Coordinated beating of algal flagella is mediated by basal coupling Kirsty Y. Wana and Raymond E. Goldsteina,1 aDepartment of Applied Mathematics and Theoretical Physics, University of Cambridge, Cambridge CB3 0WA, United Kingdom Edited by Peter Lenz, Philipps University of Marburg, Marburg, Germany, and accepted by the Editorial Board March 14, 2016 (received for review September 30, 2015) Cilia and flagella often exhibit synchronized behavior; this includes However, not all flagellar coordination observed in unicellular phase locking, as seen in Chlamydomonas, and metachronal wave organisms can be explained thus. The lineage to which Volvox formation in the respiratory cilia of higher organisms. Since the ob- belongs includes the common ancestor of the alga Chlamydo- servations by Gray and Rothschild of phase synchrony of nearby monas reinhardtii (CR) (Fig. 1B), which swims with a familiar IP swimming spermatozoa, it has been a working hypothesis that syn- breaststroke with twin flagella that are developmentally posi- chrony arises from hydrodynamic interactions between beating fila- tioned to beat in opposite directions (Fig. 1 C and D). However, a ments. Recent work on the dynamics of physically separated pairs of Chlamydomonas mutant with dysfunctional phototaxis switches flagella isolated from the multicellular alga Volvox has shown that stochastically the actuation of its flagella between IP and AP hydrodynamic coupling alone is sufficient to produce synchrony. modes (15, 16). These observations led us to conjecture (15) that However, the situation is more complex in unicellular organisms bear- a mechanism, internal to the cell, must function to overcome ing few flagella. We show that flagella of Chlamydomonas mutants hydrodynamic effects. deficient in filamentary connections between basal bodies display Pairs of interacting flagella evoke no image more potent than markedly different synchronization from the wild type. We perform Huygens’ clocks (17): Two oscillating pendula may tend toward micromanipulation on configurations of flagella and conclude that a synchrony (or antisynchrony) if attached to a common support, mechanism, internal to the cell, must provide an additional flagellar whose flexibility provides the necessary coupling. Here we present coupling. In naturally occurring species with 4, 8, or even 16 flagella, a diverse body of evidence for the existence of a biophysical we find diverse symmetries of basal body positioning and of the equivalent to this mechanical coupling, which, in CR and related flagellar apparatus that are coincident with specific gaits of flagellar algae, we propose is provided ultrastructurally by prominent fibers actuation, suggesting that it is a competition between intracellular connecting pairs of basal bodies (BB) (18) that are known to have coupling and hydrodynamic interactions that ultimately determines contractile properties. Such filamentary connections are absent in the precise form of flagellar coordination in unicellular algae. configurations of two pipette-held uniflagellate cells and defective in a class of CR mutants known as vfl (variable number of flagella) green algae | flagella | synchronization | basal fibers | internal coupling (Fig. 1B). We show, in both cases, that the synchronization states are markedly different from the wild-type breaststroke. ossession of multiple cilia and flagella bestows significant Seeking evidence for the generality of putative internal control Pevolutionary advantage upon living organisms only if these of flagellar coupling in algal unicells, we use light microscopy, organelles can achieve coordination. This may be for purposes of high-speed imaging, and image processing to elucidate the re- swimming (1, 2), feeding (3), or fluid transport (4, 5). Multiciliation markable coordination strategies adopted by quadriflagellates, may have evolved first in single-celled microorganisms due to the octoflagellates, and hexadecaflagellates, which possess networks of propensity for hydrodynamic interactions to couple their motions, basal, interflagellar linkages that increase in complexity with fla- but it was retained in higher organisms, occurring in such places as gella number. The flagellar apparatus, comprising BBs, connecting the murine brain (6) or human airway epithelia (7). Since Sir James Gray first noted that “automatic units” of flagella beat in “an orderly sequence” when placed side by side (8), others have Significance observed the tendency for nearby sperm cells to undulate in unison or aggregate (9, 10), and subsequently the possible hydrodynamic In areas as diverse as developmental biology, physiology, and origins of this phenomenon have been the subject of extensive biomimetics, there is great interest in understanding the mecha- theoretical analyses (2, 5, 11). Despite this, the exclusiveness and nisms by which active hair-like cellular appendages known as fla- universality of hydrodynamic effects in the coordination of neigh- gella or cilia are brought into coordinated motion. The prevailing boring cilia and flagella remains unclear. theoretical hypothesis over many years is that fluid flows driven by We begin by considering one context in which hydrodynamic beating flagella provide the coupling that leads to synchronization, interactions are sufficient for synchrony (12). The alga Volvox but this is surprisingly inconsistent with certain experimentally carteri (VC) is perhaps the smallest colonial organism to exhibit observed phenomena. Here we demonstrate the insufficiency of cellular division of labor (13). Adult spheroids possess two cell hydrodynamic coupling in an evolutionarily significant range of types: Large germ cells interior of an extracellular matrix grow to unicellular algal species bearing multiple flagella, and suggest that form new colonies, whereas smaller somatic cells form a dense the key additional ingredient for precise coordination of flagellar surface covering of flagella protruding into the medium, enabling beating is provided by contractile fibers of the basal apparatus. swimming. These flagella generate waves of propulsion, which, “ ” Author contributions: K.Y.W. and R.E.G. designed research, performed research, analyzed despite lack of centralized or neuronal control ( coxless ), are data, and wrote the paper. coherent over the span of the organism (14). In addition, somatic The authors declare no conflict of interest. cells isolated from their embedding colonies (Fig. 1A)beattheir This article is a PNAS Direct Submission. P.L. is a guest editor invited by the Editorial flagella in synchrony when held sufficiently close to each other (12). Board. Pairwise configurations of these flagella tend to synchronize in Freely available online through the PNAS open access option. phase (IP) when oriented with power strokes in the same direction, 1To whom correspondence should be addressed. Email: [email protected]. but antiphase (AP) when oriented in opposite directions, as This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. predicted (15) if their mutual interaction were hydrodynamic. 1073/pnas.1518527113/-/DCSupplemental. E2784–E2793 | PNAS | Published online May 2, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1518527113 Downloaded by guest on October 1, 2021 A C CR cells turn by modulation of bilateral symmetry. During pho- PNAS PLUS multicellular IP AP totaxis (33), photons incident on the eyespot activate voltage-gated cis calcium channels, which alter levels of intracellular calcium, leading BB 2+ 5 μm to differential flagellar responses. Ionic fluctuations (e.g., Ca )alter not only the flagellar beat but also the synchrony of a pair. Gait 1. parallel 2. anti-parallel trans changes involving transient loss of synchrony (called “slips”) occur BB stochastically at rates sensitive to such environmental factors (15, 27, 34) as temperature, light, chemicals, hydrodynamics, and age of cell culture. In free-swimming cells, slips can alter the balance of hy- drodynamic drag on the cell body, producing a rocking motion that promotes subsequent resynchrony of flagella (31), but this does not B D cis BB explain the robust IP synchrony in cells held immobilized on mi- unicellular cropipettes (16, 29), nor the motility of isolated and reactivated flagellar apparatuses (35). The altered beat during slips is analogous to the freestyle gait (AP in Fig. 1) characterized in the phototaxis mutant ptx1, which stochastically transitions between IP and AP gaits 5 μm (15, 16). The dependence of CR flagellar synchronization state on or physiology through temperature or ionic content of the medium (27) trans BB leads us now to the possibility for intracellular coupling of flagella. no robust IP synchrony Early work (18) identified thick fibers connecting the two Chla- × × 3 Fig. 1. Flagellar synchronization in multicellular vs. unicellular algae. (A)Pairsof mydomonas BBs, including a 300 250 75 nm bilaterally sym- isolated, somatic flagella of VC tend to synchronize either in IP or AP depending metric distal fiber (DF), bearing complex striation patterns with a on their relative orientation. (B) CR flagella maintain position 2 yet swim a robust periodicity of ∼ 80 nm (Fig. 1B). Two or more parallel proximal IP breaststroke that is lost (i) by mutation of the DF, and (ii) in pairs of nearby fibers (PF) also connect the BB at one end (Fig. 1B). Striation uniflagellate cells. (C and D) Ultrastructure comprising BBs, rootlets, and eyespot periodicity varies across species, and is