Harunori Ishikawa

Formation of arrowhead complexes with heavy meromyosin in a variety of cell types.

The parasitic protozoan Toxoplasma gondii has been examined with the electron microscope in order to study the fine structure and the formation of the membranes surrounding the cell. The study of the ultrastructure of the membranes covering the parasite shows the existence of a three-membraned complex. Only the outer membrane is considered to be the plasma membrane; the two membranes below it form an inseparable whole of changeable molecular architecture (modifications in appearance depending on the methods of fixation, local differentiation). During reproduction, which takes place by fission or more often by endogeny, the membranes of the daughter individuals are formed from the membranes of the parent. At first the middle and inner membranes of the parent extend, separating the cytoplasm of the daughter cells from that of the parent. The three-membrane complex of the endozoites is completed at the time of their liberation; the external membrane of the parent covers the leaving endozoites; thus, the plasma membrane of the daughter cells derives also from that of the parent. These findings on the origin and role of limiting membranes during reproduction differ entirely from those described so far for other cells.

Association of the actin cytoskeleton with glass-adherent proteins in mouse peritoneal macrophages

Summary— When mouse peritoneal macrophages adherent to glass surface were removed by treatment with triethanolamine and Nonidet P‐40, fine thread structures of unique loops were left behind on glass at the sites of cell adhesion. To examine the ultrastructural relationship between such looped threads and cytoskeletal components in glass‐adherent macrophages, we successfully used the ‘zinc method’ to remove most of the cytoplasm including nuclei and to expose the cytoskeleton associated with the ventral plasma membrane. The cytoskeleton was seen to be mainly composed of actin filaments forming dense networks. The network contained scattered star‐like foci from which actin filaments radiated. When the ventral plasma membrane‐cytoskeleton complex was further treated with Nonidet P‐40, the membrane was dissolved to expose the glass surface with actin foci persisting on glass. When the complex was removed by further treatment with Nonidet P‐40 and DNase I, the looped threads became visible. Confocal laser microscopy of glass‐adherent macrophages stained with fluorescent phalloidin showed the preferential distribution of F‐actin in the ventral cytoplasm along the plasma membrane, where intense fluorescent spots were also scattered. Confocal interference reflection microscopy revealed densely populated dark dots and striae of focal contact, which corresponded in overall distribution to actin foci and looped threads. These observations suggest that actin cytoskeleton is closely associated with looped threads to reinforce cell adhesion to glass.

Identification of novel adhesion proteins in mouse peritoneal macrophages

Summary— When mouse peritoneal macrophages were made to adhere firmly on glass surface and then removed by sequential treatment with hypotonic triethanolamine and Nonide P‐40, a set of proteins were found to be left behind at the sites of adherent cells. Such glass‐adherent proteins were detected as round or ellipsoidal patches of autofluorescence under a confocal laser microscope, and visualized ultrastructurally as aggregates to narrow threads of unique loop structures which were composed of linearly aligned particles of 22 ± 2 nm in diameter. Lithium dodecylsulfate‐polyacrylamide gel electrophoresis of the glass‐adherent proteins showed two major bands, 12 kDa and 14 kDa, which always co‐existed in any different sample. The polyclonal antibody raised against these two proteins specifically stained the glass‐adherent proteins in situ. The adhesion of macrophages to glass was significantly blocked with Fab fragments of the antibody. The in situ cross‐linking experiment suggested that these two proteins might be closely associated with each other to form complexes. Hence, these proteins can be reasonably considered to be responsible for non‐specific adhesion of macrophages to glass.

The association of microtubules with the plasmalemma in epidermal tendon cells of the river crab

The mode of association of microtubules (MTs) with the plasmalemma in epidermal tendon cells of the river crab, Potamon dehaani was studied by thin‐section electron microscopy. In the leg muscle, the tendon cells connect striated muscle cells with the cuticle, forming specialized junctions at both ends. At the muscle‐tendon cell junction, the apposed plasmalemmas are interdigitated in a zig‐zag pattern separated by a uniform space of about 50 nm, where the basal lamina is shared by two cells. At the tendon cell‐cuticle junction, the plasmalemma of the tendon cell forms many conical invaginations, into which dense fibrous material extends from the cuticle. Inside the tendon cell, numerous microtubules run parallel to the direction of tension transmission and are arranged into parallel bundles of various sizes. Within such bundles, fine filamentous structures cross‐link adjacent MTs. MTs span the entire length of the cell and attach at their both ends to the junctional domains of the plasmalemma. The junctional plasmalemma is characterized by formation of an electron‐dense undercoat, through which MTs are connected with the plasmalemma proper. The ultrastructural features of MT association with the plasmalemma are basically the same at both junctions. At the junctions, MTs usually terminate with free ends and are linked laterally to the plasmalemmal undercoat with fine filamentous structures. These observations emphasize the role of the plasmalemmal undercoat as a device of the attachment of MTs to the plasmalemma.

Some ultrastructural observations on the mode of association of microtubules with the plasmalemma in epidermal tendon cells of the river crab

Summary— The ultrastructural aspects of the association of microtubules (MTs) with the plasmalemma in epidermal tendon cells of the river crab, Polamon dehaani, were studied by thin‐section electron microscopy combined with detergent treatment. In the tendon cell, MTs were linked laterally by anchoring filaments to the plasmalemma via a submembranous electron‐dense layer called the plasmalemmal undercoat. To further clarify how such anchoring filaments are spatially related to the plasmalemma through the undercoat, we carefully examined and compared thin‐section images obtained from various specimen preparations using saponin and Triton X‐100. When the tissues were treated with saponin or Triton, electron‐dense materials in the undercoat were extracted in varying degrees to expose internal substructures. The undercoat appeared to show a two‐layer organization, the inner and outer layers. In more extracted samples, filamentous networks became prominent in the outer layer. Anchoring filaments were seen to attach to such filamentous networks, which in turn were linked to the plasmalemma proper. Thus, it may be reasonable to consider that the filamentous network constitutes the core structure of the plasmalemmal undercoat which is structurally reinforced by extractable electron‐dense materials.