Cell Biology International 28 (2004) 387e396 www.elsevier.com/locate/cellbi Analysis of the contraction of an organelle using its birefringency: the R-fibre of the Ceratium (Dinoflagellate) flagellum* ) Hidemi Satoa, Claude Greuetb, Monique Cachonb, Jacky Cossonb, aNagano University, Shimonogo, Ueda-shi, Nagano 386-12, Japan bU.M.R. 7009 du CNRS, Biologie du De´veloppement, Observatoire Oce´anologique de Villefranche-sur-Mer, 06230 Villefranche-sur-mer, France Received 23 September 2003; revised 19 January 2004; accepted 15 March 2004 Abstract Some organelles responsible for contraction consist of bundles of 2e4 nm filaments called nanofilaments. Such organelles are present in the longitudinal flagellum of Ceratium (Dinoflagellate): the R-fibre is the motor system for contraction and parallels the axoneme, which is responsible for wave generation. We used a highly sensitive polarization microscope developed by one of the authors to measure the birefringence of these nanofilament bundles during contraction in vivo. Our results show that the R-fibre gives a highly birefringent signal, retarding the polarization to much the same extent irrespective of the direction of polarization. By rotating the axis of the microscope compensator we confirmed that the birefringence is positive, suggesting that the bundles run parallel to the longitudinal axis of the flagellum. Conversely, when the compensator was rotated contrary to the direction of retardation, the bundle appeared dark (except when the organelle was in a fully contracted state). Experiments performed on detergent-treated and ATP-reactivated flagella show that a portion of the flagella regained activity with the addition of ATP in the presence of low Ca2C concentrations. This demonstrates the ability to reactivate flagellar motility after permeabilization and that axonemal microtubules were not responsible for the strong flagellar birefringence. Combined with complementary data from DIC microscopy of demembranated flagella and electron microscopy, these findings have led to the development of a model of the R-fibre and a comparison with other types of birefringent nanofilament bundles, such as the myoneme of Acantharia. Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Polarizing microscopy; Nanofilaments; Myoneme; Flagellar motility 1. Introduction (Cachon et al., 1983) and myonemes (Febvre, 1971) of Acantharia. Some protists are able, by virtue of special organelles, All these organelles have 2e4 nm nanofilament to contract parts of their cell bodies. Examples are bundles in their structure. Optical anisotropy has been found in ciliates: the stalk of Vorticella (Amos, 1971), studied by polarizing microscopy in only a few such the appendix of Tontonia (Greuet et al., 1986), the organelles (the Acantharian myonemes, Febvre et al., flagellar rootlets of Chlamydomonas (Salisbury and 1990): they are birefringent and the sign of birefringence Floyd, 1978), and in Dinoflagellates the peduncule can be either positive or negative. Therefore polarization of Noctiluca (Soyer, 1968, 1970), Erythropsidinium microscopy appears as a technique of choice for in vivo (Greuet, 1967, 1981) and Leptophyllus (Cachon and quantitative measurements of the degree of compaction Cachon, 1964). Other examples are the flagellar rootlets of the flagellar organelles made of arrays of very small filaments (2e4 nm) which parallel the axoneme, in the longitudinal flagellum of Dinoflagellates of the Ceratium * This publication is presented in honor of Prof. Hidemi Sato on the occasion of his 77th birthday. group. The aim of polarization microscopy is mostly to ) Corresponding author. quantify low levels of local birefringence, so-called E-mail address: [email protected] (J. Cosson). ‘retardance’ of light in a specimen observed at very 1065-6995/$ - see front matter Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2004.03.007 388 H. Sato et al. / Cell Biology International 28 (2004) 387e396 high magnification. Retardance is evaluated in radians a Panasonic CCD camera and a Super VHS (NTSC) by rotation of the polarization filter (compensator) in- video tape recorder. The nature and sign of birefrin- cluded in the optical path. As these measurements need gence in flagella were determined by altering the re- also highly sensitive detection (video camera or film) we fractive indices of the surrounding media (Sato et al., used a highly sensitive polarizing microscope to obtain 1975). Images were obtained either from video monitor information about the contraction state of the bundles photographs, or by digitalisation of still frames recorded of nanofilaments. So far, most studies of birefringent on S-VHS and enhanced using MacIntosh software. organelles were devoted to chromosomal organisation (Cachon et al., 1989) or mitotic spindles (Sato et al., 2.3. Electron microscopy 1975). In our present study on the Ceratium flagellum, the latter always appeared to be birefringent, either Fixation was carried out according to Maruyama while it contracts or during its contractionerelaxation (1981,1982). The unicells were fixed in 0.1 M phosphate cycles (Cachon et al., 1991, 1992). In the present paper buffer containing 5% glutaraldehyde, 0.8e1.0 M glu- we describe additional results obtained after demem- cose, pH 7.4e7.8 at room temperature for 1 h, washed branation and other treatments of the flagellum, as well in 0.3 M phosphate and 0.8 M glucose, post-fixed in 2% as ultrastructural features by electron microscopy. OsO4 in phosphateeglucose buffer, progressively dehy- drated, then finally embedded in Spurr’s low viscosity medium (Spurr, 1969). Sections were cut with a MT2 2. Materials and methods Porter Blum microtome, stained with 9% uranyl acetate in methanol followed by lead citrate, then examined 2.1. Biological materials with a Hitachi H 603 electron microscope. Two species of Ceratium, C. furca (Ehrenberg) and C. 2.4. Detachment of flagella, permeabilization limulus (Gourret), were collected by filtration with thin and reactivation nylon nets (5e20 mm mesh) in Villefranche Bay (France) and Calvi Bay (STARESO, Corsica), and during the 1991 We intended to solubilize the microtubules of the autumn in Toba Bay (Marine Biological Laboratory of axoneme to see if the bundle of nanofilaments was still Sugashima, Nagoya University, Japan). Individual speci- positively birefringent. In some experiments flagella mens were isolated with a Pasteur pipette under a binocular were detached from the cells by an increase of the Ca2C microscope and maintained at room temperature for several concentration up to 20 mM in seawater containing hours in Petri dishes containing Millipore-filtered seawater. 20 mM TriseCl pH 8.2 to prevent any pH shift. A medium, commonly used for dynein coupled activity 2.2. Polarizing microscopy and DIC microscopy (Cosson et al., 1988) was used to permeabilize in situ or detached flagella. This medium is composed of: 0.1 M The specimens were suspended in seawater on a glass potassium glutamate, 0.5 M glucose, 20 mM TriseCl, slide with a coverslip and a thin layer of spacer (Valap, 2 mM EGTA, 4e10 mM Mg acetate, 1 mM dithiothrei- a mixture of Vaseline, Lanoline and Paraffin 2:2:1). A tol, 0.007% Triton X-100, pH adjusted to 7.8 with highly sensitive polarizing microscope, designed by Sato KOH (chemicals were from Sigma, St Quentin, 38297 et al. (1991), specially constructed by Nikon Engineering France). Most flagella remained contracted inside the Co. and located in the Sugashima Marine Biological flagellar pocket, but in a few cases isolated flagella were Laboratory, was used for all observations. All optical observed. These were reactivated by addition of 1 mM lenses including Apochromatic 60! objective lenses ATP, as previously observed for Oxyrrhis flagella (K.K. Nikon, Tokyo and Olympus Optical K.K. Tokyo) (Cosson et al., 1988). For assays of microtubule sliding, and a rectifier (Nikon, K.K.) were selected for high optical the axonemes were briefly digested in the same buffer as quality. The system was set on a vertical optical bench and above with trypsin (0.1 mg/ml) at room temperature. images were collected with a Hamamatsu C.1 100 type SIT camera (Hamamatsu Photonics, Hamamatsu), which can be switched to a photomicrographic recording system 3. Results by 90( rotation of the supporting device (Sato et al., 1991). In addition, this microscope arrangement can 3.1. Behaviour of the flagellum of native easily be converted to DIC (Differential Interference cells observed with DIC optics (Fig. 1) Contrast) by inserting two Wollaston prisms into the optical path. All retardation measurements were obtained The longitudinal flagellum of Ceratium issues from with a BraceeKo¨hler type compensator. a long basal body located at the bottom of a deep The flagellar movement patterns and associated cylindrical flagellar pocket about 6 mm in diameter and changes of birefringence were routinely recorded with 16 mm in length (Cachon et al., 1992). This flagellum H. Sato et al. / Cell Biology International 28 (2004) 387e396 389 Fig. 1. Photographs obtained with Differential Interference Contrast (DIC in aec) and Dark Field (DF in dei) microscopy of three different states of contraction of the longitudinal flagellum of C. furca in aec where only the flagellated half of the cell is visible and of C. limulus in dei only the flagellum is visible, arrow head at origin. In d and in gei, multistrobe illumination at 150 Hz flash frequency. a and d Z relaxed state, with sine waves; b and e Z beginning of contraction (20 ms); c and f Z fully contracted at 40 ms (arrow). gei Z decontraction (1, 2 & 3 s after contraction, respectively). Bar scale in lower part of i panel Z 100 mm. Cell body located on top left of each photograph. reaches 200e300 mm length in some individuals and it state of the elements present in the flagellum, the beats in pseudo-sinusoidal waves, which are quasi-planar axoneme itself, which is paralleled by fibres (see details and circular arc shaped (Brokaw and Wright, 1963).