Fig 2: Cell-type specific effects of bet-1B overexpression. (a) Life spans of N2 (blue) and bet-1B overexpression in muscle (myo-3p, red), intestine (gly-19p, green), and hypodermis (col-19p, purple) grown on empty vector (EV) from hatch. p-Values versus N2 control: myo-3p::bet-1B (red) = p < 0.0001; gly-19p::bet-1B (green) = p < 0.0001; col-19p::bet-1B (purple) = p < 0.0001. See Tables S6 and S7 for complete life span statistics. (b) Representative fluorescent images of adult animals expressing LifeAct::mRuby from an intestine-specific promoter, gly-19p. N2, wild-type animals were grown on EV or bet-1 RNAi from hatch, and bet-1B overexpression animals (bet-1p::bet-1B) were grown on EV from hatch. All animals were imaged at day 1, 5, and 9 of adulthood. Images were captured on a Leica Stellaris 5 confocal. Scale bar is 10 µm. (c) Representative fluorescent images of adult animals grow on mCherry expressing bacteria as described in Materials and Methods. Wild-type N2 and bet-1B overexpression (bet-1B) animals were grown on empty vector (EV) or a 90/10 mix of ev/act-1 RNAi mixed with 20% mCherry bacteria from hatch. Animals were clarified on OP50 for 2 h prior to imaging at day 1, 5, and 9 of adulthood. Scale bar is 500 µm. (d) Quantification was performed across 2 technical replicates each of 3 biological replicates for a total of 6 replicates. The % of animals exhibiting gut colonization of fluorescent bacteria were quantified per replicate and each dot represents a technical replicate. Lines represent mean and interquartile range. **p < 0.01, n.s. = p > 0.05 using student t-test. (e) Representative fluorescent images of adult animals expressing LifeAct::mRuby from a hypodermis-specific promoter, col-19p. N2, wild-type animals were grown on EV or bet-1 RNAi from hatch, and bet-1B overexpression animals (bet-1p::bet1B) were grown on EV from hatch. All animals were imaged at day 1, 5, and 9 of adulthood. Images were captured on a Leica Thunder Imager. Scale bar is 10 µm. (f) Brood assay was measured by counting number of live progeny born by a single parent as described in Materials and Methods in N2 wild-type (blue) and bet-1B overexpressing (green) animals. Each dot represents a single animal and lines represent median and interquartile range. ***p < 0.001 measured by non-parametric Mann–Whitney testing. Data is representative of 3 independent trials.

Fig 3: Quantitative and morphological differences between early endosomes in ECFCs and HEK293T cells.A) Representative images of ECFC (top) and HEK293T (bottom) cells where each panel shows the raw confocal image (left) and the identified object in Cellprofiler (right). Every different color indicates a different object. Cells were stained with DAPI (blue) and antibodies against ß-catenin (red) and EEA1 (green). Scale bar represents 50 µm. Images were taken with a Leica Stellaris 5 LIA fitted with a 63x objective. Per cell type, 3 tile scans (each 754630 µm2 in size) were analyzed with the OrganelleProfiler pipeline. B) Cell count per image. C) The cell area (µm2) per cell of all 3 tile scans pooled (n = 1078 ECFCs and n = 4618 HEK293T cells). D) Number of endosomes per cell. E) Mean relative distance of the endosome to the nucleus per cell. F) Mean eccentricity of endosomes per cell. Data is shown as median with min/max boxplot. Dashed red line represents mean eccentricity of WPBs as determined in Fig 3G. Mann-Whitney U test was performed on not normally distributed data (C,D,E,F). Unpaired T test with Welch’s correction was performed on normally distributed data (B); **p<0.01, ***p<0.001, ****p<0.0001.