Serial section electron microscopy of hemolysing erythrocytes (fixed at 12 s after the onset of osmotic hemolysis) revealed long slits and holes in the membrane, extending to around 1 ~m in length. Many but not all of the slits and holes (about 100-1000 A wide) were confluent with one another. Ferritin and colloidal gold (added after fixation) only permeated those cells containing membrane defects. No such large holes or slits were seen in saponintreated erythrocytes, and the membrane was highly invaginated, giving the ghost a scalloped outline. Freeze-etch electron microscopy of saponin-treated membranes revealed 40-50 A-wide pits in the extracellular surface of the membrane. If these pits represent regions from which cholesterol was extracted, then cholesterol is uniformly distributed over the entire erythrocyte membrane.

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A B S T R A C TIt is known that there are 100 A-wide circular structures associated with the erythrocyte membrane in immune lysis. To determine whether these structures were functional holes extending through the membrane, freeze-etch electron microscopy was carried out. Sheep erythrocytcs incubatcd with either rabbit complement or rabbit antibody (anti-sheep erythrocyte antibody) did not hemolyzc and did not reveal any abnormalitics in freezectch or negative-stain clcctron microscopy. Erythrocytes incubated with both complement and antibody revealed rings on the extracellular surface (etch face) of the cell membrane. o Allowing for the 30 A-thick Pt/C replica, the dimensions of the surface rings were similar to those seen by negative staining. The ring's central depression was level with the plane of the membrane; some rings were closed circles, othcrs were crescent shaped. The cleavage face of the cxtraccllular leaflet revealed globule aggregates, each aggregate appearing to be composed of about four fused globules. The cleavage face of the cytoplasmic leaflet was normal. When immune lysis was carried out in the presence of ferritin, fcrritin was subsequently detected in all lysed erythrocytes. If ferritin was added after immune lysis was complete, only 15 % of the cells were pcrmcatcd by fcrritin, indicating that transien[ openings exist in the cell membrane during immune lysis. No abnormal structures were detected when C6-deficicnt rabbit serum was used as a source of complement. It is concluded that antibody and complement produce surface rings, prelytic leakage of K +, colloid osmotic swelling, membrane disruption, and membrane resealing; the surface rings persist after these events.

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