Detergent-Extracted Volvox Model Exhibits an Anterior–Posterior Gradient in Flagellar Ca2+ Sensitivity

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 flagella 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 developmental 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. rousseletii, 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 axonemes of V. rousseletii were not clearly visible by dark-field microscopy 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

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