Attachment strips: a new type of hemidesmosome-like structure in the protozoon Katablepharis ovalis Skuja

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1 Attachment strips: a new type of hemidesmosome-like structure in the protozoon Katablepharis ovalis Skuja ROBERT EDWARD LEE Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, CO 80533, USA and PAUL KUGRENS Department of Biology, Colorado State University, Fort Collins, CO 80523, USA Summary Katablepharis ovalis Skuja is a free-living unicellular flagellate with a phagocytic mode of nutrition. Each cell has one or two hemidesmosome-like attachment strips consisting of electron-dense ridges that pass through the plasma membrane to end at the inner wall. Each attachment strip occurs over a group of microtubules of the cytoskeleton and, like the cytoskeleton, is oriented along the long axis of the cell. The attachment strips occur only over the medial area of the cell and are not found over the anterior one-third or posterior one-sixth of the cell. The attachment strips appear to function like hemidesmosomes, attaching the cell to the extracellular matrix. However, the attachment strips in Katablepharis do not have the cytoplasmic component of hemidesmosomes, e.g. fibrils and electron-dense plaques. Among unicellular organisms, hemidesmosomes have been previously reported only in trypanosomes, where hemidesmosomes on an expanded flagellum attach the parasite to the cuticle of the alimentary canal of the insect host. The possible relationship between the attachment strips in Katablepharis and the hemidesmosomes in trypanosomes is discussed. Key words: Katablepharis, attachment strip, trypanosome, hemidesmosome Introduction A hemidesmosome i8 a specialized area of the plasma membrane that serves to attach a cell to the extracellular matrix. Hemidesmosomes are common in higher animals, where they secure the cell to the basal lamina (Staehelin, 1974; Staehelin and Hull, 1978). Among unicellular organisms, hemidesmosomes and desmo9omes have only been found in trypanosomes (Killick-Kendrick et al. 1974; Molyneux, 1977; Gardner and Molyneux, 1988; Walters et al. 1989). During an ultrastructural investigation of Katablepharis ovalis, a free-living, phagocytic flagellate, we observed a specialized area of the plasma membrane that appeared to secure the cell wall to the plasma membrane. While the specialized area of the plasma membrane in Katablepharis appeared to be functionally similar to a hemidesmosome, it differed somewhat structurally. The following report describes this structure, which we have called an attachment atrip. Materials and methods Katablepharis ovalis was isolated from pond A-l of the Depart- Journal of Cell Science 98, (1991) Printed in Great Britain The Company of Biologista Limited 1991 ment of Energy's Rocky Flats Nuclear-Weapons Plant near Golden, Colorado. Katablepharis was isolated into a bialgal culture with Chrysochromuhna, on which Katablepharis feeds. The organisms were grown in sterilized water from Dowdy Lake, Red Feather, Colorado, which had 40 ml of Algal-Gro (Carolina Biological Supply) added per liter. Micrographs of thin-sectioned material fixed by conventional glutaraldehyde-osmium tetroxide fixation were obtained using previously described methods (Kugrens and Lee, 1988). Freezefracture replicas were obtained by 'slamming' living cells against a liquid nitrogen-cooled copper block, followed by freeze fracture according to the method of Kugrens and Lee (1987). Results Katablepharis ovalis is a biflagellate unicell that has a two-layered wall surrounding the cell body and flagella (Fig. 1A). The cell has an anterior mouth and feeding apparatus, a central nucleus, and one or more posterior food vacuoles (Fig. 1A). An outer cytoskeleton, composed of groups of four to eight microtubules occurs beneath the plasma membrane, arranged along the long axis of the cell. Each cell has one or two attachment strips composed of thin ridges of electron-dense material that pass through the plasma membrane to terminate at the inner layer of the wall (Figs 1B,C, 2). The ridges of electron-dense 245

2 v mt * i \ w as ' B Fig. 1. Micrographs of thin sections of Katablepharis. (A) Longitudinal section of a cell showing the flagella (f), the anterior mouth (m), the two-layered wall (w) surrounding the cell, the microtubules (mt) of the cytoskeleton, the nucleus and the posterior food vacuole (fv). Bar, 1 /an. (B) A portion of a cross-section of a cell showing an attachment strip (as) occurring outside of a group of four microtubules of the cytoskeleton. n, nucleus; p, plasma membrane; rer, cisternum of rough endoplasmic reticulum; w, wall. Bar, 0.5 /an. (C) A fairly thick cross-section of a cell at the level of the nucleus. The two attachment strips (as) appear as electrondense material between the plasma membrane and the inner wall (w) in areas outside the microtubules (mt) of the cytoskeleton. Bar, 0.5 /an. material are 12 nm apart. A 'thick' thin section (Fig. 1C) shows that the inner wall is closely pressed to the electrondense material of the attachment strip. The attachment strip is always oriented directly over one of the micro tubular groups of the cytoskeleton (Figs 1B,C, 2, 3A). Attachment strips occur only over the medial area of the cell and do not extend into the anterior one-third of the cell (Fig. 3A) or into the posterior one-sixth of the cell. The exact microtubular group of the cytoskeleton with an attachment strip varies; attachment strips have been seen on ventral, lateral and dorsal surfaces. In freeze-fracture replicas, an attachment strip is visible as rows of projections of irregular size on the protoplasmic face (P-face) of the plasma membrane (Fig. 3A-C). The irregular nature of the projections and ridges is probably due to distortion during the fracture, a common phenomenon. The microtubular groups of the cytoskeleton commonly occur as indentations in the P face of the plasma membrane, with the ridges and projections of the attachment strip occurring over the microtubular group (Fig. 3A). The extracellular face (E face) of the plasma membrane at the attachment strip has four to eight linear 246 R. E. Lee and P. Kugrens slits, 12 nm apart (Fig 3D,E). Sometimes, the E-face slits have a cross-hatched appearance, with the slits having a regular structure parallel and perpendicular to the long axis of the attachment strip (Fig. 3E). Occasionally, the projections of the attachment strip pull off a P face during fracture, revealing slits in the P face similar to the slits in the E face (Fig. 3C). Discussion Drosophila wing epidermal cells have microtubules associated with hemidesmosomes (Mogensen and Tucker, 1987, 1988; Tucker et al. 1986). The hemidesmosomes in Drosophila wing epidermal cells have electron-dense material on each side of the plasma membrane, as do the attachment strips of Katablepharis. In Drosophila wing epidermal cells, the exterior electron-dense material of the hemidesmosome appears to attach the plasma membrane to the cuticle (see Fig. 11, Mogensen and Tucker, 1987), similar to the situation in Katablepharis. However, in

3 OUTER WALL INNER WALL ATTACHMENT STRIP o o ooo o PLASMA MEMBRANE CYTOPLASM MICRQTUBULES Fig. 2. A schematic drawing of the structure of an attachment strip and associated structures. Drosophila wing epidermal cells, the electron-dense material is not organized into distinct ridges spanning the plasma membrane, as it is in Katablepharis. Also in Drosophila wing epidermal cells, the microtubules terminate near, and at right angles to, the hemidesmosome. Mogensen and Tucker (1987) postulate that some of the electron-dense material of the Drosophila hemidesmosome is responsible for the formation of the microtubules adjacent to the hemidesmosome. This is probably not the case in Katablepharis where the microtubules associated with the attachment strips are oriented parallel to, and do not terminate near, the attachment strips. Among unicellular organisms, hemidesmosomes have only been reported in the trypanosomes. In the trypanosomes, hemidesmosomes on an expanded flagellum attach the cell to the cuticle of the alimentary canal of the insect host (Killick-Kendrick et al. 1974; Molyneux, 1977; Gardner and Molyneux, 1988; Walters et al. 1989). The attachment strips in Katablepharis do not have the welldeveloped cytoplasmic system of electron-dense fibrils and plaques that occur in hemidesmosomes in the trypanosomes and multicellular organisms (Staehelin, 1974; Staehelin and Hull, 1978; Gipson et al. 1983). However, an attachment strip in Katablepharis is closely associated with a microtubular group of the cytoskeleton. The linearly arranged microtubules of the cytoskeleton may provide the mechanical support given by electron-dense fibrils and plaques of hemidesmosomes in other organisms. Also, the wall of Katablepharis may not be bound very tightly to the plasma membrane, so the fibrils and electron-dense plaques associated with the stronger hemidesmosome may not be necessary. More robust hemidesmosomes may be required to attach a trypano- some to the cuticle of the alimentary canal of an insect, or to attach epithelial cells to a basal lamina in multicellular organisms. Indeed, one wonders why Katablepharis has an attachment strip at all. There are a large number of organisms, both unicellular and multicellular, motile and non-motile, that have an extracellular matrix in the form of a cell wall or theca. An attachment strip or hemidesmosome has never been reported in these organisms. It, therefore, seems unlikely that the simple presence of a wall requires an attachment strip. There must be a unique situation in Katablepharis that requires the presence of an attachment strip. It is our belief that this unique situation in Katablepharis is the phagocytic mode of nutrition coupled with the presence of a wall. We believe that the mouth of Katablepharis attaches the cell to its prey and that the contents of the prey are taken into food vacuoles in Katablepharis, much like the ingestion of prey in suctorians. Attachment of Katablepharis to a prey organism would require an opening in the anterior end of the extracellular matrix to expose the mouth to the prey. The active ingestion would result in a rearrangement of the inner wall. The positioning of the opening in the anterior portion of the extracellular matrix may not be possible unless the inner wall is anchored to the plasma membrane by attachment strips in the medial portions of the cell. It is interesting to speculate on the possible relationship between the attachment strip in Katablepharis and the hemidesmosome. Katablepharis is probably a very old organism in terms of evolution. It is possible that the relatively simple structure of the attachment strip evolved into the more complex hemidesmosome as evolution progressed. Such an evolutionary progression would have resulted in the loss of the association of microtubules with Attachment strips 247

4 3A Fig. 3. Transmission electron micrographs of freeze-fracture replicas of cells rapidly frozen by 'slamming*, showing the structure of the attachment strips in the plasma membrane. Bars, 0.1 jnn. (A) This fracture of the protoplasmic face of the plasma membrane shows the mouth (m) at the anterior end of the cell. Three groups of microtubules of the cytoskeleton are represented by indentations of the plasma membrane (arrowheads). The attachment strip (as) is seen at the bottom of the micrograph as a strip of projections over one of the microtubular groups of the cytoskeleton. (B,C) Replicas of the P face of the plasma membrane in the area of the attachment strip. The projections arising out of the P face are the electron-dense ridges that traverse the plasma membrane in thin sections. Some of the ridges are missing over the P face of the plasma membrane in C, revealing the linear slits in the P face through which the ridges project (arrowhead). (D,E) Replicas of the extracellular face (E face) of the plasma membrane, showing the structure of the attachment strip. (D) The usual appearance of the E face with linear ridges and a few intra-membrane particles interdispersed along the strip. (E) Occasionally the E face of the plasma membrane in the attachment strip has a cross-hatched appearance. 248 R. E. Lee and P. Kugrens

5 the attachment strip, and the development of cytoplasmic filaments with a cytoplasmic plaque to form the hemidesmosome. We thank Dr Andrew Staehelin for reading the manuscript and making helpful suggestions. References GARDNER, R. A. AND MOLYNEUX, D. H. (1988). Trypanosoma {Megatrypanum) mcertum from Pipistrellus: development and transmission by cimicid bugs. Parasitology 98, GIPSON, I. K., GRILL, S. M., SPURH, S. J. AND BRENNAN, S. J. (1983). Hemidesmosome formation in vitro. J. Cell Biol. 97, KILUCK-KKNDRJCK, R., MOLYNEUX, D. H. AND ASHFORD, R, W. (1974). Leishmania in phlebotomid sandflies. I. Modifications of the flagellum associated with the attachment to the mid-gut and oesophageal valve of the sandfly. Proc. ft. Soc. hand. B, 187, KUGRBNS, P. AND LEE, R. E. (1987). An ultrastructural survey of cryptomonad periplasts using quick-freezing freeze-fracture techniques. J. Phycol 23, KUORENS, P. AND LEE, R. E. (1988). Ultrastructure of fertilization in a cryptomonad. J. Phycol. 24, MOCENSEN, M. M. AND TUCKER, J. B. (1987). Evidence for microtubule nucleation at plasma membrane-associated sites in Drosophila J. Cell Sci. 88, MOGENSEN, M. M. AND TUCKER, J B. (1988). Intermicrotubular actin filaments in the transalar cytoskeletal arrays of Drosophila. J. Cell Sci. 91, MOLYNEUX, D. H. (1977). Vector relationships in the Trypanosomatidae. Adv. ParasU. 15, STAEHELIN, L. A. (1974). Structure and function of intercellular junctions. Int. Rev. Cytol. 39, STABHEUN, L A. AND HULL, B. E. (1978). Junctions between living cells. Sci Amer. 238 (May), TUCKER, J. B., MILNKR, M. J., CURRIE, D. A., MUIR, J. W., FORREST, D. A. AND SPENCER, M. J. (1986) Centrosomal microtubule-organizing centres and a switch in the control of protofilament number for cell surface-associated microtubules during Drosophila wing morphogenesis. Eur. J. Cell Bwl. 41, WAI/TEBS, L. L., CHAPLIN, G. L., MODI, G. B. AND TESH, R. B. (1989). Ultrastructural biology of Leishmania (Viannia) panamensis (=Leishmania braziliensis panamensis) in Lutzomyia gomezi (Diptera:Psychodidae): A natural host-parasite association. Am. J. trop. Med. Hyg. 40, (Received 29 August Accepted 10 October 1990) Attachment strips 249

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