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Chapter 46 Preparation and Reactivation of Spermatozopsis Cytoskeletons
CHAPTER 46
Preparation and Reactivation of Spermatoxomis Cvtoskeletons Karl-Ferdinand Lechtreck and Michael Melkonian Botanisches Insitut Universitat zu Koln D-50931 Koln Federal Republic of Germany
I. Introduction 11. Methods A. Materials B. Isolation of Spermatozopris Cytoskeletons C. Reactivation of Isolated Cytoskeletons References
I. Introduction The flagellategreen alga Spermatozopsis similis F’reisig et Melkonian (Chlorophyceae) was originally isolated from a small pond near Madingley, Cambridge, England, in April 1980. The crescent and spirally twisted cells are naked and 4-6 pm long. They bear two flagella of subequal length and contain a single chloroplast with an eyespot but lacking a pyrenoid (Preisig and Melkonian, 1984). Cells reproduce asexually in the flagellate state by longitudinal division into two progeny cells; sexual reproduction is unknown. S . similis can be grown in mass cultures in a modified Waris medium to cell density of 1-3 x lo7 cells/ ml (McFadden and Melkonian, 1986). The basal apparatus resembles that of Chlumydornonas reinhardtii in comprising two basal bodies interconnected by distal and proximal connecting fibers and four microtubular flagellar roots. In addition, two types of fibrous flagellar METHODS IN CELL BIOLOGY VOL. 47 Copyright 0 1995 by Academic Press. Inc. AII rights of reproduction in any form reserved.
335
336
Karl-Ferdinand Lechtreck and Michael Melkonian
roots occur in S. similis: a single small nucleus-basal body connector (NBBC), which contains the calcium-modulated EF-hand protein centrin (also known as caltractin), and two striated microtubule-associated fibers (SMAFs or system I fibers) which accompany the two-membered microtubular flagellar roots over most of their length (Melkonian and Preisig, 1984; McFadden et al., 1987; Pate1 et al., 1992). The complete absence of a cell wall or glycocalyx allows the isolation of structurally intact cytoskeletons from S. sirnilis by extraction with nonionic detergents (Nonidet P-40, Triton X-100) using minimal shear forces. The cytoskeletons retain the unique shape of intact cells and consist of the two axonemes, the interconnected basal bodies, the fibrous roots, the four microtubular flagellar roots, and 6-12 secondary cytoskeletal microtubules (SCMTs). The root microtubules and most of the SCMT are bound together posteriorly by a fibrous cap-like structure, the rhizosyndesmos (Fig. 1) (Lechtreck et al., 1989). The addition of 5 mM MgSO, to the isolation buffer yields nucleoflagellar apparatus complexes in which a stable nuclear remnant (the karyoskeleton) remains attached to the basal bodies by the NBBC (Figs. 2,3). Isolated cytoskeletons can be used as a source for biochemical analysis of cytoskeletal proteins such as tubulin and centrin (Hohfeld et al., 1994) or to isolate subcomponents of the flagellar apparatus such as basal apparatuses and SMAFs (Lechtreck et al., 1989). The coiled-coil forming protein SF-assemblin, which is the major component of SMAF in various green flagellates, was first isolated from the cytoskeletons of S. similis (Lechtreck and Melkonian, 1991; Weber et al., 1993; see also Chapter 44 in this volume). Some flagellar apparatus functions of S. similis have been reconstituted in uitro, namely, axonemal shedding, axonemal motility, and reorientation of the basal bodies (McFadden et al., 1987). Cytoskeletons isolated and reactivated at
46. Reactivation of Spermatozopsis Cytoskeletons
337
Fig. 1 Negatively stained whole-mount preparation of cytoskeletons of Spermatozopsis similis
isolated in MT buffer, illustrating the antiparallel basal body orientation. Cytoskeletons were allowed to adhere to pioloform-coated copper grids for 5 minutes and then stained with 1% (wh) uranyl acetate for 2 minutes. Bar = 5 pm. Adapted with permission from Lechtreck and Melkonian (1991, Fig. la, p. 707).
Fig. 2 Phase-contrast light micrograph of isolated cytoskeleton in reactivation buffer (without ATP) at M Ca2+.Bar = 5 pm. Fig. 3 At IO-’ M Ca2+,the basal bodies and axonemes are reoriented to the parallel orientation. Phase-contrast light micrograph of isolated cytoskeleton. Bar = 5 pm.
Fig. 4 Phase-contrast light micrograph of isolated cytoskeleton resuspended in lo-’ M Ca2+.
Bar 3b
=
-
5 pm. Figures 2 - 4 are adapted with permission from McFadden et al. (1987, Figs.
d, p. 906).
338
Karl-Ferdinand Lechtreck and Michael Melkonian
11. Methods
A. Materials Spermatozopsis similis (strain SAG 1.85; Schlosser, 1986) may be obtained from Sammlung von Algenkulturen, Pflanzenphysiologisches Institut , Nikolausberger Weg 18, D-37073 Gottingen, Federal Republic of Germany.
1. Equipment
Low-speed bench-top centrifuge Light microscope with phase-contrast optics Perfusion chamber (Hyams and Borisy, 1978)
2. Chemicals ATP CaC1, Dithiothreitol (DTT) Ethylene glycol bis(P-aminoethyl ether)-N,N’-tetraacetic acid (EGTA) 4-(2-Hydroxyethyl)- 1-piperazineethanesulfonicacid (Hepes) KCl KOH MgSo, Disodium ethylenediaminetetraacetic acid (Na,EDTA) Nonidet P-40 (NP-40) or Triton X-100 Paraformaldehyde Polyethylene glycol (PEG, M , 20,000) Sigmacote
3. Buffers MT buffer: 30 mM Hepes, 5 mM MgSO,, 25 mM KCI, 5 mM EGTA, adjusted with KOH to pH 7 Lysis buffer: MT buffer plus 0.2-2% (viv) NP-40 Reactivation buffer: 30 mM Hepes, 5 mM MgSO,, 25 mM KCl, 0.5% ( w h ) PEG, 1 mM DTT, 0.5 mM Na,EDTA, 1 mM ATP, solution adjusted to pH 7.3 at 22°C [A detailed description of Ca2+-bufferedreactivation solutions is given in Bessen et al. (1980). Buffers containing defined Ca2+concentrations can also be calculated using various programs (e.g., the program EQCAL, L. Backman, Biosoft, Cambridge, U .K.).l
46. Reactivation of Spermtotopsis Cytoskeletons
339
B. Isolation of Sperrnatozopsis Cytoskeletons 1. Concentrate cells by centrifugation at 500g in a bench-top centrifuge for 5 minutes. 2. Wash cells once in MT buffer and centrifuge as above. 3. Resuspend cells in ice-cold MT buffer and after 5 minutes add an equal volume of lysis buffer [0.2-2% NP-40 (v/v) in MT buffer]. Use MT buffer lacking MgS04 to isolate cytoskeletons without a karyoskeleton (Fig. 1). 4. For ultrastructural or immunological studies of isolated cytoskeletons, fix cytoskeletons with 3% ( v h ) formaldehyde in MT buffer for at least 20 minutes at room temperature. 5. For mass isolation of cytoskeletons, lyse cells with 1% NP-40 or Triton X-100(v/v, final concentration) in MT buffer and pellet the cytoskeletons at 3000g for 20 minutes. Wash the cytoskeletons several times with decreasing concentrations of detergent (0.5,0.25,0.1%, v/v) and twice without detergent. C. Reactivation of Isolated Cytoskeletons 1. Precool all buffers and equipment to 4°C. 2. Concentrate, wash, and lyse cells as described in Section II,B, steps 1-3, but use MT buffer plus 1% ( w h ) PEG and 1 mM DTT. 3. Centrifuge lysate at lOOOg in microtubes for 5 minutes. 4. Discard the green supernatant and resuspend the upper milky portion of the biphasic pellet in MT buffer plus PEG and DDT. 5 . Reactivate cytoskeletons within 6 hours of isolation by suspending a 5-pI aliquot of cytoskeletons in 2 ml reactivation buffer. 6. Observe reactivated cytoskeletons in phase contrast in a perfusion chamber such as that described by Hyams and Borisy (1978) using siliconized glass slides. 7. To induce forward swimming (Fig. 2), use calcium concentrations at or below 10 - 8 M in the standard reactivation buffer. 8. To induce basal body reorientation and backward swimming of cytoskeletons, adjust the calcium concentration to M (Fig. 4). Calcium M lead to shedding of axonemes from the cytoskelconcentrations 2 etons.
References Bessen, M.,Fay, R. B., and Witman, G . B. (1980). Calcium control of waveform in isolated axonemes of Chlamydomonas. J. Cell Biol. 86,446-455.
340
Karl-Ferdinand Lechtreck and Michael Melkonian
Hohfeld, I., Beech, P. L., and Melkonian, M. (1994). Immunolocalization of centnn in Oxyrrhis marina Dujardin (Dinophyceae). J . Phycol. 30, 474-489. Hyams, J. S . , and Borisy, G. G. (1978). Isolated flagellar apparatus of Chlamydomonas: characterization of forward swimming and alteration of waveform and reversal by calcium in uitro. J . Cell Sci. 33, 235-253. Kreimer, G. (1994). Cell biology of phototaxis in flagellate algae. Int. Rev. Cytol. 148, 229-310. Kreimer, G., Brohsonn, U., and Melkonian, M. (1991a). Isolation and partial characterization of the photoreceptive organelle for phototaxis of a flagellate green alga. Eur. J . Cell Biol. 55, 318-327. Kreimer, G., Marner, F.-J., Brohsonn, U., and Melkonian, M. (1991b). Identification of 11-cis and all-trans-retinal in the photoreceptive organelle of a flagellate green alga. FEBS Lett. 293, 49-52. Kreimer, G., and Witman, G. B. (1994). A novel touch-induced, Ca2+-dependentphobic response in a flagellate green alga. Cell Motil. Cyroskel. 29, 97-109. Lechtreck, K.-F., McFadden, G. I., and Melkonian, M. (1989).The cytoskeleton of the naked green flagellate Spermatoiopsis sirnilis: Isolation, whole mount electron microscopy, and preliminary biochemical and immunological characterization. Cell Motil. Cyroskel. 14,552-561. Lechtreck, K.-F., and Melkonian, M. (1991). Striated microtubule-associated fibers: Identification of assemblin, a novel 34-kD protein that forms paracrystals of 2-nm filaments in vitro. J. Cell Biol. 115, 705-716. McFadden, G. I., and Melkonian, M. (1986). Use of Hepes buffer for microalgal culture media and fixation for electron microscopy. Phycologia 25, 551-557. McFadden, G. I., Schulze, D., Surek, B., Salisbury, J. L., and Melkonian, M. (1987). Basal body reorientation mediated by a Ca2+-modulatedcontractile protein. J . Cell B i d . 105, 903-912. Melkonian, M., and Preisig, H. R. (1984). Ultrastructure of the flagellar apparatus in the green flagellate Sperrnatoiopsis similis. Plant Syst. Euol. 146, 145-162. Patel, H.,Lechtreck, K.-F., Melkonian, M., and Mandelkow, E. (1992). Structure of striated microtubule-associated fibers of flagellar roots. Comparison of native and reconstituted states. J . MoI. Biol. 227, 698-710. Preisig, H. R., and Melkonian, M. (1984). A light and electron microscopical study of the green flagellate Spermatozopsis similis sp. nov. Plant Sysr. Evol. 146, 57-74. Schlosser, U.G. (1986). Sammlung von Algenkulturen Gottingen: additions to the collection since 1984. Ber. Dtsch. Bot. Ges. 99, 161-168. Weber, K . , Geisler, N.,Plessman, U., Bremerich, A., Lechtreck, K.-F., and Melkonian, M. (1993). SF-assemblin, the structural protein of the 2-nm filaments from striated microtubule associated fibers of algal flagellar roots, forms a segmented coiled coil. J . Cell Biol. 121, 837-845.
Preparation and Reactivation of Spermatoxomis Cvtoskeletons Karl-Ferdinand Lechtreck and Michael Melkonian Botanisches Insitut Universitat zu Koln D-50931 Koln Federal Republic of Germany
I. Introduction 11. Methods A. Materials B. Isolation of Spermatozopris Cytoskeletons C. Reactivation of Isolated Cytoskeletons References
I. Introduction The flagellategreen alga Spermatozopsis similis F’reisig et Melkonian (Chlorophyceae) was originally isolated from a small pond near Madingley, Cambridge, England, in April 1980. The crescent and spirally twisted cells are naked and 4-6 pm long. They bear two flagella of subequal length and contain a single chloroplast with an eyespot but lacking a pyrenoid (Preisig and Melkonian, 1984). Cells reproduce asexually in the flagellate state by longitudinal division into two progeny cells; sexual reproduction is unknown. S . similis can be grown in mass cultures in a modified Waris medium to cell density of 1-3 x lo7 cells/ ml (McFadden and Melkonian, 1986). The basal apparatus resembles that of Chlumydornonas reinhardtii in comprising two basal bodies interconnected by distal and proximal connecting fibers and four microtubular flagellar roots. In addition, two types of fibrous flagellar METHODS IN CELL BIOLOGY VOL. 47 Copyright 0 1995 by Academic Press. Inc. AII rights of reproduction in any form reserved.
335
336
Karl-Ferdinand Lechtreck and Michael Melkonian
roots occur in S. similis: a single small nucleus-basal body connector (NBBC), which contains the calcium-modulated EF-hand protein centrin (also known as caltractin), and two striated microtubule-associated fibers (SMAFs or system I fibers) which accompany the two-membered microtubular flagellar roots over most of their length (Melkonian and Preisig, 1984; McFadden et al., 1987; Pate1 et al., 1992). The complete absence of a cell wall or glycocalyx allows the isolation of structurally intact cytoskeletons from S. sirnilis by extraction with nonionic detergents (Nonidet P-40, Triton X-100) using minimal shear forces. The cytoskeletons retain the unique shape of intact cells and consist of the two axonemes, the interconnected basal bodies, the fibrous roots, the four microtubular flagellar roots, and 6-12 secondary cytoskeletal microtubules (SCMTs). The root microtubules and most of the SCMT are bound together posteriorly by a fibrous cap-like structure, the rhizosyndesmos (Fig. 1) (Lechtreck et al., 1989). The addition of 5 mM MgSO, to the isolation buffer yields nucleoflagellar apparatus complexes in which a stable nuclear remnant (the karyoskeleton) remains attached to the basal bodies by the NBBC (Figs. 2,3). Isolated cytoskeletons can be used as a source for biochemical analysis of cytoskeletal proteins such as tubulin and centrin (Hohfeld et al., 1994) or to isolate subcomponents of the flagellar apparatus such as basal apparatuses and SMAFs (Lechtreck et al., 1989). The coiled-coil forming protein SF-assemblin, which is the major component of SMAF in various green flagellates, was first isolated from the cytoskeletons of S. similis (Lechtreck and Melkonian, 1991; Weber et al., 1993; see also Chapter 44 in this volume). Some flagellar apparatus functions of S. similis have been reconstituted in uitro, namely, axonemal shedding, axonemal motility, and reorientation of the basal bodies (McFadden et al., 1987). Cytoskeletons isolated and reactivated at
46. Reactivation of Spermatozopsis Cytoskeletons
337
Fig. 1 Negatively stained whole-mount preparation of cytoskeletons of Spermatozopsis similis
isolated in MT buffer, illustrating the antiparallel basal body orientation. Cytoskeletons were allowed to adhere to pioloform-coated copper grids for 5 minutes and then stained with 1% (wh) uranyl acetate for 2 minutes. Bar = 5 pm. Adapted with permission from Lechtreck and Melkonian (1991, Fig. la, p. 707).
Fig. 2 Phase-contrast light micrograph of isolated cytoskeleton in reactivation buffer (without ATP) at M Ca2+.Bar = 5 pm. Fig. 3 At IO-’ M Ca2+,the basal bodies and axonemes are reoriented to the parallel orientation. Phase-contrast light micrograph of isolated cytoskeleton. Bar = 5 pm.
Fig. 4 Phase-contrast light micrograph of isolated cytoskeleton resuspended in lo-’ M Ca2+.
Bar 3b
=
-
5 pm. Figures 2 - 4 are adapted with permission from McFadden et al. (1987, Figs.
d, p. 906).
338
Karl-Ferdinand Lechtreck and Michael Melkonian
11. Methods
A. Materials Spermatozopsis similis (strain SAG 1.85; Schlosser, 1986) may be obtained from Sammlung von Algenkulturen, Pflanzenphysiologisches Institut , Nikolausberger Weg 18, D-37073 Gottingen, Federal Republic of Germany.
1. Equipment
Low-speed bench-top centrifuge Light microscope with phase-contrast optics Perfusion chamber (Hyams and Borisy, 1978)
2. Chemicals ATP CaC1, Dithiothreitol (DTT) Ethylene glycol bis(P-aminoethyl ether)-N,N’-tetraacetic acid (EGTA) 4-(2-Hydroxyethyl)- 1-piperazineethanesulfonicacid (Hepes) KCl KOH MgSo, Disodium ethylenediaminetetraacetic acid (Na,EDTA) Nonidet P-40 (NP-40) or Triton X-100 Paraformaldehyde Polyethylene glycol (PEG, M , 20,000) Sigmacote
3. Buffers MT buffer: 30 mM Hepes, 5 mM MgSO,, 25 mM KCI, 5 mM EGTA, adjusted with KOH to pH 7 Lysis buffer: MT buffer plus 0.2-2% (viv) NP-40 Reactivation buffer: 30 mM Hepes, 5 mM MgSO,, 25 mM KCl, 0.5% ( w h ) PEG, 1 mM DTT, 0.5 mM Na,EDTA, 1 mM ATP, solution adjusted to pH 7.3 at 22°C [A detailed description of Ca2+-bufferedreactivation solutions is given in Bessen et al. (1980). Buffers containing defined Ca2+concentrations can also be calculated using various programs (e.g., the program EQCAL, L. Backman, Biosoft, Cambridge, U .K.).l
46. Reactivation of Spermtotopsis Cytoskeletons
339
B. Isolation of Sperrnatozopsis Cytoskeletons 1. Concentrate cells by centrifugation at 500g in a bench-top centrifuge for 5 minutes. 2. Wash cells once in MT buffer and centrifuge as above. 3. Resuspend cells in ice-cold MT buffer and after 5 minutes add an equal volume of lysis buffer [0.2-2% NP-40 (v/v) in MT buffer]. Use MT buffer lacking MgS04 to isolate cytoskeletons without a karyoskeleton (Fig. 1). 4. For ultrastructural or immunological studies of isolated cytoskeletons, fix cytoskeletons with 3% ( v h ) formaldehyde in MT buffer for at least 20 minutes at room temperature. 5. For mass isolation of cytoskeletons, lyse cells with 1% NP-40 or Triton X-100(v/v, final concentration) in MT buffer and pellet the cytoskeletons at 3000g for 20 minutes. Wash the cytoskeletons several times with decreasing concentrations of detergent (0.5,0.25,0.1%, v/v) and twice without detergent. C. Reactivation of Isolated Cytoskeletons 1. Precool all buffers and equipment to 4°C. 2. Concentrate, wash, and lyse cells as described in Section II,B, steps 1-3, but use MT buffer plus 1% ( w h ) PEG and 1 mM DTT. 3. Centrifuge lysate at lOOOg in microtubes for 5 minutes. 4. Discard the green supernatant and resuspend the upper milky portion of the biphasic pellet in MT buffer plus PEG and DDT. 5 . Reactivate cytoskeletons within 6 hours of isolation by suspending a 5-pI aliquot of cytoskeletons in 2 ml reactivation buffer. 6. Observe reactivated cytoskeletons in phase contrast in a perfusion chamber such as that described by Hyams and Borisy (1978) using siliconized glass slides. 7. To induce forward swimming (Fig. 2), use calcium concentrations at or below 10 - 8 M in the standard reactivation buffer. 8. To induce basal body reorientation and backward swimming of cytoskeletons, adjust the calcium concentration to M (Fig. 4). Calcium M lead to shedding of axonemes from the cytoskelconcentrations 2 etons.
References Bessen, M.,Fay, R. B., and Witman, G . B. (1980). Calcium control of waveform in isolated axonemes of Chlamydomonas. J. Cell Biol. 86,446-455.
340
Karl-Ferdinand Lechtreck and Michael Melkonian
Hohfeld, I., Beech, P. L., and Melkonian, M. (1994). Immunolocalization of centnn in Oxyrrhis marina Dujardin (Dinophyceae). J . Phycol. 30, 474-489. Hyams, J. S . , and Borisy, G. G. (1978). Isolated flagellar apparatus of Chlamydomonas: characterization of forward swimming and alteration of waveform and reversal by calcium in uitro. J . Cell Sci. 33, 235-253. Kreimer, G. (1994). Cell biology of phototaxis in flagellate algae. Int. Rev. Cytol. 148, 229-310. Kreimer, G., Brohsonn, U., and Melkonian, M. (1991a). Isolation and partial characterization of the photoreceptive organelle for phototaxis of a flagellate green alga. Eur. J . Cell Biol. 55, 318-327. Kreimer, G., Marner, F.-J., Brohsonn, U., and Melkonian, M. (1991b). Identification of 11-cis and all-trans-retinal in the photoreceptive organelle of a flagellate green alga. FEBS Lett. 293, 49-52. Kreimer, G., and Witman, G. B. (1994). A novel touch-induced, Ca2+-dependentphobic response in a flagellate green alga. Cell Motil. Cyroskel. 29, 97-109. Lechtreck, K.-F., McFadden, G. I., and Melkonian, M. (1989).The cytoskeleton of the naked green flagellate Spermatoiopsis sirnilis: Isolation, whole mount electron microscopy, and preliminary biochemical and immunological characterization. Cell Motil. Cyroskel. 14,552-561. Lechtreck, K.-F., and Melkonian, M. (1991). Striated microtubule-associated fibers: Identification of assemblin, a novel 34-kD protein that forms paracrystals of 2-nm filaments in vitro. J. Cell Biol. 115, 705-716. McFadden, G. I., and Melkonian, M. (1986). Use of Hepes buffer for microalgal culture media and fixation for electron microscopy. Phycologia 25, 551-557. McFadden, G. I., Schulze, D., Surek, B., Salisbury, J. L., and Melkonian, M. (1987). Basal body reorientation mediated by a Ca2+-modulatedcontractile protein. J . Cell B i d . 105, 903-912. Melkonian, M., and Preisig, H. R. (1984). Ultrastructure of the flagellar apparatus in the green flagellate Sperrnatoiopsis similis. Plant Syst. Euol. 146, 145-162. Patel, H.,Lechtreck, K.-F., Melkonian, M., and Mandelkow, E. (1992). Structure of striated microtubule-associated fibers of flagellar roots. Comparison of native and reconstituted states. J . MoI. Biol. 227, 698-710. Preisig, H. R., and Melkonian, M. (1984). A light and electron microscopical study of the green flagellate Spermatozopsis similis sp. nov. Plant Sysr. Evol. 146, 57-74. Schlosser, U.G. (1986). Sammlung von Algenkulturen Gottingen: additions to the collection since 1984. Ber. Dtsch. Bot. Ges. 99, 161-168. Weber, K . , Geisler, N.,Plessman, U., Bremerich, A., Lechtreck, K.-F., and Melkonian, M. (1993). SF-assemblin, the structural protein of the 2-nm filaments from striated microtubule associated fibers of algal flagellar roots, forms a segmented coiled coil. J . Cell Biol. 121, 837-845.