On the Localization of Voltage-sensitive Calcium Channels in the Flagella of Chlamydomonas reinhardtii

This study was undertaken to prove that voltage-sensitive calcium channels controlling the photophobic stop response of the unicellular green alga Chlamydomonas reinhardtii are exclusively found in the flagellar region of the cell and to answer the question as to their exact localization within the flagellar membrane. The strategy used was to amputate flagella to a variable degree without perturbing the electrical properties of the cell and measure flagellar currents shortly after amputation and during the subsequent regeneration process. Under all conditions, a close correlation was found between current size and flagellar length, strongly suggesting that the channels that mediate increases in intraflagellar calcium concentration are confined to and distributed over the total flagellar length. Bald mutants yielded tiny flagellar currents, in agreement with the existence of residual flagellar stubs. In the presence of the protein synthesis inhibitor cycloheximide, flagellar length and flagellar currents also recovered in parallel. Recovery came to an earlier end, however, leveling off at a time when in the absence of cycloheximide only half maximal values were achieved. This suggests the existence of a pool of precursors, which permits the maintenance of a constant ratio between voltage-sensitive calcium channels and other intraflagellar proteins. C HLAMYDOMONAS reinhardtii, a unicellular green freshwater alga, possesses a photosensory system and two flagella, which enable it to translate information about intensity and direction of the ambient light into defined orientational responses (Boscov and Feinleib, 1979). During normal forward swimming the two flagella move in a breast stroke-like fashion 50 times per second. An apparent asymmetry in the beating pattern leads to a rotation around the longitudinal axis with a frequency of 2 Hz. Thereby, one revolution takes between 20 and 25 strokes, i.e., 500 ms. This allows the alga to screen the ambient light and to correct for changes in its direction (Foster and Smyth, 1980; Riiffer and Nultsch, 1985, 1987, 1990). Depending on the brightness of the actinic light, three classes of behavioral responses can be distinguished: (a) in weak light, Chlamydomonas swims towards the light (positive phototaxis); (b) in stronger light, it swims away from it (negative phototaxis); and (c) in very strong light, particularly when there are sudden and pronounced changes in intensity, it stops transiently. In the course of this "stop response; Chlamydomonas changes from its normal forward swimming to a phase of slower backward swimming, caused by an undulation movement of the flagella. Afterwards it resumes forward swimming in a new direction. Stop responses start within 50 ms of the stimulus and last for about 300 ms (Uhl and Hegemann, 1990). The authors' present address is Botanisches Institut der Ludwig Maximilians Universitit, Menzinger Strasse 67, D-80638 Miinchen, Germany. There is good evidence that the different flageUar beating patterns are all governed by the internal calcium concentration (Schmidt and Eckert, 1976). This has been concluded from in vitro experiments with isolated flagella (Hyams and Borisy, 1978) and the flagella of detergent-extracted cell models (Bessen et al., 1980; Kamiya and Witrnan, 1984), where it was found that all naturally occurring beating patterns can be mimicked simply by changing the free calcium concentration in the medium. In vivo changes in [Cal] can arise from calcium release from internal stores or from a change in the balance between calcium influx through channels in the cell membrane and calcium expulsion through calcium pumps or exchange carriers. While evidence for the former is still lacking, it has been established that rapid lightinduced changes in internal calcium can be brought about by two classes of ion channels: one is located in the eyespot region of the cell, where the photoreceptor molecules reside, and the other is confined to the flagellar region. According to the electrical model of Harz and Hegemann (1991) the immediate action of light is to open the channels in the eyespot region, leading to an inward current with concomitant cell depolarization. Current amplitude is graded with photon exposure and when its size exceeds a critical level opens the presumably voltage-sensitive channels in the flageUar region. Under physiological conditions both currents are carried by calcium ions. The threshold for the opening of flagellar channels coincides with the threshold for the stop response, suggesting that the undulation movement of the flagella is caused by a massive local calcium influx (Harz et al. 1992). © The Rockefeller University Press, 0021-9525/94/06/1119/7 $2.00 The Journal of Cell Biology, Volume 125, Number 5, June 1994 1119-1125 1119 on July 7, 2017 jcb.rress.org D ow nladed fom So far an exact localization of the voltage-sensitive calcium channels in the flagellar region has not been possible. On the basis of freeze-etch electron microscopic studies it has been suggested that the calcium conductances may reside in the ciliary necklace, with the ciliary necklace particles being the calcium channels (Gilula and Satir, 1972; Fisher et al., 1976). This communication, however, which uses an electrophysiological assay and a gentle procedure for producing healthy cells with flagella that have been amputated to various degrees, comes to a different conclusion. Under all conditions tested the size of the flagellar currents was correlated with the flagdlar length, indicating that the voltagesensitive calcium channels are equally distributed over the total length of both flagella. Materials and Methods Culturing Vegetative and Gametic Cells Chlaraydomonas reinhardtii cells of the cell wall-deficient mutant CW-2 were grown for 10-14 d on high salt acetate medium plates (HSM according to Sueoka et ai., 1967, supplemented with 15 mM sodium acetate, 250 mM sorbitol as osmoticum, 0.3 % yeast-extract, and 0.9 % agar IMA, Niirnberg, Germany) in the presence of continuous white light of 2 Wm -2. While vegetative cells and gametes yield photocurrents of similar shape and size, the photosensitivity is higher in the case of gametes (Hegemann, P., personal communication). Therefore, cells were allowed to differentiate into gametes by transforring them from one dish into 10-12 ml nitrogen-deficient minimal medium (NMM) (Hegemann et al., 1988), containing 125 mM sorbitol. After 3 h the cells were pelleted and resuspended into 10-12 ml of NMM without sorbitol. By this time the cells had stabilized to such an extent that only a minor fraction was lysed due to the osmotic shock. Differentiation into flagellated cells was completed after 24 h. Tests for mating competence were not carried out, however. The phototactically most active cells were separated by photoselection in a low salt (freshwater-like) medium containing 3 mM K2HPO4, 100 #M CaCI2, adjusted to pH 6.8 with HC1. They were subsequently resuspended in the same medium and dark adapted for 1 h before an experiment. All incubations were carried out in 50-ml polypropylen tubes at a concentration of 1.5 x 10 s cells m1-1. To allow for optimal aeration, cell suspensions were shaken at 190 rpm on an orbit shaker (Bachofer GmbH, Reutingen, Germany). Mechanical Removal of the Flagella Immediately before an experiment, cells were pelleted and resuspended into 5 mi "quasi-intracellular" medium (3.5 x l0 s cells ml-t), mimicking the proposed intracellular milieu (Nichols and Rikmenspoel, 1978). It contained 10 mM K+-ATP, 3 mM MgC12, and 60 mM K +, adjusted to pH 6.8 by mixing suitable amounts of 30 mM K2HPO4 and 60 mM KI-I2PO4. A free calcium concentration of 100 nM was achieved by titrating 200 ttM BAPTA (1,2,-bis(2-aminophennxy)ethane N,N,/V,/V-tetraacetic acid) with CaC12 according to Tsien (1980). Flagella were removed by sheafing forces in a 10 ml potter homogenizer. The amputated cells were immediately diluted 10-fold, using a medium which brought the concentration of the various salts back to freshwater conditions. Owing to the experimental procedure the medium contains 100/tM Ca 2+, 8.5 mM K +, 1.5 mM CI-, 1 raM ATE 300 ttM Mg 2+, and 20 tiM BAPTA. The presence of Mg 2+ and ATP has no measurable effect on the electrical currents. To keep the cells dark adapted the whole procedure was performed under red light and at room temperature. Photoreceptor currents with maximal amplitude could be recorded a few minutes following this treatment. Flngellar regeneration kinetics of a CW-2 population were measured under the same conditions, except that they were not exposed to repetitive actinic flashes. Aliquots were taken at given time intervals and the cells were fixed by the addition of 0.37 % formaldehyde (final concentration). Flagellar length was determined using a 100x oil immersion objective (NA = 1.3) and an ocular micrometer. Values from 10-15 cells were averaged for each data point, t = 0 marks the beginning of the amputation. In the case of the long-zero experiments the kinetics of flagellar regeneration were measured from a single cell by frame to frame analysis of a video film. Electrical Recording from DeflageUated Cells Photocurrents were recorded using the suction pipette technique (Litvin et al., 1978; Baylor et al., 1979), as previously applied to Ch/amydomonas by Harz and Hegernann (1991). In order to suck a substantial fraction of the cell into the pipette in a most gentle manner, pipettes with nearly parallel tip walls were used. They were pulled in two steps from borosilicat capillaries (OD 1.6 raM, wall thickness 0.5 mm, Hilgenberg, Malsfold, Germany). Since the openings were too small after pulling, the tips were broken with the help of a small, heated glass sphere, and subsequently polished such that an opening of 2-4/~m resulted. Pipette resistances of 60-80 M r were obtained. Cells were sucked into the pipette under microscopic control, using infrared illumination from an IR-LED (HLP-40; Hitachi Instrs., Inc., San Jose, CA), emitting 40 mW of radiation at 780 nm), a 40x PlanNcofluoar objective (Zeiss, Oberkochen, Germany) and an IR-sensitive CCD camera (i2S; Bordeaux-Cedex, France). Suction was applied until about one third of the cell was in the pipette and the resistance had increased to 100-150 Mr . This was accomplished with the help o f t low pressure application after Baylor et al. (1979). Photostimulation was achieved through the epifluorescence attachment of the inverted microscope, using the objective as condensor system. A xenon-flashlamp (EG&G Electro-Optics, Salem, MA) produced flashes of 40/~s duration, which were filtered through a broadband interference filter transmitting maximally at 500 rim, the absorption maximum of Chlamydomonas rhodopsin (Foster et ai., 1984; Uhl and Hegemann, 1990; Beckmann and Hegemann, 1991; Harz and Hegemarm, 1991). Electrical signals were recorded with a holding potential of 0 mV, using an EPC-7 patch damp amplifier (List, Darmstadt, Germany). Signals were passed through a 3-kHz low-pass Bessel filter and fed into the A/Dconverter of a TL-1 DMA computer interface, used in a 486 IBMcompatible computer. Data acquisition and analysis was performed under pClamp 5.5.1 (Axon Instruments, Foster City, CA). Single current traces were filtered with a digital gaussian filter to 500 Hz. Autolysin Treatment of Bald Mutants Bald mutants were cultured in the same way as the CW-2, except that their HSM plates contained no sorbitol. Recording electrical currents from these cells, which possess a cell wall, requires its removal. It was achieved using cell wnil-degrading enzyme released by mating gametes (Matsuda et al., 1985) (Jaenicke et al., 1987), kindly provided by Dr. Walfenschmidi (University of Cologne, Germany). Samples of lyophylized enzyme were dissolved in distilled water, bringing the concentration of Ca 2+ and Mg 2+ to 100/~M and I mM, respectively, and mixed with 2 vol of the cell suspension. Cycloheximide at 5 #g/ml (final concentration) was supplemented to inhibit the synthesis of new ceil wall proteins. After incubation for 1 h at 180C, the mixture contained cells that had shed main components of their cell wall (Imam and Snell, 1988; Monk, 1988; Walfenschmidt et ai., 1988), as judged from the fact that they could be sucked into the pipette without complications, and yielded seal resistances comparable to those obtained with the CW-2 mutant. Results and Discussion Experimental Strategy There are two fast, light-dependent calcium currents in Chlamydomonas: a primary calcium inward current in the eyespot region, termed photoreceptor current (P), and a secondary calcium inward current in the flagellar region, termed flagellar current (F)). (P) follows the stimulus with virtually no delay, while F follows P with a variable delay of 5-100 ms (data not shown). Both currents are transient. They can be recorded as negative currents when the respective source regions are inside the pipette and as positive currents when the source regions are outside, a configuration which is much more frequently encountered (Fig. 1 a). Mixed constellations, i.e., flagella inside and eyespot outside the pipette (Fig. 1 b) or eyespot inside and flagella outside the pipette (Fig. 1 c), yield current traces with a positive and a negative component. The fact that the time course of both The Journal of Cell Biology, Volume 125, 1994 1120 on July 7, 2017 jcb.rress.org D ow nladed fom