Type II phosphatidylinositol 4-kinase regulates trafficking of secretory granule proteins in Drosophila

Abstract

Type II phosphatidylinositol 4-kinase (PI4KII) produces the lipid phosphatidylinositol 4-phosphate (PI4P), a key regulator of membrane trafficking. Here, we generated genetic models of the sole Drosophila melanogaster PI4KII gene. A specific requirement for PI4KII emerged in larval salivary glands. In PI4KII mutants, mucin-containing glue granules failed to reach normal size, with glue protein aberrantly accumulating in enlarged Rab7-positive late endosomes. Presence of PI4KII at the Golgi and on dynamic tubular endosomes indicated two distinct foci for its function. First, consistent with the established role of PI4P in the Golgi, PI4KII is required for sorting of glue granule cargo and the granule-associated SNARE Snap24. Second, PI4KII also has an unforeseen function in late endosomes, where it is required for normal retromer dynamics and for formation of tubular endosomes that are likely to be involved in retrieving Snap24 and Lysosomal enzyme receptor protein (Lerp) from late endosomes to the trans-Golgi network. Our genetic analysis of PI4KII in flies thus reveals a novel role for PI4KII in regulating the fidelity of granule protein trafficking in secretory tissues.

Fig. 1.

Deletions in PI4KII are protein null. (A) Physical map of Drosophila PI4KII (CG2929, Pi4KIIα) showing somatic and testis-specific transcripts and predicted exons (dark blue, coding regions; light blue, untranslated regions; red, stop codons). P-element GE28807 (triangle) was excised to generate a homozygous lethal deletion Df(3R)730 (red) that removes PI4KII and CG14671. Lethality can be rescued with genomic DNA encoding CG14671 (light gray) but not PI4KII (dark gray). (B) Drosophila (Dm) PI4KII C-terminal kinase domain (dark gray) is homologous to human (Hs) PI4KIIα and PI4KIIβ, whereas N-terminal regions (white) are not conserved. Percentage sequence identity (and similarity) is shown for homologous portions relative to the somatic isoform. (C) RT-PCR detects a somatic transcript in males (M) and females (F) (left panel, arrowhead) and a testis-specific transcript in males (right panel, arrowhead). (D) Immunoblot probed with anti-PI4KII (top) and anti-tubulin (bottom). Anti-PI4KII recognizes a single ∼90 kDa band in wild type (lanes 1-4), but not in the PI4KIIΔ mutant (lanes 5, 6). (E) Scanning confocal micrograph of late L3 salivary gland stained for endogenous PI4KII shows a punctate distribution that is absent from a PI4KIIΔ cell (marked by absence of GFP and outlined by white dashed line).

Fig. 2.

PI4KII is required for proper glue granule biogenesis. (A-D) Spinning-disk confocal micrographs of late L3 salivary cells expressing the glue granule marker Sgs3-DsRed. Compared with wild type (A) or fwd mutant (D), PI4KIIΔ exhibits strikingly small glue granules (B) that can be rescued by a wild-type PI4KII genomic transgene (C). (E) Scanning confocal micrograph showing that the small granule phenotype of the PI4KIIΔ mutant is cell-autonomous (mutant cell marked by absence of GFP and outlined by white dashed line). (F,G) Transmission electron micrographs of wild-type (F) and PI4KIIΔ mutant (G) late L3 salivary glands. In wild type, granules are large and well organized, with electron-dense material near the membrane and filamentous material near the center of each granule. PI4KIIΔ salivary glands exhibit small granules, as well as vacuolated structures that contain filamentous material (arrows).

Fig. 3.

PI4KII localizes to the Golgi and LEs in live salivary gland cells. Spinning-disk confocal micrographs of live L3 salivary cells expressing mCherry-PI4KII (red) and YFP-Golgi or YFP-Rab7 (green). Boxed regions are magnified in insets. (A,B) In mid-L3 (stage 0) salivary cells, mCherry-PI4KII localizes adjacent to YFP-Golgi (A, arrows). mCherry-PI4KII also colocalizes with YFP-Rab7 at late endosomes (LEs) in stage 0 salivary cells (B, arrows). (C,D) In late L3 (stage 2) salivary cells, mCherry-PI4KII localizes adjacent to YFP-Golgi (C, arrows) and to tubular structures lacking YFP-Golgi (C, arrowhead). mCherry-PI4KII colocalizes with YFP-Rab7 at LEs (D, arrows) and to endosomal tubules (D, arrowhead) lacking YFP-Rab7 (inset). See supplementary material Movies 1 and 2.

Fig. 4.

PI4KII localizes to dynamic tubular endosomes. Spinning-disk confocal micrographs of live L3 (stage 2) salivary cells expressing mCherry-PI4KII alone (B,C,F) or with GFP-LAMP (A) or β-tubulin-GFP (D,E,G). (A) Projection constructed from a z-stack of 52 confocal sections. The y-axis was cropped and the resulting slice turned 90°. mCherry-PI4KII and GFP-LAMP are enriched near the cell cortex and colocalize on LEs (arrows). mCherry-PI4KII is also seen deeper in the cell. (B) xy projection generated from a z-stack of seven optical sections acquired starting from the basal surface. mCherry-PI4KII is present in tubules (arrowheads) linking LEs (arrows). (C) xz projection generated from a z-stack of 60 optical sections. The y-axis was cropped and the slice turned 90°. mCherry-PI4KII is enriched at the cell cortex (arrow) and on tubules (arrowheads) that extend perpendicular to the cell surface. (D) Projection of nine optical sections. mCherry-PI4KII-containing tubules colocalize with microtubules (β-tubulin-GFP, arrowheads). (E-G) Time-lapse fluorescence micrographs (elapsed time in seconds). (E) Single optical section. Tubules containing mCherry-PI4KII (arrowheads) extend and retract from endosomes (arrow) along microtubules. See supplementary material Movie 3. (F) Projections of five optical sections. mCherry-PI4KII-containing tubules rapidly form (frames 2 and 3, green arrowheads) and break (frame 4, red arrowhead). Retracting tubules appear to exert a pulling force, displacing endosomes (frames 4-6, arrows). See supplementary material Movie 4. (G) Projections of three optical sections. mCherry-PI4KII-containing vesicles move rapidly along microtubules (arrowheads). See supplementary material Movie 5.

Fig. 5.

PI4KII is dispensable for recruiting the clathrin adaptor proteins AP-1 and EpsinR to the Golgi. Scanning confocal micrographs of L3 salivary glands stained for AP-1γ (A) or EpsinR (B). Localization of these clathrin adaptors appears normal in PI4KIIΔ cells (marked by the absence of GFP and outlined by yellow dashed lines).

Fig. 6.

PI4KIIΔ mutants accumulate granule proteins in enlarged LEs. (A-H) Spinning-disk confocal micrographs of live late L3 salivary cells. Arrows point to regions magnified in insets (A-F) or shown on right (G,H). (A,B) Projections of 32 (A) or 101 (B) slices. YFP-Rab7-positive endosomes are larger in the PI4KIIΔ mutant (B) than in wild type (A). (C,D) Projections of 19 optical slices. Acidic LEs or lysosomes stained with Lysotracker are larger in the PI4KIIΔ mutant (D) than in wild type (C). (E,F) Single optical sections. GFP-Lerp localizes to enlarged organelles in the PI4KIIΔ mutant (F) as compared with wild type (E). (G,H) Single optical sections of cells expressing YFP-Rab7 and Sgs3-DsRed. YFP-Rab7-positive LEs lacking luminal Sgs3-DsRed are present near the cell cortex in wild type (G). Enlarged YFP-Rab7-positive LEs accumulate luminal Sgs3-DsRed and are scattered throughout the cell in the PI4KIIΔ mutant (H). (I) Scanning confocal micrographs of a fixed salivary gland showing altered distribution of the SNARE Snap24 (blue) on organelles lacking Sgs3-DsRed (red) in a PI4KIIΔ cell (marked by absence of GFP and outlined by yellow dashed lines). Arrows indicate Snap24 accumulation on enlarged organelles lacking Sgs3.

Fig. 7.

Retromer localization and dynamics are altered in PI4KIIΔ mutants. Spinning-disk confocal micrographs of live L3 (stage 2) salivary cells. (A) GFP-PI4KII colocalizes with mCherry-Vps29 on endosomes (arrows), but mCherry-Vps29 is largely excluded from mCherry-PI4KII-containing tubules (arrowheads). See supplementary material Movie 6. (B,C) Projections of 22 optical slices. Vps29-GFP is uniformly distributed around enlarged endosomes in the PI4KIIΔ mutant (C) relative to wild type (B). (D) Time-lapse fluorescence micrographs (elapsed time in seconds) reveal that Vps29-GFP localizes to dynamic foci associated with the limiting membranes of LEs in wild type (left), but is more uniformly and stably distributed around the periphery of an enlarged LE in the PI4KIIΔ mutant (right). See supplementary material Movie 7. (E,F) Projections of 13 optical slices. Garnet-GFP-labeled endosomes are larger in the PI4KIIΔ mutant (F) than in wild type (E). (G,H) mCherry-PI4KII tubular endosomes are of normal morphology in retromer (vps35¹) (G) and AP-3δ (g¹) (H) mutant salivary cells.

Fig. 8.

PI4KII catalytic activity is required for granule biogenesis and normal endosome size. (A) Catalytic activity of Drosophila PI4KII. Untransfected control (ctrl, lane 1) or transfected (lanes 2-4) COS-7 cells expressing FLAG-tagged wild-type (lane 2), ATP mutant (lane 3) or CAT mutant (lane 4) PI4KII. Immunoblot shows membrane extracts probed with anti-PI4KII antibody. PI4K assay shows an autoradiograph of radiolabeled PI4P analyzed by TLC. PI4K catalytic activity is shown relative to that of control cells (0%) and cells transfected with wild-type PI4KII (100%). PI4KII activity [counts per minute (cpm)] is shown in the bar chart (identical results were obtained in two independent experiments). (B,C) Spinning-disk confocal micrographs of late L3 salivary cells expressing Sgs3-GFP and mCherry-PI4KII (B) or mCherry-PI4KII^CAT (C) in a PI4KIIΔ background. mCherry-PI4KII (B) but not mCherry-PI4KII^CAT (C) fully rescues the glue granule and endosome defects of the PI4KIIΔ mutant. mCherry-PI4KII^CAT localizes to enlarged organelles that can contain Sgs3-GFP (insets).

Fig. 9.

PI4KII catalytic activity is required for endosomal tubule dynamics and endosome morphology. Spinning-disk confocal micrographs of late L3 salivary cells expressing mCherry-PI4KII (A,C) or mCherry-PI4KII^CAT (B,D) in a PI4KIIΔ background. (A,B) Time-lapse fluorescence micrographs (elapsed time in seconds). mCherry-PI4KII localizes to dynamic endosomal tubules (A, arrowheads), whereas mCherry-PI4KII^CAT shows minimal localization to tubules (B, arrowheads). See supplementary material Movies 8 and 9. (C,D) YFP-Rab7 and mCherry-PI4KII show strong overlap on LEs (C, insets), whereas YFP-Rab7 and mCherry-PI4KII^CAT localize to different endosomes (D) or to distinct domains on the same endosomes (D, insets).

Fig. 10.

Model of PI4KII function during glue granule biogenesis. (A) At least six trafficking steps (1-6) contribute to normal granule biogenesis in wild-type salivary gland cells. Vesicles containing granule cargo (red shading) and SNAREs (blue) traffic to immature secretory granules (1) that fuse with each other to form mature granules (2). SNAREs are recycled for future rounds of granule biogenesis, potentially via AP-1/clathrin-dependent trafficking to Rab7- and PI4KII-positive LEs (3), followed by retrieval to the trans-Golgi network (TGN) (4). Lerp and its associated lysosomal enzymes traffic from the TGN to LEs (5), from which Lerp is subsequently recycled to the TGN (4). PI4KII-dependent tubular endosomes might be involved in retrograde trafficking from LEs to the TGN. Any granule cargo aberrantly missorted (5) or retrieved (3) to LEs is rapidly degraded in lysosomes (6). (B) In the absence of PI4KII, initial stages of granule formation are normal (1). However, granules fail to reach full size (2), presumably owing to reduced availability of SNARES for homotypic fusion of immature granules, a consequence of a defect in the retrieval of SNAREs from LEs (4) or due to missorting of SNAREs from the TGN to LEs (5). Additionally, missorted granule cargo accumulates in LEs, presumably owing to impaired lysosomal degradation (6) caused by a defect in recycling of Lerp (4). Red crosses indicate trafficking steps that appear compromised in PI4KIIΔ mutant salivary gland cells.