i i “coupling100316˙full” — 2016/3/10 — 18:12 — page 1 — #1 i i Coordinated Beating of Algal Flagella is Mediated by Basal Coupling Kirsty Y. Wan ∗ and Raymond E. Goldstein ∗ ∗Department of Applied Mathematics and Theoretical Physics, University of Cambridge, Wilberforce Road, Cambridge CB3 0WA, UK Submitted to Proceedings of the National Academy of Sciences of the United States of America Cilia and flagella often exhibit synchronized behavior; this includes (VC) is perhaps the smallest colonial organism to exhibit cellular divi- phase-locking, as seen in Chlamydomonas, and metachronal wave sion of labor (13). Adult spheroids possess two cell types: large germ formation in the respiratory cilia of higher organisms. Since the ob- cells interior of an extracellular matrix grow to form new colonies, servations by Gray and Rothschild of phase synchrony of nearby swim- while smaller somatic cells form a dense surface covering of flag- ming spermatozoa, it has been a working hypothesis that synchrony ella protruding into the medium, enabling swimming. These flag- arises from hydrodynamic interactions between beating filaments. Recent work on the dynamics of physically separated pairs of flagella ella generate waves of propulsion which despite lack of centralized isolated from the multicellular alga Volvox has shown that hydrody- or neuronal control (“coxless”) are coherent over the span of the or- namic coupling alone is sufficient to produce synchrony. However, ganism (14). In addition, somatic cells isolated from their embedding the situation is more complex in unicellular organisms bearing few colonies (Fig. 1A) beat their flagella in synchrony when held suf- flagella. We show that flagella of Chlamydomonas mutants deficient ficiently close to each other (12). Pairwise configurations of these in filamentary connections between basal bodies display markedly dif- flagella tend to synchronize in-phase (IP) when oriented with power ferent synchronization from the wildtype. We perform micromanip- strokes in the same direction, but antiphase (AP) when oriented in ulation on configurations of flagella and conclude that a mechanism, opposite directions, as predicted (15) if their mutual interaction were internal to the cell, must provide an additional flagellar coupling. hydrodynamic. Yet, not all flagellar coordination observed in unicel- In naturally-occurring species with 4, 8 or even 16 flagella, we find diverse symmetries of basal-body positioning and of the flagellar ap- lular organisms can be explained thus. The lineage to which Volvox paratus that are coincident with specific gaits of flagellar actuation, belongs includes the common ancestor of the alga Chlamydomonas suggesting that it is a competition between intracellular coupling reinhardtii (CR) (Fig. 1B), which swims with a familiar in-phase and hydrodynamic interactions that ultimately determines the pre- breaststroke with twin flagella that are developmentally positioned cise form of flagellar coordination in unicellular algae. to beat in opposite directions (Fig. 1C,D). Yet, a Chlamydomonas mutant with dysfunctional phototaxis switches stochastically the ac- Chlorophyte j Prasinophyte j eukaryotic algae j internal cou- tuation of its flagella between IP and AP modes (15, 16). These ob- pling j flagellar synchronization j basal fibers servations led us to conjecture (15) that a mechanism, internal to the cell, must function to overcome hydrodynamic effects. Pairs of interacting flagella evoke no image more potent than Huy- Significance Statement gens’ clocks (17): two oscillating pendula may tend towards syn- In areas as diverse as developmental biology, physiology and chrony (or anti-synchrony) if attached to a common support, whose biomimetics there is great interest in understanding the mechanisms flexibility providing the necessary coupling. Here we present a di- by which active hair-like cellular appendages known as flagella or verse body of evidence for existence of a biophysical equivalent to cilia are brought into coordinated motion. The prevailing theoret- this mechanical coupling, which in CR and related algae we propose ical hypothesis over many years is that fluid flows driven by beat- is provided ultrastructurally by prominent fibers connecting pairs of ing flagella provide the coupling that leads to synchronization, but basal bodies (BB) (18) that are known to have contractile proper- this is surprisingly inconsistent with certain experimentally observed ties. Such filamentary connections are absent in configurations of two phenomena. Here we demonstrate the insufficiency of hydrodynamic pipette-held uniflagellate cells and defective in a class of CR mutants coupling in an evolutionarily significant range of unicellular algal known as vfl (Fig. 1B). We show in both cases that the synchroniza- species bearing multiple flagella, and suggest the key additional in- tion states are markedly different from the wildtype breaststroke. gredient for precise coordination of flagellar beating is provided by Seeking evidence for the generality of putative internal control of contractile fibers of the basal apparatus. flagellar coupling in algal unicells, we use light microscopy, high- speed imaging and image-processing to elucidate the remarkable co- ordination strategies adopted by quadri-, octo-, and hexadecaflagel- Introduction lates, which possess networks of basal, interflagellar linkages that ossession of multiple cilia and flagella bestows significant evo- increase in complexity with flagella number. The flagellar appara- Plutionary advantage upon living organisms only if these or- tus, comprising BBs, connecting fibers, microtubular rootlets and the ganelles can achieve coordination. This may be for purposes of swim- transition regions of axonemes, is among the most biochemically and ming (1, 2), feeding (3), or fluid transport (4, 5). Multiciliation may morphologically complex structures occurring in eukaryotic flagel- have evolved first in single-celled microorganisms due to the propen- sity for hydrodynamic interactions to couple their motions, but was retained in higher organisms, occurring in such places as the murine Reserved for Publication Footnotes 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 observed the tendency for nearby sperm cells to undulate in unison or aggregate (9, 10), and subsequently the possible hydrodynamic origins of this phenomenon have been the subject of extensive theoretical analyses (2, 5, 11). De- spite this, the exclusiveness and universality of hydrodynamic effects in the coordination of neighboring cilia and flagella remains unclear. We begin by considering one context in which hydrodynamic in- teractions are sufficient for synchrony (12). The alga Volvox carteri www.pnas.org/cgi/doi/10.1073/pnas.0709640104 PNAS Issue Date Volume Issue Number 1{16 i i i i i i “coupling100316˙full” — 2016/3/10 — 18:12 — page 2 — #2 i i 2+ multicellular IP AP ing to differential flagellar responses. Ionic fluctuations (e.g. Ca ) cis alter not only the flagellar beat, but also the synchrony of a pair. BB Gait changes involving transient loss of synchrony (called ’slips’), 5 µm occur stochastically at rates sensitive to such environmental factors (15, 27, 34) as temperature, light, chemicals, hydrodynamics, and 1. parallel 2. anti-parallel trans BB age of cell culture. In free-swimming cells, slips can alter the balance of hydrodynamic drag on the cell body, producing a rocking motion V. carteri that promotes subsequent resynchrony of flagella (31), but this does not explain the robust IP synchrony in cells held immobilized on mi- cropipettes (16, 29), nor the motility of isolated and reactivated flag- ellar apparatuses (35). The altered beat during slips is analogous to cis BB the freestyle gait (AP in Fig. 1) characterized in the phototaxis mu- unicellular tant ptx1, which stochastically transitions between IP and AP gaits (15, 16). The dependence of CR flagellar synchronization state on physiology through temperature or ionic content of the medium (27) leads us now to the possibility for intracellular coupling of flagella. 5 µm Early work (18) identified thick fibers connecting the two Chlamy- or 3 C. reinhardtii domonas BBs, including a 300×250×75 nm bilaterally symmetric trans BB distal fiber (DF), bearing complex striation patterns with a periodic- no robust IP synchrony ity of ∼ 80 nm (Fig. 1B). Striation periodicity varies across species, Fig. 1: Flagellar synchronization in multi- vs uni- cellular algae. A) Pairs and is changeable by chemical stimuli – indicating active contractility of isolated, somatic flagella of V. carteri (VC) tend to synchronize either in (36). The DF contains centrin, also found in NBBCs (Fig. 1B) which in-phase (IP) or anti-phase (AP) depending on their relative orientation. B) C. are involved in localization of BBs during cell division (37). The two reinhardtii (CR) flagella maintain position 2 yet swims a robust IP breaststroke BBs have an identical structure of 9 triplet microtubules which form that is lost i) by mutation of the distal fiber (DF), and ii) in pairs of nearby uni- a cartwheel arrangement (38). Importantly, the DF lies in the plane flagellate cells. C+D) Ultrastructure comprising basal bodies (BBs), rootlets, of flagellar beating, and furthermore attaches to each BB at the same and eyespot in VC and CR. [PF - proximal fiber(s); NBBC - nuclear-basal- body connectors; t1 − 9: numbered microtubule triplets.] site relative to the beating