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Mutants Deficient in Ca -Dependent Control of Flagellar Dominance O Research Article 529 Phototactic activity in Chlamydomonas ‘non- phototactic’ mutants deficient in Ca2+-dependent control of flagellar dominance or in inner-arm dynein Noriko Okita1, Nahoko Isogai1, Masafumi Hirono1, Ritsu Kamiya1 and Kenjiro Yoshimura2,* 1Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0063, Japan 2Doctoral Course of Structural Biosciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan *Author for correspondence (e-mail: [email protected]) Accepted 4 November 2004 Journal of Cell Science 118, 529-537 Published by The Company of Biologists 2005 doi:10.1242/jcs.01633 Summary In the mechanism underlying the phototactic behavior of Both were found to retain reduced phototactic abilities. Chlamydomonas, Ca2+ has been thought to control the These results indicate that both Ca2+-dependent flagellar dominance between the two flagella so as to steer the cell dominance control and inner-arm dynein subspecies f are to correct directions. A newly isolated mutant, lsp1, that important for phototaxis, but are not absolutely necessary. displays weak phototaxis was found to be defective in Analysis of the flagellar beat frequency in lsp1 cell models this Ca2+-dependent shift in flagellar dominance; in showed that both of the flagella beat at the frequency of the demembranated and reactivated cell models, the trans cis flagellum in wild type. In addition, lsp1 and ptx1 were flagellum (the flagellum farthest from the eyespot) beat found to be deficient in determining the sign of phototactic more strongly than the other (the cis flagellum) in about migration. Hence, the Ca2+-dependent flagellar dominance half of the cells regardless of the Ca2+ concentration control detected in demembranated cells might be involved between <10–9 M and 10–6 M, a range over which wild-type in the determination of the sign of phototaxis. The gene cell models display switching of flagellar dominance. This responsible for the lsp1 mutation was identified by is unexpected because ptx1, another mutant that is also phenotype rescue experiments and found to have sequences deficient in flagellar dominance control, has been reported for phosphorylation. to lack phototactic ability. We therefore re-examined ptx1 and another reportedly non-phototactic mutant, ida1, Journal of Cell Science Key words: Chlamydomonas, Phototaxis, Flagella, Calcium ion, Cell which lacks inner arm dynein subspecies f (also called I1). model Introduction direction of light by scanning the surrounding light conditions Almost all kinds of motile protist can sense ambient conditions (Foster and Smith, 1980; Schallar and Uhl, 1997; Isogai et al., and move toward environments suitable for their survival. How 2000). Upon light reception, the photoreceptor induces an they display such purposeful behavior is of general interest for inward current at the eyespot [photoreceptor current (PRC)] the control of motility and behavior in eukaryotic cells. The and depolarizes the membrane (Litvin et al., 1978; Harz and Hegemann, 1991). Depolarization then produces a Ca2+ influx biflagellate green alga Chlamydomonas detects surrounding 2+ light conditions and swims to where the light intensity is best through voltage-dependent Ca channels on the flagellar membrane, resulting in an increase in the intraflagellar Ca2+ suited for photosynthesis. Depending on the cell’s condition concentration (Beck and Uhl, 1994; Pazour et al., 1995; and the light intensity, cells swim either towards the light Yoshimura et al., 1997). source (positive phototaxis) or away from the light source The change in the intraflagellar Ca2+ concentration is (negative phototaxis). Both kinds of phototaxis are triggered believed to change the balance of the forces produced by the by the light reception by a rhodopsin-related molecule in the two flagella, to control the cell’s swimming direction. Evidence eyespot, a discrete membranous structure located on the for such a Ca2+-dependent flagellar dominance control came equator of the cell body (Foster et al., 1984; Sineshchekov et from a study using demembranated and reactivated cells al., 2002; Nagel et al., 2002). The eyespot is most sensitive to (Kamiya and Witman, 1984). The flagellum closest to the the light coming perpendicular to the eyespot; this is because eyespot (the cis flagellum) was found to beat more strongly the layers of carotenoid granules lining the eyespot reflect the than the one farthest from the eyespot (the trans flagellum) at light coming from the front and the chloroplast in the cell body <10–8 M Ca2+, whereas the trans flagellum beats more strongly shields the light coming from behind. This directional than the cis flagellum at 10–7 M to 10–6 M Ca2+. Isolation of sensitivity of the receptor, in combination with cellular rotation the mutant ptx1 supported the importance of the Ca2+- around the longitudinal axis, enables the cell to detect the dependent flagellar dominance regulation in the phototaxis 530 Journal of Cell Science 118 (3) mechanism because, in this non-phototactic mutant, the cis A more objective determination of the phototactic ability in a given flagellum was more active than the trans flagellum in about half sample of cells was carried out as follows. Cells grown to a density of the total cell models, irrespective of the Ca2+ concentration of 1×106 cells ml–1 were placed in 10×10×10 mm polystyrene between 10–9 M and 10–7 M (Horst and Witman, 1993). A later chambers and irradiated for about 5 minutes by a 30-W fluorescent study using mutants lacking flagellar inner-arm dynein lamp placed ~20 cm behind the chamber. Under these conditions, subspecies f (also called dynein I1) (ida1, ida2, ida3 and ida7) whereas wild-type cells swam to one side of the chamber and formed a tight band on the bottom of chamber, phototaxis-deficient cells suggested that the change in the flagellar dominance is brought swam in random directions. The difference in the distribution of cell about through a mechanism involving phosphorylation of a density was clearly identified by the naked eye. subunit of this dynein. These inner-arm dynein mutants and the mutants mia1 and mia2, which display aberrant phosphorylation forms of the f subspecies, were found to Motility assays exhibit no phototaxis (King and Dutcher, 1997). However, The phototactic abilities of various mutants were quantified by whether the phototaxis loss in these mutants is in fact due to recording and analysing their behavior under continuous an abnormality in the Ca2+-dependent control of the flagellar unidirectional illumination of various intensities. For this experiment, balance remains to be studied. cells were first washed with an experimental solution {1 mM KCl, In this study, we aimed to obtain clues to the mechanism of 0.3 mM CaCl2, 0.2 mM EGTA, 5 mM 2-[4-(2-hydroxyethyl)-1- piperazinyl]ethanesulfonic acid (HEPES)} and kept under red light phototaxis by isolating and characterizing mutants deficient in for more than 20 minutes before phototaxis assays. Cells were then phototaxis, following previous studies that showed the put in a 10×10×10 mm polystyrene trough placed on the stage of an effectiveness of such an approach (Horst and Witman, 1993; inverted microscope (IX70, Olympus, Tokyo, Japan). After further Pazour et al., 1995). Of about ten mutants obtained, one (lsp1) adaptation under red light (λ>600 nm) for a few minutes, the cells showed a novel feature: it is weakly phototactic even though were illuminated by blue-green light through a filter (λ=500 nm). To its cell models do not show a Ca2+-dependent change in avoid adaptation, successive light stimulation was applied after an flagellar dominance. This finding prompted us to re-examine interval of 1.5 minutes. The behavior of the cells was video recorded the relationship between the flagellar dominance control and with a CCD camera. The angle (θ) between the light direction and the phototactic response in other mutants that have been reported swimming direction of each cell was measured for 1.3 seconds to be non-phototactic. Our results indicate that these mutants beginning 5 seconds after the actinic light was turned on. As a measure of the phototactic activity in mutant and wild-type cells, the retain residual phototactic activities, suggesting that there are average of |cosθ| was calculated. The average is normalized such that multiple pathways for generation of phototactic behavior. it is 0 when all cells are moving randomly (average=2/π) and it is 1 when all cells are moving towards the light source. For the assay of photophobic responses, a flash (λ=500 nm) was Materials and Methods applied to the cells while their swimming tracks were being video Strains and culture conditions recorded under red light. The proportion of cells that transiently A Chlamydomonas reinhardtii mutant lacking nitrate reductase, nit1, displayed backwards swimming upon the flash application was was used as the parental cell for insertional mutagenesis. An arginine quantified at various light intensities. autotrophic mutant, arg7, was used to make double mutants. ptx1, 2+ Journal of Cell Science ptx5, ptx6 and ptx7 are deficient in phototaxis and Ca -dependent changes in flagellar dominance (Horst and Witman, 1993; Pazour et Demembranation and reactivation of Chlamydomonas cells al., 1995). ida1 is a mutant that lacks the subspecies f of inner-arm For examination of flagellar dominance at various Ca2+ dynein (Kamiya et al., 1991) and has been reported to exhibit no concentrations, cells were demembranated and reactivated as phototaxis (King and Dutcher, 1997). For physiological experiments, described previously (Kamiya and Witman, 1984). Briefly, cells were cells were cultured in TAP medium (Gorman and Levine, 1965) on a washed three times in HES (10 mM HEPES pH 7.3, 0.5 mM EGTA, 12 hour light, 12 hour dark cycle for 2-3 days. 4% sucrose) and demembranated by the addition of NP40 (to a final concentration of 0.2%) on ice.
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