Drosophila and Humans
(A) Vps13D contains a UBA domain with a conserved hydrophobic patch known to mediate the interaction with poly-ubiquitin.
(B) Interaction of the Vps13D UBA domain or a phenylalanine-to-alanine mutant with tetra-ubiquitin chains. Results are representative of at least three binding assays.
(C–E) TEM highlighting the mitochondria of Vps13D UBA domain (ΔUBA) heterozygous (C) and homozygous mutants (D) as well as Vps13DΔUBA/Df(3L)BSC613 (E) midguts. Scale bars represent 1 μm.
(F and G) Percent area occupied by mitochondria (F) and average mitochondrion size (G) quantitated from at least five fields from three intestines per sample. n.s., not significant. Error bars, mean ± SEM; statistical significance: Student’s t test.
(H and H′) ATP5A staining in Vps13DΔUBA/+ control guts 2 hr after puparium formation. Results are representative of six biological replicates. Scale bars represent 25 μm.
(I and I′) ATP5A staining in Vps13DΔUBA/Vps13DΔUBA mutant guts 2 hr after puparium formation. Results are representative of six biological replicates. Scale bars represent 25 μm.
(J) ATP5A staining is quantitated from three intestines each from heterozygous control and mutant midguts. Error bars, mean ± SEM; statistical significance: Student’s t test.
(K and L) TOM20 labeling of control (K) and Vps13D KO (L) HeLa cells. Scale bars represent 5 μm.
(M) Quantitation of percent mitochondria representing tubular, short, or large and round phenotypes in control HeLa or Vps13D KO cell lines 12, 19, and 45. Results are representative of three biological replicates. For tubular mitochondria, p values are all < for 12, 19, and 45 versus control HeLa. For short mitochondria, p = 0.85, 0.07, and 0.98, respectively, for 12, 19, and 45 versus control HeLa. For large and round mitochondria, p values are all < for 12, 19, and 45 versus control HeLa. Error bars, mean ± SEM.
(N–O) Vps13D tagged internally with GFP (Vps13D-I-GFP) rescues the large and round mitochondrial phenotype seen in Vps13D KO cells (N and N′). Scale bars represent 5 μm. Results are quantitated in (O) and are representative of three biological replicates. Error bars, mean ± SEM; statistical significance: Student’s t test.
See also Figures S3 and S4.
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6. Figure 4
Decreased Vps13D Function Leads to Enlarged Mitochondria Because of a Defect in Mitochondrial Dynamics
(A and B) TEM of Drp1 heterozygous (A) versus transheterozygous (B) mutant intestines. Scale bars represent 0.5 μm.
(C) Homozygous Drp1 mutant intestine cells fail to clear mitochondria compared to a control Drp1/+ intestine (results are quantitated from at least four fields from two [controls] or three [mutant] intestines). Error bars, mean ± SEM; statistical significance: Student’s t test.
(D) Drp1 mutant intestine cells exhibit a defect in programmed cell size reduction (quantitated from 41 cells from 4 guts/genotype). Error bars, mean ± SEM; statistical significance: Student’s t test.
(E–F′′) Drp1 localization in control (E–E′′) versus Vps13D KO (F–F′′) HeLa cells co-stained with Tom20.
(G) Western blot analysis of Drp1 accumulation on mitochondria in Vps13D KO versus wild-type HeLa cells. Actin, COXII, and Tom20 were used to identify the cytosolic (cytosol) and mitochondrial (mito) fractions. WCL, whole-cell lysate.
(H) Western blot analysis of Drp1 and MFF phosphorylation in Vps13D KO versus control HeLa cells. Western results are representative of at least three independent experiments.
(I and J) Quantitation of three replicates of the western blot in (H) for analyses of Drp1 (I) and Mff (J) levels. Error bars, mean ± SEM.
(K–M) TEM of Marf IR (K), Vps13D IR (L), and Vps13D, Marf double-knockdown (M) intestines. Scale bars represent 1 μm.
(N–P) Percent mitochondrial area (N), average mitochondrion size (O), and cell size (P) in Marf, Vps13D, and Marf Vps13D double-knockdown intestines. Results are representative of at least three biological replicates. Error bars, mean ± SEM; statistical significance: Student’s t test.
See also Figure S4.
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