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Studies on the mechanism of cell elongation in Blepharisma japonicum
Europ .], Prot isto\. 27 , 46- 54 (199 1) Ma rch 28, 1991
European Jour na l of
PROTISTOLOGY
Studies on the Mechanism of Cell Elongation in Blepharisma japonicum 4.Three Dimensional Construction of the Kinetosomal Complex and its Functional Role on Cell Elongation Masaki Ishida, Toshinobu Suzaki and Yoshinobu Shigenaka Laboratories of Cell Biology, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan
SUMMARY The kinetosomal complex of a heterotrich ciliate Blepharisma japonicum was investigated at ultrastru cturallevcl, with special reference to its cell elongation in response to light stimulation. In serial sections, vacuole-associated microtub ules were found to be originated from both anterior fiber sheet and left surface of the anterior kinetoso me. These microtu bules form a bundle and extend towa rd the ant erior end of the organism. The postciliary microtubular sheet is atta ched to the prox imal half of the posterior kinetosome. Two types of transverse microtubules are present: the anterior 8-9 microtubul es arising at the base of the anterior kinetosome and the posterior 2-3 microtu bules arising between the paired kinetoso mes. Based on these results, a th ree dimension al model of the kinetosomal complex was pro posed and the mechanism of cell elongation in Blepbarisma was discussed.
Abbreviations A AFS AT M My P PA PMS PT SM V VAM
= = = = = =
= = = = = =
alveolus anterior fiber sheet anterior tran sverse microtubular ribbon mitochondrion myoneme pigment granule postciliary accessory fiber postciliary microtubular sheet posterior transverse microtubular ribbon subpellicular microtubules vacuole vacuole associated microt ubules
Introduction In protozoan ciliates, a certai n degree of contraction and elongation is popul ar and can be observed as direct responses again st externa l stimuli. Although the movements are more or less different from species to species, 0932-4739/91/0027-0046$3.5010
most of these movements are concerned with avoidin g reactions from various kind s of inadequate conditions [17]. In heterotrich ciliates such as Stentor and Spirostomum, two types of filamentous systems, myonem es and postciliary microtubular sheets are present beneath the plasma membrane [2, 7, 16, 18, 19]. It was demonstrated earlier that contraction of myonemes resulted in roundingup of the cell body, and sliding between adjacent microtubular sheets occurred during contraction [8, 21]. The contraction of myonemes resembles that of spasmo nemes in peritrich ciliates such as Vorticella, Carchesium and Z oothamnium, where the contraction takes place ATPindependently by using calcium ions [1,20]. On the ot her hand , little is kno wn about the mechanism of elongation of the cell body which immediately follows the rapid cell contraction. In Stentor, the pr esence of microtubular components is suggested to be indispensable for cell elongation . Accordin g to Hu ang & Pitelka [8], the mot ive force for cell elongation may be attributed to active sliding between adjacent, overlapping sheets of postciliary microtubule s in a similar manner to the elongation pro cess of a © 1991 by Gustav Fischer Verlag, Stuttga rt
Kinetosomal Complex of Blepharisma . 47
portable telescope. Our earlier report suggested that ATP is required for such elongation process in Spirostomum [9]. Blepharisma japonicum is a unique freshwater heterotrich ciliate which shows cell elongation in response to light stimulation without preceding rapid contraction of the cell body [12, 13]. This ciliate is also capable of body bending in food uptake [14]. Peculiar to this ciliat~, Matsuoka & Shigenaka [13] found bundles of cytoplasmic microtubules (vacuole-associated microtubules) which run in parallel with the longitudinal axis of the cell body along the surface of cortical vacuoles. They suggested that these microtubules are involved in cell elongation as the functional components, since cell elongation was inhibited when these microtubules were disassembled by colchicine treatment while myonemes and postciliary microtubules remained intact. In addition to the vacuole-associated microtubules, pairs of basal bodies (kinetosomes) and their fibrous associates containg microtubular sheets and bundles have been considered to be closely related to cell elongation [10, 12]. In attempting to understand the contribution of these microtubular systems to cell elongation, it is necessary to investigate the exact distribution and origin of these structures. In the present study, we examined detailed ultrastructure of the kinetosomal complex of Blepharisma japonicum and found that the vacuole-associated microtubules are derived from either the anterior fiber sheet or the anterior kinetosome. In addition, three dimensional construction of the kinetosomal complex including its associated microtubules, myonemes and other fibrillar components is discussed with respect to cell shortening and elongation mechanism in Blepharisma japonicum.
Material and Methods
Cell Culture and Light Stimulation Blepharisma japonicum strain R-13 was provided by Dr. K. Taneda, Kochi University, Kochi, Japan, and was cultured at 23 ± 1 °C in a lettuce infusion containing small amount of CaC0 3 • All cultures were kept in darkness, and the subculture was carried out at intervals of about 3 weeks. As the light source for stimulation (1,000 lux), we employed a halogen lamp equipped in a light microscope (Nikon, DIAPHOT). The light intensity was determined with a photoelectric illuminometer (Tokyo Photo-Electric, ANA-999). In order to eliminate the effect of heat rays, an infrared-absorbing filter was placed between the lamp and the cell suspension.
Electron Microscopy Prior to fixation, the cells were washed in a test solution (1 mM KCl, 1 mM CaCh, 1 mM Tris-HCl buffered at pH 7.2) and adapted in darkness for 1 h in a test tube. To examine the ultrastructural changes between elongated and contracted states, the cell suspension was divided into two aliquots under dim red light « 20 lux). One was kept in darkness for 15 min and the other was stimulated with light for 15 min. The cells were then fixed for 10 min with a mixture of 2.5 % glutaraldehyde and 1 % OS04 in cacodylate buffer (pH 7.2) at room temperature. After postfixation with 1 % aqueous uranyl acetate for 10 min, they
were dehydrated through a graded series of ethanol, and embedded in Spurr's low viscosity resin. Ultrathin sections were cut on a LKB 2088 ultramicrotome with a diamond knife, and stained with 3 % uranyl acetate for 7 min and lead citrate stain for 3 min. Specimens were then examined in a transmission electron microscope (JEOL, JEM-I00S) operating at 80 kV.
Results As shown in Fig. 1, four kinds of intracellular fibrous components can be recognized in the cross section of Blepharisma japonicum: vacuole-associated micro~ubules (VAM, Fig. Ib), subpellicular microtubules (SM, Fig. lc), postciliary microtubular sheets (PMS, Fig. ld), and myonemes (arrows in Fig. la). Of these four elements, vacuole-associated micro tubules are unique to Blepharisma and have not been described for other heterotrich ciliates, These microtubules are located deeper in the cytoplasm in association with vacuoles which are usually concentrated near the cortical region. A flat alveolus lies below the plasma membrane, under which subpellicular microtubules (Fig. lc) and postciliary microtubular sheets (Fig. Id) are located. A sheet of postciliary microtubules is attached to the proximal half of the posterior kinetosome, which usually extends over at least six kinetids. The most proximal part of the postciliary microtubular sheet is connected to the postciliary accessory fibers on both side of their sheet. The accessory fiber on the right side of the microtubular sheet originates at triplets 7 and 8 of the posterior kinetosot,ne, and the left accessory fiber arises at triplets 1 and 9 (Figs. Id, 6 and 9). When the organism is cross-sectioned at the level of anterior region of the cell body, about 15 sheets of microtubules can be observed in either elongated or normal dark-adapted organisms (Fig. l d), Figs. 2 and 3 are electron micrographs of tangential sections through the posterior ectoplasm of a normal, dark-adapted cell and an elongated cell, respectively. The distance between neighboring dikinetids (Figs. 2 and 3, arrow) altered considerably, which became approximately two or three-fold longer in the elongated organism (Fig. 3) in contrast to the normal one (Fig. 2). At normal and dark-adapted state, the anterior fiber sheet arises between kinetosomes of a pair and its distal end attaches to the postciliary microtubular sheet of the next, anterior kinetosomal complex (Fig. 2, arrowhead). In contrast, at the elongated state, the anterior fiber sheet was found to be detached from the postciliary microtubular sheet (Fig. 3, arrowhead), which was also confirmed in serial sections (data not shown here). Figs. 4 to 6 were obtained from serial sections of dark-adapted organims cut in different planes, showing the orientation of two kinetosomes and their associates containing vacuole-associated microtubules (VAM), a postciliary microtubular sheet (PMS), an anterior fiber sheet (AFS) and the myoneme (My). The postciliary microtubular sheet originates from the posterior kinetosome and tapers as it extends longitudinally and posteriorly. The postciliary ribbons overlap one another along the right side of the kinety and collectively from the Km
48 . M. Ishida, T. Suzaki and Y. Shigenaka
Kinetosomal Complex of Blepharisma . 49
Fig. 2. Longitudinal section through posterior region of a darkadapted organism. Arrows show posterior kinetosomes. The tip of the anterior fiber sheet (AFS) is attached to the postciliary microtubular ribbon at the position shown by an arrowhead. Scale bar = 1.0 urn.
fiber as in other heterotrich ciliates. The vacuoleassociated microtubules, which extend anteriorly from the kinetosomes, arise from both the anterior side of the anterior fiber sheet and the left lateral surface of the anterior kinetosome (Figs. 3-5 and 7). Two kinds of transverse microtubules originate near each kinetosomal pair. The anterior ribbon of 8 or 9 microtubules arises near triplets 3-5 of the anterior kinetosome, while a group of 2 or 3 microtubules (posterior transverse microtubules) originates between kinetosomes of a pair (Figs. 8 and 9). The myoneme is associated with posterior kinetosomes at their internal ends (Fig. 5), interconnecting adjacent kinetosomal complexes. As diagramed in Fig. 10, many other fibrous materials are also observed to join the kinetosomes of a pair. These include a fiber linking triplets 1 and 2 of the anterior kinetosome (Ai and A2 ) with the triplet 3 of the posterior kinetosome (P3), and fibers linking A9 with P4 and As with P, (kinetosomal triplets are numbered according to Lynn [11]).
Fig. 3. Longitudinal section through the posterior region of an elongated organism, showing wider distance between two neighboring kinetosomal pairs. Arrows show posterior kinetosomes. The tip of the anterior fiber sheet (arrowhead) is detached from the postciliary microtubules. Scale bar = 1.0 urn.
... Fig. 1. Electron micrographs of a cross section of Blepharisma japonicum showing typical structures in the anterior part of the cell cortex. a. A low magnification picture showing the locations of the vacuole-associated microtubules (VAM), subpellicular microtubules (SM) and postciliary microtubular ribbons (PMS). They are enclosed in black boxes and are shown in higher magnifications in b, c and d. b. A bundle of vacuole-associated microtubules consists of approximately 17 microtubules. c. Subpellicular microtubules can be observed as bundles of about 9-11 microtubules, located just beneath the pellicular alveolus (A). d. Approximately 16 postciliary microtubular ribbons are seen to overlap with each other. The view is from anterior toward posterior end of the cell. Scale bars = 1.0 (a), 0.2 (b, c and d) urn,
50 . M. Ishida, T. Suzaki and Y. Shigenaka
1· .
c
d
Fig. 4. Serial sections cut through a kinetosomal complex showing orientation of the vacuole-associated microtubules (arrow heads), anterio r fiber sheet (AFS), and postciliary accessory fibers (PA) on both sides of the postciliary microtubular ribbon (PMS). The organism is longitudinally sectioned at the level of the kinetosomes. The vacuole associated microtubules (arrowheads) originate from either the anterior fiber sheet (AFS) or the lateral surface of the anterior kinetosom e, extending deeper inside the anterior cytopla sm. The view is from base toward tip of the kinetosomes. Scale bar = 1.0 urn.
Fig. S. Serial sections showing association between the kinetosomal pair and the anterior fiber sheet, vacuole-associated microtubules and myoneme. The anterior fiber sheet (AFS) originates between the paired kinetosomes. The vacuole-associated microtubules extending from the anterior kinetosome are indicated by an arrowhead in Fig. Sa, while thos e from the anteri or fiber sheet can be seen in Figs. Sb-e (arrowheads). The contractile myoneme (My) is associated with the most pro ximal part of the posterior kinetosome. Scale bar = 1.0 urn.
Kinetosornal Complex of Blepharisma . 51
7
8
Fig. 6. Serial sections of a kinetosomal complex show ing the association of a paired kinetosomes with various fibrou s component such as vacuole-as sociated microtubules (VAM), anterior fiber sheet (AFS), postciliary microtubular sheet (PMS), postciliary accessory fibers (PA), anterior transverse microtubular ribbon (AT) and short fibrils which link the kinetosome s of a pair . The view is from base tow ard tip of the kineto some. The proximal part of the anterior fiber sheet is divergent and conn ected with both anterior and po sterior kinetosomes. Scale bar = 0.2 11m.
Discussion Giese [5] has described many years ago that Blepharisma is a photosensitive ciliate. It was reported in his mono graph [5] that the contractile vacuole slowed or stopped functioning after expo sure to light, and enlargement of the contractile vacuole was induced by stronger illumination. Recently Matsuoka [12] reported that Blepharisma showed cell elongation in response to light stimulus. M atsuoka and Shigenaka [13] have examined the process
Figs. 7- 9. Longitudinal and obliqu e sections of kinetosomal complexes and associated microtubular components such as vacuole-associated microtubules (VAM) and transverse microtubuies (AT and PT). Approximately 7- 9 vacuole-a ssociated microtubules are seen in close associati on with the lateral surface of the proximal part of the anterior kinetosome (Fig. 7). The posterior (PT) and anteri or (AT) transverse microtubular ribbon s are show n in Figs. 8 and 9. Scale bar = 0.2 11m.
of cell elongation and found that this movement was completely inhibited by 1 h treatment with 10 mM colchicine or 1 mM erythro-9-[3-2-(h ydroxynon yl)]adenine (EHNA), suggesting that active microtubule sliding might be involved in this movement. They also suggested that the vacuole-associated micro tubules may be the functional components of cell elongation since these microtubules disassembled by treatm ent with colchicine, while other microtubules rema ined morphologically unchanged.
52 . M. Ishida, T. Suzaki and Y. Shigenaka
10a
c
b
d
Fig. 10. Schematicfigures of a kinetosomal pair and their fibrous associates at different levels in transverse section. Sections of four differentlevels of a kinetosomal pair, from its middleto basal level,are represented from top to bottom. A kinetosomal pair is associated with microtubular components such as a postciliary microtubular sheet (PMS), vacuole-associated microtubules (VAM), postciliary accessory fibers (PA), an anterior transverse microtubular ribbon (AT),a posterior transverse microtubular ribbon (PT), and an anterior fiber sheet (AFS). The kinerosornal triplets are numbered according to Lynn [11].
Our observations have revealed that the vacuoleassociated microtubules originate at either the anterior surface of the anterior fiber sheet or the lateral side of the anterior kinetosome. In cross sections, the most proximal regions of the vacuole-associated microtubules sometimes appeared as short projections protruded from the surface of the anterior fiber sheet (Figs. 2, 6 and 9, and Fig. 3.6 in [5]). Although vacuole-associated microtubules have not been described for other ciliates, similar projections of the anterior fiber sheet are known to be present in other heterotrichs such as Climacostomum [15], Stentor [2, 8] and Spirostomum [6]. Therefore, it seems likel y that similar microtubules exist also in other species. Whether these microtubules are involved in generating the motive force for cell elongation in Blepharisma is a difficult question which must be examined. A possible mechanism for cell elongation is that some of the vacuole-associated microtubules in a bundle may slide against the microtubules which originate from the next kinetosomal pair, thus increasing the distance between adjacent kinetosomal pairs. The vacuole-associated microtubules could also be involved in releasing the distal end of the anterior fiber sheet from the postciliary microtubular ribbon, allowing
for the displacement of the adjacent kinetosomal complexes. A detailed model of the arrangement of microtubules and paired kinetosomes is shown in Fig. 11. Like in other heterotrichs [2, 6, 8, 15], Blepharisma [aponicum has a postciliary sheet of microtubules which originates from the posterior kinetosome of each kinetosomal pair, and two transverse microtubular ribbons arise near each kinetosomal pair. The anterior ribbon of 8 or 9 microtubules arises at the base of the ant erio r kinetosome near triplets 3-5, extending upward into the ectoplasmic ridges just beneath the pellicle. These microtubules seem to be connected with the subpellicular microtubules (Fig. l c, SM) as suggested earlier by Gerassimova and Seravin [4]. The other ribbon of 2-3 microtubules originates between kinetosomes of a pair. Although these microtubules are newly found here for Blepharisma, similar structures have been described for other heterotrichs such as Spirostomum [21], Condylostoma [3], Climacostomum [15], and Stentor (Fig. 15 in [2]). Whether these microtubulcs are also connected with the subpellicular microtubules is not certain.
Kinetosomal Complex of Blepbarisma . 53
Fig. 11. A three-dimensional model constructed with polyethylene tubing depicting the organization of the kinetosomal complex of Blepharisma [aponicum at dark-adapted (a) and elongated (c) states. The anterior fiber sheet (AFS) is attached at the postciliary microtubule s of the next, anterior kineto somal complex in dark-adapted cells. In the elongated state, distance between neighboring kinetosomal pairs becomes wider and the tip of the anterior fiber sheet is detached from the postciliary microtubular ribbon. Enlarged figures (b, d) show bottom and oblique views of the paired kinctosornes and their fibrous associates. A large arrow indicates the posterior direction.
References 1 Amos W. B (1975): Contraction and calcium binding in the vorticcllid ciliates. In: Inoue S. and Stephens R. E. (eds.): Molecules and cell movement, pp. 411-436. Raven Press, New York. 2 Bannister L. N. and Tatchel E. C. (1968 ): Contractility and the fiber systems of Stentor coeruleus. J. Cell. Sci., 3, 295- 308. 3 Bohatier ] . (1978): Morphologie ultra structurale de Condylostoma, cilie polyhymenophora. Protistologica , 14, 433-450.
4 Gerassimova Z. P. and Seravin L. N . (1976): Ectoplasmic fibrillar system of infusoria and its role for the understanding of their phylogeny. Zoo!' Zh., 55, 645-656 (in Russian with English summa ry). 5 Giese A. C. (1973): Blepbarisma. The biology of lightsensitive protozoan. Stanford University Press, Stanford , California. 6 Grain J. (1968): Les systemes fibrillaires chez Stentor igneus Ehrenberg et Spirostomum ambiguum Ehrenberg. Prot istologica, 4, 27-36.
54 . M. Ishida, T. Suzaki and Y. Shigenaka 7 Huang B. and Mazia D. (1975): Ciliate contractility. In: Inoue S. and Stephens R. E. (eds.): Molecules and cell movement, pp. 389-409. Raven Press, New York. 8 Huang B. and Pitelka D. R. (1973): The contractile process in the ciliate Stentor coeruleus. 1. The role of microtubules and filaments. J. Cell Biol., 57, 704-728. 9 Ishida H. and Shigenaka Y. (1988): Cell model contraction in the ciliate Spirostomum. Cell Motil. Cytoskel., 9, 278-282. 10 Ishida M., Shigenaka Y. and Taneda K. (1989): Studies on the mechanism of cell elongation in Blepharisma japonicum. 1. A physiological mechanism how light stimulation evokes cell elongation. Europ. J. Protistol., 25, 182-186. 11 Lynn D. H. (1981): The organization and evolution of microtubular organelles in ciliated protozoa. Biol. Rev., 56, 243-292. 12 Matsuoka T. (1983): Negative phototaxis in Blepharisma iaponicum, J. Protozool., 30, 409-414. 13 Matsuoka T. and Shigenaka Y. (1985): Mechanism of cell elongation in Blepharisma japonicum, with special reference to the role of cytoplasmic microtubules. Cytobios, 42, 215-226. 14 Nilsson J. R. (1967): An African strain of Blepharisma [aponicum (SUZUKI). A study of the morphology, gigantism
15 16 17 18 19 20 21
and cannibalism, and macronuclear aberration. Compt. Rend. Trav. Lab. Carlsberg, 36, 1-24. Peck R., Pelvat B., Bokivar I. and De Haller G. (1975): Light and electron microscopic observations on the heterotrich ciliate Climacostomum virens. J. Protozool., 22, 368-385. Randall J. T. and Jackson S. F. (1985): Fine structure in Stentor polymorphus. J. Biophys. Biochem. Cytol., 4, 807-830. Stebbings H. and Hyams J. S. (1979): Cell motility. Longman Group Lrd., London. Yagiu R. and Shigenaka Y. (1960): Electron microscopical studies on the fibrillar system in the protozoan ciliates. Jpn. J. Exp. Morphol., 14, 1-52. Yagiu R. and Shigenaka Y. (1963): Electron microscopy on the longitudinal fibrillar system in Spirostomum ambiguum. J. Protozool., 10,364-369. Yamada-Horiuchi K. and Asai H. (1985): Circular dichroism of Ca2+-binding protein from the spasmoneme of Carchesium. Compo Physiol., 81B, 927-931. Yogosawa-Ohara R., Suzaki T. and Shigenaka Y. (1985): Twisting contraction mechanism of a heterotrichous ciliate, Spirostomum ambiguum. 2. Role of longitudinal microtubular sheet. Cytobios, 44, 215-230.
Key words: Ciliate - Kinetosomal complex - Microtubule - Electron microscopy - Motility Yoshinobu Shigenaka, Laboratories of Cell Biology, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima 730, Japan
European Jour na l of
PROTISTOLOGY
Studies on the Mechanism of Cell Elongation in Blepharisma japonicum 4.Three Dimensional Construction of the Kinetosomal Complex and its Functional Role on Cell Elongation Masaki Ishida, Toshinobu Suzaki and Yoshinobu Shigenaka Laboratories of Cell Biology, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan
SUMMARY The kinetosomal complex of a heterotrich ciliate Blepharisma japonicum was investigated at ultrastru cturallevcl, with special reference to its cell elongation in response to light stimulation. In serial sections, vacuole-associated microtub ules were found to be originated from both anterior fiber sheet and left surface of the anterior kinetoso me. These microtu bules form a bundle and extend towa rd the ant erior end of the organism. The postciliary microtubular sheet is atta ched to the prox imal half of the posterior kinetosome. Two types of transverse microtubules are present: the anterior 8-9 microtubul es arising at the base of the anterior kinetosome and the posterior 2-3 microtu bules arising between the paired kinetoso mes. Based on these results, a th ree dimension al model of the kinetosomal complex was pro posed and the mechanism of cell elongation in Blepbarisma was discussed.
Abbreviations A AFS AT M My P PA PMS PT SM V VAM
= = = = = =
= = = = = =
alveolus anterior fiber sheet anterior tran sverse microtubular ribbon mitochondrion myoneme pigment granule postciliary accessory fiber postciliary microtubular sheet posterior transverse microtubular ribbon subpellicular microtubules vacuole vacuole associated microt ubules
Introduction In protozoan ciliates, a certai n degree of contraction and elongation is popul ar and can be observed as direct responses again st externa l stimuli. Although the movements are more or less different from species to species, 0932-4739/91/0027-0046$3.5010
most of these movements are concerned with avoidin g reactions from various kind s of inadequate conditions [17]. In heterotrich ciliates such as Stentor and Spirostomum, two types of filamentous systems, myonem es and postciliary microtubular sheets are present beneath the plasma membrane [2, 7, 16, 18, 19]. It was demonstrated earlier that contraction of myonemes resulted in roundingup of the cell body, and sliding between adjacent microtubular sheets occurred during contraction [8, 21]. The contraction of myonemes resembles that of spasmo nemes in peritrich ciliates such as Vorticella, Carchesium and Z oothamnium, where the contraction takes place ATPindependently by using calcium ions [1,20]. On the ot her hand , little is kno wn about the mechanism of elongation of the cell body which immediately follows the rapid cell contraction. In Stentor, the pr esence of microtubular components is suggested to be indispensable for cell elongation . Accordin g to Hu ang & Pitelka [8], the mot ive force for cell elongation may be attributed to active sliding between adjacent, overlapping sheets of postciliary microtubule s in a similar manner to the elongation pro cess of a © 1991 by Gustav Fischer Verlag, Stuttga rt
Kinetosomal Complex of Blepharisma . 47
portable telescope. Our earlier report suggested that ATP is required for such elongation process in Spirostomum [9]. Blepharisma japonicum is a unique freshwater heterotrich ciliate which shows cell elongation in response to light stimulation without preceding rapid contraction of the cell body [12, 13]. This ciliate is also capable of body bending in food uptake [14]. Peculiar to this ciliat~, Matsuoka & Shigenaka [13] found bundles of cytoplasmic microtubules (vacuole-associated microtubules) which run in parallel with the longitudinal axis of the cell body along the surface of cortical vacuoles. They suggested that these microtubules are involved in cell elongation as the functional components, since cell elongation was inhibited when these microtubules were disassembled by colchicine treatment while myonemes and postciliary microtubules remained intact. In addition to the vacuole-associated microtubules, pairs of basal bodies (kinetosomes) and their fibrous associates containg microtubular sheets and bundles have been considered to be closely related to cell elongation [10, 12]. In attempting to understand the contribution of these microtubular systems to cell elongation, it is necessary to investigate the exact distribution and origin of these structures. In the present study, we examined detailed ultrastructure of the kinetosomal complex of Blepharisma japonicum and found that the vacuole-associated microtubules are derived from either the anterior fiber sheet or the anterior kinetosome. In addition, three dimensional construction of the kinetosomal complex including its associated microtubules, myonemes and other fibrillar components is discussed with respect to cell shortening and elongation mechanism in Blepharisma japonicum.
Material and Methods
Cell Culture and Light Stimulation Blepharisma japonicum strain R-13 was provided by Dr. K. Taneda, Kochi University, Kochi, Japan, and was cultured at 23 ± 1 °C in a lettuce infusion containing small amount of CaC0 3 • All cultures were kept in darkness, and the subculture was carried out at intervals of about 3 weeks. As the light source for stimulation (1,000 lux), we employed a halogen lamp equipped in a light microscope (Nikon, DIAPHOT). The light intensity was determined with a photoelectric illuminometer (Tokyo Photo-Electric, ANA-999). In order to eliminate the effect of heat rays, an infrared-absorbing filter was placed between the lamp and the cell suspension.
Electron Microscopy Prior to fixation, the cells were washed in a test solution (1 mM KCl, 1 mM CaCh, 1 mM Tris-HCl buffered at pH 7.2) and adapted in darkness for 1 h in a test tube. To examine the ultrastructural changes between elongated and contracted states, the cell suspension was divided into two aliquots under dim red light « 20 lux). One was kept in darkness for 15 min and the other was stimulated with light for 15 min. The cells were then fixed for 10 min with a mixture of 2.5 % glutaraldehyde and 1 % OS04 in cacodylate buffer (pH 7.2) at room temperature. After postfixation with 1 % aqueous uranyl acetate for 10 min, they
were dehydrated through a graded series of ethanol, and embedded in Spurr's low viscosity resin. Ultrathin sections were cut on a LKB 2088 ultramicrotome with a diamond knife, and stained with 3 % uranyl acetate for 7 min and lead citrate stain for 3 min. Specimens were then examined in a transmission electron microscope (JEOL, JEM-I00S) operating at 80 kV.
Results As shown in Fig. 1, four kinds of intracellular fibrous components can be recognized in the cross section of Blepharisma japonicum: vacuole-associated micro~ubules (VAM, Fig. Ib), subpellicular microtubules (SM, Fig. lc), postciliary microtubular sheets (PMS, Fig. ld), and myonemes (arrows in Fig. la). Of these four elements, vacuole-associated micro tubules are unique to Blepharisma and have not been described for other heterotrich ciliates, These microtubules are located deeper in the cytoplasm in association with vacuoles which are usually concentrated near the cortical region. A flat alveolus lies below the plasma membrane, under which subpellicular microtubules (Fig. lc) and postciliary microtubular sheets (Fig. Id) are located. A sheet of postciliary microtubules is attached to the proximal half of the posterior kinetosome, which usually extends over at least six kinetids. The most proximal part of the postciliary microtubular sheet is connected to the postciliary accessory fibers on both side of their sheet. The accessory fiber on the right side of the microtubular sheet originates at triplets 7 and 8 of the posterior kinetosot,ne, and the left accessory fiber arises at triplets 1 and 9 (Figs. Id, 6 and 9). When the organism is cross-sectioned at the level of anterior region of the cell body, about 15 sheets of microtubules can be observed in either elongated or normal dark-adapted organisms (Fig. l d), Figs. 2 and 3 are electron micrographs of tangential sections through the posterior ectoplasm of a normal, dark-adapted cell and an elongated cell, respectively. The distance between neighboring dikinetids (Figs. 2 and 3, arrow) altered considerably, which became approximately two or three-fold longer in the elongated organism (Fig. 3) in contrast to the normal one (Fig. 2). At normal and dark-adapted state, the anterior fiber sheet arises between kinetosomes of a pair and its distal end attaches to the postciliary microtubular sheet of the next, anterior kinetosomal complex (Fig. 2, arrowhead). In contrast, at the elongated state, the anterior fiber sheet was found to be detached from the postciliary microtubular sheet (Fig. 3, arrowhead), which was also confirmed in serial sections (data not shown here). Figs. 4 to 6 were obtained from serial sections of dark-adapted organims cut in different planes, showing the orientation of two kinetosomes and their associates containing vacuole-associated microtubules (VAM), a postciliary microtubular sheet (PMS), an anterior fiber sheet (AFS) and the myoneme (My). The postciliary microtubular sheet originates from the posterior kinetosome and tapers as it extends longitudinally and posteriorly. The postciliary ribbons overlap one another along the right side of the kinety and collectively from the Km
48 . M. Ishida, T. Suzaki and Y. Shigenaka
Kinetosomal Complex of Blepharisma . 49
Fig. 2. Longitudinal section through posterior region of a darkadapted organism. Arrows show posterior kinetosomes. The tip of the anterior fiber sheet (AFS) is attached to the postciliary microtubular ribbon at the position shown by an arrowhead. Scale bar = 1.0 urn.
fiber as in other heterotrich ciliates. The vacuoleassociated microtubules, which extend anteriorly from the kinetosomes, arise from both the anterior side of the anterior fiber sheet and the left lateral surface of the anterior kinetosome (Figs. 3-5 and 7). Two kinds of transverse microtubules originate near each kinetosomal pair. The anterior ribbon of 8 or 9 microtubules arises near triplets 3-5 of the anterior kinetosome, while a group of 2 or 3 microtubules (posterior transverse microtubules) originates between kinetosomes of a pair (Figs. 8 and 9). The myoneme is associated with posterior kinetosomes at their internal ends (Fig. 5), interconnecting adjacent kinetosomal complexes. As diagramed in Fig. 10, many other fibrous materials are also observed to join the kinetosomes of a pair. These include a fiber linking triplets 1 and 2 of the anterior kinetosome (Ai and A2 ) with the triplet 3 of the posterior kinetosome (P3), and fibers linking A9 with P4 and As with P, (kinetosomal triplets are numbered according to Lynn [11]).
Fig. 3. Longitudinal section through the posterior region of an elongated organism, showing wider distance between two neighboring kinetosomal pairs. Arrows show posterior kinetosomes. The tip of the anterior fiber sheet (arrowhead) is detached from the postciliary microtubules. Scale bar = 1.0 urn.
... Fig. 1. Electron micrographs of a cross section of Blepharisma japonicum showing typical structures in the anterior part of the cell cortex. a. A low magnification picture showing the locations of the vacuole-associated microtubules (VAM), subpellicular microtubules (SM) and postciliary microtubular ribbons (PMS). They are enclosed in black boxes and are shown in higher magnifications in b, c and d. b. A bundle of vacuole-associated microtubules consists of approximately 17 microtubules. c. Subpellicular microtubules can be observed as bundles of about 9-11 microtubules, located just beneath the pellicular alveolus (A). d. Approximately 16 postciliary microtubular ribbons are seen to overlap with each other. The view is from anterior toward posterior end of the cell. Scale bars = 1.0 (a), 0.2 (b, c and d) urn,
50 . M. Ishida, T. Suzaki and Y. Shigenaka
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c
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Fig. 4. Serial sections cut through a kinetosomal complex showing orientation of the vacuole-associated microtubules (arrow heads), anterio r fiber sheet (AFS), and postciliary accessory fibers (PA) on both sides of the postciliary microtubular ribbon (PMS). The organism is longitudinally sectioned at the level of the kinetosomes. The vacuole associated microtubules (arrowheads) originate from either the anterior fiber sheet (AFS) or the lateral surface of the anterior kinetosom e, extending deeper inside the anterior cytopla sm. The view is from base toward tip of the kinetosomes. Scale bar = 1.0 urn.
Fig. S. Serial sections showing association between the kinetosomal pair and the anterior fiber sheet, vacuole-associated microtubules and myoneme. The anterior fiber sheet (AFS) originates between the paired kinetosomes. The vacuole-associated microtubules extending from the anterior kinetosome are indicated by an arrowhead in Fig. Sa, while thos e from the anteri or fiber sheet can be seen in Figs. Sb-e (arrowheads). The contractile myoneme (My) is associated with the most pro ximal part of the posterior kinetosome. Scale bar = 1.0 urn.
Kinetosornal Complex of Blepharisma . 51
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Fig. 6. Serial sections of a kinetosomal complex show ing the association of a paired kinetosomes with various fibrou s component such as vacuole-as sociated microtubules (VAM), anterior fiber sheet (AFS), postciliary microtubular sheet (PMS), postciliary accessory fibers (PA), anterior transverse microtubular ribbon (AT) and short fibrils which link the kinetosome s of a pair . The view is from base tow ard tip of the kineto some. The proximal part of the anterior fiber sheet is divergent and conn ected with both anterior and po sterior kinetosomes. Scale bar = 0.2 11m.
Discussion Giese [5] has described many years ago that Blepharisma is a photosensitive ciliate. It was reported in his mono graph [5] that the contractile vacuole slowed or stopped functioning after expo sure to light, and enlargement of the contractile vacuole was induced by stronger illumination. Recently Matsuoka [12] reported that Blepharisma showed cell elongation in response to light stimulus. M atsuoka and Shigenaka [13] have examined the process
Figs. 7- 9. Longitudinal and obliqu e sections of kinetosomal complexes and associated microtubular components such as vacuole-associated microtubules (VAM) and transverse microtubuies (AT and PT). Approximately 7- 9 vacuole-a ssociated microtubules are seen in close associati on with the lateral surface of the proximal part of the anterior kinetosome (Fig. 7). The posterior (PT) and anteri or (AT) transverse microtubular ribbon s are show n in Figs. 8 and 9. Scale bar = 0.2 11m.
of cell elongation and found that this movement was completely inhibited by 1 h treatment with 10 mM colchicine or 1 mM erythro-9-[3-2-(h ydroxynon yl)]adenine (EHNA), suggesting that active microtubule sliding might be involved in this movement. They also suggested that the vacuole-associated micro tubules may be the functional components of cell elongation since these microtubules disassembled by treatm ent with colchicine, while other microtubules rema ined morphologically unchanged.
52 . M. Ishida, T. Suzaki and Y. Shigenaka
10a
c
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Fig. 10. Schematicfigures of a kinetosomal pair and their fibrous associates at different levels in transverse section. Sections of four differentlevels of a kinetosomal pair, from its middleto basal level,are represented from top to bottom. A kinetosomal pair is associated with microtubular components such as a postciliary microtubular sheet (PMS), vacuole-associated microtubules (VAM), postciliary accessory fibers (PA), an anterior transverse microtubular ribbon (AT),a posterior transverse microtubular ribbon (PT), and an anterior fiber sheet (AFS). The kinerosornal triplets are numbered according to Lynn [11].
Our observations have revealed that the vacuoleassociated microtubules originate at either the anterior surface of the anterior fiber sheet or the lateral side of the anterior kinetosome. In cross sections, the most proximal regions of the vacuole-associated microtubules sometimes appeared as short projections protruded from the surface of the anterior fiber sheet (Figs. 2, 6 and 9, and Fig. 3.6 in [5]). Although vacuole-associated microtubules have not been described for other ciliates, similar projections of the anterior fiber sheet are known to be present in other heterotrichs such as Climacostomum [15], Stentor [2, 8] and Spirostomum [6]. Therefore, it seems likel y that similar microtubules exist also in other species. Whether these microtubules are involved in generating the motive force for cell elongation in Blepharisma is a difficult question which must be examined. A possible mechanism for cell elongation is that some of the vacuole-associated microtubules in a bundle may slide against the microtubules which originate from the next kinetosomal pair, thus increasing the distance between adjacent kinetosomal pairs. The vacuole-associated microtubules could also be involved in releasing the distal end of the anterior fiber sheet from the postciliary microtubular ribbon, allowing
for the displacement of the adjacent kinetosomal complexes. A detailed model of the arrangement of microtubules and paired kinetosomes is shown in Fig. 11. Like in other heterotrichs [2, 6, 8, 15], Blepharisma [aponicum has a postciliary sheet of microtubules which originates from the posterior kinetosome of each kinetosomal pair, and two transverse microtubular ribbons arise near each kinetosomal pair. The anterior ribbon of 8 or 9 microtubules arises at the base of the ant erio r kinetosome near triplets 3-5, extending upward into the ectoplasmic ridges just beneath the pellicle. These microtubules seem to be connected with the subpellicular microtubules (Fig. l c, SM) as suggested earlier by Gerassimova and Seravin [4]. The other ribbon of 2-3 microtubules originates between kinetosomes of a pair. Although these microtubules are newly found here for Blepharisma, similar structures have been described for other heterotrichs such as Spirostomum [21], Condylostoma [3], Climacostomum [15], and Stentor (Fig. 15 in [2]). Whether these microtubulcs are also connected with the subpellicular microtubules is not certain.
Kinetosomal Complex of Blepbarisma . 53
Fig. 11. A three-dimensional model constructed with polyethylene tubing depicting the organization of the kinetosomal complex of Blepharisma [aponicum at dark-adapted (a) and elongated (c) states. The anterior fiber sheet (AFS) is attached at the postciliary microtubule s of the next, anterior kineto somal complex in dark-adapted cells. In the elongated state, distance between neighboring kinetosomal pairs becomes wider and the tip of the anterior fiber sheet is detached from the postciliary microtubular ribbon. Enlarged figures (b, d) show bottom and oblique views of the paired kinctosornes and their fibrous associates. A large arrow indicates the posterior direction.
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Key words: Ciliate - Kinetosomal complex - Microtubule - Electron microscopy - Motility Yoshinobu Shigenaka, Laboratories of Cell Biology, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima 730, Japan