Cell Invasion In Vivo via Rapid Exocytosis of a Transient Lysosome-Derived Membrane Domain

Abstract

Invasive cells use small invadopodia to breach basement membrane (BM), a dense matrix that encases tissues. Following the breach, a large protrusion forms to clear a path for tissue entry by poorly understood mechanisms. Using RNAi screening for defects in Caenorhabditis elegans anchor cell (AC) invasion, we found that UNC-6(netrin)/UNC-40(DCC) signaling at the BM breach site directs exocytosis of lysosomes using the exocyst and SNARE SNAP-29 to form a large protrusion that invades vulval tissue. Live-cell imaging revealed that the protrusion is enriched in the matrix metalloprotease ZMP-1 and transiently expands AC volume by more than 20%, displacing surrounding BM and vulval epithelium. Photobleaching and genetic perturbations showed that the BM receptor dystroglycan forms a membrane diffusion barrier at the neck of the protrusion, which enables protrusion growth. Together these studies define a netrin-dependent pathway that builds an invasive protrusion, an isolated lysosome-derived membrane structure specialized to breach tissue barriers.

Keywords: basement membrane; cell invasion; dystroglycan; exocytosis; invasive protrusion; lysosome; membrane diffusion barrier; membrane dynamics; netrin signaling; vesicle trafficking.

Figure 1. The invasive protrusion locally increases AC area and volume

(A) AC invasion in C. elegans (top, lateral view schematic; bottom, ventral view of laminin::GFP-labeled BM). Left: During the early L3 larval stage, the AC (blue) sits atop the BM (purple) and the two P6.p VPC descendants (1° VPCs, grey). Actin-rich invadopodia (red) form, and one breaches the BM (arrow top, arrowhead bottom). Center: At the time the underlying P6.p descendants divide, the invasive protrusion expands and the BM hole widens. Right: By completion of next P6.p descendant division, the BM opening has expanded beyond the border of the AC, the invasive protrusion has retracted, and the AC contacts the central P6.p descendants. (B) Time-lapse of AC invasive protrusion formation. AC membrane labeled by cdh-3>GFP::CAAX, blue and greyscale; BM (laminin::mCherry, magenta) position shown by orange dotted lines. Wild type (top) and unc-40 (e271) (bottom) animals. Invasive protrusion isosurfaces (the portion of the AC below the BM, purple). (C) AC volume before protrusion formation (P6.p 2-cell stage), during protrusion expansion (P6.p 2/4-cell transition), and after retraction (P6.p 4-cell stage) for wild type (white) and unc-40 (e271) mutants (blue). Volume is normalized to AC volume of corresponding genotype before invasion – see Methods (n > 10 each category; **p<0.01, n.s. = not significant (p>0.05), One-way ANOVA with Tukey-HSD post-hoc test). (D) AC protrusion volume over time in wild type (blue) and unc-40 (e271) (red) animals from the time of BM breach until time-lapse ended (time points shown every 10 minutes; see Methods). In this and all subsequent figures, protrusion volume growth was compared over the first 30 minutes of protrusion growth, the time of maximum growth in wild type animals. Average expansion rates (dotted lines) ± SD are shown (n=10 animals for each group; ***p<0.001, n.s. = not significant (p>0.05), Student’s t-test). Scale bars = 5µm. See also Figure S1.

Figure 2. UNC-6 (netrin) polarizes lysosomes to build the invasive protrusion

(A) Lysosomes (LMP-1::GFP) in the ACs of wild type (top), unc-40 (e271) (middle), and unc-6 (ev400) (bottom) animals before protrusion formation (P6.p 2-cell stage), during protrusion expansion (P6.p 2/4-cell transition), and after retraction (P6.p 4-cell stage). Arrowheads indicate lysosome localization. BM position, orange. (B) Polarity of LMP-1::GFP at the AC invasive membrane before BM breach. (n≥11 animals per genotype; ***p<0.001, One-way ANOVA with Tukey HSD post-hoc test). (C) Time-lapse of AC invasive protrusion formation in a uterine-specific RNAi background. AC membrane labeled with cdh-3>mCherry::PLCδ^PH, blue and greyscale, purple isosurface below; BM (laminin::GFP, magenta) position shown by orange dotted lines. (D) AC protrusion volume over time in control (blue) and ppk-3 (RNAi) (red) animals. Average expansion rates (dotted lines) ± SD for the first 30 minutes of protrusion growth (n=14 animals for wild type, n=10 animals for ppk-3 (RNAi); *p<0.05, Student's t-test). (E) Lysosomes (LMP-1::mCherry, magenta) polarize within the invasive protrusion (arrowhead) toward UNC-6::mNG (green, unc-6 (cp190)), which is expressed in the VPCs and localizes in extracellular punctae (arrow). An unc-6 (ev400) mutant with membrane-tethered UNC-6 (zmp-5>unc-6::nlg-1::TM::GFP) expressed in a dorsal uterine cell (orange arrowhead). The AC extends a small protrusion with the lysosome marker LMP-1::mCherry (white arrowhead) polarized toward the ectopic UNC-6::GFP. Scale bars = 5µm. See also Figure S2.

Figure 3. The t-SNARE SNAP-29 facilitates invasive protrusion expansion

(A) A snap-29 transcriptional reporter (snap-29>GFP; top right) expressed in the AC. An AC expressed translational reporter (cdh-3>mCherry::SNAP-29; bottom) is polarized (arrowheads) to the invasive membrane before invasion (left) and within the protrusion (right); BM position, orange. (B) Colocalization of mCherry::SNAP-29 and LMP-1::GFP during protrusion formation. 0.5µm confocal z-slices of the AC body (top,) and invasive protrusion (middle); sum projection of the AC (bottom). Yellow line (top inset, right) indicates point of fluorescence measurement shown in (C). (C) Fluorescence measurement shows colocalization of peak SNAP-29 (red) and LMP-1 (blue) signal. (D) Time-lapse of AC protrusion formation in a uterine specific RNAi strain. AC membrane labeled by cdh-3>mCherry::PLCδ^PH, blue and greyscale, purple isosurface below; BM (laminin::GFP, magenta) position shown by orange dotted lines. (E) AC protrusion volume over time for wild type (blue) and snap-29 (RNAi) (red) animals. Average expansion rates (dotted lines) ± SD for first 30 minutes of protrusion growth (n=14 animals for control, n=10 animals for snap-29 (RNAi); *p<0.05, Student's t-test). Wild type controls were the same as in Figure 2D. Scale bars = 5µm. See also Figure S3.

Figure 4. The exocyst complex promotes invasive protrusion formation

(A) Time-lapse of AC protrusion formation. AC membrane labeled by cdh-3>mCherry::PLCδ^PH, blue and greyscale, purple isosurface below; BM (laminin::GFP, magenta) position shown by orange dotted lines. (B) AC protrusion volume over time for wild type (blue) and exoc-8 (ok2523) (red). Average expansion rates (dotted lines) ± SD for first 30 minutes of protrusion growth (n=10 animals for wild type, n=13 animals for exoc-8 (ok2523); ***p<0.001, Student's t-test). Asterisks indicate statistical significance – see Methods. Scale bars = 5µm. See also Figure S4.

Figure 5. The invasive protrusion is a segregated membrane domain

(A) A reporter of ZMP-1 localization (zmp-1>GFP::ZMP-1-GPI) (top) and corresponding spectral representation of fluorescence intensity (bottom) shows that ZMP-1 becomes highly concentrated in the invasive protrusion during its formation. (B) Quantification of GFP::ZMP-1-GPI polarity before invasion (P6.p 2-cell stage, n=29 animals) and in the invasive protrusion (P6.p 2/4-cell transition, n=28 animals; n.s. = not significant (p>0.05), Student's t-test). (C) Fluorescence loss in photobleaching (FLIP) of AC membrane reporter (cdh-3>GFP::CAAX). Red dot indicates target of photobleaching (left). A smoothed spectral intensity map of percentage of GFP lost (center). The white dotted lines outline the AC. FLIP ratios (right) were determined by dividing the average loss in the region of photobleaching (X) by the average loss in the region of the AC not targeted (Y). (D) FLIP ratios before invasion (P6.p 2-cell stage, n=5 animals), during protrusion formation (targeting the invasive protrusion or the AC body, P6.p 2/4-cell transition, n=20 animals), and after AC invasion (P6.p 8-cell stage, n=6 animals) (*p<0.05, ***p<0.001, One-way ANOVA with Tukey HSD post-hoc test). Scale bars = 5µm. See also Figure S5.

Figure 6. The BM receptor dystroglycan (DGN-1) forms a membrane diffusion barrier that promotes protrusion formation

(A) ACs expressing dgn-1::mNG (dgn-1 qy18) (left) and spectral representations of fluorescence intensity (center), with BM (laminin::GFP, right). DGN-1::mNG protein localizes to the invasive membrane before invasion (top), concentrates at the AC-BM interface (arrowheads) at the neck of the protrusion (middle), and is lost at this location (bottom) as the BM moves beyond the AC (brackets). The protrusion also retracts at this time. (B) F-actin (cdh-3>mCherry::moesinABD) similarly concentrates at the neck of the invasive protrusion at the AC-BM interface (arrowheads) during protrusion formation. (C) Ventral view of the AC-BM interface shows Factin localization at the site of initial BM breach (top, arrowhead), in a ring (arrowheads) at the AC-BM contact site as the BM hole expands (middle), which is then lost (arrowheads) after the BM gap extends beyond the AC (bottom). (D) Time-lapse of AC protrusion formation in a uterine-specific RNAi strain with the AC membrane labeled by cdh-3>mCherry::PLCδ^PH, blue and greyscale, purple isosurface below; BM (laminin::GFP, magenta) position shown by orange dotted lines. (E) AC protrusion volume over time for wild type (blue) and dgn-1 (RNAi) (red). Average expansion rates (dotted lines) ± SD for first 30 minutes of protrusion growth (n=14 animals for wild type, n=16 animals for dgn-1 (RNAi); *p<0.05, Student's t-test). Wild type controls were the same as in Figure 2D. (F) Fluorescence loss in photobleaching (FLIP) of AC membrane reporter (cdh-3>GFP::CAAX). Red dot indicates target of photobleaching (left). A smoothed spectral intensity map of percentage of GFP lost (center). The white dotted lines outline the AC. FLIP ratios (right) were determined by dividing the average loss in the region of photobleaching (X) by the average loss in the region of the AC not targeted (Y). (G) FLIP ratios for wild type and dgn-1 (RNAi). Controls were shared with the 2/4-cell transition data in Figure 5D (n=20 animals for wild type, n=10 animals for dgn-1 (RNAi); **p<0.01, Student's t-test). Scale bars = 5µm. See also Figure S6.

Figure 7. The BM is a scaffold for protrusion growth

(A) F-actin (cdh-3>mCherry::moesinABD; left) marks the invasive protrusion (purple isosurface), UNC-6 (center), and overlay on DIC (right, BM position, orange). Endogenous UNC-6 (top) and dorsal uterine cell membrane tethered UNC-6 (bottom, arrowhead). Scale bars = 5µm. (B) Maximum protrusion volume (n=12 animals for wild type, n=8 animals for unc-6 (ev400) with ectopic dorsal UNC-6; ***p<0.001, Student's t-test). (C) Following invadopodial breach, the netrin receptor UNC-40 localizes to the breach site where it is activated by UNC-6 (netrin), which is secreted from in the central vulval cells (P6.p and its descendants). UNC-40 polarizes lysosomes, which add membrane to form the invasive protrusion through SNAP-29-mediated exocytosis. During protrusion growth GPI-anchored ZMP-1 concentrates in the invasive protrusion, and the BM receptor dystroglycan and F-actin localize to a ring at the ACBM interface, forming a membrane diffusion barrier. This diffusion barrier allows focused membrane addition and expansion of the protrusion that clears an opening in the BM and vulval tissue allowing the AC to enter the vulval tissue.