Autophagosomes move towards the ncMTOC in fat cells.

A: Schematic drawing of the experimental design for screening. B: Non-fused autophagosomes accumulate in the perinuclear region upon Vps16A silencing. C: The cis-Golgi compartment remains unchanged upon the expression of vps16a RNAi. D, E: The accumulation of autophagosomes is not perinuclear in α or β tubulin; vps16a double RNAi cells. F-I: Quantification of data shown in B-E; n=10 cells. J, K: Autophagosomes position at an ectopic MTOC (yellow arrows) formed upon Shot silencing. (29 out of 41 cells exhibited an ectopic MTOC = 70.73%). L: Autophagosomes are near microtubule minus-ends, marked by Khc-nod-LacZ. The boxed area in the main panels, marked by cyan, is enlarged in L’’’ (proximity sites indicated by cyan arrowheads). M, N: Correlative ultrastructural analysis shows autophagosomes accumulating near the nucleus upon Vps16A silencing (border of control and silencing cells marked by green) (M). Shot knockdown causes aggregation of autophagosomes in ectopic foci in vps16a RNAi cells (N). An ectopic cleavage furrow (hallmark of Shot depletion (Sun et al., 2019)) is also visible (cyan arrows in N’). Note: the magnification of N is higher to better show this structure. Nuclei are encircled in blue in J’ and L’, L’’’. The boundaries of RNAi cells are highlighted in magenta in the grayscale panels.

A dynein-dynactin complex is required for minus-end directed autophagosome transport.

A-G: Knockdown of dynein (A-D) and dynactin subunits (E-G) results in the peripheral redistribution of autophagosomes in vps16a RNAi cells (red arrows). H, I: Kinesin silencing does not affect the perinuclear accumulation of autophagosomes in vps16a RNAi cells. J: Proposed model of the suggested dynein-dynactin complex responsible for autophagosome positioning in fat cells. DHC: dynein heavy chain; DIC: dynein intermediate chain; DLIC: dynein light intermediate chain; DLC: dynein light chain. K: Quantification of data shown in A-I; n=10 cells. The boundaries of RNAi cells are highlighted in magenta in the grayscale panels.

The proper dynein/kinesin ratio determines the directionality of autophagosome transport.

A, B: Overexpression of kinesin motors blocks the minus-end transport of autophagosomes, leading to their accumulation in the cell periphery in vps16a RNAi cells (red arrows). C: In dynactin-kinesin-vps16a triple RNAi cells, autophagosomes are distributed throughout the cytoplasm. D: Overexpression of the recombinant minus-end motor Khc-nod-LacZ does not affect minus-end directed autophagosome transport in vps16a RNAi cells. E: Khc-nod-LacZ expression partially rescues the dynactin KD-induced peripheral redistribution of autophagosomes in vps16a RNAi cells. F: Quantification of data shown in A-E; n=10. Data for dctn1-p150 RNAi is included as a positive control for peripheral distribution (shown in Figure 2E, K). The boundaries of RNAi or kinesin overexpressing cells are highlighted in magenta in the grayscale panels.

Rab7 and Rab39 small GTPases and their interactors are responsible for bidirectional movement of autophagosomes.

A-H: Knockdown of Rab7 (A), its interactor Epg5 (B), the subunits of its guanine nucleotide exchange factor Mon1 (C) and Ccz1 (D), as well as Rab39 (E) and its interactor ema (F), inhibits the perinuclear positioning of autophagosomes in vps16a RNAi cells. In contrast, other factors such as Plekhm1 (G) and prd1 (H) do not affect autophagosome positioning. I: Proposed model of Rab small GTPases with their adaptors involved in autophagosome positioning. J: Quantification of data shown in A-H; n=10 cells. The boundaries of RNAi cells are highlighted in magenta in the grayscale panels.

Epg5 is responsible for bidirectional movement of autophagosomes.

A-B: The distribution of Atg8a (A) or Rab7 (B) positive autophagosomes becomes dispersed upon the expression of epg5 and vps16a RNAi. C: Overexpression of YFP-tagged Rab7 does not rescue the scattered distribution of mCherry-Atg8a positive autophagosomes in the absence of Epg5 in vps16a RNAi cells, even though the colocalization of YFP-Rab7 with mCherry-Atg8a remains unaffected. Cyan arrowheads within insets (marked by a cyan box in panel C) point to YFP-Rab7 and mCherry-Atg8a double positive dots. D: The localization of Arl8 positive lysosomes remains perinuclear in epg5, vps16a double RNAi cells. The boundaries of RNAi and YFP-Rab7 expressing cells are highlighted in magenta in the grayscale panels. E-H: Quantification of data shown in A-D; n=10 cells. I, J: Epg5-9xHA colocalizes with endogenous Rab7 (I) or Atg8a (J) positive structures in S2R+ cells. Cyan arrowheads within insets (marked by cyan boxes in panels I and J) point to Epg5-9xHA and Rab7 or Atg8a double positive structures, respectively. I’ and J’ show scatter plots generated from the images of cells in panels I and J, respectively, depicting the intensity correlation profiles of Epg5-9xHA with Rab7 or Atg8a. Pearson correlation coefficients (R) are indicated, with the average R (n=10 cells) also shown, indicating colocalization in both cases. K, L: Coimmunoprecipitation experiments show that Epg5-9xHA binds to Rab7-FLAG (K) and endogenous Dhc64C (L) in cultured Drosophila cells. The asterisk in K marks immunoglobulin light chain.

Epg5 regulates autolysosome maturation.

A, B: Epg5 knockdown results in a significant reduction in the size of 3xmCherry-Atg8a positive autolysosomes (B) compared to control RNAi (luciferase RNAi) expressing cells (A). C, D: Epg5 RNAi cells lack large Rab7 (C) and Arl8 (D) positive autolysosomes, which are present in surrounding control cells (cyan arrowheads in insets point to Rab7 and Arl8 positive autolysosomes in control cells). The boundaries of RNAi cells are highlighted in magenta in the grayscale panels. E-H: Quantification of data shown in A-D; n=10 cells.

Minus-end directed transport is required for autolysosome maturation.

A-E: Autolysosome size is significantly reduced upon the loss of dynein (A-D) or dynactin (E) function. Red arrows point to autolysosomes at the cell periphery in A’-E’ and in the inset of E. F, G: Kinesin knockdowns do not significantly influence autolysosome size. H: Autolysosome size increases at ectopic foci (yellow arrows) in shot RNAi cells. The boundaries of RNAi-expressing cells are highlighted in magenta in the grayscale panels. The outlines of nuclei are drawn in blue in the inset of E’ and in H’. I-L: Quantification of data shown in A-H; n=10 cells.

Loss of the autophagosome positioning machinery decreases autophagosome-lysosome fusion.

A-G: In starved control RNAi (luciferase) expressing cells, mCherry-Atg8a overlaps with endogenous Lamp1 (A), indicating normal autophagosome-lysosome fusion and autolysosome formation. Autolysosomes still form in shot (B) or khc RNAi (C) cells, as mCherry-Atg8a colocalizes with endogenous Lamp1 similar to controls, but these are found in ectopic foci (yellow arrows) in shot RNAi cells. The outlines of nuclei are drawn in blue in B’ and B’’. Conversely, RNAi-s targeting factors responsible for minus-end directed autophagosome transport (D-G) decrease this overlap, suggesting less effective autophagosome-lysosome fusion. The GFP signal of RNAi-expressing cells is false-colored blue in composite images. The boundaries of RNAi-expressing cells are highlighted in magenta. Cyan arrowheads point to mCherry-Atg8a/Lamp1 double-positive structures, while magenta and green arrows indicate mCherry-Atg8a or Lamp1 single-positive dots, respectively. H: Quantification of data shown in A-G; n=10 cells.

Model of the transport of autophagosomes and lysosomes in starved fat cells.

Before fusion, autophagosomes and lysosomes are transported towards the perinuclear ncMTOC by a cytosolic dynein complex in starved fat cells to ensure proper fusion and effective degradation. This process requires Rab7, Rab39, and their interactors Epg5 and Ema on autophagosomes, and Rab2, Rab7, Rab39, and the Rab39 interactor Ema, but not Epg5, on lysosomes. After fusion, Arl8 mediates the plus- end transport of autolysosomes, while Rab39 promotes dynein-regulated minus-end directed transport. Thus, the motility of pre-fusion and post-fusion organelles is differently regulated: pre-fusion organelles generally move towards the MTOC, while post-fusion organelles exhibit bidirectional motility. This spatial regulation ensures proper fusion rates and degradation efficiency.