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Dual function for Tango1 in secretion of bulky cargo and in ER-Golgi morphology

  1. M. Leptina,b,4
  1. aDirectors' Research Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany;
  2. bInstitute of Genetics, University of Cologne, 50674 Cologne, Germany
  1. Edited by Kai Simons, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, and approved October 16, 2017 (received for review June 27, 2017)

  1. Fig. 2.

    Function of cargo proteins and their distribution upon loss of Tango1. (AE) Terminal cells were visualized by expressing mCD8mCherry under the terminal-cell–specific driver SRF-gal4. (E) Manual quantification of branch numbers in terminal cells expressing different RNAi; cells expressing tango1 RNAi (B) have fewer branches than control cells (A). Neither dpy RNAi (C) nor pio RNAi (D) affect branch numbers. Control, n = 8; tango1-IR, n = 9; dpy-IR, n = 8; pio-IR, n = 9. Bars represent mean ± SEM. Significance was determined using two-tailed t test. (F and G) Confocal projections of control (F) and tango1-IR (G) terminal cells expressing SRF > GFP and stained for βPS integrin (βInt). Arrowheads point to βInt localization. (H and I) Confocal projections of control (H) and tango1-IR (I) terminal cells expressing SRF > GFP and stained for Crb. Arrowheads point to Crb localization. (J) Airyscan images of details of tracheal dorsal trunk cells expressing Dpy-YFP and stained for Tango1 and Golgi marker GM130. White squares indicate the magnified, representative regions in J′J?. (J′) Magnification of the area marked in J. (J′′) Orthogonal views of a single plane from J′. The white arrowhead points to GM130 signal and the green arrowhead points to Dpy-YFP signal associated to Tango1. (J?) Magnification of the area marked in J′. Asterisks in J′ and J″ show the Tango1 ring magnified in J?. (K and L) Confocal projections of control (K) and tango1-IR (L) terminal cells expressing SRF > mCD8mCherry (K and L) and Dpy-YFP (K′ and L′) and stained for Pio (K″ and L″). [Scale bars: AD, 40 μm; FI, K, and L, 10 μm; J, 5 μm; J′, 2 μm (also applies to J″); J′′′, 1 μm.]

  2. Fig. 3.

    Effect of tango1 and dpy knockdown on Crb and the ER stress response. (AD) Terminal cells expressing mCD8mCherry (AD) and Xbp1-GFP (A″D″) under SRF-gal4. Xbp-1-GFP is translated and accumulated in the nucleus only after activation of the ER-stress response. (A′D′) In control (A′ and A″) and dpy-IR cells (C′ and C″), Crb localizes to the luminal membrane and Xbp1-GFP is not detectable. In tango1-IR cells (B′ and B″), Crb does not reach the membrane and Xbp1-GFP accumulates in the nucleus. These defects can be suppressed by additionally knocking down dpy (D′ and D″). (EG) Terminal cells expressing GFP and the ER stress response target Xbp1 under SRF-gal4. (E and E′) Bright field (BF) imaging shows lack of air in the tracheal branches. (F) Quantification of number of branches in tracheal cells expressing Xbp1 under SRF-gal4. Bars represent mean ± SD. Control, n = 5, SRF > Xbp1spliced, n = 8. Significance was assessed using Student’s t test. (G) Terminal cells expressing Xbp1spliced where stained against βInt and Crb. Arrowheads point to the normal distribution of both proteins. (Scale bars: A–D and G, 10 μm; E, 40 μm.)

  3. Fig. 4.

    Rescue of cell morphology in tango1-depleted cells by removal of Dpy. (AD) Bright field (BF) and mCD8mCherry expression under the terminal cell-specific driver SRF-gal4. In control (A and A′) and dpy-IR cells (C and C′), branches are filled with gas, whereas the absence of tango1 leads to failure of air-filling and reduced branching (B and B′). Both defects are suppressed by additionally knocking down dpy (D and D′). (E) Quantification of branching in AD. Bars represent mean ± SEM. Control, n = 4; tango1-IR, n = 9; dpy-IR, n = 8; tango1-IR+dpy-IR, n = 8. Significance was determined using one-way ANOVA and Tukey’s multiple comparisons test.

  4. Fig. 5.

    Distribution of cargo proteins and Sec16 in cells lacking Tango1. White boxes in AD are representative fields magnified in A′–A?, B′–B″, C′C?, and D′–D?, respectively. (A and B) Confocal projections of terminal cells expressing mCD8mCherry under SRF-gal4, endogenously tagged Dpy-YFP (A′ and B′) and stained for Sec16 (A′′ and B′′). While in control cells Dpy-YFP is seen at the luminal membrane (AA′′′), in cells lacking tango1 Dpy-YFP shows a cytoplasmic distribution that partially overlaps with Sec16 (BB′′′). (C and D) Confocal projections of terminal cells expressing GFP under SRF-gal4 and stained for Sec16 and Crb. While in control cells Crb localizes to the luminal membrane (CC′′′), in cells lacking tango1 Crb shows a cytoplasmic distribution that partially overlaps with Sec16 (DD′′′). (Scale bars: A–D, 10 μm; A?D?, 2 μm.)

  5. Fig. 6.

    Effect of loss of Tango1 and Dpy on Sec16 distribution. (AD) Tango1 (A′D′) and Sec16 (A″D″) in terminal cells expressing GFP under SRF-gal4. In control (AA?) and dpy-IR cells (CC?), Sec16 particles are homogeneous in size and fluorescence intensity; in tango1-IR cells, Sec16 particle size and fluorescence intensity is variable (BB?), and this variability is not altered by simultaneously removing Dpy (DD?). Insets in (A″D″) are magnifications of representative regions, indicated by the white squares. (E and F) Variance of Sec16 particle size (E) and Sec16 fluorescence intensities (F). Control, n = 5; tango1-IR, n = 4; dpy-IR, n = 4; tango1-IR+dpy-IR, n = 5. Significance was determined using one-way ANOVA and Sidak’s multiple comparisons test. (Scale bars: 10 μm; Insets, 2 μm.)

  6. Fig. 7.

    Effect of loss of Tango1 and Dpy on GM130 distribution. (AD) Terminal cells expressing GFP under SRF-gal4, and stained for the Golgi marker GM130 (A′–D′). In control (A–A″) and dpy-IR cells (C–C″), the distribution and size of GM130-labeled structures is homogeneous, whereas in tango1-IR cells, GM130 is seen in heterogeneous aggregates (B–B″). Knocking down dpy in tango1-IR cells does not rescue GM130 distribution (DD″). Insets in A′D′ are magnifications of representative regions, indicated by the white squares. (Scale bars: 10 μm; Insets, 2 μm.) (E) Model showing the role of Tango1 in Dpy trafficking and the indirect consequences of Dpy blockage. In the absence of Tango1, the structure of the ER, COPII, and Golgi apparatus are changed, ERGIC53 is retained in the ER, and the ER-stress response is activated. Additionally, neither Dpy nor Crb reach the plasma membrane. If Dpy levels are reduced, ERGIC53 shuttles out of the ER, the ER-stress response is no longer active and Crb can be secreted. However, the ER, COPII, and Golgi morphology are not restored.

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