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First published online 2 December 2008
doi: 10.1242/jcs.033027

Journal of Cell Science 122, 25-35 (2009)
Published by The Company of Biologists 2009
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Interplay between Rab5 and PtdIns(4,5)P2 controls early endocytosis in the Drosophila germline

Julien Compagnon1, Louis Gervais1,*, Mabel San Roman1,2, Sophy Chamot-Bœuf1 and Antoine Guichet1,{ddagger}

1 Institut Jacques Monod, UMR 7592, CNRS, Université Paris 7, 2 Place Jussieu, 75005, France
2 Electron Microscopy Facility, Institut Jacques Monod, UMR 7592, CNRS, Université Paris 7, 2 Place Jussieu, 75005, France


Figure 1
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  		Fig. 1. Rab5 is required for endocytosis in the Drosophila oocyte. (A) Schematic representation of a stage 9 egg chamber. Anterior is up, posterior is down. (B-E) Yolk protein internalization in control (B,D) and in rab52 GLC (C,E) stage 10 oocytes. (B,C) GFP autofluorescence; (D,E) Yolk autofluorescence. (C) The absence of nuclear GFP in the germline cells marks the presence of a GLC. (D,E) In rab52 GLCs, no yolk granule autofluorescence was visible (E) when compared with control oocytes (D). (F,G) Quantification of autofluorescence along the broken line in D and E. Note that, unlike in the control, there is no detectable increase in autofluorescence intensity in the rab52 oocyte (o) when compared with the adjacent follicle cells (fc), which are heterozygous for rab52 in both cases. (H,I) Yl localization in wt control (H) and in rab52-GLCs (I) at stage 9. Blue, DNA; red, Yl. In rab52-GLCs, Yl is found in abnormal cytoplasmic compartments (arrows in I) and, as in control (J), at the oocyte PM. Scale bars: 50 µm.

 

Figure 2
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  		Fig. 2. Ultrastructure of the endocytic compartment in rab52 mutant oocytes. (A) Schematic representation of the morphology of intermediates in the clathrin-dependent vitellogenins endocytic pathway. FCCP, forming clathrin-coated pit; CCP, clathrin-coated pit (arrowheads in C,E,G); EEV, early endocytic vesicles (double arrowheads in C,E,G); CCV, clathrin-coated vesicles; NEEV, naked early endocytic vesicles; T, tubular intermediates (arrows in C,E,G); Y1 and Y2, forming yolk granules; Y3, condensed yolk granules. (B,C) Electron micrographs of late stage 9 wt egg chambers showing the oocyte (B) and a representative region of the lateral cortex (C). Note the large quantity of dark yolk granules in the oocyte cytoplasm in B and that all intermediates in the clathrin-dependent vitellogenin endocytic pathway are found in C. (D,E) Electron micrographs of rab52 GLC late stage 9 egg chambers showing the oocyte (D) and a representative region of the lateral cortex (E). Note the absence of dark yolk granules and the presence of endocytic intermediate aggregates (red circles) in the oocyte cytoplasm (D). Green arrowhead indicates the aggregate shown in supplementary material Fig. S1G,H. (E) Note that EEV density is increased, and that tubules and forming yolk granules are very rare in this region. (F) Top: the density of endocytic intermediates ~3.5 µm below the oocyte PM per µm2 of oocyte cytoplasm is shown. The numbers in brackets in the first column represent the surface oocyte cytoplasm considered for each genotype. The numbers in brackets in the remaining columns represent the number of structures counted. Middle: the density of endocytic intermediates found deeper than 3.5 µm below the oocyte PM per µm2 of oocyte cytoplasm is shown. The numbers in brackets in the first column represent the surface of oocyte cytoplasm considered for each genotype. The numbers in brackets in the remaining columns represent the number of structures counted. Schematic representation of the oocyte showing, in orange, the subcortical region ~3.5 µm below the oocyte PM and, in pink, the remaining oocyte cytoplasm. Bottom: the density of endocytic intermediates per µm2 of oocyte cytoplasm calculated with the densities determined in the tables above, and considering that the region 3.5 µm below the PVS represents 18% of the oocyte cytoplasm. CCP, clathrin-coated pit; EEV, early endocytic vesicles; Y1-Y2, forming yolk granules; Y3, condensed yolk granules. (G) High magnification of the early endocytic intermediate aggregate indicated by the red arrowhead in B. oo, oocyte; fc, follicle cells; nc, nurse cells; v, vitelline bodies. The larger size of vitelline bodies and longer follicle-cell microvilli in E compared with C correspond to the fact that this egg chamber, although in stage 9 (similar to the control), is slightly older than that shown in C. Scale bars: (B,D) 2.5 µm; (C,E,G) 500 nm.

 

Figure 3
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  		Fig. 3. PtdIns(4,5)P2 is required for Rab5 localization and yolk endocytosis. (A-C) Rab5 localization during oogenesis in wt egg chambers. (A) From germarium (upper left) to stage 6 (bottom right); (B) stage 8; (C) stage 10. Note the recruitment of Rab5 in the oocyte cortical region during the vitellogenic stages (B,C). (D-G) Rab5 and PH(PLC{delta})-GFP localization in wt stage 10 egg chambers. (F; red in D,G) Rab5; (E; green in D,G) PH(PLC{delta})-GFP. (E-G) Correspond to the region inside the boxed area in D. Rab5 is visible at and in a narrow region below the oocyte PM (F,G; arrow). (H-S) PH(PLC{delta})-GFP, Lycopersicon esculentum (L.e.) Lectin and Rab5 localization in control (H-M) and sktl{Delta}5/sktl2.3 (N-S) stage 10 egg chambers. (I,K,O,Q; green in H,J,N,P) PH(PLC{delta})-GFP; (L,R; red in H,J,N,P) L.e. lectin; (M,S) Rab5. At the oocyte PM, when compared with wt, the PH(PLC{delta})-GFP signal is reduced in sktl{Delta}5/sktl2.3 mutants, whereas L.e. lectin, highlighting the PM, is unaffected (arrows in I,J-L,O,P-R). The recruitment of Rab5 in the oocyte cortical region is impaired in sktl{Delta}5/sktl2.3 mutants when compared with controls (compare M and S). (T,U) Yl localization in control (T) and sktl{Delta}5/sktl2.3 mutant (U) stage 10 egg chambers. Green, PH(PLC{delta})-GFP; red, Yl. The localization of Yl in sktl{Delta}5/sktl2.3 is similar to control. (V,W) Electron micrographs of stage 9 egg chambers. (V) wt; (W) sktl2.3 GLCs. Note that, in sktl2.3 GLCs, unlike in the control, no dark yolk granules are visible in the oocyte cytoplasm (W). oo, oocyte; fc, follicle cells; nc, nurse cells.

 

Figure 4
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  		Fig. 4. Ultrastructure of the endocytic compartment in Sktl-deficient oocytes. (A) Electron micrograph of a stage 9 wt egg chamber, showing a representative region of the lateral cortex. Note the presence of: CCP, clathrin-coated pits (black arrowheads in A-C); EEVs, early endocytic vesicles (double arrowheads in A-C); T, tubular intermediates (black arrows in A-C); Y3, condensed yolk granules. (B,C) Electron micrographs of a stage 9 sktl2.3 GLC egg chamber, showing two regions of the lateral cortex in this context. (B) Note the presence of CCPs on long abnormal PM invaginations (red arrowhead) and the presence of abnormal yolk-containing profiles apparently not connected with the PM (red arrows). (C) Note the presence of large accumulations of yolk proteins at the oocyte PM (green arrowhead); the presence of yolk-containing profiles apparently not connected with the PM are very rare. oo, oocyte; fc, follicle cells; Y1, forming yolk granule. Scale bars: 500 nm.

 

Figure 5
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  		Fig. 5. Rab5 controls the removal of PtdIns(4,5)P2 from early endocytic intermediate membrane. (A-D) PH(PLC{delta})-GFP localization in wt (A) and rab52-GLCs (B-D) stage 9 egg chambers. (C) Inset on the anterior margin. (D) Cross-section through the broken line in B. Note the presence of large PLC(PLC{delta})-GFP-positive structures in the oocyte cytoplasm when Rab5 is absent which are never seen in control (A) (projection of optical sections). Scale bars: 50 µm, except in C, 25 µm. (E-H) YP1 and PH(PLC{delta})-GFP localization in wt (E) and rab52 GLC (F-H) stage 10 egg chambers. (G; red in E,H) YP1; (F; green in E,H) PH(PLC{delta})-GFP; (blue in E,H) DNA. (Inset, E) In controls, YP1 is found throughout the cytoplasm. (H) When Rab5 is absent, YP1 discrete staining inside the oocyte cytoplasm is lost and it is found on large PH(PLC{delta})-GFP-positive structures in the oocyte cytoplasm. Scale bars: 50 µm. (I-L) {alpha}-adaptin and PH(PLC{delta})-GFP localization in wt (I) and rab52 GLC (J-L) stage 9 egg chambers. (K; red in I,L) {alpha}-adaptin; (J; green in I,L) PH(PLC{delta})-GFP. Note that, when Rab5 is absent, large PH(PLC{delta})-GFP-positive structures in the oocyte cytoplasm are associated with {alpha}-adaptin. Scale bars: 30 µm. (M-T) F-actin and PH(PLC{delta})-GFP localization in wt (M,Q) and rab52 GLC (N-P,R-T) stage 9 egg chambers. (O,S; red in M,P,Q,T) F-actin; (N,R; green in M,P,Q,T) PH(PLC{delta})-GFP. Note that the large PH(PLC{delta})-GFP-positive structures in the oocyte cytoplasm observed when Rab5 is absent contain F-actin (projection of optical sections).

 

Figure 6
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  		Fig. 6. Sktl overexpression impairs endocytosis. (A-B''') {alpha}-adaptin and PH(PLC{delta})-GFP localization in control (A) and RFP-Sktl-overexpressing (B-B''') stage 9 egg chambers. (B; green in A,B''') PH(PLC{delta})-GFP; (B'; red in A,B''') {alpha}-adaptin; (B'', blue in B''') RFP-Sktl. Note that, when RFP-Sktl is overexpressed, large PH(PLC{delta})-GFP/RFP-Sktl-positive structures in the oocyte cytoplasm are present and are associated with {alpha}-adaptin. (C-D''') Rab5 and PH(PLC{delta})-GFP localization in control (C) and RFP-Sktl-overexpressing (D-D''') stage 9 egg chambers. (D; green in C,D''') PH(PLC{delta})-GFP; (D'; red in C,D''') Rab5; (D'', blue in D''') RFP-Sktl. Note that, when RFP-Sktl is overexpressed, large PH(PLC{delta})-GFP/RFP-Sktl-positive structures in the oocyte cytoplasm are present and are associated with Rab5. (E-F''') Yl and PH(PLC{delta})-GFP localization in control (E) and RFP-Sktl-overexpressing (F-F''') stage 9 egg chambers. (F; green in E,F''') PH(PLC{delta})-GFP; (F'; red in E,F''') Yl; (F''; blue in F''') RFP-Sktl. Note that, when RFP-Sktl is overexpressed, large PH(PLC{delta})-GFP/RFP-Sktl-positive structures in the oocyte cytoplasm are present and are associated with Yl. (G-I'') YP1 localization in control (G) and RFP-Sktl-overexpressing (H-I'') stage 9 egg chambers. (I'; blue in H-I) RFP-Sktl; (I''; red in G-I) YP1. Note that, when RFP-Sktl is overexpressed, large RFP-Sktl-positive structures in the oocyte cytoplasm are present and are associated with YP1. (J-K''') F-actin and PH(PLC{delta})-GFP localization in control (J) and RFP-Sktl-overexpressing (K-K''') stage 9 egg chambers. (K; green in J,K''') PH(PLC{delta})-GFP; (K'; red in J, K''') F-actin; (K''; blue in K''') RFP-Sktl. Note that, when RFP-Sktl is overexpressed, large PH(PLC{delta})-GFP/RFP-Sktl-positive structures in the oocyte cytoplasm are present and are associated with F-actin. (L-N) yolk internalization in control (L,L') and RFP-Sktl-overexpressing (M,M') stage 10 egg chambers. (L',M'; green in L,M) Yolk autofluorescence; (red in M) RFP-Sktl. When RFP-Sktl is overexpressed, yolk granule autofluorescence is lower when compared with the control oocyte. (N) Quantification of autofluorescence along the broken lines in L and M. Note that, when RFP-Sktl is overexpressed, the increase of yolk-granule autofluorescence in the oocyte (oo) is lower when compared with the adjacent follicle cells (fc) not expressing RFP-Sktl in both cases. (O-P'') Sktl and PH(PLC{delta})-GFP localization in control (O) and rab52 GLCs (P-P'') stage 9 egg chambers. (P'; green in O,P) PH(PLC{delta})-GFP (P''; red in O,P) Sktl. Note that, in rab52 GLCs, large PH(PLC{delta})-GFP-positive structures in the oocyte cytoplasm are present and are associated with Sktl. Scale bars: 50 µm.

 

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