Fig 1.
Lamellipodia and blebs in Dictyostelium cells.
(A and B) Phase contrast and fluorescence images of a typical cell expressing GFP-ABD, an actin filament probe. The cell extended lamellipodia (arrow) followed by blebs (large arrowheads). (C) Kymographs generated from the rectangles in panel A. Note that actin assembly advanced as the lamellipodium extended. Here, the white lines indicate the leading edge of the lamellipodium. (D) Kymographs generated from the rectangles in panel B. A bleb showing a sudden extension (asterisks). Note that actin was not detected along the edge of the initial extension of the bleb and that the actin cortex remained at the basal region of the bleb (small arrowheads in panel B). Approximately 2 seconds later, actin gradually appeared along the edge (arrow). (E) The velocity of lamellipodia and bleb extension (n = 21). (F) The frequency of the appearance of lamellipodia and blebs. Note that lamellipodia were frequently observed (14.4 ± 7.9 times / 5 min), whereas blebs were rarely observed (0.7 ± 1.8 times / 5 min). Bars, 2.5 μm.
Fig 2.
(A) A typical microsurgery experiment. A cell expressing GFP-ABD was cut using a microneedle under confocal microscopy. The nucleate fragment continued to migrate, extending a lamellipodium even after cutting. Here, the asterisks indicate the nucleus. The anucleate fragment did not migrate and instead repeatedly extended blebs (arrows). Bar, 10 μm. (B) Time course of bleb-like extension in a typical anucleate fragment. Bar, 2.5 μm. (C) Kymographs generated from the rectangles in panel B. Note that the bleb-like structure extended very quickly and that actin did not localize to its leading edge immediately after extension, suggesting that these extensions from the anucleate fragments are ‘blebs.’ Bar, 2.5 μm. (D) The cytoplasm was disconnected in the middle of the cell by placing a microneedle into the cell under DIC microscopy. After disconnecting the cytoplasm, the half without a nucleus (arrowhead) vigorously extended blebs (arrows), and the other half with a nucleus did not extend them. Both sides of the cytoplasm rejoined when the microneedle was removed, showing that the cytoplasm was not actually cut. After the microneedle was removed, the blebbing ceased. These disconnection experiments could be repeated in the same cells and reversibly induced blebbing. Bar, 10 μm.
Fig 3.
(A) The frequency of blebbing in various knockout mutants. Knockout mutants in signal proteins, membrane trafficking proteins, and cytoskeleton-related proteins were subjected to the microsurgery-based blebbing assay. Detailed information regarding these mutants is provided in S1 Table. The blebs were counted for 5 min after the mutant cells were cut by microsurgery. (B) Mapping of the assayed genes onto the chemotactic signal network. The binding of cAMP to the cAMP receptor on the membrane activates multiple intracellular signaling cascades, transmits signals to cytoskeletal proteins, and finally drives cell migration toward the chemoattractant. Red, mutants that showed increased blebbing; blue, mutants that showed reduced blebbing; green, no detectable difference compared to wild type cells (as determined by the t-test statics).
Fig 4.
Cortical tension powered by myosin II is required for blebbing.
(A) A cell expressing GFP-myosin II was cut, and the dynamics of myosin II in the anucleate fragments were observed under confocal microscopy. Myosin II was not observed along the leading edges of the blebs immediately after extension (arrows), and cortical myosin II remained in the basal region. (B) Kymographs of the dynamics of phase contrast and fluorescence images in the rectangles in panel A. (C) The frequency of blebbing in the anucleate fragments of wild-type (AX2), myosin II-null (HS1), and blebbistatin-treated wild-type cells. (D) A wild-type cell expressing GFP-ABD was observed under pressure applied via an agar block overlay. The cell frequently extended blebs under pressure (arrow). (E) Kymographs of the dynamics of phase contrast and fluorescence images in the rectangles shown in D. (F) Frequency (times per 5-min period) of blebbing with and without an agar overlay (n = 20). (G) After a myosin II-null cell was cut (14.1 sec), the anucleate fragment was pressed with an agar block (293.9–317.7 sec). DIC microscopy shows that the blebs extended under the pressure of the agar (arrows). Bars, 5 μm.
Fig 5.
Disassembly of microtubules induced blebbing.
(A) When cells expressing GFP-tubulin were cut, the microtubules disassembled within 6.6 ± 4.9 sec (n = 19) in the anucleate fragments, whereas the nucleate fragments retained their microtubule networks (arrowheads). (B) When uncut cells were treated with thiabendazole, most of the microtubules disappeared except at MTOC (arrowheads). These cells frequently extended blebs (arrows). (C) The frequency of blebbing in untreated cells and cells treated with thiabendazole. Bar, 10 μm.
Fig 6.
PIP2 is a key regulator of blebbing.
(A) Comparison of the blebbing frequencies of anucleate fragments of wild-type, PI3K-null, and PTEN-null cells after cutting. Note that the blebbing frequency was significantly increased in PI3K-null cells and decreased in PTEN-null cells. When wild-type cells were cut in the presence of LY294002, the number of blebs increased (*, significant difference compared with AX2, n = 22, p < 0.01). (B) Cells expressing the GFP-PH domain, a marker of PIP3, were cut under confocal microscopy. Time course of phase contrast and fluorescence images before and after cutting. PIP3 was localized along the edges of the lamellipodia (arrow). When the cell was cut into two fragments, the nucleate fragments continued to migrate, and PIP3 was localized along the leading edge (arrow). However, in the anucleate fragment, PIP3 delocalized and became evenly distributed in the cytoplasm. The anucleate fragment frequently extended blebs, but PIP3 did not localize to the blebs (arrowheads). The fluorescence in the blebs appears slightly higher than that in the cytoplasm, which was caused by the exclusion volume [58]. (C) Enlarged images of the fragment shown in panel B. (D) The time course of fluorescence intensity in the fragment (rectangle in panel C), demonstrating that the fluorescence signal increases in the cytoplasm after cutting (arrow). (E) When microtubules were disassembled in cells expressing the GFP-PH domain in the presence of thiabendazole (TB), the cells frequently extended blebs (arrowheads). Note that GFP-PH diffused in the cytoplasm and did not localize to the bleb cortex. (F) Uncut cells expressing GFP-PTEN (G129E), a marker of PIP2, were observed in the presence of thiabendazole (TB). (G) Kymograph of the rectangle in panel F. Note that PIP2 did not localize along the leading edge of the bleb (arrowheads) immediately after extension but rather gradually accumulated there, followed by the retraction of the bleb. Bars, 10 μm.
Fig 7.
Blebs can be induced in leukocytes by microsurgery.
(A) Human leukocytes were individually cut into two fragments under DIC microscopy. The arrow shows the nucleus. After cutting, the nucleate fragment continued normal migration by extending lamellipodia. However, the anucleate fragment frequently extended blebs (arrowheads). (B) Sequential images of lamellipodium extension (arrowhead) and a kymograph generated from the rectangle. (C) Sequential images of bleb extension (arrowhead) and a kymograph generated from the rectangle. (D) Velocities of lamellipodia and blebs. Note that blebs extend much faster than lamellipodia. (E) Bleb frequencies in untreated and nocodazole-treated uncut cells. Note that leukocytes extend more blebs in the presence of nocodazole. Bars, 10 μm.
Fig 8.
Current model for blebbing signaling.
(A) The results of our present study indicate that microtubules can be positioned upstream of bleb extension. In the presence of microtubules, PIP3 localizes to the anterior membrane and induces lamellipodia formation by enhancing the assembly of actin at that location. Without microtubules, PIP3 delocalizes from the cell membrane. PIP2 recruits myosin II along the cortex, generating more internal pressure inside of the cell. A bleb will extend at the weakest part of the cortex (where the levels of PIP2 and myosin II proteins are low) because myosin II contributes to the rigidity of the cortex. (B) A model of lamellipodium and bleb extension in uncut cells. When microtubules extend to the cortex, unknown signals are conveyed along the microtubules that locally induce PIP3 accumulation and actin assembly in the cortex, resulting in the extension of lamellipodium (left). However, when microtubules retract or detach from the cortex, PIP3 delocalizes from the cortex. In the presence of intracellular pressure, based on the power of myosin II, the cell membrane detaches from the weakest part of the actin cortex, resulting in bleb formation. Blue double arrows, myosin II; red, actin filaments; green, PIP3.