Desmosomal coupling in apoptotic cell extrusion

Abstract

The mechanical coupling of epithelia enables coordination of tissue functions and collective tissue movements during different developmental and physiological processes. This coupling is ensured by cell-cell junctions, including adherens junctions (AJs) and desmosomal junctions (DJs) [1, 2]. During apoptosis, or programmed cell death, a dead cell is expelled from the tissue by coordinated processes between the dying cell and its neighbors. Apoptotic cell extrusion is driven by actomyosin cable formation and its contraction, and lamellipodial crawling of the neighboring cells (Fig. S1A-A, Movie S1) [3-6]. Throughout cell extrusion, the mechanical coupling of epithelia needs to be maintained in order to preserve tissue homeostasis [3]. Although much is known about the regulation of AJs in apoptotic cell extrusion [6-9], the role and dynamics of DJs during this process remains poorly understood. Here, we show that DJs stay intact throughout and are crucial for apoptotic cell extrusion. Pre-existing DJs between the apoptotic cell and neighboring non-dying cells remain intact even during the formation of de novo DJs between non-dying cells, suggesting that the neighboring cells possess two DJs in the middle of apoptotic cell extrusion. We further found that an actomyosin cable formed in the vicinity of DJs upon apoptosis, and subsequently deviated from DJs during its constriction. Interestingly, the departure of the actomyosin cable from DJs coincided with the timing when DJs lost their straightness, suggesting a release of junctional tension at DJs, and a mechanical coupling between DJs and actomyosin contractility. The depletion of desmoplakin, which links desmosomes and intermediate filaments, resulted in defective apical contraction and an inability to form de novo DJs, leading to a failure of apoptotic cell extrusion. Our study provides a framework to explain how desmosomes play pivotal roles in maintaining epithelial sheet integrity during apoptotic cell extrusion.\n\nO_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC=\"FIGDIR/small/816280v1_figs1.gif\" ALT=\"Figure 1\">\nView larger version (49K):\norg.highwire.dtl.DTLVardef@454179org.highwire.dtl.DTLVardef@cd7e75org.highwire.dtl.DTLVardef@1295ed1org.highwire.dtl.DTLVardef@1102c8f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure S1:C_FLOATNO Desmosomal junctions at the interface during apoptotic extrusion. A) Confocal images showing actin (Lifeact-Ruby) dynamics during apoptotic cell extrusion in both apical (top panel) and basal (bottom panel) sections of the neighboring cell. Blue-filled and open arrows show the actomyosin cable (top panel) and the lamellipodia (bottom panel) respectively. A) Illustration showing the definition of interface ROI during extrusion. A) Graphs plotting normalized apoptotic cell area (black) and actin (blue) intensity at the dying cell interface during extrusion (n=5 independent experiments). B) Confocal images showing the localization of overexpressed Dsg-FLAG-EGFP (green) and endogenous desmoplakin (red) in MDCK monolayers (n=3 independent staining experiments). B) Magnified images of the white-filled and dotted boxes from B showing co-existence of dotted and continuous junctional clusters in the cell. C) Confocal images showing mCherry-E-cadherin dynamics at the interface during extrusion. C) Graphs plotting normalized apoptotic cell area (black) and E-Cadherin (blue) intensity during constriction (n=5 independent experiments). D) Confocal images showing desmoglein 2 and membrane (mCherry-delta-PH) dynamics in both apical (top panel) and basal (bottom panel) sections of the cell. White arrows (bottom panel) highlights formation of de novo junctions (n=8 independent experiments). E) Confocal images of endogenous desmosomes in the apical (top panel) and basal (bottom panel) sections during different phases of cell extrusion. Yellow and white arrows indicate pre-existing and de novo junctions, respectively (n=182 extrusion events from 7 independent experiments). F) Confocal images showing desmosome (green), killer red (mem-Killer red, cyan) and caspase 3 (magenta) before and after photo-irradiation. White arrow denotes extent of the dying cell. F) Confocal images showing desmosome dynamics (black) in the apical (top panel) and basal (bottom panel) sections of the neighboring cell. Red and blue arrows highlight pre-existing and de novo junctions respectively. F) Transverse view of a neighboring cell showing DJ dynamics after apoptosis induction through Killer Red. Red and blue arrows indicate pre-existing and de novo junctions respectively (n=4 independent experiments). The data from A and C represent mean {+/-}SEM. Scale bars, 10 m except C (5 m).\n\nC_FIG