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. 2003 Nov;5(11):987-93.
doi: 10.1038/ncb1055. Epub 2003 Oct 19.

Discs-Large and Strabismus are functionally linked to plasma membrane formation

Ok-Kyung Lee  1 , Kristopher K Frese, Jennifer S James, Darshana Chadda, Zhi-Hong Chen, Ronald T Javier, Kyung-Ok Cho
Affiliations

Discs-Large and Strabismus are functionally linked to plasma membrane formation

Ok-Kyung Lee et al. Nat Cell Biol. 2003 Nov.

Abstract

During early embryogenesis in Drosophila melanogaster, extensive vesicle transport occurs to build cell boundaries for 6,000 nuclei. Here we show that this important process depends on a functional complex formed between the tumour suppressor and adaptor protein Discs-Large (Dlg) and the integral membrane protein Strabismus (Stbm)/Van Gogh (Vang). In support of this idea, embryos with mutations in either dlg or stbm displayed severe defects in plasma membrane formation. Conversely, overexpression of Dlg and Stbm synergistically induced excessive plasma membrane formation. In addition, ectopic co-expression of Stbm (which associated with post-Golgi vesicles) and the mammalian Dlg homologue SAP97/hDlg promoted translocation of SAP97 from the cytoplasm to both post-Golgi vesicles and the plasma membrane. This effect was dependent on the interaction between Stbm and SAP97. These findings suggest that the Dlg-Stbm complex recruits membrane-associated proteins and lipids from internal membranes to sites of new plasma membrane formation.

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Figures

Figure 1
Figure 1
Dlg interacts with Stbm. (a) Domain structures of Dlg and Stbm. (b) Stbm antiserum, but not pre-immune serum, recognizes a 75K band in embryonic extracts (left). Western blot of two individual embryos (stage 16) derived from Df(2R)NP4/CyO parents (right). All control CS embryos expressed the 75K protein (data not shown). (c, d) Stbm and Dlg (arrow) co-immunprecipitate from embryo extracts. (e, f) Stbm and Dlg also interact in COS-7 cells. Combinations of transfected expression plasmids are indicated. Stbm recovered from the immunocomplex, as shown in e and f, or expressed endogenously in COS-7 cells, as shown in e, is indicated by arrows or an arrowhead, respectively. The size of fly Stbm expressed in the COS-7 cells (asterisk in f) is shown for comparison.
Figure 2
Figure 2
Epithelial cells of stbm mutant embryos exhibit defects in membrane formation. The source of specimen and visualized proteins are indicated at the upper or lower right corners, respectively. All panels are single confocal images, except a and b, which are composites from multiple confocal sections spanning the vertical cell boundary. (a, b) Cross-section showing membrane pattern of CS and stbm7-6 embryos during cellularization. Arrows indicate absence of Dlg and Dlt. (c-f) Tangential sections (c, e) and cross-sections (d, f) of CS (c, d) and stbm7-6 (e, f) embryos. Arrows in c and e mark the cleavage furrow where confocal sections in d and f were taken, respectively. Arrowheads indicate Dlt pattern. (g) stbm7-6 /stbm153 embryo with similar phenotype as stbm7–6 in e. Abnormal Dlt pattern is marked with an arrowhead. (h, i) Visualization of membrane structure with conA in CS (h) and stbm7–6 (i) embryos. Arrowheads in h and i indicate the presence and absence, respectively, of Dlg, Dlt and conA. Scale bar represents 10 µm.
Figure 3
Figure 3
Expression pattern of Stbm and Dlg in fly and mammalian cells. (a) Localization of Stbm in plasma membrane (arrowhead) and vesicle-like structures (arrow) from wing imaginal disc. (b) Expression of Dlg and Stbm in numerous vesicles of salivary gland duct cells. The basolateral plasma membrane slightly below the septate junction is marked with an arrowhead. (c) Localization of Stbm, Dlg and Hrs in cellularizing embryos. (d) Colocalization of Stbm and Dlg in vesicle-like structures (arrowheads) and patches of the plasma membrane (arrow). (e, f) Colocalization of Stbm and GM130 in vesicular structures in embryos before cellularization. Dashed line indicates surface of embryo. The region marked with a rectangle is magnified in f. (g) Vesicles in an cellularizing embryo containing Stbm (arrow), GM130 (asterisk), or both (arrowhead). (h) Detection of endogenous Stbm in human cell lines TE85 and 293, mouse cell line NIH3T3, and fly embryos. (i) Vesicles containing Stbm, GM130 or both in a TE85 cell lacking Golgi stack structure. Nucleus (N) is outlined. (j, k) TE85 cells with extensive Golgi stacks stained for endogenous GM130 and endogenous Stbm (j) or for exogenous YFP–p58 and endogenous Stbm (k). Nuclei were stained with DAPI. Scale bar represents 5 µm in a and b, 10 µm in c, 7 µm in d and e, 4 µm in f–I and 25 µm in j and k.
Figure 4
Figure 4
Dlg and Stbm must interact to colocalize and promote membrane formation. Various cDNAs transfected into TE85 cells are indicated at upper right corners. Nuclei (N) were visualized with DAPI (data not shown). (a) Vesicular localization of ectopically expressed fly Stbm. (b) Cytoplasmic localization of ectopically expressed YFP–SAP97. (c–f) Pattern of YFP–SAP97 in cells cotransfected with YFP–SAP97 and stbm. The regions marked with arrows in c and arrowhead in e are enlarged in d and f, respectively. Vesicles containing both YFP–SAP97 and fly Stbm are marked with arrows. (g–i) Colocalization of SAP97 and Stbm in cells transfected with fly stbm and SAP97 (g), but not with fly stbmPBM and SAP97 (h) or fly stbm and SAP97ΔPDZ1–2 (i). (j–m) Tangential views of the epithelial cells from wing disc expressing GFP–SAP97 alone (j) or together with Stbm (k). The UAS and Gal4 lines used to drive overexpression of Stbm or GFP–SAP97 are indicated at the upper right corners. The regions marked with rectangles in j and k are magnified and shown with the Nrx pattern in l and m, respectively. The extent of basolateral membrane visualized by Nrx is marked by brackets (l, m). One example of aggregated Dlg structures is indicated by an arrow (j). Scale bar represents 10 µm in a–c, e and g–k, 2.5 µm in d and f, and 5 µm in l and m.
Figure 5
Figure 5
Dlg is required for plasma membrane formation in both embryos and larvae. (a) Tangential view of a dlgHF321 embryo cultured at restrictive temperature lacks Dlg at the membrane. (b) Composite images of multiple cross-sections spanning the vertical cell boundary of the same embryo, revealing mis-localization of Dlg and Dlt. (c) A similar dlgHF321 embryo, showing cytoplasmic vesicle-like structures containing Stbm. Dlg localizes diffusely in the cytoplasm or in vesicle-like structures. (d, e) dlgm52 mutant clones are marked by the absence of Myc marker. Myc is expressed on the dlg+ chromosome. A mutant clone (dlg−/dlg−) and wild-type clones (twin spots; dlg+/dlg+) are marked by yellow and red lines, respectively. Unmarked cells are dlg+/dlg−. Localization of Nrx at the septate junction is more diffuse (arrowhead) or absent (arrow) in the dlg− clone. Note the higher level of Nrx in twin spot than in dlg+/dlg− cells (d). Membrane structure of the dlg− clone is visualized with Dlt (e), and a portion is magnified at the bottom left corner (e′). Scale bar represents 10 µm in a–c, and 20 µm in d and e.
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