Membrane discontinuities in C. elegans embryonic cells.

A, Schematic drawing of a metaphase cell and the classical membrane invagination model. B, Schematic drawing of a metaphase cell with a ruptured plasma membrane and a cytokinesis model showing that the detached sister membranes mediate membrane extension in C. elegans embryonic cells. C, A membrane opening (MO, indicated by white dashed line) in an embryonic cell at the embryo’s periphery. D, Membrane openings in the embryo’s central regions. Yellow arrows point to single plasma membranes (SPM) traversing the cytoplasm. Plasma membranes are in blue. E, Summary of the single EM section survey of plasma membrane continuity. Orange lines outline the plasma membranes. Solid gray circles/ellipses enclosed by dashed lines represent evenly stained nucleoid regions. Condensed chromosome structures are colored gray-blue. Light brown areas represent cytoplasmic regions.

Metaphase membrane rupture.

A, Membrane discontinuities or openings (MO) (white dashed line) in a metaphase cell (MC). Enlarged views of the boxed regions (1, 2 and 3) are shown. Yellow arrows indicate the plasma membrane (PM). B, MOs (marked by 1 and 2) are detected in anaphase (AC) and other early embryonic cells; enlarged views of the MOs are shown (white dashed lines). All yellow arrows point from the interior of the cell toward the plasma membrane. C and D, Three-dimensional (3D) reconstruction of a metaphase cell. Representative EM images are shown in (C). Purple lines outline the plasma membranes. White dashed lines outline the exposed cytoplasmic region. Blue lines outline the condensed chromosome region. D, The 3D reconstruction of the metaphase cell. The plasma membrane (PM) and condensed chromosome region are displayed.

The detached severed sister membranes.

A, Representative image of a classical back-to-back membrane arrangement. B, Representative image of detached severed sister membranes. Diagrams of the corresponding cytokinesis models are displayed on the right. C, Tips of two detached sister membranes. D, Cytosol-immersed single membranes separating the cytoplasmic regions of two adjacent cells. Enlarged views of regions 1 and 2 are shown below. Plasma membranes (PM) are indicated by yellow arrows (A, B and D) or blue lines. Asterisks indicate membrane tips.

Cellular vesicles on detached sister membranes.

A, A vesicle localized at the tip of a cytosol-immersed membrane. B, Vesicles clustering near the cytosol-immersed region of sister membranes. Contact or fusion between vesicles and membranes is indicated. The rectangular inset shows an enlarged view of a gap junction (GJ) plaque. C, Putative fusion events between vesicles and free-ended membranes. D, Vesicles found near the separated sister membranes. Plasma membranes are in blue.

Membrane rupture on a tri-nucleated cell.

A, The 3D model of a tri-nucleated cell showing the membrane rupture (black). B, Reconstruction of the membrane ruptures and adjacent plasma membranes. Individual membranes, each associated with a distinct nucleus, are color-coded (blue, yellow, and purple). C and D, Membrane openings (MO, dashed white lines) arise either from separation between sister membranes (C) or between the severed membrane edges of the same cell (D). E, In a single EM section, the multiple openings originate from the same rupture. F and G, The opposing severed edges are close to each other in the yellow, purple and blue cells. Plasma membranes (PM) are indicated in blue. Asterisks indicate membrane tips. H, Schematic diagram of the formation of cells with three or four nuclei.

Detection of membrane disruptions by optical microscopy.

A, The putative membrane rupture and extension (*). B, Two extending free-ended sister membranes (*). Insets are enlarged views. C, Possible free-ended edges of detached membranes, probably from multiple cells (*). Below are proposed models illustrating the membrane disruptions that could explain the anomalous GFP signals. Images were taken at 4-second intervals for (A and B) and 8-second intervals for (C). Green signal is GFP::PH(PLC1δ1).

The exposed cytoplasmic regions are covered by extracellular matrix layers.

A-C, The exposed cytoplasmic region is covered by the permeability barrier layer (PBL, red triangles and red dashed lines). PM, plasma membrane (yellow arrows and blue lines). The boxed region in (A) is enlarged in (B), and the boxed region in (B) is enlarged in (C). D, The exposed cytoplasmic region can also be confined by the peri-embryonic layer (PEL).

Membrane discontinuities in externally laid C. elegans embryos.

A, An externally laid embryos with a membrane discontinuity (white box). B, Higher-magnification view of the membrane opening (MO) (white dashed line). Yellow arrow, plasma membrane; asterisk (*), condensed chromosome.

The wide distribution of cells with membrane discontinuities in early C. elegans embryos.

A, Schematic diagram of C. elegans embryogenesis. B-N, Representative low-magnification EM images of early embryos within adult hermaphrodites. Red lines outline cells contain membrane openings. In (F), a tri-nucleated cell is marked with &. (L) and (M) show embryos during the transition from the one-cell to the two-cell stage.

Membrane discontinuities in cells during mitosis.

A-C, Membrane discontinuities or openings (MO, white or black dashed lines) in cells at metaphase (A), anaphase (B) or telophase (C). Yellow arrows points to plasma membranes (PMs).