Adhesion-mediated heterogeneous actin organization governs apoptotic cell extrusion

Background

Epithelial tissues experience substantial turnover through cell division and cell death (Anti et al., 1998; Creamer et al., 1961; Liang et al., 2017). Importantly, exposure to pathogens, UV radiation, as well as mechanical and chemical insults increases apoptosis and damage to the epithelium. Remarkably, despite these toxic stresses, epithelial tissues manage to preserve their integrity and barrier function. They do so by employing a process known as apoptotic cell extrusion (Duszyc et al., 2017; Nanavati et al., 2020). During this process, the dying cell signals to healthy neighbouring cells to assemble a contractile actomyosin ring to expel the apoptotic cell while preventing gap formation and maintaining tissue barrier function (Andrade and Rosenblatt, 2011; Gu et al., 2011; Lubkov and Bar-Sagi, 2014; Rosenblatt et al., 2001).

Notably, recent studies have shown that apart from assembling contractile actomyosin rings, epithelial cells can expel dying cells by generating lamellipodia protrusions (Kocgozlu et al., 2016). Whether these cellular and supracellular structures work together and how they do so remains poorly understood. In this new preprint, Le et al. used a combination of laser-induced cell death, traction force microscopy, micro-patterning, and genetic manipulations to investigate the contributions of cell-cell adhesions, cellular protrusions, and contractility to apoptotic cell extrusion.

 

Key findings

To study the effects of lamellipodia protrusions and actomyosin ring formation during apoptotic cell extrusion, the authors used mosaic cultures of Wild-Type (WT) and GFP-actin or Ruby-Lifeact-expressing MDCK cells. In both naturally-occurring and laser-induced cell death, neighbouring cells assembled 1) thin membrane ruffles containing actin indicative of lamellipodia protrusions at the basal region and 2) actomyosin cables at the apico-lateral domain. Both lamellipodia protrusions and actomyosin cable formation are required to drive extrusion. This is indicated by the decrease in successful cell extrusion events when either lamellipodia protrusions are inhibited using the Rac1 inhibitor (NSC-23766) or when actomyosin cable formation is perturbed when cells are treated with either blebbistatin (Myosin II inhibitor) or SMIFH2 (formin inhibitor). Although basal lamellipodia protrusions precede the formation of actomyosin cables, these processes are mutually co-regulated. Disrupting lamellipodia protrusions enhanced actomyosin cable assembly and perturbing actomyosin cable assembly increased lamellipodia protrusions.

Figure 1. Extrusion of a wild-type cell (asterisk) undergoing apoptosis surrounded by a majority of healthy neighbouring cells expressing GFP-actin. Adapted from Figure 1A from Le et al. 2020.

 

Interestingly, the authors noticed that even though neighbouring cells were assembling actomyosin cables during extrusion, these cables contained irregular patterns of myosin IIA localization and were slanted along the z-axis. Uniform myosin distribution was only observed at the final stages of extrusion at the peak of caspase-3 (a protease activated during apoptosis) expression. This is in stark contrast to the conventional notion that uniform, supracellular actomyosin cables were engaged in cell extrusion.

Cell-cell adhesions play an integral role in facilitating apoptotic cell extrusion (Lubkov and Bar-Sagi, 2014; Michael et al., 2016; Teo et al., 2020). The authors noted that modulating cell-cell adhesion strength alone was sufficient to persuade neighbouring cells to adopt either a dominant lamellipodia-driven or actomyosin cable assembly approach. In α-catenin knocked down cells, which were unable to form adherens junctions and displayed reduced junctional tension, extrusion relied predominantly on lamellipodia protrusions. Expression of αcat-WT or αcat-ΔMod mutant (constitutively recruits vinculin) in α-catenin knocked down cells led to pronounced actomyosin cable assembly around the dying cell. In contrast, expression of αcat-L344P (unable to recruit vinculin) formed prominent lamellipodia protrusions around the dying cell instead.

Apart from cell-cell adhesions, substrate geometry is also involved in promoting cell extrusion (Saw et al., 2017). In this vein, the authors developed micropatterns containing both a positive curvature (∩) and a negative curvature (∪) on non-adhesive patches. Indeed, they noticed the preferential assembly of actomyosin cables around negative curvatures before positive curvatures. In addition, lamellipodia protrusions formed predominantly around positive curvatures before converting to actomyosin cables when the protruding front encounters the edge of the patch.

 

What I like about this preprint

The authors have nicely adopted interdisciplinary approaches to address a fundamental biological question: How do epithelia remove dying cells while maintaining tissue barrier function? This study uncovers the cooperation between different cellular (lamellipodia protrusions) and heterogenous supracellular (actomyosin cables) structures in facilitating cell extrusion. In addition, despite the challenges involved in dissecting how mutually co-regulated pathways are modulated and I think the authors have done a great job here using appropriate molecular handles and techniques to approach the question.

 

Questions for the authors

1. The observation that actomyosin cables in neighbouring cells are heterogeneous and tilted is fascinating! Since inhibiting Rac1 disrupts the heterogeneity, are there any speculations on how Rac1-driven lamellipodia protrusions might be involved?
2. Considering that early basal sealing driven by persistent lamellipodia protrusion is important for apoptotic cell extrusion, could this be a phenomenon that is adopted by other types of cell extrusion as well? For instance, live-cell extrusion. If Rac1 is opto-genetically activated in surrounding wild-type cells to induce persistent lamellipodia protrusions, would that result in the extrusion of its neighbouring cell independent of caspase activation?
3. Using non-adhesive patches to identify forces generated by actomyosin cables is very interesting. Would you expect to see similar forces generated by dominant-negative Rac1-expressing cells mosaically cultured with wild-type cells that are dying?

 

References:

Andrade, D., and Rosenblatt, J. (2011). Apoptotic regulation of epithelial cellular extrusion. Apoptosis 16, 491-501.

Anti, M., Armuzzi, A., and Gasbarrini, G. (1998). Epithelial cell turnover and apoptosis. Ital J Gastroenterol Hepatol 30 Suppl 3, S276-278.

Creamer, B., Shorter, R.G., and Bamforth, J. (1961). The turnover and shedding of epithelial cells. I. The turnover in the gastro-intestinal tract. Gut 2, 110-118.

Duszyc, K., Gomez, G.A., Schroder, K., Sweet, M.J., and Yap, A.S. (2017). In life there is death: How epithelial tissue barriers are preserved despite the challenge of apoptosis. Tissue Barriers 5, e1345353.

Gu, Y., Forostyan, T., Sabbadini, R., and Rosenblatt, J. (2011). Epithelial cell extrusion requires the sphingosine-1-phosphate receptor 2 pathway. J Cell Biol 193, 667-676.

Kocgozlu, L., Saw, T.B., Le, A.P., Yow, I., Shagirov, M., Wong, E., Mege, R.M., Lim, C.T., Toyama, Y., and Ladoux, B. (2016). Epithelial Cell Packing Induces Distinct Modes of Cell Extrusions. Curr Biol 26, 2942-2950.

Liang, J., Balachandra, S., Ngo, S., and O’Brien, L.E. (2017). Feedback regulation of steady-state epithelial turnover and organ size. Nature 548, 588-591.

Lubkov, V., and Bar-Sagi, D. (2014). E-cadherin-mediated cell coupling is required for apoptotic cell extrusion. Curr Biol 24, 868-874.

Michael, M., Meiring, J.C.M., Acharya, B.R., Matthews, D.R., Verma, S., Han, S.P., Hill, M.M., Parton, R.G., Gomez, G.A., and Yap, A.S. (2016). Coronin 1B Reorganizes the Architecture of F-Actin Networks for Contractility at Steady-State and Apoptotic Adherens Junctions. Dev Cell 37, 58-71.

Nanavati, B.N., Yap, A.S., and Teo, J.L. (2020). Symmetry Breaking and Epithelial Cell Extrusion. Cells 9.

Rosenblatt, J., Raff, M.C., and Cramer, L.P. (2001). An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism. Curr Biol 11, 1847-1857.

Saw, T.B., Doostmohammadi, A., Nier, V., Kocgozlu, L., Thampi, S., Toyama, Y., Marcq, P., Lim, C.T., Yeomans, J.M., and Ladoux, B. (2017). Topological defects in epithelia govern cell death and extrusion. Nature 544, 212-216.

Teo, J.L., Tomatis, V.M., Coburn, L., Lagendijk, A.K., Schouwenaar, I.M., Budnar, S., Hall, T.E., Verma, S., McLachlan, R.W., Hogan, B.M., et al. (2020). Src kinases relax adherens junctions between the neighbors of apoptotic cells to permit apical extrusion. Mol Biol Cell, mbcE20010084.

 

 

Posted on: 23 September 2020

doi: https://doi.org/10.1242/prelights.24816

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