EMBO/soda-nel huggingface dataset:
Dynamic Assembly of the Exomer Secretory Vesicle Cargo Adaptor Subunits (2015)
Figure 4

Figure 4. Formation of the exomer tetramer is dependent on ChAP concentration.

(A-B) Overexpression of Bch2 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN. Fluorescence images of cells expressing Chs5-GFP (A) and binding kinetics of Chs5-GFP at the TGN (B) in WT, BCH2 and GPD-BCH2 strains. Bar, 5 µm. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. The schematic cartoons depict the predicted majority of the analyzed complexes; Chs5 dimer:violet ovals, Bch2:dark blue circles.

(C-D) Overexpression of Chs6 in absence of other ChAPs results in more efficient Chs5 recruitment and stabilization at the TGN. (C) Fluorescence images of cells expressing Chs5-GFP in WT, CHS6 and GPD-CHS6 strains. Bar, 5 µm. (D) Binding kinetics of Chs5-GFP at the TGN in WT, , CHS6 and GPD-CHS6 strains. Data processing was carried out as in (A-B). The schematic cartoons depict the predicted majority of the analyzed complexes; Chs5 dimer: violet ovals, Chs6: light blue circles.

(E-G) Overexpression of Chs6 rescues Chs3 export in absence of other ChAPs. (E) Fluorescence images of cells expressing Chs3-GFP in WT, CHS6 and GPD-CHS6 strains. Arrows point to Chs3-GFP signals at the bud neck. Bar, 5 µm. The inset represents a twofold magnification.

(F) Quantification of phenotypes in (E); 100 small budded and 100 large budded cells were quantified per experiment. Average and SD of three independent biological experiments is shown.

(G) Chs3 GFP GPD-CHS6 strain was sensitive to calcofluor to a similar extent as the WT strain. Plates were incubated at 30°C for 2-3 days. A representative drop test of three independent biological experiments is shown.
Dynamic Assembly of the Exomer Secretory Vesicle Cargo Adaptor Subunits (2015)
Figure 5

Figure 5. ChAPs cooperate during the assembly of the exomer tetramer in vivo.

Fluorescence images of cells expressing Chs5-GFP and TGN marker Sec7 DsRed (A, C) and binding kinetics of Chs5-GFP at the TGN (B, D) in WT, CHS6 and CHS6 BUD7 (A, B) or CHS6 BCH2 (C, D) strains. The brightness and contrast were adjusted differently in the merged images. Bar, 5 µm. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.
Dynamic Assembly of the Exomer Secretory Vesicle Cargo Adaptor Subunits (2015)
Figure 6

Figure 6. ChAPs compete and cooperate for binding to Chs5 in vivo.

(A-D) ChAPs are differently dependent on each other in terms of binding at the TGN. Binding kinetics of GFP-tagged Chs6 (A), Bud7 (B), Bch2 (C) and Bch1 (D) at the TGN in the presence (WT) or absence of the other ChAPs (). The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.
Dynamic Assembly of the Exomer Secretory Vesicle Cargo Adaptor Subunits (2015)
Figure 7

Figure 7. The relative expression levels of ChAPs are set to fine-tune the exomer assembly pathway.

(A-B) Overexpression of Bch1 displaces Chs6 from the TGN and impairs plasma membrane localization of Chs6-dependent cargo Chs3. Fluorescence images of Chs6-GFP (A) and Chs3-GFP (B) in WT and GPD-BCH1 strains. Bar, 5 µm. Arrows point to Chs3-GFP signals at the bud neck. The inset represents a twofold magnification.

(C) Quantification of the phenotypes in (B); 100 small budded and 100 large budded cells were quantified per experiment. Average and SD of three independent biological experiments is shown.

(D) Chitin production is reduced upon Bch1 overexpression. Fluorescence images of chitin stained with calcofluor in Chs3-GFP WT, chs5 and GPD-BCH1 strains.

(E) Overexpression of Bch1 does not impair plasma membrane localization of the Bch1-dependent cargo Pin2. Fluorescence images of Pin2-GFP in WT and GPD-BCH1 strains.

(F-I) Overexpression of Chs6 displaces Bch1 and Chs5 from the TGN. Fluorescence images (F-G) and binding kinetics at the TGN (H-I) of cells expressing Bch1-GFP (F, H) and Chs5-GFP (G, I) upon overexpression of Chs6 under GPD promoter from 2μ plasmid. The insets in (F, G) represent two-fold magnifications. The arrows in (F) point to residual Bch1 staining at the TGN. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. Size bars represent 5 µm.
Dynamic Assembly of the Exomer Secretory Vesicle Cargo Adaptor Subunits (2015)
Figure 8

Figure 8. Cargo promotes assembly of the exomer tetramer.

(A) Overexpression of Pin2 enhances Chs5 stabilization at the TGN. Binding kinetics of Chs5 GFP in WT, bud7bch1 and bud7bch1 strain expressing Bch2-dependent cargo Pin2 from a centromeric or a 2μ plasmid. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.

(B) Comparison of the expression levels of the endogenous Pin2 with the levels of Pin2 expressed from a centromeric or a 2μ plasmid. Pgk1 serves as loading control. A representative immunoblot of three independent biological experiments is shown.
Tissue tension and not interphase cell shape determines cell division orientation in the Drosophila follicular epithelium (2018)
Figure 1

Fig 1: Epithelial topology becomes increasingly regular as the early egg chamber matures.

A) Egg chambers expand and elongate through development. This is acheieved through collective migration (A') of the FE.

B) Egg chambers flatten along the coverslip, allowing for follicle cell morphologies to be compared in two dimensions. In (B), three live w1118 (control) egg chambers expressing the membrane marker Basigin::YFP are shown in multiple planes of focus, with a z-reconstruction at the bottom. Because the egg chambers flatten at the coverslip, the basal follicle cell morphologies can be revealed in a single focal plane. To account for differences in follicle cell height between the three egg chambers, which increase in maturity from left to right, separate focal planes are shown for the apical morphologies. A diagram illustrating the position of the three focal planes with respect to the flattened egg chamber is shown in (B'). Scale bars = 20 μm.

C-E) Follicle cell cross-sectional area decreases (C) and cell shape becomes more regular (D) and hexagonal (E) as early-stage egg chambers mature. These measurements were derived from at least three egg chambers from at least three flies per AR range. Bars in (C) and (D) represent mean and standard deviation. Statistical significance for cell side distributions was calculated using a chi-squared test. Number of cells used for morphological analysis: 1.0-1.2 apical n=23, basal n=27; 1.2-1.4 apical n=114, basal n=61; 1.4-1.6, apical n=160, basal n=149. Vertex counts: 1.0-1.2 apical n =38, basal n=33; 1.2-1.4 apical n=73, basal n=70; 1.4-1.6, apical n=284, basal n=259.
Tissue tension and not interphase cell shape determines cell division orientation in the Drosophila follicular epithelium (2018)
Figure 2

Fig 2: Tissue regularity is a function of cell proliferation.

A, B) Egg chamber elongation is unaffected by reduced cell proliferation. String-shRNA expressed for 24 hours alters epithelial shape and cell number (A), but not the relationship between egg chamber aspect ratio and cross-sectional area, which is quantified in (B). Chambers shown in (A) have approximately equal widths, with the cross-sectional area corresponding to developmental stage 6. Scale bars = 20 μm. Number of chambers analyzed: w1118 1.0-1.2 n=24,1.2-1.4 n=14,1.4-1.6 n=18 String-shRNA 1.0-1.2 n= 11 ,1.2-1.4 n=10,1.4-1.6 n=15. Bars represent mean and standard deviation.

C, D) Cell proliferation is necessary for normal tissue morphology. Follicle cell shape regularity (C) and polygonality (D) measurements in control and String-shRNA egg chambers of different aspect ratios. These measurements were derived from at least three egg chambers from at least three flies per AR range. Bars represent mean and standard deviation. Statistical significance for cell side distributions was calculated using a chi-squared test. Number of cells used for analysis: w1118 1.0-1.2 n= 23, 1.2-1.4 n=114, 1.4-1.6 n=160; String-shRNA 1.0-1.2, n=6, 1.2-1.4 n=19, 1.4-1.6 n=17.
Tissue tension and not interphase cell shape determines cell division orientation in the Drosophila follicular epithelium (2018)
Figure 3

Fig 3: The follicular epithelium is under anisotropic apical tension.

A-C) Epithelial cell size and shape is affected by decreased proliferation. Representative images of epithelial cells taken from egg chambers with aspect ratios of approximately 1.4 (A). Cell outlines are marked with Basigin::YFP. Scale bars =­ 5 μm. Cells become significantly larger (B) and more aligned with the tissue elongation axis when proliferation is stopped using String-shRNA (C). Cell area shown on a log4 scale in (B). Number of cells analyzed: w1118 1.0-1.2 n= 14, 1.2-1.4 n=114, 1.4-1.6 n=160; String-shRNA 1.0-1.2, n=10, 1.2-1.4 n=19, 1.4-1.6 n=17. Statistical significance calculated using a Mann-Whitney test.

D, E) The follicular epithelium 'thins out' over the egg chamber equator when proliferation is blocked. Cartoon demonstrates the tissue cell shape observed in the follicular epithelium in control (w1118) and String-shRNA treated egg chambers. Quantification of the phenomenon shown in (D) reveals that cells are shorter over the equator than at the anterior pole in elongated egg chambers when proliferation is blocked. Statistical significance calculated using unpaired t-test with Welch's correction. Number of cells analyzed: w1118 1.0-1.2 n= 21, 1.2-1.4 n=11, 1.4-1.6 n=10; String-shRNA 1.0-1.2, n=9, 1.2-1.4 n=11, 1.4-1.6 n=13.

F) Recoil is observed when cuts are made in apical cortex with a 2-photon laser with egg chambers in saggital view. Images show cortex before and after cutting. Scale bars = 10 μm. Yellow arrows indicate edges of ablated region.

G) Recoil is higher when cuts are made at the apical cortex at the equator of egg chambers, rather than at the poles. Quantification of experiment shown in (F). Statistical significance tested by Mann-Whitney test. Number of laser cuts: equator n=30, pole n=20.

H) Recoil is observed when larger cuts are made in apical cortex with a UV laser. Images show cortex before and after cutting. Red lines indicate the lines over which recoil was calculated, parallel to the axes of the egg chamber. Scale bars = 5 μm. Yellow arrows indicate edges of ablated region.

I) Recoil tends to be higher towards the AP (elongation) axis compared to the the UD (rotation) axis. Graph shows the ratio of recoil speed plotted on a log2 scale in the AP versus the UD axis. Red line at x=1 indicates equal recoil in both axes. Values above 1 indicate higher recoil speeds in the AP axis lower than 1 indicate higher recoil in the UD axis. AP/UD Ratio Median = 1.242 ; Mean = 2.1. Wilcoxon Signed Rank Test to test for significance of the recoil ratio AP/UD shows that the median is significantly different from 1 to p = 0.0172. Number of cuts = 20. Bar represents median.

Data information: Bars in (B, C, E) represent mean and standard deviation. Laser ablation experiments in (F-I) performed on tissue from sqhAX3, sqh::GFP flies.
Tissue tension and not interphase cell shape determines cell division orientation in the Drosophila follicular epithelium (2018)
Figure 4

Fig 4: Follicle cell divisions are biased towards tissue expansion but not interphase cell shape.

A) Cartoon demonstrating how the tissue expansion angle is determined.

B) i) Planar division orientation is initially random in the follicular epithelium, with a significant bias towards the egg chamber elongation axis evident at stage 6 (asterisk, p = 0.0031, Wilcoxon Signed Rank Test, Theoretical Mean of 45; p = 0.0218 when comparison to a randomly generated list of the same size). Number of cells analyzed: stage 2/3 n=16, stage 4 n=27 stage 5 n=30, stage 6 n=40. ii) Quantification of the angle of tissue expansion as calculated by vector addition of the minor and major axis of egg chambers. Number of egg chambers analyzed: stage 2/3 n=10, stage 4 n=15, stage 5 n=15, stage 6 n=10. iii) Quantification of cell elongation angles (as shown in E) with respect to the chamber elongation axis by stage from live egg chambers. Number of cells analyzed: stage 2/3 n=18, stage 4 n=49 stage 5 n=64, stage 6 n=97. Bars represent mean and standard deviation.

C) Planar division orientation at Stage 6 correlates with tissue expansion in Fat2-shRNA and Pak-shRNA egg chambers. Mann Whitney test w1118 versus Pak shRNA p = 0.0094. Neither the Fat2-shRNA nor Pak-shRNA distributions are stastically different from random (Wilcoxon Signed Rank Test, Theoretical Mean of 45). Number of cells analyzed: w1118 n =97, Fat2-shRNA n=44, Pak-shRNA n=39. Egg chambers analyzed: w1118 n=40, Fat2-shRNA n=21, Pak-shRNA n=21.

D) Neighboring cells can form new attachments underneath (basal to) a mitotic cell that has moved apically. Scale bars = 5 μm. Thin yellow lines in the XZ reconstructions indicate the z-position of the two XY focal planes.

E) Workflow for determining follicle shape shapes. Segmentation of an egg chamber with an aspect ratio of 1.5 reveals that follicle cell elongation is usually perpendicular to the elongation axis of the egg chamber. Cell outlines were marked with Basigin::YFP. Scale bars = 5 μm.

F) Live confocal imaging shows that the long axis of a follicle cell at interphase does not predict the orientation of division. Long axis orientations at the timepoints shown were determined as in (E). Scale bars = 5 μm.

G) The interphase long axis does not correlate with the division angle in both the follicular epithelium (FE) and embyronic neuroectoderm (ENE). n=10 cells (FE), 18 cells (ENE).
Tissue tension and not interphase cell shape determines cell division orientation in the Drosophila follicular epithelium (2018)
Figure 5

Fig 5: Mud does not determine planar spindle orientation in the FE.

A) The vertex long axis of cells does not correlate with the division angle in both the FE or ENE. The cell vertex distribution at interphase using both the vertex bipolarity and a modified method that takes into account the distance that vertices lie from cell barycentres (modified vertex bipolarity) does not predict the division angle. n=10 cells (FE), n=18 cells (ENE). Bars represent mean and standard deviation.

B, C) Pins and Mud are symmetric around the entire cell cortex in (B) mitotic follicle cells and (C) mitotic cells in the ENE. (B' and C') Confocal imaging of immunostained fixed tissue. Scale bars = 5 μm.

D) Planar division orientation in the follicular epithelium is independent of Mud. Division angles were calculated relative to the elongating axis of the egg chamber using an unbiased analysis tool. Number of cells analyzed: w1118 n=52; mud3/mud4 n=43; Random number generator n=50; Mean division angle w1118= 31.6o, mud3/mud4= 30.9o, Random number generator = 46o; Wilcoxon Signed Rank Test, Theoretical Mean of 45, w1118 p < 0.0001, mud3/mud4 p = 0.0003, Random number generator p = 0.7779. Spindle angles in mud3/mud4 egg chambers are not significantly different from w1118 (p = 0.7859, Mann Whitney test). Spindle angles in w1118 and mud3/mud4 egg chambers are statistically different from the randomly generated list (p = 0.006 and p = 0.0044, Mann Whitney test).

E) Live imaging reveals a mitotic spindle rotating in the plane of the FE until anaphase at timepoint 0. Timepoints shown in minutes relative anaphase. Scale bars = 5 μm.

F) The variance in mitotic spindle angle in the ENE reduces as cells approach metaphase. Graph shows root mean square of spindle angles approaching metaphase (defined at t = 0 secs). Number of cells analyzed= 22.
Tissue tension and not interphase cell shape determines cell division orientation in the Drosophila follicular epithelium (2018)
Figure 6

Fig 6: Apical actomyosin shows a dynamic relocalization during early egg chamber development.

A, B) Live confocal imaging of Myosin-II shown by Zipper::YFP (A) and Spaghetti squash-mCherry (B) at the apical cortex of follicle cells during egg chamber elongation showing a transition from cell to tissue-level organization during stage 6. (B) The localization Sqh-mCherry is shown relative to adherens junction marked by Shg::GFP.
Tissue tension and not interphase cell shape determines cell division orientation in the Drosophila follicular epithelium (2018)
Figure 7

Fig 7: Canoe disruption disrupts spindle orientation, Myosin organization and tissue packing.

A) Apical Myo-II localization as marked by Sqh-GFP is disrupted in round (stage 2/3) and elongated (stage 6) egg chambers. Red arrows point to the seperation of Myo-II at cell junctions, indicating that actomyosin is no longer correctly linked to adherens junctions. Live confocal imaging. Scale bars = 5 μm.

B) Planar division orientation in the follicular epithelium is dependent on apical tension in a Canoe-dependent manner, with Canoe-shRNA treated egg chambers showing a randomization of division orientation. Division angles were calculated relative to the elongating axis of the egg chamber using an unbiased analysis tool. Number of cells analyzed: w1118 n=52; Canoe-shRNA n=58; Random Number Generator n = 50. Mean division angle w1118= 31.6o, Canoe-shRNA= 43.3o, Random number generator = 46o; Mann Whitney test versus w1118: Canoe-shRNA p = 0.0345 ; Wilcoxon Signed Rank Test, Theoretical Mean of 45, w1118 p < 0.0001, Canoe-shRNA p = 0.7411, Random number generator p = 0.7779. Bars represent mean and standard deviation.

C) Sidedness is affected by planar division orientation in an in silico simulated epithelium. One round of doubling extending over a six-hour period was simulated in a stretched tissue under different conditions for division orientation. The three time points shown reflect cell geometry in the first two hours (early), second two hours (mid), and last two hours (late).

D, E) Tissue topology is affected by disruption of Canoe, but not Mud. (D) Statistical significance for cell side distributions was calculated using a chi-squared test. Number of cells analyzed: w1118 1.4-1.6 n=284; Canoe-shRNA 1.4-1.6 n=310; mud3/mud4 1.4-1.6 n=251. (E) Representative image of a Canoe-shRNA follicle epithelium. Cell outlines are marked with Basigin::YFP. This egg chamber has no mitotic cells and AR of 1.58. Scale bars = 5 μm.
Selective autophagy degrades DICER and AGO2 and regulates miRNA activity (2012)
figf1

(a) Western blot analysis of HeLa cells treated with siRNA for 84 h. (b) Western blot analysis of HeLa cells treated with a second independent siRNA targeting ATG5, ATG7 or NDP52. GAPDH and total protein stained with Coomassie blue serve as loading controls. (c) Western blot analysis of HeLa cells cultured in Hank’s buffered salt solution (Starved) or DMEM+ 10% FBS (Serum). (d) Western blot analysis of HeLa cells treated (48 h) with an inhibitor of mTORC1 (RAP, 20 nM) or control (DMSO). (e) Western blot analysis comparing the levels of DICER in MDA-231, T47D and MDA-435 cells. (f) Western blot analysis of MDA-231, T47D and MDA-435 cells treated (48 h) with inhibitors of mTORC1 (RAP, 20 nM), mTORC1 and 2 (PP242, 100 nM), or DMSO (control). (g) Western blot analysis of HeLa cells treated with CQ or DMSO for 12 h. (h) Western blot analysis of HeLa cells treated with BAF (200 nM, 12 h), RAP (20 nM, 24 h), or RAP and BAF. The results in a–h are representative of three experiments and use
α-tubulin (TUBA) as a loading control. (i) Western blot analysis of DICER (monoclonal antibody 13D6) or control (EEF1A1) in HeLa cells treated with control siRNA or siRNA targeting DICER. (j) Confocal analysis of the signal from an anti-DICER monoclonal antibody (clone 13D6) or an isotype control monoclonal antibody in HeLa cells. Scale bar, 10 μm. (k,l) RT–qPCR analysis of AGO1, AGO2 and DICER mRNA in HeLa cells treated for 12 h with DMSO, BAF or CQ (k) or siRNA for 84 h (l). Error bars show s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.
Selective autophagy degrades DICER and AGO2 and regulates miRNA activity (2012)
figf2

(a) Localization using confocal microscopy of endogenous DICER (monoclonal antibody 13D6) and NDP52 in HeLa cells treated with RAP or control. The arrows highlight some examples of co-localization. The lower left insets show higher magnifications of the areas outlined in the main panel. Scale bars, 5 μm. (b) Quantification of DICER co-localization with NDP52. Total fluorescence intensity of DICER co-localized with punctae (>0.2 μm3) of NDP52 was quantified with Volocity software in RAP-treated cells/control-treated cells over several
Z-stacked microscope fields per experiment (n = 4, error bars s.e.m.). (c) Localization using confocal microscopy of endogenous DICER (monoclonal antibody 13D6) and HcRed–LC3 in HeLa cells treated with BAF or control. The arrows highlight some examples of co-localization. Insets as in a. Scale bar, 5 μm. (d) Quantification of DICER co-localization with HcRed–LC3 was performed as for NDP52 in b. (e) Confocal microscopy analysis of GFP–DCP1A and HcRed–LC3wt in HeLa cells. Scale bar, 10 μm. (f–i) Localization of DICER (monoclonal antibody 13D6) detected with anti-mouse IgG (10 nm gold beads) by electron microscopy in CQ- (20 μM, 12 h) treated HeLa cells. Mito, mitochondria; MVB, multivesicular body. The black arrows highlight all gold beads. (j) Western blot analysis of fractions from a discontinuous gradient of Histodenz (15%, 20%, 24%, 26%) described to enrich autophagosomes (light AV) and autophagolysosomes (heavy AV; ref. ). HEK293T cells were treated with CQ (20 μM, 16 h). Fractions enriched in multivesicular body and lysosomal markers are indicated. Material that was pelleted by centrifugation at 100,000g (100 K pellet) or that remained in solution after the 100,000g spin (soluble) was not added to the gradient. (k) Western blot analysis of UB and p62 in discontinuous Histodenz gradients (as in j) of HEK293T cells treated with control (DMSO) or CQ. Uncropped images of blots are shown in Supplementary Fig. S3.