Figure 4.
LPIN-1 promotes lysosome stores for invasive protrusion formation. (A) Left: Maximum intensity z-projected fluorescence images showing the AC (cyan, mCherry::PLCδPH) protrusion (arrowheads) in a control and an AC-specific lpin-1 RNAi treated animal (BM, orange dashed lines) with a small BM breach observed corresponding to the reduced protrusion after loss of lpin-1 (middle, bracket, observed in n = 5/5 lpin-1-1 RNAi time-lapses). Isosurfaces of the AC invasive protrusion (below, arrowheads) at the time of maximum protrusion volume (see Video 2). Right: Boxplot of the maximum invasive protrusion volume in control and lpin-1 RNAi treated animals (n = 7 control and 5 lpin-1 RNAi animals, * P ≤ 0.05, Mann–Whitney U test). (B) Max intensity z-projected time-lapse fluorescence images (h:min, above) and isosurface images showing AC lysosomes (LMP-1::mNG) accumulating at the site of BM breach and entering the protrusion (observed in n = 3/3 time-lapses). (C) Left: Max intensity z-projected fluorescence and isosurface images showing the AC lysosomes (LMP-1::mNG) in control and a lpin-1 RNAi treated animal at the initiation of protrusion formation (late P6.p 2-cell stage and 2–4-cell stage). Right: Boxplot of AC lysosome volume in control and lpin-1 RNAi treated animals (n = 21 control and 23 lpin-1 RNAi animals, **** P ≤ 0.0001, unpaired two-tailed Student’s t test). (D) Left: Sum intensity z-projected images show the polarized localization of GFP::CED-10 (Rac) and GFP::MIG-2 (Rac-like) at the AC invasive plasma membrane (arrow) in control and lpin-1 RNAi treated animals at the late P6.p 2-cell stage at the initiation of protrusion formation. Right: Boxplot showing the basal/apical ratio of GFP::CED-10 and GFP::MIG-2 ([CED-10] n = 12 control and 13 lpin-1 RNAi animals; [MIG-2] n = 8 control and 11 lpin-1 RNAi animals, ns [not statistically significant], P > 0.05, Mann–Whitney U test). (E) Left: Sum intensity z-projections of ZMP-1::mNG fluorescence showing the invasive membrane enrichment (arrow) in a control and a lpin-1 RNAi treated animal at the initiation of invasive protrusion formation (late P6.p 2-cell stage and 2–4 cell stage). Right: Boxplot showing the AC basal/apical ratio of ZMP-1::mNG fluorescence intensity in control and lpin-1 RNAi treated animals (n = 10 control and 10 lpin-1 RNAi animals, ns [not statistically significant], P > 0.05, unpaired two-tailed Student’s t test). All data are from two or more replicates. Scale bars, 5 µm.

LPIN-1 promotes lysosome stores for invasive protrusion formation. (A) Left: Maximum intensity z-projected fluorescence images showing the AC (cyan, mCherry::PLCδPH) protrusion (arrowheads) in a control and an AC-specific lpin-1 RNAi treated animal (BM, orange dashed lines) with a small BM breach observed corresponding to the reduced protrusion after loss of lpin-1 (middle, bracket, observed in n = 5/5 lpin-1-1 RNAi time-lapses). Isosurfaces of the AC invasive protrusion (below, arrowheads) at the time of maximum protrusion volume (see Video 2). Right: Boxplot of the maximum invasive protrusion volume in control and lpin-1 RNAi treated animals (n = 7 control and 5 lpin-1 RNAi animals, * P ≤ 0.05, Mann–Whitney U test). (B) Max intensity z-projected time-lapse fluorescence images (h:min, above) and isosurface images showing AC lysosomes (LMP-1::mNG) accumulating at the site of BM breach and entering the protrusion (observed in n = 3/3 time-lapses). (C) Left: Max intensity z-projected fluorescence and isosurface images showing the AC lysosomes (LMP-1::mNG) in control and a lpin-1 RNAi treated animal at the initiation of protrusion formation (late P6.p 2-cell stage and 2–4-cell stage). Right: Boxplot of AC lysosome volume in control and lpin-1 RNAi treated animals (n = 21 control and 23 lpin-1 RNAi animals, **** P ≤ 0.0001, unpaired two-tailed Student’s t test). (D) Left: Sum intensity z-projected images show the polarized localization of GFP::CED-10 (Rac) and GFP::MIG-2 (Rac-like) at the AC invasive plasma membrane (arrow) in control and lpin-1 RNAi treated animals at the late P6.p 2-cell stage at the initiation of protrusion formation. Right: Boxplot showing the basal/apical ratio of GFP::CED-10 and GFP::MIG-2 ([CED-10] n = 12 control and 13 lpin-1 RNAi animals; [MIG-2] n = 8 control and 11 lpin-1 RNAi animals, ns [not statistically significant], P > 0.05, Mann–Whitney U test). (E) Left: Sum intensity z-projections of ZMP-1::mNG fluorescence showing the invasive membrane enrichment (arrow) in a control and a lpin-1 RNAi treated animal at the initiation of invasive protrusion formation (late P6.p 2-cell stage and 2–4 cell stage). Right: Boxplot showing the AC basal/apical ratio of ZMP-1::mNG fluorescence intensity in control and lpin-1 RNAi treated animals (n = 10 control and 10 lpin-1 RNAi animals, ns [not statistically significant], P > 0.05, unpaired two-tailed Student’s t test). All data are from two or more replicates. Scale bars, 5 µm.

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