Supplementary Materials1. the linker cell to control its death have been identified (Blum et GB-88 al., 2012; Kinet et al., 2016; Malin et al., 2016), here we examined the mechanism of linker cell clearance. We find evidence that the linker cell is removed by entosis, a cell-cell-adhesion-based mechanism originally discovered in cancers (Overholtzer et al., 2007). RESULTS Linker Cell Clearance Results in Separation of a Lobe Structure To investigate linker cell clearance, we examined the temporal dynamics by time-lapse imaging in 3 dimensions (4D imaging) utilizing a strain with linker cell GFP expression (promoter::GFP) (Abraham et al., 2007). After completing migration, linker cells rounded and moved left or right of the midline and anterior, presumably due to engulfment by either the left or right U cell (U.lp or U.rp) (Abraham et al., 2007). We noted that as linker cells moved left or right, a subcellular piece extended from the cell body and detached, remaining at the midline (Figures 1A and S1A; Video S1). This separating lobe was 2.1 0.74 m in diameter and was detected in 65 out of 67 worms examined. To determine the relative timing of lobe separation and engulfment, worms were generated with expression of GFP in engulfing U cells (promoter::GFP) and a marker of cortical actin in the linker cell, the calpoinin homology domain of the actin-binding protein Utrophin (UtrCH) (Morris et al., 1999) fused to mCherry (promoter::mCherry::UtrCH). By 4D imaging, we found that a lobe formed from the linker cell and separated as it became engulfed, detaching from the back, opposite the direction of engulfment (Figure S1B). Open in a separate window Figure 1. Linker Cell Engulfment and Entotic Cell Death Involve Separation of a Lobe Structure(A) 4D imaging of linker cell engulfment shows the formation and separation of a lobe (arrowhead). Images are maximum projections, times are h:min. See Video S1. (B) Entotic cells form lobes. Images show MCF-7 cells labeled with green and red Cell Tracker dyes GB-88 imaged by 4D microscopy; times are h:min. Arrowhead indicates lobe that undergoes cleavage. See Video S3A. (C) Lobe cleavage is a feature of entotic cell death. Top graph shows percent entotic MCF-7 cells imaged for 20 h that exhibit lobe cleavage (black bars) and one of three possible fates: remaining inside of hosts without dying (no change), escape from hosts, or cell death. Gray bars show the percentage of cells without lobe cleavage. For no change, n = 16; escape, n = 34; and cell death, n = GB-88 14; n represents the total number Mouse monoclonal to Chromogranin A of cells imaged from more than three biological replicates. Bottom graph shows five representative lobe cleavages and entotic cell death events; relative times start at lobe cleavage (blue bars, arrow), and cell deaths are indicated by black bars. Scale bars, 10 m. (D)Graph shows cortical to cytoplasmic ratio of GFP::UtrCH (blue line, left y-axis) in a linker cell from the time of engulfment marked by lobe formation (arrowhead). Green line shows GFP intensity over time; black line (righty axis) shows distance of lobe separation from linker cell. Hatched box represents timing of linker cell death (arrow) determined by cortical actin ratio and GFP intensity (see Figure S1D for additional examples). Right images GB-88 show linker cell quantified in graph. Top rows show maximum projections of GFP::UtrCH fluorescence; arrowhead indicates lobe. Bottom row shows the x-y confocal plane of GFP::UtrCH fluorescence from the same cell..