Detergent-extracted Volvox model exhibits an PNAS PLUS + anterior–posterior gradient in flagellar Ca2 sensitivity

Noriko Uekia and Ken-ichi Wakabayashia,1

aLaboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama-shi, Kanagawa 226-8503, Japan

Edited by Krishna K. Niyogi, Howard Hughes Medical Institute, University of California, Berkeley, CA, and approved December 8, 2017 (received for review September 1, 2017) Volvox rousseletii is a multicellular spheroidal green alga contain- suggested by studies using demembranated and reactivated cells ing ∼5,000 cells, each equipped with two flagella (cilia). This or- and flagella (5, 6). ganism shows striking photobehavior without any known Multicellular spheroidal species of Volvocales, including Volvox intercellular communication. To help understand how the behav- species, have often been regarded as colonial Chlamydomonas. ior of flagella is regulated, we developed a method to extract the However, alignment of Chlamydomonas cells on the surface of a whole organism with detergent and reactivate its flagellar motil- spheroid, with each cell displaying breaststroke-like flagellar ity. Upon addition of ATP, demembranated flagella (axonemes) in beating, would result in spheroids unable to swim in one direction. the spheroids actively beat and the spheroids swam as if they Unlike a C. reinhardtii cell, each cell in a Volvox spheroid has two + were alive. Under Ca2 -free conditions, the axonemes assumed flagella beating in the same direction. A Volvox spheroid has an planar and asymmetrical waveforms and beat toward the poste- anterior–posterior (A–P) axis, and its ∼10,000 flagella beat toward rior pole, as do live spheroids in the absence of light stimulation. In the posterior pole with a slight tilt from the A–P axis (7, 8) (Fig. 1 −6 2+ the presence of 10 MCa , however, most axonemes beat three- A and B, Top). Flagellar beating causes the surrounding fluid to dimensionally toward the anterior pole, similar to flagella in flow posteriorly around the spheroid so that the spheroid swims 2+ photostimulated live spheroids. This Ca -dependent change in forward while rotating bodily. flagellar beating direction was more conspicuous near the anterior Volvox spheroids and C. reinhardtii cells differ in their regu- pole of the spheroid, but was not observed near the posterior lation of flagellar beating during photoresponses. While a 2+ pole. This anterior–posterior gradient of flagellar Ca sensitivity spheroid swims with bodily rotation, the light signal sensed by a PLANT BIOLOGY may explain the mechanism of V. rousseletii photobehavior. single cell increases when it faces the light source and decreases when it faces the opposite side because of light reflection by the flagella | calcium | phototaxis | Volvox carotenoid layers of the eyespot. Phototactic turning occurs when the flagella on Volvox carteri and Volvox aureus cells facing otile photosynthetic organisms require photobehavioral the light source stop beating (9, 10) (Fig. S1A). In large-spheroid Mresponses (photoresponses) to inhabit environments suit- species, such as Volvox rousseletii and Volvox barbari, the di- able for photosynthesis. Green algae of the order Volvocales rection of flagellar beating changes when light is perceived by the swim with flagella protruding from each cell and display appro- anterior hemisphere in a posterior-to-anterior direction, while its priate behaviors in response to surrounding light. Organisms in ciliary waveform is retained (3, 10) (Fig. 1B, Bottom and Figs. the section Volvox (= Euvolvox, a genus of Volvocales) (Fig. S1A and S2). This flagellar response is called ciliary reversal. S1A) have unique characteristics, such as large cell number, a Importantly, the magnitude of this response has a gradient along complex developmental process (1), high swimming velocity (2), the A–P axis, in that the response is more pronounced near the and marked photoresponses (3). They display two kinds of anterior pole of the spheroid and is rarely observed near its photoresponses: phototaxis and photoshock. Phototaxis is di- posterior pole (3). This gradient may be produced by differences rectional swimming toward or away from a light source, whereas in eyespot size, which is greater in cells near the anterior pole photoshock is a transient stop or deceleration in response to sudden changes in light intensity. Each cell of both unicellular Significance and multicellular species of the order Volvocales commonly uses its eyespot to sense light and generates propulsive force by The multicellular green alga Volvox rousseletii displays photo- beating two flagella. The eyespot, an orange spot in the cell, taxis by changing its flagellar beating pattern in response to consists of layers of carotenoid-rich granules in the chloroplast photoreception. However, the molecular mechanism underlying and photoreceptor proteins (channel rhodopsins) localized in the flagellar regulation is unknown. This study describes a method plasma membrane right above these granules. The carotenoid to demembranate whole spheroids using a nonionic detergent, layers function as a light reflector, making the eyespot a highly with the addition of ATP reactivating flagellar motility. These reactivated spheroids swam like live spheroids. Flagellar beating directional photoreceptive organelle (4). 2+ Regulation of flagellar beating in photoresponses has been direction was altered in a Ca -dependent manner, with a extensively studied in a unicellular species of Volvocales, Chla- greater change in the anterior hemisphere than in the posterior hemisphere. These findings indicate that V. rousseletii has an mydomonas reinhardtii.AC. reinhardtii cell swims by beating its + anterior–posterior gradient of flagellar sensitivity to Ca2 , which two flagella in asymmetrical/cilia-type waveforms and in opposite likely plays a key role in V. rousseletii phototaxis. directions (Fig. S2). Because the beating planes of the two fla-

gella are slightly skewed from each other, the cell rotates while Author contributions: N.U. and K.W. designed research, performed research, analyzed swimming. During photoshock responses, the waveform tran- data, and wrote the paper. siently changes to a symmetrical/flagella-type pattern and the cell The authors declare no conflict of interest. swims backward for a short period (Fig. S2). In phototaxis, the This article is a PNAS Direct Submission. force balance between the two flagella is dependent on the di- Published under the PNAS license. rection of light, causing the cell to turn toward or away from the 1To whom correspondence should be addressed. Email: [email protected]. light source (Fig. S2). Both kinds of photoresponse are likely to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 2+ be mediated by changes in intraflagellar Ca concentration, as 1073/pnas.1715489115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1715489115 PNAS Early Edition | 1of8 Downloaded by guest on October 1, 2021 method was not successful, we found that several modifications of the original protocol resulted in successful demembranation and reactivation (details are provided in Materials and Methods). (i) Because V. rousseletii spheroids cannot be efficiently con- centrated by centrifugation, buffer exchange entailed trapping (Fig. 2A) or a strainer-scooping method (see Fig. 5A) rather than centrifugation. (ii) The concentration of nonionic detergent used for C. reinhardtii (0.1%) demembranation was too high for V. rousseletii, resulting in deflagellation during perfusion. The optimal concentration for V. rousseletii, as determined by the degree of deflagellation and the efficiency of reactivation, was much lower, in the range of 0.01–0.03%, and varied by de- velopmental stage (Figs. 1C and 2B). (iii) Polyethylene glycol (PEG) was omitted from the reactivation buffer. The buffer used to reactivate C. reinhardtii cell models contained 0.5% PEG [molecular weight (Mw) = 20,000] to mimic the molecular crowding effect in vivo (6), thereby increasing the reactivation rate. Although PEG increased the reactivation rate in V. rous- seletii, it tended to distort the spheroids (Fig. S3). PEG was therefore added only when the trapping method was used (see Fig. 4). (iv) Although dark-field microscopy is often used to monitor demembranated C. reinhardtii cells, the flagellar axo- nemes of V. rousseletii were not clearly visible by dark-field mi- croscopy because of strong halation from the spheroid. Thus,

Fig. 1. Life cycle and swimming style of V. rousseletii. (A) Schematic diagrams showing the directions of flagella-generated fluid streams (blue arrows) around a spheroid. The A–P current with a slight tilt causes the spheroid to swim while rotating bodily in a counterclockwise direction when viewed from the posterior pole. The dark gray circles in the spheroid represent gonidia that localize mainly in the posterior hemisphere. (B) Schematic diagrams showing flagellar beating in the somatic cells on the spheroid surface in an anterior region (box in A). Fla- gellar waveforms in posterior (Top) and anterior (Bottom) beating modes are shown. The blue and red arrows indicate the current generated in the two modes. Modified from ref. 3. Note that the cell closest to the anterior pole is depicted with a large eyespot (a red spot), with the eyespot size gradually de- creasing with distance from the pole. (C) Asexual life cycle of V. rousseletii cul- tured on a 16-h/8-h light/dark cycle. Spheroids in three stages (termed stages I–III in this study) were used: Stage I represents newly hatched spheroids, in which gonidia (next-generation embryos) just start cell division (Left); stage II represents spheroids, in which gonidia are undergoing cleavage (Center); and stage III represents expanded spheroids, in which daughter spheroids have undergone inversion and are ready to hatch (Right). A, anterior pole; P, posterior pole. (Scale bar: 200 μm; note that the bar length differs in the three photographs.)

and smaller in cells near the posterior pole (3). However, the signal(s) that causes ciliary reversal is not known. In vitro reactivation of the motility of detergent-extracted cili- ated organisms, such as Paramecium, C. reinhardtii, and sea urchin Fig. 2. Detergent sensitivity of flagella along the A–P axis of spheroids at sperm, is a powerful method for studying the regulatory mecha- the three developmental stages. (A) Schematic diagrams showing the trap- nisms of cilia and flagella (6, 11–13). However, application of this ping method for demembranation of Volvox spheroids. (i–iii) Drop of cul- method to whole Volvox spheroids has been difficult, since these ture containing several spheroids was placed on a glass slide. The medium spheroids are much larger in size (500–1,000 μm) than those of was drained with a pipette, and washing solution (HMDEK; Materials and other organisms studied to date. This study was designed to elu- Methods) was added. (iv) After a few minutes, spheroids were withdrawn cidate the mechanism underlying ciliary reversal and its gradient with a pipette and (v and vi) placed in a perfusion chamber. (vii) Top view of – the chamber. Solution containing either Igepal or ATP was perfused. along the A PaxisinV. rousseletii. Whole spheroids were (B) Degree of demembranation in the three stages of spheroids treated with demembranated by detergent extraction and subsequently reac- different concentrations of detergent. The demembranation index was cal- tivated. Our results suggest that ciliary reversal in V. rousseletii is culated for the anterior or posterior hemisphere as follows: If visual in- + + dependent on Ca2 concentration, with the Ca2 responsiveness spection showed that all flagella in the examined area were stopped after of demembranated flagella having a gradient along the A–Paxis. detergent perfusion, the index was 1; if any flagella were moving, the index was 0. Index values were counted in three to 26 spheroids, with the average Results defined as the demembranation index. The arrowhead in stage I indicates V. rousseletii the conditions used for Fig. 5, and the arrows in stage II indicate the con- Reactivation of the Motility of Axonemes. To reactivate ditions used for Figs. 3 and 4. The filled arrow and squares are located in the the flagellar motility of detergent-extracted V. rousseletii, we first anterior hemisphere, and the open arrow and circles are located in the tested the method developed for C. reinhardtii. Although this posterior hemisphere.

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1715489115 Ueki and Wakabayashi Downloaded by guest on October 1, 2021 demembranated V. rousseletii was monitored by phase-contrast PNAS PLUS microscopy. The optimal concentration of a nonionic detergent, Igepal CA-630 (a substitute for Nonidet P-40; hereinafter referred to as Igepal) was determined by washing spheroids with a buffer and trapping them between a glass slide and a coverslip. The sample was then gently perfused with Igepal-containing buffer for demembranation (Fig. 2A). Determinations of the demembra- nation rate (defined as 1 when no flagella moved and 0 when at least one flagellum moved after demembranation in a given sector of a spheroid) indicated the presence of an A–P gradient in Igepal sensitivity throughout the developmental stages (Fig. S4). Cells at the anterior pole were the most sensitive to Igepal (i.e., their flagella stopped beating at lower concentrations), and those at the posterior pole were the least sensitive (Fig. 2B and Fig. S4). To reproducibly repeat experiments, we selected three easily distinguishable stages in development, termed stages I–III (Fig. 1C). Stage I consisted of newly hatched spheroids, with flagella most sensitive to Igepal (Fig. 2B). Stage II consisted of middle-aged spheroids whose gonidia were in the midst of cell division, with cells in the posterior hemisphere becoming less sensitive than in stage I. Stage III consisted of the expansion stage, when gonidia finished inversion, with the Igepal concen- tration necessary to demembranate posterior flagella being the highest among the three stages. We then determined the optimal conditions to reactivate the motility of demembranated flagella and spheroids. Because high concentrations of Igepal cause deflagellation, lower concentra- PLANT BIOLOGY tions of Igepal were desirable. Stage II spheroids are best suited for monitoring flagella because the distance between flagella on the spheroidal surface is longer than in stage I, which allows monitoring of their waveforms, and because their gonidia are smaller than in stage III, making the A–P axis clearly identifiable. Fig. 3. ATP-dependent beat frequency in the axonemes of detergent- Stage I, however, is optimal for monitoring whole spheroids, extracted Volvox (DEV). (A) Beat frequency of axonemes near the anterior because the same low concentration of Igepal (0.01%) effectively and posterior poles in DEVs at different ATP concentrations. Three to six demembranates both anterior and posterior hemispheres (Fig. axonemes were measured. (B) Beat frequency of flagella in live cells near the 2B, arrowhead). Thus, high-speed monitoring of axonemes anterior and posterior poles. The average in six flagella is shown for each. in trapped spheroids was performed using stage II spheroids (C) Double-reciprocal plot of the data in A. Intercepts yielded apparent max- demembranated with 0.01% (for anterior flagella) or 0.03% imal beat frequencies of 43.5 Hz (anterior region) and 48.8 Hz (posterior re- (for posterior flagella) Igepal (Fig. 2B, closed and open arrows, gion) and apparent Michaelis constants of 0.10 mM (anterior) and 0.22 mM ATP (posterior). respectively). Reactivation of whole detergent-extracted spher- oids was assessed using stage I spheroids demembranated with + 0.01% Igepal. various organisms. We therefore examined the effect of Ca2 on The axonemal motility of detergent-extracted V. rousseletii axonemal motility in the DEV. spheroids (DEVs) was successfully reactivated by treatment with We first assessed flagellar motion in live spheroids that an ATP-containing buffer. The average beat frequencies of responded to photostimulation. Under continuous light, all fla- reactivated axonemes in DEVs, as determined using a high- gella on a spheroid beat in a posterior direction in a ciliary speed camera, increased with ATP concentration in a manner waveform [Fig. 4 A, Left (setup A), B, Center (setup B), and C, consistent with Michaelis–Menten kinetics (Fig. 3 A and C). The Right (setup C) and Movie S1]. After photostimulation (Materials average beat frequencies of DEVs reactivated with >1 mM ATP and Methods), flagella in the anterior hemisphere showed ciliary were higher than those of flagella in live spheroids, which were reversal, with flagella beating in an anterior direction for a short 29.2 ± 2.7 Hz at the anterior pole and 25.1 ± 1.9 Hz at the period of time, usually 2–3s(Movie S2). The beating direction of posterior pole (Fig. 3B). Under all conditions tested in this study, a ciliary-type waveform can be determined from the appearance the average beat frequencies of flagella/axonemes were slightly of a typical hook shape in a series of waveforms. The change in higher near the anterior pole than near the posterior pole (Fig. 3 beating direction from before to after photostimulation was most A and B). For example, the beat frequencies in DEVs at 1 mM clearly observed near the anterior pole, with none observed near ATP were 41.8 ± 3.1 Hz near the anterior pole and 33.8 ± 0.9 Hz the posterior pole (Fig. 4 B and C and Movies S3 and S4). A near the posterior pole. Using double-reciprocal plots, the surface view of a region close to the anterior pole, as observed maximal beat frequencies at the anterior and posterior poles from within the spheroid [Fig. 4 A, Center (setup B) and D and were 43.5 Hz and 48.8 Hz, respectively, and the apparent Movie S5], showed that, in response to photostimulation, the Michaelis constants were 0.10 mM and 0.22 mM ATP, respec- beating direction of flagella gradually changed toward the an- tively (Fig. 3C). terior end after one or two strokes toward the posterior end. The direction of beating was almost completely reversed, but did not Calcium Control of Flagellar Stroke Direction. Our primary interest last long, returning to the original direction in a few seconds. was the identification of the factor that causes ciliary reversal in Similar to anterior flagella, flagella near the spheroid’s equator photostimulated V. rousseletii flagella. The optimal candidate (as observed from outside) [Fig. 4 A, Right (setup C) and E and + was Ca2 , a major factor that modulates flagella/cilia beating in Movie S6] continued beating in a posterior direction for one or

Ueki and Wakabayashi PNAS Early Edition | 3of8 Downloaded by guest on October 1, 2021 Fig. 4. Ca2+-dependent changes in the direction of axonemal beating. (A) Experimental setups for observation of live or demembranated spheroids in a chamber. Glass slides and coverslips (gray), spacers (orange), the A–P(A↔P) direction of the spheroids, and the regions observed (red boxes) are indicated. (Left) In setup A, a spheroid was sandwiched between a glass slide and a coverslip, and regions near the anterior pole (A) and posterior pole (P) were ob- served. (Center) In setup B, a spheroid, after gentle pressing, was placed between the glass slide and a coverslip separated by thick spacers, and the region near the anterior pole was observed. (Right) In setup C, a spheroid was attached to the glass slide with 1% polyethyleneimine, and the region near the equator was observed. (B) Frames from high-speed recordings of regions near the anterior (Top) and posterior (Bottom) poles of a live spheroid. The ob- servation using setup A was under stationary conditions in continuous light (Left) and after photostimulation (Right). The direction of the flagellar effective stroke (arrows) was determined by the direction of the typical hook shapes (red arrowheads) in the recovery stroke. After photostimulation, the direction of the flagellar effective stroke reversed in the anterior region. (Scale bar: 100 μm.) (C) Typical sequential flagellar waveforms in a single beating cycle under each condition. Waveforms recorded as in B were traced (time interval of 1/500 s). The typical hook-shaped waveforms appearing in recovery strokes are traced in magenta. (Scale bar: 10 μm.) Photographs show the time course of directional change in the flagellar effective stroke in live spheroids, near the anterior pole in setup B (D, Top) and near the equator in setup C (E, Top). (D and E, Bottom) Photographs show the time series of the change after pho- tostimulation in the boxed cell. The direction of ciliary beating and the time after onset of illumination are shown. (Scale bars: Top, 100 μm; Bottom,10μm.) In D, the direction of the effective stroke was almost reversed at 0.27 s and recovered at 4.44 s. The red asterisk indicates a presumptive anterior pole. In E,the direction of the effective stroke rotated ∼90° at 0.72 s and recovered at 1.52 s. The two-headed arrow indicates the approximate A–P direction. (F) High-speed − + recording of regions near the anterior (Top) and posterior (Bottom) poles of DEVs reactivated in the presence of 0 or 10 6 MCa2 in setup A. The ATP + concentration was 1 mM. In the presence of Ca2 , the axonemes in the anterior region showed anteriorly directed effective strokes, opposite to those in the + absence of Ca2 . However, this change was not observed in the posterior region. Arrowheads indicate the typical hook-shaped waveforms. (Scale bar: 100 μm.) (G) Typical sequential axonemal waveforms traced for a single beating cycle under each condition (time interval of 1/500 s). The hook-shaped waveforms are shown in magenta. (Scale bar: 10 μm.)

two strokes after photostimulation, but gradually rotated stroke We next examined the beating direction of reactivated axo- + direction by ∼90°. These flagella returned to their original pos- nemes in DEVs in the absence and presence of Ca2 . Under + terior direction more rapidly than the anterior flagella. Ca2 -free conditions, axonemes in reactivated DEVs beat in a

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.1715489115 Ueki and Wakabayashi Downloaded by guest on October 1, 2021 + PNAS PLUS posterior direction (Fig. 4 F and G and Movie S7), similar to onemes on spheroids show a Ca2 sensitivity gradient along the flagella in live spheroids in response to continuous light. In the A–P axis. − + presence of 10 6 MCa2 , however, the axonemes beat in an Motility Reactivation of Whole Multicellular Spheroids of V. rousseletii. anterior direction, similar to flagella in the anterior regions of 2+ photostimulated live spheroids (Fig. 4 F and G and Movie S8). In Finally, we examined whether the Ca effect on axonemal addition, the reactivation rate and the beating amplitude became beating direction contributes to the changes in spheroid swim- − + + smaller in the presence of 10 6 MCa2 than under Ca2 -free ming velocities that occur during the photoshock response. After photostimulation, live V. rousseletii spheroids show photoshock conditions. Strokes in the anterior direction were most clearly response, with their swimming velocity decreasing to 50–60% visible around the anterior pole, with axonemes near the poste- (3) (Fig. 5D). To test if this response can be reproduced in rior pole beating in a posterior direction, similar to those under 2+ + vitro by changing the Ca concentration, stage I spheroids were Ca2 -free conditions (Fig. 4 F and G and Movies S9 and S10). −3 2+ demembranated with 0.01% Igepal by the strainer-scooping Perfusion with a buffer containing 10 MCa resulted in ax- method, which demembranates spheroids while preserving their onemes that displayed waveforms of smaller amplitude and overall morphology. No spheroids displayed movement in this lower beat frequency (Fig. S5). These waveforms, however, were demembranation solution (Fig. 5A and Movies S11 and S12). not observed in photostimulated live spheroids, suggesting that Subsequently, the spheroids in the demembranation solution + − the maximum in vivo Ca2 concentration after photostimulation were transferred to a buffer solution containing 0 M or 10 6 M − − + is in the range of 10 6–10 4 M. Ca2 for reaction. Upon the addition of 1 mM ATP, most of the Overall, our in vitro experiments using DEVs showed that (i) DEVs started to swim like live spheroids. The average swimming ve- + − ciliary reversal is a Ca2 -dependent event and (ii) cells and ax- locities, as calculated from the swimming tracks, were 588 ± 99 μm·s 1 PLANT BIOLOGY

Fig. 5. DEV swimming. (A) Schematic diagrams showing the strainer-scooping method for demembranation and reactivation. Spheroids were successively transferred from the culture medium to the washing, demembranation, and reactivation solutions, with ATP added during the final step to reactivate motility. (B) Swimming trajectories of spheroids in live and demembranated/reactivated spheroids. Movements of the spheroids were video recorded and tracked for 5 s. (Left) Live spheroids under continuous light before demembranation. (Center and Right) DEVs reactivated with 1 mM ATP in the absence and − + presence of 10 6 MCa2 . (Scale bars: 5 mm.) (C) Swimming velocities (n = 20 for live spheroids, n = 30 for DEVs under each condition) calculated from the swimming trajectories for 5 s. DEVs that did not move at all were not counted. (D) Swimming velocities of live spheroids before and right after photo- stimulation calculated from the swimming trajectories for 3 s.

Ueki and Wakabayashi PNAS Early Edition | 5of8 Downloaded by guest on October 1, 2021 + Table 1. Ca2 -buffered reactivation solutions Components, M Calculated concentrations, M

2+ Approximate free [Ca ] [CaCl2] [EGTA] [EDTA] [MgATP]

− − − − 10 3 M 2.992 × 10 3 0 2.00 × 10 3 8.86 × 10 4 10−4 M 1.507 × 10−3 0 2.00 × 10−3 9.56 × 10−4 10−5 M 0.490 × 10−3 0 2.00 × 10−3 9.54 × 10−4 − − − − 10 6 M 4.742 × 10 3 5.00 × 10 3 0 9.70 × 10 4 − − − − 10 7 M 3.277 × 10 3 5.00 × 10 3 0 9.67 × 10 4 10−8 M 0.837 × 10−3 5.00 × 10−3 0 9.61 × 10−4 − − Ca-free 0 1.00 × 10 3 0 9.69 × 10 4

Final concentrations in reactivated samples. Components other than CaCl2, EGTA, and EDTA: 30 mM Hepes, 5 mM MgSO4, 1 mM DTT, 50 mM potassium acetate, 1% PEG (average Mw = 20,000) (for experiments in Fig. 4) and 1 mM ATP. The pH of all reactivation solutions was adjusted to pH 7.4.

− for live spheroids, 499 ± 97 μm·s 1 for DEVs reactivated in study has shown that ciliary reversal in V. rousseletii is (iv) rotation + Ca2 -free solution (∼85% of live spheroids), and 285 ± of beating direction with various rotation angles. In the presence − − + + 83 μm·s 1 for DEVs reactivated in buffer containing 10 6 MCa2 of Ca2 , V. rousseletii flagella near the anterior pole show almost (∼49% of the velocity of live spheroids before photostimulation full reversal (rotation of ∼180°) of beating direction. The degree and ∼94% of the velocity after photostimulation) (Fig. 5 B and C of rotation of beating direction decreases with distance from the and Movies S11 and S12). These findings suggest that the pho- anterior pole, and becomes ∼90° at the equator and 0° near the + toshock response is caused by Ca2 -mediated ciliary reversal on posterior pole. The mechanism of rotation of beating direction has the anterior hemisphere. been suggested to be different from that of reversal of beating direction in sea urchin larvae, and is not well understood to date Discussion (21). We may expect that identification of molecular/structural To understand how changes in flagellar waveform are regulated differences between anterior and posterior flagella in V. rousseletii in V. rousseletii upon photostimulation, we developed a method will provide clues to understand the molecular mechanism. to demembranate whole spheroids with a detergent and reac- + tivate their motility by addition of ATP. This method enabled us The A–P Gradient in Spheroids. The amplitude of Ca2 -dependent to examine the motility of “dead” multicellular spheroids in vitro directional change in axonemal beating showed a gradient along + and to assess the effects of Ca2 . We found that the flagellar the A–P axis, being high in cells close to the anterior pole and + axonemes show ciliary reversal in a Ca2 -dependent manner and almost zero near the posterior pole. The A–P gradient in fla- + that these axonemes are sensitive to Ca2 with a gradient along gellar photoresponse has been attributed to the gradient in the A–P axis, such that the cells near the anterior pole show the eyespot size, with eyespots larger in cells near the anterior pole greatest changes in axonemal motility. (3, 8, 15, 23). In V. rousseletii, our results clearly showed that the + Ca2 sensitivity of the flagellar axonemes also displays an A–P + + Ca2 Mediates Light-Induced Ciliary Reversal in V. rousseletii. The gradient. To date, several Ca2 -binding proteins have been beating patterns of Volvox flagella change upon photostimulation. identified in C. reinhardtii axonemes, although none has been + In V. aureus and V. carteri (Eudorina group), the most sensitive attributed as responsible for Ca2 sensitivity in axonemal beat- cells are localized at the anterior pole, at which flagella stop ing. However, a promising candidate for the flagellar regulator is + beating in response to photostimulation (9, 10, 14–16). In LC4, a Ca2 -binding subunit in outer arm dynein (18, 24). This V. rousseletii, cells near the anterior pole show ciliary reversal (3). protein may show a gradient in concentration in properties along However, the intracellular signals that trigger these flagellar re- the A–P axis of V. rousseletii axonemes. sponses have remained unclear. Our results using DEVs strongly The light sensitivity of an eyespot may increase with size, such + suggest that ciliary reversal in V. rousseletii is induced by Ca2 that a larger eyespot more readily promotes light-induced in- + influx into the flagella in response to photoreception. At intra- crease in cytoplasmic Ca2 concentration. If so, V. rousseletii + − flagellar Ca2 concentrations <10 6 M, flagella beat in a posterior spheroids may have a strong A–P gradient in photosensitivity, + − direction, whereas at intraflagellar Ca2 concentrations ≥10 6 M, resulting from the combined effects of two kinds of gradients: + flagella near the anterior pole beat in an anterior direction. Sim- one in the mechanism that regulates light-induced Ca2 influx ilar to C. reinhardtii, photoreception by channelrhodopsin at the and the other in the flagellar mechanism to change the waveform + + eyespot may cause membrane depolarization, followed by Ca2 in a Ca2 -dependent manner. In addition, our study showed an + influx through voltage-dependent Ca2 channels localized at the A–P gradient in detergent sensitivity (i.e., the Igepal concen- flagellar tips (17). tration required for complete cessation of flagellar beating), with + Ca2 has been shown to modulate flagellar/ciliary beating in the detergent sensitivity in the posterior region decreasing with various organisms. The modulation includes (i) waveform con- age. These findings indicate that flagellar membrane properties, version, (ii) reversal of bend propagation, (iii) reversal of beating such as the contents of particular lipids or membrane proteins, direction, (iv) rotation of beating direction, (v)increaseinbeat also differ along the A–P axis and with age. All of these features frequency, and (vi) arrest of beating (18) (Fig. S1B). These + of V. rousseletii spheroids may jointly constitute a robust system Ca2 -dependent modulations are responsible for various im- that enables efficient photobehavior of this organism. portant biological events, such as (i) waveform conversion in sperm flagella for sperm chemotaxis toward eggs during fertiliza- Volvox Evolution for Phototactic Steering. Results obtained in this tion and (ii) increase in beat frequency in mammalian tracheal and previous studies indicate how flagellar regulatory mecha- epithelial cilia for airway clearance (19, 20). Ciliary reversal has nisms for phototactic steering have changed during evolution been found in several organisms, including Ctenophora, Parame- from Chlamydomonas-like unicellular organisms to multicellular cium, and sea urchin, and is regarded as (iii) reversal of beating Volvox. At the cellular level, the perception of a sudden increase direction in these organisms (11, 21, 22) (Fig. S1B). Our present in light intensity by the eyespot and the increase in intraflagellar

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1715489115 Ueki and Wakabayashi Downloaded by guest on October 1, 2021 + PNAS PLUS Ca2 seem to be common to C. reinhardtii and V. rousseletii. Materials and Methods 2+ However, the process that follows the intracellular Ca increase Strain and Culture Conditions. The wild-type V. rousseletii strain MI01 (NIES- seems to differ significantly. In C. reinhardtii, flagellar beat fre- 4029) was grown in Volvox thiamin acetate (VTAC) medium (27, 28) at 28 °C + quency and amplitude of bending increase with increasing Ca2 on a 16-h/8-h light/dark cycle under white fluorescent light at 120 μmol of − −2· −1 concentrations up to 10 6 M, causing phototactic turning photons m s . through a change in the beating balance between the two flagella Volvox −4 2+ Reactivation of Detergent-Extracted Spheroids. DEVs were prepared (6, 25, 26), and at 10 MCa , when the photophobic response similar to the method used to produce C. reinhardtii cell models (6). Spheroids takes place, flagellar waveform is converted from a ciliary type to were obtained 12–24 h after transfer to a new medium. For experiments in + a flagellar type causing backward swimming (5). In V. rousseletii, Ca2 -free conditions, spheroids were washed in HMDEK solution [30 mM −6 2+ the direction of ciliary-type beating is reversed at ≥10 MCa Hepes, 5 mM MgSO4, 1 mM DTT, 1 mM EGTA, and 50 mM potassium acetate (Fig. 4 and Figs. S1A and S2). An important feature of at pH 7.4], demembranated in HMDEK solution containing an appropriate concentration (as described in main text) of Igepal (no. 18896; Sigma–Aldrich), + V. rousseletii behavior is that both photoshock response and pho- and reactivated in HMDEK with ATP. For experiments in the presence of Ca2 , 2+ totaxis involve a Ca -induced ciliary reversal (3) (Fig. S2). spheroids were washed in HMDEK or HMDKP solution [HMDEK without EGTA When photostimulation is sufficiently strong for all cells in a containing 1% PEG (average Mw = 20,000) at pH 7.4], demembranated in spheroid, all flagella in the anterior hemisphere show ciliary re- HMDEK or HMDKP solution containing Igepal, and reactivated in one of the 2+ 2+ versal with a gradient of amplitude that halts or greatly slows Ca -buffered reactivation solutions (Table 1) containing ATP. The free Ca spheroid swimming. By contrast, when a spheroid is stimulated by concentration of each sample was calculated using CALCON software, written by Shinji Kamimura of Chuo University (www.bio.chuo-u.ac.jp/nano/ continuous light coming from the side, only the flagella on the calcon.html). illuminated side in the anterior hemisphere should undergo ciliary Two methods were used for buffer exchange. First, in the trapping method reversal, causing the spheroid to turn to the light source (3) (Fig. for monitoring of flagellar motion, washed spheroids were placed in a S2). Thus, the gradient along the A–P axis is very important: If perfusion chamber made of a glass slide, spacers (0.2-mm-thick vinyl tape), flagella on the illuminated side in the posterior hemisphere also and a coverslip. The thickness of the spacer was adjusted to match the size (developmental stage) of the spheroid (single layer for stage I, double layer reverse their beating direction, then the entire spheroid would for stage II, and triple layer for stage III). The sample was perfused first with rotate rather than gradually turn to the light source. the demembranation solution and then with the reactivation solution. The A–P gradient in V. rousseletii may have other advantages Second, in the strainer-scooping method for monitoring of swimming in photobehavior. For example, the gradient may enable a spheroids, spheroids swimming in medium in a Petri dish (35 mm in diameter, spheroid to continue rotation around the A–P axis even during 10 mm thick) were scooped with a 40-μm nylon cell strainer (BD Falcon PLANT BIOLOGY photoshock response; such continuous bodily rotation should be 352340; BD Biosciences), sequentially dipped in washing solution and demembranation solution, and soaked in reactivation solution. important for a spheroid to promptly resume phototactic steer- ing. In addition, the gradient may contribute to fine-tuning of the Beat Frequency Analysis and Waveform Monitoring of Flagella/Axonemes. amplitude of photoresponse. Because of the A–P gradient, the Flagellar and axonemal beating were video recorded at 500 frames per number of cells (or the surface area of a spheroid) responding to second with a phase-contrast microscope (BX-53; Olympus) equipped with a photostimulation changes depending on the light intensity. This high-speed CCD camera (HAS-L2; DITECT Corporation). Beat frequency was might help the spheroid to respond to a wide range of light in- calculated from the average time interval required for 20 strokes. Flagellar/ axonemal waveforms were traced frame by frame on transparency films on tensities with varying amplitude of photoshock response or the monitor screen and digitized using Adobe Illustrator software. phototaxis. In conclusion, our DEV experiments showed that, in addition Tracking of Reactivated DEVs. Reactivated DEVs were monitored under a to eyespot size, the properties of flagella in V. rousseletii,including stereomicroscope (SMZ1000; Nikon) and recorded using a Macromax Scope + their Ca2 sensitivity, have a gradient along the A–Paxis.During MVC-DU CCD camera (GOKO Camera). To analyze swimming velocity, evolution from Chlamydomonas-like unicellular organisms to swimming paths of live spheroids and DEVs were tracked for 3 s and 5 s, Volvox-like multicellular organisms, Volvocales have acquired a respectively, using Image Hyper software (Science Eye). The swimming ve- locities were measured from the trajectories. spheroidal shape with a functional gradient along the A–P axis. As the spheroid increased in size, it may have differentiated into an ACKNOWLEDGMENTS. We thank Dr. Toru Hisabori (Tokyo Institute of anterior hemisphere for steering or braking after photoreception Technology) for fruitful discussions, Akinori Koitabashi and Asuka Tanno and a posterior hemisphere for constant propulsion of the (Tokyo Institute of Technology) for maintenance of Volvox cultures, and Dr. Ritsu Kamiya (Gakushuin University) for critical reading of this manu- spheroid. This division of roles in the two hemispheres may be script. This work was supported by Japan Society for the Promotion of Sci- important for the effective photobehavior of spheroids. ence KAKENHI Grants 15H01206, 15H01314, and 16K14752 (to K.W.).

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