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An endosome-associated actin network involved in directed apical plasma membrane growth - PubMed

  • ️Sat Jan 01 2022

An endosome-associated actin network involved in directed apical plasma membrane growth

Luis Daniel Ríos-Barrera et al. J Cell Biol. 2022.

Abstract

Membrane trafficking plays many roles in morphogenesis, from bulk membrane provision to targeted delivery of proteins and other cargos. In tracheal terminal cells of the Drosophila respiratory system, transport through late endosomes balances membrane delivery between the basal plasma membrane and the apical membrane, which forms a subcellular tube, but it has been unclear how the direction of growth of the subcellular tube with the overall cell growth is coordinated. We show here that endosomes also organize F-actin. Actin assembles around late endocytic vesicles in the growth cone of the cell, reaching from the tip of the subcellular tube to the leading filopodia of the basal membrane. Preventing nucleation of endosomal actin disturbs the directionality of tube growth, uncoupling it from the direction of cell elongation. Severing actin in this area affects tube integrity. Our findings show a new role for late endosomes in directing morphogenesis by organizing actin, in addition to their known role in membrane and protein trafficking.

© 2022 Ríos-Barrera and Leptin.

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Figures

Figure 1.
Figure 1.

Distribution and role of late endosomes in subcellular tube guidance. (A–D) Terminal cells expressing CD4::mIFP under btl-gal4; see also Video 1. (A) Control. (B) Cell mutant for shrb. (C) Cell expressing Vha100R755A (V100R755A), a dominant negative construct that blocks vATPase function. (D) Proportion of cells with the indicated phenotypes. Number of cells analyzed: control, n = 37; shrbO3/G5, n = 33; Vha100R755A, n = 42. Significance was assayed with a χ2 test; ****, P < 0.0001. (E–J) Terminal cells expressing CD4::mIFP and Par3::YFP under btl-gal4. (E’–J’) CD4::mIFP. (E’’–J’’) Manual tracings of the contours of the terminal cells (dark gray), subcellular tube (black), endosomes (red), and the adjacent dorsal branch cells (light gray). (E–G) Control. (H–J) Cells expressing a constitutively active FGFR, λbtl. Asterisks in A–C mark the tips of the subcellular tubes, and black and white arrowheads point to CD4 vesicles. Ventral is down.

Figure S1.
Figure S1.

Endosome maturation and distribution during subcellular tube growth. (A and B) Terminal cells expressing FYVE::GFP (A' and B') and CD4::mIFP (A'' and B'') under btl-gal4. (A) Control. (B) Cells additionally expressing Vha100R755A (V100R755A). (C–E) Terminal cells expressing CD4::mIFP, Par3::YFP, and constitutively active FGFR, λbtl, under btl-gal4. (C’ and D’) CD4::mIFP. (C’’–E’’) Manual tracings of the cell contours (dark gray), subcellular tube (black), endosomes (red), and dorsal branch cells (light gray). Black and white arrowheads point to CD4 vesicles. Ventral is down.

Figure 2.
Figure 2.

Mislocalization of late endosomes and subcellular tube guidance. (A and B) Terminal cells expressing CD4::mIFP under btl-gal4 (A’–B’) together with GrabFP-BInt (A–A’’) and endogenously labeled Rab7::YFP (B–B’’); see also Video 2. (C) Diagram of the GrabFP-BInt nanobody in vesicles and associating with Rab7::YFP around the vesicle. (C’) Venn diagram of vesicles containing CD4::mIFP, GrabFP-BInt, or Rab7::YFP. n = 395 vesicles, from six different cells homozygous for Rab7::YFP and collected across seven time points each. ND, not detected. (D–G) Cells expressing GrabFP-BInt and CD4::mIFP (D’–G’) under btl-gal4 in a Rab7::YFP homozygous (D’’–F’’) and heterozygous background (G’’). The outline of the cells was traced using the CD4::mIFP signal and is shown as a red dashed line. Black arrowheads point to vesicles containing CD4::mIFP, GrabFP-BInt, and Rab7::YFP, and white arrowheads point to vesicles that do not contain Rab7::YFP. Asterisks mark the tips of the subcellular tubes in D’, E’, and G’. (H) Proportion of cells with the indicated genotypes. Number of cells analyzed: control, n = 28; heterozygous YRab7 background, n = 26; homozygous YRab7 background, n = 59. Significance was assayed with χ2 tests; ****, P < 0.0001. Ventral is down.

Figure 3.
Figure 3.

Actin organization at late endosomes. (A and B) Terminal cell expressing Rab7::YFP and stained with phalloidin and a CBP fused to Alexa647. (A–A’’’) Channels shown in pairwise combinations. (B–B’’’) The region marked in A is magnified in B–B’’’. Arrowheads point to the contact between a Rab7::YFP ring, actin, and the tip of the subcellular tube. (C–F) Terminal cells expressing CD4::mIFP and Utr::GFP under btl-gal4. (C and D) High temporal–resolution imaging. The square shown in C–C’’ is magnified in D and D’ over four different time points (see also Video 3). Arrowheads point to CD4 vesicles. (E and F) High spatial–resolution imaging. Overview of the cell (E–E’’) and four successive time points (F–F’’) of the tip of the same cell. Arrowheads point to actin structures ahead of the subcellular tube.

Figure S2.
Figure S2.

Actin enrichment around late endosomes. (A and B) Terminal cells expressing Rab7::YFP and stained with phalloidin and a CBP fused to Alexa647. (A–A’’’) Channels shown in pairwise combinations. Arrowheads point to the contact between Rab7::YFP rings, actin, and the tip of the subcellular tube. (B) Close-ups of the same cells as in A, but shown as single Z-planes. (B') Phalloidin. (B'') Rab7::YFP. (C) Correlation between pixel fluorescence intensities in the phalloidin and YRab7 channels, in maximum-intensity projections (as shown in A panels) versus single Z-planes (as shown in B panels). Significance was assessed using two-tailed paired t test, n = 8. Ventral is down.

Figure S3.
Figure S3.

Actin reporters and their association with late endosomes. (A and B) Terminal cells expressing CD4::mIFP (A' and B'') under btl-gal4 and MoeRFP under direct control of the btl promoter (A'' and B'''); ventral is down. (A) Control. (B) Cell also expressing Shrb::GFP under btl-gal4 (B'). Arrowheads point to CD4 and MoeRFP puncta. (C–E) Larval terminal cells stained with phalloidin. (C) Control larva expressing endogenously labeled Rab7::YFP. (D) Cell expressing Rab7Q67L::YFP. (E) Cell expressing Rab7T22N::YFP. Regions marked in C–E are shown in C’–E’ but as single z-slices instead of projections. (C'') Endogenously labeled Rab7::YFP. (D'') Rab7Q67L::YFP. (E'') Rab7T22N::YFP. (C'''–E''') Phalloidin.

Figure 4.
Figure 4.

Infrared laser ablation at the growing tip of terminal cells. (A and B) Terminal cell expressing CD4::mIFP and Utr::GFP under btl-gal4. (A) Cell immediately before laser cut. The white box marks the laser-ablated region. (B) Long-term recovery, z-projections. (C–F) Terminal cells expressing Utr::GFP under btl-gal4. (C and D) Cell with normal, ventrally guided tube. (E and F) Cell with a bifurcated tube. C and E are single z-plane images of cells immediately before laser cuts; black boxes mark the laser-ablated regions. D and F are z-projections of the recovery. See also Video 4. (G) Quantification of the recoil as determined by PIV. As control, we analyzed PIV in two successive time points before laser cuts. Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. Number of cells analyzed: cells with ventrally guided tube, n = 7; cells with a bifurcated tube, n = 5. Significance was assessed using t test. Ventral is down.

Figure S4.
Figure S4.

Infrared laser ablation at the tip of terminal cells with bifurcated tubes. (A and B) Terminal cell expressing Utr::GFP (A' and B') and CD4::mIFP (A'' and B'') under btl-gal4. White arrowheads point to CD4 vesicles; black arrowheads point to actin vesicles. (A) Cell immediately before laser cut. Black and white boxes mark the laser-ablated region. (B) Long-term recovery, z-projections. Red arrowheads show the tip and the base of the ventral tube. (C and D) Quantification of the recoil as determined by length displacement over time, using manual tracking. (C) Time course. (D) Initial velocity. Box plots represent median, IQR, and IQR*1.5 below and above the IQR. Number of cells analyzed: cells with properly guided tube, n = 7; cells with bifurcated tube, n = 5. Significance was assessed using t test. Ventral is down.

Figure 5.
Figure 5.

The role of Wash in terminal cell development. (A) Terminal cell expressing CD4::mIFP and Wash::GFP under btl-gal4. The region marked in A is shown in A’–A’’’ at higher magnification over eight time points. (B–F) Cells expressing PH::GFP under btl-gal4; control (B) and wash knockdowns (C and D). (E) Proportion of cells with the indicated phenotypes. Number of cells analyzed: control, n = 29; wash-IR, n = 46. Significance was assessed with χ2 test; ****, P > 0.0001. (F) Distance between the tip of the tube to the tip of the cell in cells with a normal, ventrally guided tube. Number of cells analyzed: control, n = 28; wash-IR, n = 35. Significance was assessed with a Mann-Whitney U test. (G–J) shrbG5 heterozygous embryos expressing CD4::mIFP under btl-gal4; shrbG5 heterozygote as control (G) and shrbG5 heterozygote expressing wash-IR (H and I). (H) Cell with a misguided tube. (I and I’) Cell lacking a tube is shown at two time points. (J) Proportion of cells with the indicated phenotypes. Number of cells analyzed: shrbG5 heterozygotes (controls), n = 30; shrbG5 heterozygotes expressing wash-IR, n = 34. Significance was assessed with χ2 test; ****, P > 0.0001. (K–M) Cells expressing CD4::mIFP and Wash::GFP under btl-gal4. (K and L) Control (K and K’), cell also expressing Vha100R755A (V100R755A; L and L’). Arrowheads point to CD4 vesicles. (M) Fluorescence intensity of Wash::GFP at CD4 vesicles normalized over the mean signal of Wash::GFP in the cytoplasm. Number of cells analyzed: control, n = 11; Vha100R755A, n = 17. Significance was assessed with a Mann-Whitney U test. Box plots in F and M represent median, IQR, and IQR*1.5 below and above the IQR. Ventral is down.

Figure S5.
Figure S5.

Actin organization in cells with defective actin-binding proteins. (A–D) Cells expressing CD4::mIFP (A' and C') and Utr::GFP (A'', B', C'', and D') under btl-gal4. (A and B) sn knockdown. (C and D) btsz knockdown. Regions marked in A and C are magnified and shown over four time points in B and D, respectively. (E) Mean Utr::GFP fluorescence intensity in CD4 vesicles over time. Values were normalized to fluorescence intensity at the tube to adjust for variations in the expression of the Utr::GFP reporter. Number of vesicles analyzed: control, n = 23 from nine cells; Vha100R755A (V100R755A), n = 19 from eight cells; wash-IR, n = 20 from nine cells; sn-IR, n = 10 from five cells; and btsz-IR, n = 20 from eight cells. Values at the top are significance between genotypes, assessed by ANOVA and Tukey correction for multiple comparisons. (F) Mean number of CD4 vesicles at the tip of the cell per time point (4–28 time points per cell). Control cells, n = 11; Vha100R755A, n = 18; wash RNAi, n = 17. Box plots represent median, IQR, and IQR*1.5 below and above the IQR. Ventral is down.

Figure 6.
Figure 6.

Actin organization in cells with defective late endosomes. (A–H) Cells expressing CD4::mIFP and Utr::GFP under btl-gal4. (A–F) Control (A–A’’), cell expressing Vha100R755A (V100R755A; C–C’’), wash knockdown (E–E’’). Regions marked in A, C, and E are magnified and shown over four time points in B and B’, D and D’, and F and F’, respectively. See also Video 5. (G) Mean Utr::GFP fluorescence intensity in CD4 vesicles over time. Values were normalized to fluorescence intensity at the tube to adjust for variations in the expression of the Utr::GFP reporter. Box plot represents median, IQR, and IQR*1.5 below and above the IQR. Number of vesicles analyzed: control, n = 23 from nine cells; Vha100R755A, n = 19 from eight cells; wash-IR, n = 20 from nine cells; sn-IR, n = 10 from five cells; btsz-IR, n = 20 from eight cells. Values at the bottom are significance between normal versus misguided cells within each genotype, assessed by t test. Ventral is down.

Figure 7.
Figure 7.

Graphic summary of the role of late endosomes in subcellular tube guidance. (A) Control cells. Late endosomes situated at the tip of the cell allow the organization of an actin network that extends from the tube to the growing tip of the cell. Control cells with misguided tubes result from the nucleation of actin surrounding late endosomes not located at the tip. In cells with tube bifurcations, late endosomes form two actin nucleation centers that stabilize each subcellular branch. (B) Zoom-in to the tip of the cell (regions marked with rectangles in A). Distinct actin-binding proteins (ABPs) at different subcellular compartments coordinate proper tube formation, with Wash acting in late endosomes to bridge actin pools at the apical (via Btsz) and basal (via Talin and the filopodial organizers Fascin and Espin) cortices. Also shown are microtubules that surround the subcellular tube and that are anchored to the actin meshwork at the tip via Shot. (C–C’’’) Effects of loss of actin organizers at distinct subcellular compartments. (C) Control. (C’) Upon loss of wash or in conditions with aberrant endosomal maturation, F-actin fails to form around late endosomes. (C’’) In the absence of btsz or other actin organizers at the apical cortex, F-actin fails to cross-link to the actin meshwork at the tip. (C’’’) Lack of actin regulators at the basal cortex prevents the connection of the actin meshwork at the tip to the basal plasma membrane. All these conditions decouple the growth of the tube from that of the tip of the cell.

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