Blebs in dying and injured cells

What drives blebbing in apoptotic, necrotic and injured cells, how these blebs differ from those of living cells, whether they help the cell, and when blebbing merely marks death.

Blebbing has long been read as a sign of a cell in trouble. The earliest definition of the word bleb that we found describes dying cells: Hogue (1919) applied it to clear, rounded bulges of chick fibroblasts dying in concentrated saline (M03). Today blebbing is best known as a feature of apoptosis, the programmed cell death carried out by proteases called caspases, but it also appears in cells injured by lack of oxygen, toxins or heat. This page asks what drives these blebs, whether they help or merely mark the dying cell, and how they become apoptotic bodies.

From zeiosis to apoptosis

In the 1950s and 1960s, the blebbing of cultured cells was often called zeiosis, ‘boiling over’, a term Costero and Pomerat (1951) coined for stalked blebs on the dendrites of cultured neurons. Zeiosis was usually reversible, although some cells died in it (Rose et al., 1963; Rose, 1966). One early explanation was that a fall in ATP relaxed the cell surface and let it bulge (Lettré’s hypothesis, as summarized by Dornfeld and Owczarzak, 1958). Cells treated with cytochalasin D pointed the other way: they formed knobs of cytoplasm as they contracted (Miranda et al., 1974a), and energy poisons cut this zeiosis from 70% to 2% of HeLa cells (Godman et al., 1975). These results indicate that drug-induced blebbing, at least, is an active, energy-consuming result of cortical contraction (M08).

The term passed into the study of injury (Lemasters et al., 1983), and cell death became a major setting of bleb research. It was the setting of 43% of the bleb papers we counted for 2000–2004, its peak, and since 2015 it has been the second most common after migration.

The contractile engine of apoptotic blebbing

Apoptotic blebbing is driven by the same machinery as the blebs of living cells. Myosin II motors contract the cortex, the network of actin filaments under the membrane; the resulting tension pressurizes the cytoplasm, and the pressure inflates the membrane wherever it is not held back (Tinevez et al., 2009; see Physics of blebs). In dying cells the evidence concerns the contractile step; the pressure has not been measured in the apoptosis studies we hold.

The first clue came from serum-deprived PC12 cells kept alive with caspase inhibitors, which went on blebbing for hours to days. Only inhibitors of myosin light-chain kinase (MLCK), which activates myosin II, reduced the blebbing, and the myosin light chain was 3–5-fold more phosphorylated in blebbing cultures (Mills et al., 1998). Caspases supply the activation by cleaving the Rho-associated kinase ROCK I (Coleman et al., 2001; Sebbagh et al., 2001). The truncated kinase was 7.8 ± 0.5-fold more active (s.e.m., 5 experiments) and made cells bleb even when caspases were blocked (Coleman et al., 2001). Knocking out ROCK I almost abolished apoptotic blebbing in one of three Jurkat clones and reduced it in the other two (Tixeira et al., 2020). Because the 1998 evidence rested on inhibitors with overlapping targets, it is the truncated kinase and the knockouts that make the dependence of apoptotic blebbing on actomyosin contraction strongly supported (M11).

The same activation drives stress blebbing from which cells recover, as in pancreatic acini over-stimulated with the hormone cholecystokinin (Torgerson and McNiven, 1998).

Upstream of this shared core, the signals differ. Blebbing needed Rho in caspase-inhibited PC12 cells (Mills et al., 1998) and the kinase p38 rather than caspases in cisplatin-treated Rat-1 cells (Deschesnes et al., 2001), but caspases and not Rho in other systems (Jänicke et al., 1998; Coleman et al., 2001; Sebbagh et al., 2001). These studies differ in cell type, stimulus and whether caspases were blocked, and Sebbagh et al. (2001) proposed that adherent cells may bleb through Rho as they round up. Even the role of myosin activity varies: in endothelial cells treated with combretastatin A-4-phosphate, MLCK and myosin ATPase inhibitors raised blebbing from 16 ± 4.5% (s.d., 4 experiments) to about 39–44% of cells (Kanthou and Tozer, 2002).

How apoptotic blebs differ from blebs of living cells

The blebs themselves change as apoptosis proceeds. In DLD1 colon cancer cells, the GTPase Rnd3 marked expanding blebs and active RhoA retracting ones in both migrating and apoptotic cells. Apoptotic blebs, however, grew with time, from about 5 µm² early in apoptosis to 45 µm² late, against 14 µm² in migrating cells (10 blebs each, read from plot), and late blebs retracted slowly. Because active ROCK I made the blebs of migrating cells smaller and an active lipid scramblase, which mixes the lipids of the two membrane leaflets, made them larger, the authors propose that ROCK I produces the small early blebs and lipid scrambling the large late ones (Aoki et al., 2020; one cell line).

Fixed images are less informative. Apoptotic blebs were almost free of organelles and of F-actin except at their base (Laster and Mackenzie, 1996), and lacked membrane-bound ERM proteins and the hyaluronan coat of the cell surface, but so did blebs raised by contraction in healthy cells (Le et al., 2024), and expanding blebs of living cells also lack polymerized actin at first (Cunningham, 1995). In our reading, no criterion visible in a single image yet separates apoptotic blebs from the dynamic blebs of living cells (open question).

Blebs of necrotic and injured cells

Among injured cells, blebbing has been followed most closely in hepatocytes deprived of oxygen. In perfused rat liver, anoxic hepatocytes formed organelle-poor blebs within 15 min; on reoxygenation the blebs disappeared and lactate dehydrogenase surged into the effluent with bleb-sized particles, which the authors read as enzyme release by bleb shedding (Lemasters et al., 1983). In cultured hepatocytes under chemical hypoxia, small blebs merged into large terminal blebs without a rise in cytosolic free Ca²⁺, and the rupture of a terminal bleb was proposed, from its timing, to be the point after which injury could not be reversed (Lemasters et al., 1987; Herman et al., 1988).

Injury blebs are probably not one class (proposed, our reading). Hypoxic hepatocytes blebbed even when hypertonic sucrose shrank them, which argues against swelling as the cause, although many were already dead at the time measured (Gores et al., 1989). Depleting ATP blocked the blebbing of over-stimulated pancreatic acini but raised that of EL-4 tumor cells from 5 ± 1% to 44 ± 3% within 1 h (mean ± s.e.m.; Torgerson and McNiven, 1998; Gabai et al., 1992), so some injury blebs need energy and others appear when it runs out. The balloon-like blebs of macrophages dying by pyroptosis or necroptosis, two lytic forms of cell death, persisted with ROCK or myosin II inhibitors, which the authors report blocked apoptotic blebbing in the same study (data not shown; de Vasconcelos et al., 2019).

Blebs that contain damage

Some blebs may help an injured cell survive. When HEK 293 cells were attacked with the pore-forming toxin streptolysin O, 82 ± 6% of the cells that blebbed recovered, against 38 ± 6% of those that did not (s.e.m.; n = 85 and 140), and in example cells the entry of Ca²⁺ and the loss of cytoplasm stayed within single blebs (Babiychuk et al., 2011). Pores of the same toxin gathered in small blebs that were shed, but these formed even on fixed cells, so the authors judged them unlike pressure-driven blebs (Keyel et al., 2011). Thymocytes lacking the lipid scramblase TMEM16F hardly blebbed after a toxin and leaked more (Wu et al., 2020). In our reading, the idea that blebs contain membrane damage is proposed, because it rests on few studies.

Blebbing as a sign of death

Blebbing is often scored as a sign of dying, but the link is loose. In anoxic renal tubule cells, 36 ± 8% of cells blebbed and 25 ± 3% took up trypan blue, yet only 6 ± 1% did both (mean ± s.e.m., 1,026 cells; Mills et al., 1986). In heated Chinese hamster ovary cells in the G1 phase, bleb size rather than presence predicted the outcome: no cell whose largest bleb exceeded 0.6 of the cell-body diameter formed a colony (Borrelli et al., 1986).

Apoptotic blebbing can sometimes be reversed. Rat-1 fibroblasts kept blebbing by caspase inhibitors did not recover when serum was added back (none of more than 500; McCarthy et al., 1997), but HCT116 cells lacking the protein Bax cleaved ROCK I, blebbed and re-attached within 24 h after treatment with the death ligand TRAIL (Somasekharan et al., 2013), and stressed neuronal cells recovered from profound blebbing, though only before cytochrome c had left their mitochondria (You et al., 2023). In our reading, blebbing marks a stage that some cells can still reverse until a later point of no return.

Does apoptosis need blebbing?

If blebs were needed for the rest of apoptosis, removing them should change it, but in several studies it did not. Removing apoptotic blebs with the ROCK inhibitor Y-27632 left caspase cleavage of target proteins, the release of cytochrome c and the exposure of phosphatidylserine, the ‘eat-me’ lipid of apoptotic cells, unchanged (Coleman et al., 2001). In doxorubicin-treated Jurkat cells, phosphatidylserine exposure and uptake by macrophages proceeded normally without blebs (Shiratsuchi et al., 2002). Y-27632 also left phosphatidylserine exposure intact in DLD1 cells, although it reduced the caspase cleavage of lamin A and the formation of apoptotic bodies (Aoki et al., 2020), and mouse fibroblasts whose ROCK I cannot be cleaved by caspases died without blebs but with normal caspase activity and phosphatidylserine exposure (Julian et al., 2021). In our synthesis of these four studies, from three laboratories, blebbing is not needed for caspase activation or phosphatidylserine exposure in cultured cells (strongly supported).

What contraction and blebbing do for the organism is less clear. Mice with caspase-resistant ROCK I had more liver damage and neutrophils after a liver-selective apoptotic stimulus, which blocking the danger signal HMGB1 or its receptor TLR4 reduced, but contraction and blebbing were lost together (Julian et al., 2021). Blebs may also matter for clearance, because apoptotic blebs lose the cortex-tethered hyaluronan coat that otherwise hides phosphatidylserine from macrophages (Le et al., 2024), as described in Blebs as signaling hubs.

From blebs to apoptotic bodies

Dying cells eventually break into apoptotic bodies, and blebs are one way this happens, but not the only one. With the channel pannexin 1 blocked, blebs at the tips of thin protrusions called apoptopodia became bodies of 0.9–5 µm (Poon et al., 2014), whereas beaded apoptopodia shed bodies even when blebbing was blocked (Atkin-Smith et al., 2015). Zebrafish apoptotic cells blebbed without fragmenting in culture, but in the embryo resident phagocytes split them (Santavanond et al., 2025). Which route dominates in tissues is an open question, taken up in Fragments.

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