Extracellular vesicle formation mediated by local phosphatidylserine exposure promotes efficient cell extrusion

: Numerous unwanted cells are removed from epithelial and endothelial tissues—in which cells are tightly connected to one another—without disturbing tissue integrity and homeostasis. Cell extrusion is a unique mode of cell removal from tissues, and it plays an important role in regulating cell numbers and the eliminating unwanted cells, such as apoptotic cells, cancer cells, and cells with a lower fitness in cell competition 1-3 . During this process, cells delaminate from the cell layer, to which they initially used to adhere, through communication with the neighboring cells 4,5 . Defects in cell extrusion are believed to associate with inflammation and cancer in epithelium as well as blood vessel dysfunction 6,7 . However, the correlation between them has not yet been evaluated owing to a lack of knowledge of the underlying mechanisms. In particular, the process whereby the cell exit from the tissue remains to be elucidated. Here, we report a novel and conserved execution mechanism of cell extrusion—common to mammalian cells and Drosophila epithelia—i.e., spatiotemporally regulated extracellular vesicle formation in extruding cells at a site opposite to the direction of extrusion. Particularly, we found that a lipid-scramblase‒ mediated local exposure of phosphatidylserine is responsible for extracellular vesicle formation and is crucial for the execution of cell extrusion, and inhibition of this process disrupted prompt cell delamination as well as tissue homeostasis. Furthermore, we revealed the mechanism underlying vesicle formation. Importantly, our results reveal that membrane dynamics is the driving force by a “rocket launch”-like mechanism behind the extrusion of cells from tissues, a fundamental cell behavior in multicellular organisms that is also observed in other contexts including cancer cell invasion and neural cell differentiation. Our understanding of this new mechanism of cell extrusion enables us to

knowledge of the underlying mechanisms. In particular, the process whereby the cell exit from the tissue remains to be elucidated. Here, we report a novel and conserved execution mechanism of cell extrusion-common to mammalian cells and Drosophila epithelia-i.e., spatiotemporally regulated extracellular vesicle formation in extruding cells at a site opposite to the direction of extrusion. Particularly, we found that a lipid-scramblase- 15 mediated local exposure of phosphatidylserine is responsible for extracellular vesicle formation and is crucial for the execution of cell extrusion, and inhibition of this process disrupted prompt cell delamination as well as tissue homeostasis. Furthermore, we revealed the mechanism underlying vesicle formation. Importantly, our results reveal that membrane dynamics is the driving force by a "rocket launch"-like mechanism behind the 20  .

Main Text:
Although the contraction of the actomyosin complex produces the driving mechanical 5 force for execution of cell extrusion 8,9 , how the cell delaminates from the tissue remains a fundamental yet unanswered question. We hypothesized that membrane dynamics play a key role in the execution of cell extrusion. To investigate this, first, we visualized the cell membrane with membrane-bound GFP (membrane GFP) and analyzed the detailed membrane dynamics in both extruding cells and their neighboring cells. In Mardin-Darby canine kidney II (MDCKII) 10 cell monolayers, cell extrusion occurs spontaneously after confluence to adjust cell density 4 .We found extensive budding of the membrane of extruding cells and the presence of many vesicles inside the neighboring cells via live-imaging analysis of membrane GFP-expressing MDCKII cells (92/92 extrusion events) ( Fig. 1A and Movie S1). Immunostaining results revealed that cleaved caspase3 is contained in the vesicles of the neighboring cells (110/113 extrusion events) 15 in addition to the cytoplasm of extruding cells (Fig. 1B). This suggests that these vesicles are derived from extruding apoptotic cells and engulfed by their neighbors.
To verify this, we performed mosaic analysis via the co-culture of RFP-expressing and membrane GFP-expressing cells. The deformation process in extruding cells began with a slight invagination of the apical surface, and after 15 min, the cells showed budding-like membrane 20 dynamics ( Fig. 1C and Fig. S1). Then, the budding was rapidly fragmented, and the shed fragments were engulfed by the neighboring cells, as shown by the GFP-positive vesicles containing RFP in GFP-expressing cells adjacent to RFP-expressing extruding cells (10/10 Submitted Manuscript: Confidential 4 extrusion events) ( Fig. 1D and Movie S2). Fragmentation and engulfment occurred transiently rather than continuously, and the remaining portion of the extruding cell after fragmentation delaminated from the cell layer to the apical direction ( Fig. 1C and Fig. S1). Interestingly, fragmentation and engulfment occurred preferentially at the basolateral or basal parts of the extruding cells (10/10 extrusion events) ( Fig. 1E and Movie S3). In addition, the protrusion of 5 the extruding cell from the monolayer began simultaneously as fragmentation and engulfment ( Fig. 1C and F). These results suggest that fragmentation and/or engulfment are involved in the execution of cell extrusion.
Apoptotic cells expose phosphatidylserine (PS) as an "eat-me" signal and are engulfed by phagocytes 10 . Staining results with Annexin V, a PS-binding protein, showed that extruding 10 cells exposed PS, and signals were enriched at the basal and basolateral parts with a patched pattern (Fig. 1G), strongly suggesting that the cell fragments that exposed PS were then recognized and engulfed by the neighbors. Lysotracker staining results showed that the phagocytic vesicles fused with the lysosomes 30-60 min after the engulfment (Fig. S2).
To evaluate the function of engulfment during cell extrusion, we intended to disturb the 15 engulfment process using the MFG-E8 mutant protein. Secreted MFG-E8 protein binds to both PS exposed on apoptotic cells and integrins on phagocytes to promote phagocytosis, whereas the MFG-E8-D89E mutant protein (D89E), which carries a mutation in the integrin-binding motif, inhibits PS-mediated phagocytosis in a dominant-negative manner 11 . Although we expected that D89E-overexpression in MDCKII cells prevents engulfment after fragmentation, we observed 20 that it impaired the fragmentation process. Half of the cell extrusion did not show fragmentation, even after 25 min following the deformation of the cell shape ( Fig. 2A, Fig. S3, and Movie S4).
These extruding cells with delayed fragmentation despite the continuous membrane deformation  (Table S1), did not affect fragmentation nor extrusion (Fig. S5).
Fragmentation was also observed in apoptotic cell extrusion induced by UV irradiation in cultured cells (Fig S6). To further show that fragmentation occurs in vivo, time-lapse imaging Furthermore, when Annexin V:GFP was expressed in LECs, the adult dorsal abdomen showed loss of continuity of the epidermis and bristles at the midline (Fig. 3F), a severe defect in cell 20 death or extrusion as previously reported 16 . The knockdown of the Drosophila Xk gene (Xk or CG32579), a single homolog of the mammalian Xkr family, showed abnormality in the fragmentation of extruding cells (Fig. 3G, Fig. S7). The progression of cell extrusion after the Submitted Manuscript: Confidential 7 closure of the apical surface of the extruding cells was impaired in the Xk-knockdown epidermis as evidenced by the persistence of extruding cells in the cell layer for an extended time (Fig. 3H).
The replacement of larval cells by histoblasts was delayed in the Xk-knockdown pupal epidermis (Fig. 3I).
In another physiological cell extrusion, during cell turnover in the adult intestinal 5 epithelium, fragmentation and engulfment occurred in the Drosophila midgut as indicated by the formation of some vesicles in the neighboring cells (Fig. S8), as well as in the mouse intestinal organoid as shown by time-lapse imaging results ( Fig. S9 and Movie S10). When Xk was knocked down in an enterocyte-specific manner in the Drosophila midgut, an increased tissue width and shorter lifespan were observed (Fig. S10). These results indicate that PS exposure- 10 induced fragmentation of the extruding cells is a universal process that plays a significant role in the execution of cell extrusion to ensure tissue development and homeostasis.
Lastly, we speculated that fragmentation involves the local formation and release of extracellular vesicles (EVs), which are observed in blood coagulation, intercellular signal transduction, and various pathological processes [17][18][19] . The size of fragments formed during cell 15 extrusion ranges between 0.5 and > 2 µm in diameter, and a small part of them were positive for DNA staining (a positive example: Fig. S11 and a negative example: Fig. 1B). In EVs, including exosomes, microvesicles, and apoptotic bodies, PS exposure and actomyosin accumulation at the budding sites, mediated by the function of the Arf family, Phospholipase D (PLD), and extracellular regulated kinase (ERK), have been reported in microvesicle formation 20,21 . In 20 addition to the requirement of PS exposure for the fragmentation (Fig. 2 and 3), actin or myosin accumulated around the root of the budding sites of the fragments in MDCKII cells and Drosophila LECs (Fig. 4A and B). When Arf6, Arf1, or Pld1 was knocked down in EpH4 cells, impaired fragmentation and delayed extrusion with defective protrusion were observed (Fig. 4C-E, Fig S12). Extruding EpH4 cells with Arf6 or Pld1-knockdown showed local PS exposure (control; 77%, Arf6knockdown; 55%, and Pld1-knockdown; 58% of extruding cells), consistent with that the PS exposure precedes EV formation. Arf51F (homologous to mammalian Arf6) accumulated at the 5 apical part of extruding cells in Drosophila pupa during fragmentation (6/6 extrusion events) (Fig.4F). Arf51F-knockdown in the pupal epidermis showed impaired fragmentation and delayed progression of cell extrusion ( Fig. 4G and H). Furthermore, the knockdown of Arf79F (homologous to mammalian Arf1), which is more abundant than Arf51F, in the Drosophila midgut (Table S1) in an enterocyte-specific manner resulted in enlarged midguts and shorter 10 lifespans (Fig. S13). These data indicate that the fragmentation of the extruding cells is spatiotemporally restricted EV formation governed by a similar mechanism as microvesicle formation and promotes cell protrusion, thus the execution of cell extrusion.
Taken together, our findings reveal that local PS exposure and subsequent EV formation mediated by PLD and the ARF family in extruding cells is a conserved mechanism that promotes 15 efficient cell extrusion. Prolonging this process caused defects in epithelial tissue, suggesting that prompt execution of extrusion is critical for tissue homeostasis including such as its barrier function. EV formation occurs at the site opposite the direction of extrusion in the apico-basal axis and simultaneously with cell protrusion. Thus, membrane dynamics in extruding cells produce a kind of driving force of extrusion, in addition to the actomyosin complex formed by 20 the neighboring cells and/or extruding cells 8,9 , and might control the directionality of extrusion.
The polarized formation and/or release of the vesicles may produce force for cell protrusion by touching the neighboring cell membrane or the extracellular matrix similar to the propulsion of