Intracellular calcium redistribution during mating in Chlamydomonas reinhardii

Experimental Cell Research 160 (1985) 371-379

Intracellular Calcium Redistribution During Mating in Chlamydomonas reinhardii DEBORAH D. KASKA,‘,*

IRENE C. PISCOPO’ and AHARON GIBOR’

‘Department of Biological Sciences, Uniwrsity of California, Santa Barbara, CA 93106, and 2Philips Electronic Instruments Inc. Mahwah, NJ 07430, USA

Gametes of the unicellular green alga Chlamydomonas reinhardii recognize and adhere to cells of the opposite mating type- by flagellar contact. Adhesion between these specialized organelles signals a rapid series of mating events which result in gamete fusion. The sequence of morphological changes (flagellar tip activation, cell wall loss, and mating structure elongation), which occur as a consequence of the sexual signalling, have been characterized. The signalling mechanisms have, however, not been defined. Calcium is known to be involved during fertilization of animal species. Increased intracellular free calcium, which can be achieved either by calcium influx or by mobilization of ions from intracellular stores, has been observed during activation of both eggs and sperm. A recent report by Bloodgood & Levin that gametes of C. reinhardii preloaded with 4’Ca showed a transient increase in Ca efflux following mating, suggests that intracellular Ca redistribution may also accompany mating in this algal species. We have used X-ray microanalysis to analyze the subcellular distribution of bound calcium during mating in Chlamydomonas reinhardii. X-ray maps reveal that calcium is sequestered in discrete granules within the gamete cell body prior to mating and that during activation and cell fusion, calcium is diise throughout the cell. This suggests the possibility that calcium serves as a second messenger in this species.

Mating in Chlamydomonas reinhardii, a unicellular biflagellate green alga, has been extensively studied as a model system for cellular communication [l-4]. The interaction between gametes in this species is initiated by flagellar recognition and adhesion, which elicits a specific series of responses from both cells of the mating pair. These responses include a change in flagellar morphology [5], release of an autolysin [6] which dissolves the glycoprotein cell wall matrix, and activation of characteristic mating structures [7] (mating type-specific alterations of the cell surface). When the actin-filled fertilization tubule which emerges from the surface of the mating type plus (MT+) gamete contacts a raised region on the surface of the mating type minus (MT-) gamete, membrane fusion occurs, followed by complete merging of the cell bodies. Numerous studies have been directed toward examination of the structural changes and identification of the surface molecules involved in gamete recognition [7-lo]. The intracellular responses to surface stimulation have, however, been largely unexplored. Recently studies by Bloodgood & Levin [ll] have suggested that intracellular free calcium may increase during mating. Extracellu* To whom offprint requests should be sent. 25-858340

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372 Kaska, Piscopo and Gibor lar calcium is not required for successful mating in this species; however, gametes proloaded with 45Ca showed a transient increase in calcium efflux following mating. As suggested by Bloodgood & Levin, this calcium efflux may result from release of calcium from intracellular storage sites. Another possibility, however, is that a significant amount of calcium is secreted from activated gametes during release of the wall dissolving autolysin. Snell et al. [12] reported that lidocaine, an anaesthetic which inhibits divalent cation movement across membranes, blocked cell wall loss and gamete fusion in C. reinhardii, while flagellar adhesion and tip activation remained normal. Calcium was found to antagonize the lidocaine inhibition, suggesting the involvement of calcium in sexual signalling. Calcium has been shown to be involved during gamete interactions in a number of animal species. Release of intracellular free calcium during fertilization has been directly visualized by the luminescence of aequorin introduced by microinjection [13, 141, and both sperm and eggs of several marine species are activated by the calcium ionophore A-23187 [15, 161. In the presence of external calcium, A-23187 causes sperm to undergo the acrosomal reaction in which an actin-filled filament is extruded. Ionophore activation of eggs, however, is generally independent of external calcium; the ionophore releases calcium from intracellular stores. Cortical granule exocytosis and elevation of the fertilization membrane occur as a consequence of increased cytoplasmic free calcium. Several proteins in animal cells serve as intracellular receptors for free calcium generated in response to a stimulus. These proteins, such as calmodulin, when bound to calcium, activate a number of intracellular enzymes resulting in both metabolic and structural alteration of the cells [17, 181.The presence of calmodulin in plants, including Chlamydomonas reinhardii [ 19, 201, suggests that calcium may also serve to mediate plant response to stimuli. Calcium-linked processes in algae include stimulation of chloroplast rotation in Mougeotia [21], phytochrome dependent depolarization and inhibition of cytoplasmic streaming in Nitella [22, 231, and flagellar waveform in Chlamydomonas [24, 251. Aequorin-injected cells of Chara and Nitella showed an increase in free cytosolic calcium in response to electrical stimulation [23, 261. X-ray microanalysis was used in this study to analyse the subcellular distribution of calcium during mating in Chlamydomonas reinhardii. X-ray mapping of cells at various stages in the mating process has shown a distinct change in calcium localization. In the gamete, prior to mating, calcium is sequestered in discrete intracellular granules. As mating progresses, the calcium becomes more diffuse within the cell body. MATERIALS

AND METHODS

Cultures, Induction of Gametes, and Preparation of Autolysin Chlamydomonas reinhardii, wild-type strain 137~ (MT’ and MT-) were cultured as described elsewhere [27]. Gametes were induced in liquid culture using a growth medium lacking a nitrogen Exp Cell Res 160 (1985)

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source as described by Kates & Jones [28]. Autolysin was prepared by mixing gametes (2x10’ cells/ml) for 1 h. The suspension was then centrifuged (1000 g) and the supematant clarified by Mill&ore filtration (0.45 pm). This filtrate removed the walls of unmated gametes within 30 min.

X-ray Microanalysis

Sample Preparation

Gamete protoplasts (MT+) were prepared by treatment with autolysin as described above. After fixation for 1 min with 4% glutaraldehyde, they were washed with glass distilled water. The cells were then air-dried on polylysine-carbon-Formvar-coated copper mesh grids. The Formvar substrate was carbon-coated for stability and polylysine-coated to enhance cell adhesion to the substrate. For analysis of mating cells, equal numbers of MT+ and MT- gametes were mixed and a droplet of the mixed cells immediately applied to a polylysinecarbon-Formvar-coated copper mesh grid. After 3 min, the excess medium was removed with a filter paper wick and the adhering cells were fixed 1 min with 4% glutaraldehyde. The grids were then washed with water and air-dried. Zygotes were prepared by mixing equal numbers of MT+ and MT- gametes for 15 min prior to application to the prepared grid. Zygotes were fixed and washed as described above.

Instrumentation These data were obtained on a Philips Transmission Electron Microscope (TEM), EM420, equipped with a tungsten electron gun, eucentric goniometer stage, scanning attachment (STEM), and EDAX analysing system. Mapping is produced by use of the STEM. X-ray maps represent qualitative descriptions of the elemental distribution within the scanned region of the specimen. The X-ray image intensity is related to the concentration of the element in the area being scanned. To produce these calcium maps, a window was set on the EDAX analyser at 3.64-3.74 keV. As the beam was scanned across the specimen, only X-rays produced with values of 3.64-3.74 keV were collected. Each X-ray photon detected appeared as a dot in the image, with regions of higher concentration having a brighter signal due to higher dot density. Multiple rasters produced a cleaner X-ray map by improving the signal to noise ratio. When multiple scans are used, it is unlikely that random noise bursts will occur at the same place in the image.

RESULTS Gametes of the two mating types (MT+ and MT-) of Chlamydomonas reincannot be distinguished morphologically prior to mating. The oval cell body is surrounded by a glycoprotein cell wall matrix. One of the initial events in the mating process is dissolution of this cell wall by autolysin released from both mating gametes. To obviate consideration of possible differences in ionic composition during mating due to the presence or absence of the encasing cell wall, the walls of gametes were removed by autolysin prior to X-ray microanalysis. Fig. 1a, c shows STEM micrographs of two gamete protoplasts (MT+). Within the cell body numerous granules are seen. In previous studies we determined by spot analysis that calcium appeared to be localized in intracellular compartments (data to be published elsewhere). In the present study, calcium X-ray maps of gametes confirmed these findings (fig. 1 b, d). High density intracellular granules could be observed in TEM and STEM. X-ray mapping revealed that calcium was sequestered in these granules. Between the granules, calcium was barely detectable above the background level and the cell boundary was not well defined on the calcium maps of gametes. In fig. 2a, mating gametes are shown which were arrested by fixation after activation, but prior to cell fusion. Markers of activation include, flagellar tip hardii

Exp Cell Res 160 (1985)

374 Kaska, Piscopo and Gibor

Fig. 1. (a) Dark-field STEM micrograph of Chlamydomonas reinhardii gamete. protoplast (MT+) prior to mating; (b) calcium X-ray map of (a); (c) TEM micrograph of gamete protoplast (MT+); (4 calcium X-ray map of (12).(a) X5600; (b) 5000 counts; (c) X4900; (d) 4500 counts.

activation, and the erection of a fertilization tubule by the MT+ member of the pair. The cells are held together by extensive flagellar adhesion. In fig. 2 b the cell bodies are seen at higher magnification and the X-ray map of these activated gametes (fig. 2c) reveals a calcium distribution distinctly different from that of Exp CellRes 160(1985)

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Fig. 2. (a) TEM micrograph of gametes mated 3 min. Evidence of sexual signalling includes flagellar tip activation (*), and extension of the fertilization tubule from the MT+ gamete (arroru). (6) Darkfield STEM micrograph of cell bodies; (c) calcium X-ray map of activated gametes. Bar, 1 urn. (a) x3800; (b) x9600; (c) loo00 counts.

unmated gametes. Dense regions of calcium are still present, but are not clearly defined. Calcium now appears diffuse between the denser regions. A second mating pair and the associated calcium map are illustrated in fig. 3 a-c. These gametes, showing characteristic long regions of flagellar contact (fig. 3a), were in the process of cell fusion as evidenced by the wide cytoplasmic bridge between them. Although intracellular granules are visible in the STEM micrograph (fig. 3 b), the X-ray map indicates that calcium is generally diffuse throughout the cells (fig. 3); a greater density is apparent at the periphery and in the region of fusion. The boundary of the fused cells is now partially distinguished on the calcium map.

Fig. 3. (a) TEM micrograph of fusing gametes fixed 3 min after mating; (b) STEM micrograph of

fusing cell bodies; (c) calcium X-ray map. Bar, 1 pm. (a) x3800; (b) x9600; (c) 12000 counts. E.xp Cell Res 160 (1985)

316 Kaska, Piscopo and Gibor

Fig. 4. (a, c) STEM micrographs

of quadriftagellated zygotes fixed 15 after mating; (b, d) corresponding calcium X-ray maps of xygotes. Bar, 1 pm. (a, c) x12500; (b, d) 6500 counts.

Quadriflagellated zygotes, obtained 15 min after mating, are shown in fig. 4a, c. Two gametes of opposite mating types have now merged completely. The associated calcium X-ray maps of these zygotes indicate that calcium is now diffuse throughout the cell body (fig. 4 b, d). Variations in calcium density are absent. The calcium X-ray maps clearly delineate the outline of the cell body. DISCUSSION Successful application of X-ray microanalytical techniques to biological systems is dependent on preservation of elemental composition during sample preparation. Loss of diffusible elements occurs during fixation, embedding and sectioning procedures as well as the introduction of additional and often interfering elements. In these studies the analysis of whole cells has eliminated embedExp Cd Res 160(1985)

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ding and sectioning procedures. Brief glutaraldehyde fixation of cells was necessary for satisfactory structural preservation of the algal protoplasts. While intlux of extracellular ions was minimized by the absence of ions in fixation and washing solutions, loss of free calcium undoubtedly occurred, as glutaraldehyde is known to increase membrane permeability [29]. We interpret the results of these X-ray microanalysis to refer primarily to bound calcium. This method, therefore, represents an analysis of intracellular calcium quite different from techniques which depend upon release of cytoplasmic free calcium, such as aequorin microinjection, ionophore activation, and release of 4SCa. Here we have directly observed the reservoirs of intracellular calcium and followed the changes in these reservoirs during gamete interaction. In unmated gametes (MT+), granules sequestering calcium varied in size, number, and intracellular arrangement. We have not yet identified which of the known structures within the cells correspond to these granules. Their distribution, size, and arrangement resembles that of the conspicuous starch granules in the large chloroplast of C. reinhardii gametes [30], but they appear too dense to be starch. The mobilization of calcium from the intracellular stores was rapid and appeared to be initiated by flagellar signalling, as activated gametes exhibited extensive calcium rearrangement prior to fusion. Bloodgood & Levin [ 1l] found that the transient calcium efflux occurred within the first minutes of mating, reaching a maximum after 5 min. The dispersal of intracellular calcium seen by Xray mapping during the first 3 min of mating correlates well therefore with the time of observed efflux. A transient increase in calcium efflux was also induced by stimulation of gametes with isolated flagella of the opposite mating type. Thus the response occurred in the absence of cell fusion, a result now confirmed by calcium mapping. Following release from intracellular granules, sufficient calcium remains within the cells during fusion to produce definitive calcium X-ray maps. The diffuse distribution of the element throughout the zygote cell body is presumed to be bound calcium. The previously reported transient increase in calcium efflux [ 1l] lasting approx. 8 min in a gamete population and the rapid movement of calcium shown within individual cells by X-ray mapping strongly suggests (1) an increase in intracellular free calcium follows flagellar contact; (2) the resulting free calcium is rapidly bound again following redistribution. Although the calcium-binding protein, calmodulin, has been detected in C. reinhardii, a direct role of calmodulin in mating events of algal gametes has not yet been demonstrated. It is significant that calmodulin inhibitors, such as trifluoperazine, were found by Detmers & Condeelis to interfere with gamete activation in this species [311. These observations suggest the possibility that calcium serves as a second messenger in this species. Further credence to this suggestion is enhanced by the four known consequences of activation: (1) microtubule assembly (during flagellar tip activation [5]; (2) secretion (of autolysin) [6]; (3) elongation of an actinExp Cell Res 160 (1985)

378 Kaska, Piscopo and Gibor filled fertilization tubule [7]; and (4) cell fusion, which are all known in other systems to be regulated by calcium [32, 331. Calcium ionophore activation of gametes of C. reinhardii would be expected and was indeed reported by Claes who used A-23187 to initiate autolysin release [34]. Unfortunately this observation remains controversial. Our laboratory and others have been unable to confirm this finding [ 1I]. The ability of the Philips 420 instrument to map the location of calcium in whole cells and to delineate the redistribution during cellular communication has proved to be a valuable tool in this investigation. Demonstrating the release of calcium from intracellular granules during mating in C. reinhardii leads to further questions important both to this reaction and to cellular communication in general. (1) In what form(s) is this calcium bound within the cells so that it is preserved during fixation and washing? (2) By what mechanism does flagellar membrane contact initiate intracellular calcium release? (3) What are the regulatory links between the increase in free calcium and the diverse cellular responses? Recently Pijst et al. reported a rapid transient increase in CAMP following flagellar agglutination in a closely related species, Chlamydomonas eugametos [35]. This observation in concert with the evidence for flagellar stimulated intracellular calcium release, proposes the possibility that CAMP and calcium may serve as interrelated second messengers in intercellular communication in Chlamydomonas. Such cooperation between CAMP and calcium is well documented in animal cell response to stimuli [32, 331. This work was supported in part by NSF PMC82-16753 and by the Academic Senate Committee on Research Funding of the University of California, Santa Barbara, Calif., USA.

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Exp Cell Res 160 (1985)