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. 2009 Sep 7;186(5):693-702.
doi: 10.1083/jcb.200903079. Epub 2009 Aug 31.

P115 RhoGEF and microtubules decide the direction apoptotic cells extrude from an epithelium

Gloria Slattum  1 , Karen M McGee, Jody Rosenblatt
Affiliations

P115 RhoGEF and microtubules decide the direction apoptotic cells extrude from an epithelium

Gloria Slattum et al. J Cell Biol. .

Abstract

To preserve epithelial barrier function, dying cells are squeezed out of an epithelium by "apoptotic cell extrusion." Specifically, a cell destined for apoptosis signals its live neighboring epithelial cells to form and contract a ring of actin and myosin II that squeezes the dying cell out of the epithelial sheet. Although most apoptotic cells extrude apically, we find that some exit basally. Localization of actin and myosin IIA contraction dictates the extrusion direction: basal extrusion requires circumferential contraction of neighboring cells at their apices, whereas apical extrusion also requires downward contraction along the basolateral surfaces. To activate actin/myosin basolaterally, microtubules in neighboring cells reorient and target p115 RhoGEF to this site. Preventing microtubule reorientation restricts contraction to the apex, driving extrusion basally. Extrusion polarity has important implications for tumors where apoptosis is blocked but extrusion is not, as basal extrusion could enable these cells to initiate metastasis.

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Figures

Figure 1.
Figure 1.
Hallmarks of apical and basal apoptotic cell extrusion. Stills from phase videos of apoptotic cells extruding apically (a and c) and basally (b and d) in an MDCK cell culture epithelium (a and b) and from a zebrafish larval epidermis (c and d; teal arrows denote extruding cells). Time is given in hours and minutes. (e and f) An apoptotic cell extruding apically (e) and basally (f), immunostained for active caspase-3 (green), actin (red), and DNA (blue). Note that caspase and DNA are in the top z plane and a thick actin ring is in the bottom plane when a cell is extruded apically (e), whereas the actin ring is in the top plane and caspase and DNA are in bottom plane when extruding basally (f). Bars, 10 µm.
Figure 2.
Figure 2.
Localization of active myosin IIA but not intrinsic polarity markers are altered when cells are extruded apically or basally. (a and b) Cross sections of tight junction protein Z0-1 (a), which remains apical, and β-catenin (b), which remains basal. (c) Myosin IIA and phospho-myosin II are basolateral during apical extrusion and apical during basal extrusion. Cross sections are taken from the broken line in pictures of 3D confocal reconstructions of MDCK cells. Arrows point to dying, extruded cells in each case. Bars, 10 µm.
Figure 3.
Figure 3.
The growing ends of microtubules point toward the actin/myosin ring during extrusion. (a and b) EB1 (green), a plus-end microtubule-binding protein, colocalizes with the myosin II (red) ring during apical (a) and basal (b) extrusion. Arrows point to sites where EB1 abuts the myosin II ring. Note that most EB1 is from 0–4 µm from the base during apical extrusion, whereas during basal extrusion, only limited amounts of EB1 are seen at the apex (6 µm from the base). Bars, 10 µm. (c) Quantification of EB1 localization (>170 spots for each) from 12 extrusions each of apically extruding cells, basally extruding cells, and live junctions. Error bars indicate SEM and P < 0.0001, comparing spots from apical extrusion to basal extrusion.
Figure 4.
Figure 4.
Disruption of microtubules alters the direction a cell extrudes. (a) In control (DMSO-treated) MDCK monolayers, microtubules dip down toward the basolateral surface of the actin ring, whereas nocodazole either blocks contraction of the ring (as shown) or drives extrusion basally. Stabilizing microtubules with taxol freezes microtubules at the apex compared with control and drives extrusion basally. Broken lines indicate the site of cross sections from 3D confocal reconstructions above. Bars, 10 µm. (b) Quantification of drug-treatments from six experiments, n = 1,350 extruding cells per treatment. P-values for apical and basal extrusions, respectively, were 0.0011 and 0.0066 for nocodazole and 0.0023 and 0.0023 for taxol. Error bars indicate SEM.
Figure 5.
Figure 5.
p115 RhoGEF is required for apical extrusion. (a) p115 RhoGEF (red; separate middle panel) form filaments that point toward the basolateral surface of the extruding ring during apical extrusion, whereas RhoA (green; bottom panel) is present through the apical and basal regions of the ring. Corresponding colored arrows indicate sites of interaction with the actin/myosin ring. Bar, 10 µm. (b) siRNA-mediated knockdown of p115 RhoGEF (above, lanes from one immunoblot) disrupts apical extrusion, whereas scrambled siRNA does not. The graph shows the percentage of apical versus basal extrusion from >1,000 cells from four siRNAs experiments, where P < 0.0001. (c) p115 RhoGEF localizes to the base of the extruding ring during apical extrusion, whereas it does not when microtubules are disrupted with nocodazole or taxol, or in control live junctions, where P-values are <0.0001 for control versus each treatment; n = 10 extrusions per treatment. (d) Model for how microtubules might control extrusion direction. An early apoptotic cell sends signals to its live neighboring epithelial cells, which react by reorienting microtubules toward the basolateral surfaces of the cells that contact the dying cell. p115 RhoGEF, by association with microtubules, assembles and activates Rho-mediated actin and myosin II contraction along the basolateral surface, extruding the cell apically (top). Basal extrusion occurs if the microtubules do not reorient or if p115 RhoGEF is absent (bottom).
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