Reorganisation of Complex Ciliary Flows Around Regenerating Stentor

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bioRxiv preprint doi: https://doi.org/10.1101/681908; this version posted June 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1 Reorganisation of complex ciliary flows around

2 regenerating Stentor coeruleus 1,8 3 Kirsty Y. Wan* , Sylvia K. Hürlimann2,8, Aidan M. Fenix4,5,8, 4 Rebecca M. McGillivary3,8, Tatyana Makushok3,8, Evan Burns6,9, 5 Janet Y. Sheung7,9, Wallace F. Marshall*3,8 6 1. Living Systems Institute, University of Exeter, Stocker Road, Exeter, UK 7 2. Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA 8 3. Department of Biochemistry and Biophysics, UCSF, San Francisco, CA, USA 9 4. Department of Pathology, University of Washington, WA, USA 10 5. Center for Cardiovascular Biology, University of Washington, WA, USA 11 6. Department of Biology, Vassar College, NY, USA 12 7. Department of Physics and Astronomy, Vassar College, NY, USA 13 8. Marine Biological Laboratory, Physiology Course, Woods Hole, MA, USA 14 9. Marine Biological Laboratory, Whitman Center, Woods Hole, MA, USA 15 16 Keywords: Cilia coordination, metachronal waves, regeneration, development, ciliary flows, Stentor 17 18 19 Summary 20 21 The phenomenon of ciliary coordination has garnered increasing attention in recent decades, with multiple 22 theories accounting for its emergence in different contexts. The heterotrich ciliate Stentor coeruleus is a 23 unicellular organism which boasts a number of features which present unrivalled opportunities for 24 biophysical studies of cilia coordination. With their cerulean colour and distinctive morphology, these large 25 protists possess a characteristic differentiation between cortical rows of short body cilia used for swimming, 26 and an anterior ring structure of fused oral cilia forming a membranellar band. The oral cilia beat 27 metachronously to produce strong feeding currents. In addition to this complex body plan, Stentor have 28 remarkable regenerative capabilities. Minute fragments of single cells can over the period of hours or days, 29 regenerate independently into new, proportional individuals. Certain environmental perturbations elicit a 30 unique programmed response known as oral regeneration wherein only the membranellar band is shed and 31 a new, ciliated oral primordium formed on the side of the body. Here, we target oral regeneration induced by 32 sucrose-shock to reveal the complex interplay between ciliary restructuring and hydrodynamics in Stentor, 33 which accompanies the complete developmental sequence from band formation, elongation, curling, and 34 migration toward the cell anterior. 35

36 “When the anterior part is open, one may perceive about its Edges a very lively Motion; and when the Polyps 37 presents itself in a certain manner, it discovers, on either side of these edges of its anterior part, somewhat 38 very much resembling the wheels of a little Mill, that move with great velocity.” 39

40 A. Trembley F.R.S describing the membranellar band of Stentor, 41 Phil. Soc. Trans. Royal Society (London), 1744.

42 1. Introduction 43 1.1. Stentor - the trumpet animalcule 44 The first recorded account of these fascinating funnel-shaped creatures was made by Abraham Trembley F.R.S, 45 who in a letter to the president of the Royal Society [1], wrote “I have herewith the honour of transmitting to 46 you the particulars of several observations I have made, during the course of the last summer, upon some 47 species of very minute Water-Animals…” [2]. (In fact, at 1the time Trembley had misidentified these organisms

*Authors for correspondence ([email protected], [email protected])

1 as Hydra.) These unicellular ciliates, which are easily visible to the naked eye, are named Stentor for their 2 trumpet-like morphology, in honour of a loud-voiced Greek hero who fought in the Trojan War. Stentor are 3 widely-occurring in both freshwater and marine habitats, exhibiting both free-swimming and sedentary 4 characteristics depending on environmental circumstance. Individual cells undergo dynamic and extensive 5 shape changes due to a highly-contractile cortical structure, assuming a pear or tear-drop shape when free- 6 swimming, contracting into a ball when disturbed, or else extending up 1mm in a rest state in which cells 7 become attached to substrates via a posterior holdfast (Figure 1a). In the latter state, a large number of ciliated 8 feeding organelles are visible at the anterior end of the organism, which stir the fluid collectively to produce 9 strong feeding currents, drawing food and other particulates towards the mouth of the organism. Not only is 10 the oral apparatus the site of a remarkable degree of ciliary coordination and highly-coupled fluid-structure 11 interactions, it is the most differentiated cortical landmark of a Stentor, which can also be regenerated in 12 response to injury or damage. The present study solicits this dramatic regenerative capability to investigate 13 ciliary organisation and coordination - which remains an open and confounding problem in ciliary biology. 14 Here, state-of-the-art imaging and flow field tracking is combined for the first time, to explore the extreme 15 restructuring of cilia and extracellular flows during Stentor oral regeneration. 16 While diverse species of Stentor exist, we focus exclusively on S. coeruleus Ehrenberg 1830 -- a widely 17 distributed species, notable for their striking cerulean pigmentation. S. coeruleus, also called the Blue Stentor [2, 18 3], was favoured historically for cytological studies due to its large size and moniliform macronucleus [4], and 19 has been the subject of recent genomic studies [5]. The conical surface of the organism bears longitudinal 20 stripes of distinctive colouration 21 extending from the anterior all the 22 way down to the tapered holdfast: 23 cortical rows of constant width, 24 alternate with pigmented stripes of 25 graded width – thereby breaking 26 the patterning symmetry (Figure 27 1b). The pigmented bands increase 28 in width from left to right around 29 the cell, culminating in a special 30 region called the locus of stripe 31 contrast (LSC) or “ramifying zone”. 32 This region demarcates the location 33 where the new oral primordium 34 and a new field of basal bodies will 35 form. 36 The body cilia are inserted 37 in rows along the clear stripes all 38 over the body; the anterior body 39 cilia are somewhat shorter than 40 those at the posterior [4]. 41 Contractile fibres called myonemes 42 subtend each row of body cilia, and 43 are involved in whole-body 44 contractions. The broad anterior 45 end of the organism is encircled by 46 an adoral zone of external cilia that 47 are fused into rows of transverse 48 plates termed membranelles (sensu 49 Sterki 1878). In SEM studies, Figure 1a) Schematic of a single Stentor cell with key features highlighted. Inset - 50 Randall and Jackson showed [6] ultrastructure of membranellar band [6] showing rows of oral cilia arranged in 51 that these adoral structures were parallel stacks (each approximately 7.5µm x1.5µm); b) confocal immunofluorescence 52 not individual large cilia but rather images showing the structure and organisation of the membranellar band (M), cortical 53 two or three rows of tightly-packed striation patterns (notice the sharp change in width at the locus of stripe contrast: 54 cilia (20-25 cilia per row), so that LSC), and associated rows of short body cilia, c) top view of the frontal field and gullet region (G). [Scalebars = 10 µm.] 55 each membranelle maintains 56 functional unity as an intercalated ciliary sheath (Figure 1a, inset). The entire membranellar band, also known 57 as the peristome, contains around 250 such stacked membranelles; one end terminates freely, while the other 58 end spirals into a funnel-shaped invagination or gullet (Figure 1c). The region enclosed by the membranellar 59 band is the frontal field, which is distinguished by a change in stripe orientation – the frontal field stripes are 60 also narrower since they originated as ventral stripes that have been shifted forward over the course of oral 61 primordium formation (see section 2), together with the new stripes which will go on to develop in this zone. 62 bioRxiv preprint doi: https://doi.org/10.1101/681908; this version posted June 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1 1.2. Regenerative prowess of Stentor 2 Regeneration of ciliated structures provides a unique way of probing the emergence and reorganisation of 3 ciliary interactions over developmental timescales. A hallmark of life is its ability to repair, heal wounds, or 4 replenish lost or damaged cellular structures [7, 8]. Single cells are of special interest, for regeneration must 5 necessarily take place entirely within the same cell. One classic example is flagellar regeneration in the 6 biflagellate alga Chlamydomonas, where amputated flagella can be regrown to full length in about 1.5 hours [9, 7 10], whereupon biflagellar coordination also recovers [11, 12]. Early researchers demonstrated the versatility 8 of Stentor as a model system for studies of regeneration, made possible by their large size and manoeuvrability, 9 as well as remarkable regenerative capabilities [13, 14]. Questioning the limits of cellular indivisibility led the 10 likes of F.R. Lillie and T.H. Morgan [15, 16] to conduct dissection experiments on Stentor to determine the 11 minimal ingredients for regeneration. They found that smallest of fragments capable of regenerating into new 12 individuals that would go on to divide and multiply, were spheroids of only <100 µm in diameter. Nucleated 13 fragments irrespective of whether they originate from the head or tail, are all capable of full regeneration. 14 Here, we study one particular developmental sequence of events which features consistently during 15 Stentor regeneration, in which a new membranellar band is formed de novo (Figure 2a-g). This characteristic 16 program is followed routinely by cells undertaking physiological cell division to develop a second oral 17 primordium [17], and also by cells that have been induced by diverse treatments (such as dissections, grafting 18 and chemical exposure [18]) to shed and subsequently replace an existing primordium and associated oral 19 structures. Initiation of regeneration requires the presence of at least one macronuclear node, some 20 cytoplasmic material, as well as a piece of the microtubule cortex. Selective inhibition studies have shown that 21 DNA-dependent RNA synthesis is required to supply the new protein required during intermediate stages of 22 regeneration, but not during the later stages. Depending on temperature, medium, and other unknown factors 23 [19], the total regeneration time ranges between 8-10 hours at 20-25 °C. Wherever successful, regeneration 24 proceeds through the following major stages (Figure 2a): i) within the first hour a rift forms at the site of the 25 new primordium; ii) the first cilia sprout, reaching ~ 0.5 µm, new basal bodies form in the vicinity of existing 26 somatic cilia, membranellar cilia are distributed randomly, and beating is uncoordinated at this stage; iii) at 3- 27 4 hours proliferation of basal bodies continues and the new primordium attains the definitive width of 12 µm, 28 cilia approach full length though shorter cilia are still evident, and some evidence of coordinated ciliary 29 activity is visible in SEM sections [20]; iv) cilia assemble into membranelles and the membranellar band 30 structure continues to lengthen, the moniliform nucleus condenses; v) at 6.5-7 hours, significant membranellar 31 band migration towards the anterior of the organism occurs, vi) finally, after ~8 hours, the newly formed 32 membranellar band curls all the way around the anterior end, and the nucleus renodulates. 33 For this study, we regenerated ~80 Stentor cells, and can confirm all qualitative morphological features 34 of the above staging. Oral regeneration in S. coeruleus was induced by sucrose shock, with slight modifications 35 from published protocols [21, 5], which were based on historical works (see supp. materials). Briefly, Stentor 36 were exposed to a 10% sucrose solution for 2-3 minutes, which elicited synchronous shedding of the 37 membranellar band in >80% of the cases (supp. Materials). Cells were then left to recover in fresh medium, 38 and extracellular flows and ciliary activity accompanying regeneration were analysed on a cell-by-cell basis. 39 40 2. Reorganisation of ciliary flows during regeneration 41 In order to measure the flow fields around Stentor at high spatiotemporal resolution, we developed a simple 42 but effective protocol for preventing cell body motion, adapted from conventional cell attachment techniques. 43 Glass-bottom petri dishes used for imaging were pre-treated with Poly-D-Lysine to encourage surface 44 adherence of Stentor (supp. Materials). Contrary to many other methods trialled previously, this particular 45 surface attachment protocol had minimal effect on cell viability, allowing for live-cell imaging for extended 46 periods of time, and did not adversely alter the oral regeneration dynamics. The boundary conditions 47 consisted of a free surface (a large open droplet), and a flat solid substrate. To obtain flow fields around the 48 regenerating organisms, we seeded the medium with passive tracers (1 µm polystyrene beads) and performed 49 Particle Image Velocimetry (PIV) [22]. The body cilia remained motile but beat only intermittently throughout 50 the oral regeneration process, and were capable of producing large-scale flows. This unsteady nature of ciliary 51 activity is not fully-understood, but also pertains to non-adhered organisms (supp. Figure S1). Here, in order 52 to isolate flow contributions due to membranellar cilia only, we restrict our discussion and analysis to 53 recordings which do not show excessive body cilia motion or body contractions (supp. Figure S2), and in 54 which flows remained steady over the entirety of each 30s recording. 55 56 2.1. Early regeneration and a linear membranellar band (0-5 hrs post-sucrose shock) 57 Under brightfield microscopy, the preliminary stages of regeneration were particularly difficult to discern in 58 the substrate-adhered organisms -- it is not possible to see the newly-formed rift and elongating membranellar 59 band until after a substantial structure has been formed. Sucrose-induced shedding rarely removes 100% of 60 the membranelles -- in most cases a small number of these will remain deep inside the old gullet, destined to 61 merge with the newly formed membranellar band once the latter migrates successfully all the way to the bioRxiv preprint doi: https://doi.org/10.1101/681908; this version posted June 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1 anterior. In these cases, the old membranelles can produce weak flows towards the gullet (Figure 2c). The new 2 membranellar cilia reach a perceptible length at 4-5 hours, forming a linear band on the side of the organism. 3 4 2.2. Membranellar band growth and reorientation (5-7 hrs post-sucrose shock) 5 As the membranellar cilia continue to grow, ciliary coordination increases concomitantly (see section 3). The 6 new membranellar band is usually conspicuous under DIC microscopy at 5 hours after sucrose shock, and 7 associated with localised flows that are directed posteriorly and largely parallel to the cell surface (Figure 2d, 8 supp. Video 1). Over the next hour, the membranellar cilia proceed to lengthen and reorganise [20], producing 9 flows which exhibit a greater perpendicular component that is directed away from the cell body (Figure 2e). 10 Flow magnitudes remain low during this period, on the order of 10 µm/s. After ~7 hours, the band structure 11 migrates upwards and the new gullet begins to form, the cell anterior becomes more conical and distortion of 12 streamlines is observed (Figure 2f). 13 14 2.3. Emergence of the feeding vortex and completion of regeneration (>7 hrs post-sucrose shock) 15 As the arc of the developing membranellar band continues to move upward, the two ends eventually approach 16 each other and the enclosed stripes bend into arcs in the frontal field (Figures 1, 2a). Finally, a characteristic 17 double-vortex pattern emerges – as in the control organisms (Figures 2b, g). In fully-regenerated Stentor, the 18 membranelles exhibit robust phase coordination and metachronal waves (MCW), which results in a sharp 19 transition to faster flows (order 100 µm/s) and appearance of the “alimentary vortex” sensu Maupas [2]. 20 Structurally similar flow fields were also measured in Stentor that have not been adhered to a substrate, but 21 rather were attached spontaneously by their posterior holdfast (supp. Figure S1). The morphology of these

Figure 2: a) Chronology of membranellar restructuring during induced oral regeneration in Stentor coeruleus: at time 0 the existing membranellar band was induced to shed by sucrose shock, after 1-2 hrs a rift opens in the vicinity of the indifferent zone, between 2-5 hrs membranellar cilia sprout, lengthen, and rearrange themselves into rows of stacked membranelles, after 5 hrs, the membranellar band undergoes elongation and gradual migration to finally assume a nearly-circular structure at the anterior end; b)-g) PIV measurements of the extracellular flow fields associated with the regenerating membranellar band for a control cell, and at the indicated times post-shock for different regenerating cells. [Colormaps indicate flow speed. Masked, brightfield images of the adhered organisms have been overlaid on top of the flow maps. MB demarcates the location of the membranellar band - wherever it is clearly identifiable.] bioRxiv preprint doi: https://doi.org/10.1101/681908; this version posted June 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1 flow fields compare well with feeding flows around isolated Vorticella (a contractile, stalked ciliate which bears 2 strong resemblance to Stentor) in the presence of a no-slip boundary [23, 24]. 3 4 3. Ciliary coordination and emergence of metachronal waves 5 3.1. Spatiotemporal correlations 6 Next, we use high-speed imaging (frame rate of 1 KHz) to investigate how the arrangement and synchronicity 7 of the membranellar cilia contributes to the restructuring of extracellular flows during oral regeneration. Image 8 sequences were processed to localise fast-moving ciliary structures (Figure 3a and inset). Greyscale intensities 9 measured from each region of interest were cross-correlated to determine how the spatiotemporal 10 coordination exhibited by the beating membranelles changes over time.

Figure 3: a) To measure ciliary coordination, a heatmap was used to first localise the beating cilia, e.g. to a narrow band on the surface of the organism (arrow in direction of increasing arclength from posterior to anterior). Dashed line encloses a region of interest, parallel to the ciliary band, which was used to compute intensity kymographs. Inset: kymograph can reveal local structure and coherence. b) The 2D intensity autocorrelation was used to measure changes in ciliary coordination over the course of regeneration. Sustained MCWs (parallel lines of high correlation propagating posterior-anteriorly) only emerge at very late times, here, slope = MCW speed. 11 At very early times, the short membranelles beat without proper coordination. Over the next 1-2 hours, the 12 growing ciliary band remains linear and incomplete waves of activity are propagated from the newly formed 13 oral primordium toward the anterior (Figure 3b, 6hr 30min, supp. Video 2). However, there is no long-range 14 coordination at this stage contrary to reports in historic works [20]. Instead, characteristic wave-like structures 15 appear and disappear intermittently along the membranellar band, but rarely extending beyond one or two 16 wavelengths. As the reshaping structure begins to migrate toward the anterior, the MCWs become slightly 17 more coordinated and sustained over much longer distances (Figure 3b, 7hr 30min). The individual 18 membranelles beat in a direction perpendicular to the developing peristome to propagate a diaplectic MCW. 19 Hereafter, the ciliary beat amplitude no longer changes, but the beat frequency and speed of wave propagation 20 remain subject to intracellular control [25]. Very late in regeneration, a single, highly-coordinated, MC is 21 propagated circularly from the gullet around the entire peristome - and it is only then that global coordination 22 of the membranelles is attained (supp. Video 3). Existence of a single unidirectional wave is evident from plots 23 of 2D intensity autocorrelations as a function of time and arclength along the developing membranellar band. 24 In the examples shown, both control Stentor and very late-stage regenerating cells show parallel lines of equal 25 slope (Figure 3b), corresponding to 0.58 mm/s and respectively 0.57 mm/s for the speed of MCW propagation, 26 and 16.1 Hz versus 20.8 Hz for the mean beat frequencies of individual membranelles. The timing of global 27 coordination is also coincident with the emergence of the strong feeding vortex. bioRxiv preprint doi: https://doi.org/10.1101/681908; this version posted June 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1 3.2. Infraciliature and hydrodynamic interactions 2 To explain the lack of ciliary coordination at early times and emergence of global order at late times, we 3 correlated our light microscopy data with transmission electron microscopy (TEM) sections of fixed Stentor 4 (supp. Materials). Paulin & Bussey [20] showed previously that in the early stages of oral regeneration new 5 membranellar cilia sprout from basal bodies that are arranged randomly in the anarchic field, consistent with 6 observed lack of ciliary coordination. At 4-5 hrs these undergo restructuring to assemble regularly stacked 7 rows of membranelles, with fibrillar structures connecting neighbouring membranelles also appearing at this 8 stage (Figure 32, [6]). A rotation in direction of fluid pumping from longitudinal to transverse (Figure 2d, e) is 9 fully consistent with the transition from a longitudinally directed beat pattern to a transversely directed beat 10 [20]. The intermittent coordination between nearby membranelles observed at this stage (Figure 3b) can be 11 attributed to hydrodynamic interactions: strong correlation at small spatial and temporal lags arises from 12 strong coupling between nearby 13 membranelles over distances of ~10 µm, 14 in both intermediate (cilia have already 15 reached full-length, Figure S3) and late- 16 stage regenerating cells. This lengthscale 17 is comparable to the bifurcation length 18 for hydrodynamic coupling [26]. Further 19 evidence of hydrodynamic effects can be 20 deduced from Tartar's finding that ciliary 21 coordination in grafted Stentor can be 22 immediate – long before any new cellular 23 connections could have formed between 24 the new and old membranelles [2]. 25 Though necessary, such passive 26 fluid-structure interactions alone are 27 insufficient to achieve global ciliary 28 coordination. Instead, Stentor MCWs 29 are regulated by excitatory signalling 30 via a “silver line” system of inter- 31 membranellar connections with the Figure 4: Correlating TEM sections with live-cell light microscopy imaging at the same regeneration stage -- at 6 hrs 45mins post sucrose-shock: a) the ultrastructure 32 primordium-facing membranelles acting is indistinguishable from control Stentor, fibrillar structures extend from the 33 as pacemaker [25]. As in some species of membranelles into the cytoplasm, in addition to transverse connections between 34 algal multiflagellates [27, 28], contractile neighbouring membranelles (arrows); b) cilia at the corresponding stage in live 35 elements can reverse the beating cells exhibit local but not global coordination – transient waves are propagated 36 direction of the membranelles from the new oral primordium situated at the posterior (P), to the anterior end of 37 dynamically [29]. Addition of digitoxin the organism (A). 38 and other chemicals can directly modulate intermembranellar connectivity leading to a change in MCW wave 39 velocity but affect the beat frequencies to a lesser extent [30]. Existence of a single peristomal frequency must 40 require physical continuity across entire membranellar band – indeed in non-regenerating Stentor, MCWs can 41 be stopped or altered by severing or manipulating the basal fibres between membranelles [2, 31]. 42 We conclude therefore that geometry is the final ingredient required to achieve global metachrony. In 43 particular, by the 7 hr stage, all internal, fibrillar structures are already present (Figure 4a) as in control Stentor, 44 yet coordination could not be sustained along the entire linear band of membranelles (Figure 4b). Instead, a 45 MCW with a unique, constant wave speed only emerges after the conical reshaping of the frontal field where 46 the entire band becomes nearly circular. These findings highlight the importance of topology [32], curvature 47 [33], and periodic boundary conditions [34] for the stabilisation of MCWs on ciliated surfaces. Particularly, 48 this is consistent with the notion that global ciliary coordination requires a precise orientation and localisation 49 of stacked membranelles. We suggest that for this reason oral regeneration in Stentor always proceeds in the 50 same manner in order to attain a highly-specific, ultimate ciliary configuration which can effectively exploit 51 the interciliary hydrodynamic interactions [35, 36, 37]. Further investigation of the specialised geometric 52 constraints exhibited by Stentor membranellar cilia, and their relation to the structure and dynamics of the 53 MCWs, will appear in a follow-up study. 54 55 4. Discussions and Outlook 56 The formation of a new oral apparatus in Stentor is a remarkable feat of organellar regeneration, differentiation, 57 and structural reorganisation involving the proliferation of 15,000 individual basal bodies -- their 58 reconfiguration and relocalisation into a functional entity, along with a precise arrangement and migration of 59 associated subpellicular microtubules, basal-body associated fibres and other contractile filaments. Here, this 60 process has been studied systematically in live cells for the first time with correlative flow-tracking and high- 61 speed imaging. Live-cell imaging is indispensable when inferring dynamics and behaviour, as erroneous bioRxiv preprint doi: https://doi.org/10.1101/681908; this version posted June 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1 conclusions could be drawn from interpretation of static EM images alone. Here, we developed a robust 2 methodology to track how spatiotemporal coherence in the membranellar structure evolves in substrate- 3 adhered organisms over the full course of regeneration (up to 10 hours of continuous imaging), starting from 4 incoherent random ciliary beating, to enhanced coordination and reorientation of fluid pumping, and 5 ultimately to the emergence of a single directional MCW spanning the peristome which generates and sustains 6 large-scale vortical flows. The synchronization dynamics of MCWs in Stentor add a novel dimension to a 7 growing literature on biophysical studies of interacting arrays of cilia in existing model systems [38, 39, 28]. 8 Specifically, the present study highlights the necessity of correct basal body and membranellar alignment, 9 fibrillar connections, as well as hydrodynamic interactions to achieve overall metachronal coordination. 10 Stentor is an emerging model organism that has great potential for biophysical studies of ciliary 11 regeneration and coordination, offering many novel avenues for consideration by the interested reader. Its 12 unique and multifarious regenerative capabilities can be used in conjunction with targeted physical 13 manipulations such as dissection, grafting or induced resorption to study ciliary and flow reorganisation. 14 However, the novelty of this system also presents a number of challenges. Firstly, we were unable to obtain 15 cell-cycle synchronized cultures; this coupled with the highly-contractile cell body meant that experimental 16 organisms were highly variable in size, shape, and behaviour. Secondly even though the dynamics concerned 17 are intrinsically three-dimensional, for constraints of phototoxicity and imaging speed we use 2D-imaging 18 only. As part of this study, we had also intended to investigate an alternate form of oral regeneration termed 19 in situ regeneration, in which membranellar cilia reputedly could be induced to sever at the transition zone 20 and regrown in place (as in the biflagellate alga Chlamydomonas [40]), without any morphological restructuring 21 of the cytoskeleton [41, 42]. In situ regeneration would have enabled the effective decoupling of intracellular 22 or ultrastructural changes from passive hydrodynamic interactions during cilia and membranellar regrowth, 23 but after multiple attempts the present authors were unable to reproduce this configuration - the 24 membranellar cilia were always shed together with the entire membranellar band. Finally, we suggest that 25 microsurgical manipulation can provide further insights into the ciliary coordination mechanism. 26 Despite these sources of variability, our results demonstrate a remarkable consistency in the timing 27 and dynamics of Stentor oral regeneration. Electing to use the sucrose-shock method we were able to induce 28 reversible and simultaneous shedding of membranellar bands in large sample sizes. Physiological 29 regeneration, for instance due to injury of feeding organelles or excess mechanical stresses, also elicit a similar 30 programmed response [43, 4]. Thus, membranellar regeneration appears to be a universal mechanism by 31 which unicellular protists repair and refresh their oral structures [44]. The concomitant change in topology 32 from linear to circular appears to be critical for attainment of global coordination of membranelles and the 33 inward-directed feeding flows. In this regard, protists such as Stentor that can achieve great complexity of 34 cortical organisation and internal control over locomotor appendages, could hold the key to understanding 35 the evolution of neural circuitry [45, 46] designed for deriving coherence of ciliary arrays. 36 37 Endnotes 38 Authors' Contributions 39 KYW & SKH conceived the study, performed regeneration experiments, data analysis, and wrote the 40 manuscript; AMF performed IF experiments; RMM & TM cultured organisms, assisted with microscopy, 41 contributed EM data; EB & JYS obtained flow data for unattached organisms; WFM secured funding, provided 42 supervision, and initiated the collaboration at the MBL in Woods Hole. All authors contributed to project 43 design and article revision, and have given approval for the final version. 44 Acknowledgments 45 We thank Greyson Lewis and Akanksha Thawani for discussions. We thank Kasia Hammer (Central 46 Microscopy Facility, MBL) for preparing samples for electronmicroscopy, and the Nikon Imaging Center at 47 UCSF for access to imaging facilities. We remain indebted to the staff and students of the MBL Physiology 48 Course for creating a stimulating environment for scientific research. 49 Competing Interests 50 We declare we have no competing interests. 51 Funding 52 We gratefully acknowledge financial support from the Marine Biological Laboratory at Woods Hole, MA, NIH 53 grant R35 GM097017 (WFM) and the University of Exeter, UK (KYW). 54 55 56 The following multimedia files accompany this study: 57 1. DIC imaging of flow fields around a regenerating Stentor (5.5 hrs post sucrose shock) 58 2. High-speed video of linear band (6.5 hrs post sucrose shock) 59 3. High-speed video of fully-regenerated membranellar band (8.5 hrs post sucrose shock) 60 61 bioRxiv preprint doi: https://doi.org/10.1101/681908; this version posted June 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1 References [1] A. Trembley, “Letter to the Royal Society,” Philosophical Transactions of the Royal Society (London), 1744. [2] V. Tartar, The biology of Stentor, Pergamon Press, 1961. [3] M. M. Slabodnick and W. F. Marshall, “Stentor coeruleus,” Current Biology, vol. 24, R783-4, 2014. [4] H. P. Johnson, “A contribution to the morphology and biology of the Stentors,” Journal of morphology, vol. 8, pp 467-562, 1893. [5] P. Sood, R. McGillivary and W. F. Marshall, “The Transcriptional Program of Regeneration in the Giant Single Cell, Stentor coeruleus,” bioRxiv 240788, 2017. [6] J. T. Randall and S. F. Jackson, “Fine Structure and Function in Stentor polymorphus,” The Journal of Cell Biology, vol. 4, pp. 807-830, 11 1958. [7] S. K. Y. Tang and W. F. Marshall, “Self-repairing cells: how single cells heal membrane ruptures and restore lost structures,” Science, vol 356, pp. 1022-1025, 2017. [8] P. W. Reddien and A. S. Alvarado, “Fundamentals of Planarian regeneration,” Annual Review of Cell and Developmental Biology, vol. 20, pp. 725-757, 11 2004. [9] J. L. Rosenbaum, “Flagellar elongation and shortening in Chlamydomonas: The Use of Cycloheximide and Colchicine to Study the Synthesis and Assembly of Flagellar Proteins,” The Journal of Cell Biology, vol. 41, pp. 600-619, 5 1969. [10] N. L. Hendel, M. Thomson and W. F. Marshall, “Diffusion as a Ruler: Modeling Kinesin Diffusion as a Length Sensor for Intraflagellar Transport,” Biophysical Journal, vol. 114, pp. 663-674, 2018. [11] K. Y. Wan, K. C. Leptos and R. E. Goldstein, “Lag, lock, sync, slip: the many 'phases' of coupled flagella,” Journal of the Royal Society Interface, vol. 11, p. 20131160, 2014. [12] R. E. Goldstein, M. Polin and I. Tuval, “Emergence of Synchronized Beating during the Regrowth of Eukaryotic Flagella,” Physical Review Letters, vol. 107, p. 148103, 2011. [13] N. Terra, “Leucine Incorporation into the membranellar bands of regenerating and non-regenerating Stentor,” Science, vol. 153, pp. 543-5, 1966. [14] W. Moxon, “On some points in the anatomy of Stentor and its mode of division,” Journal of Anatomy, vol 3, pp 279-293, 1869. [15] F. R. Lillie, “On the smallest parts of Stentor capable of regeneration; a contribution on the limits of divisibility of living matter,” Journal of Morphology, vol. 12, pp 239-249, 1896. [16] T. H. Morgan, “Regeneration of proportionate structures in Stentor,” Biological Bulletin, vol. 2(6), pp. 311-328, 1901. [17] V. Tartar, “Reactions of and Stentor coeruleus and to certain and substances added to the medium,” Experimental cell research, vol. 13(2), pp. 317-32, 1957. [18] E. A. James, “Regeneration and Division in Stentor coeruleus: the and Effects of Microinjected and Externally Applied actinomycin D and puromycin,” Developmental Biology, vol. 16(6), pp. 577-593, 1967. [19] B. B. Burchill, “Synthesis of RNA and Protein in Relation to Oral Regeneration in the Ciliate Stentor coeruleus,” Journal of Experimental Zoology, vol. 167, pp 427-438, 1968. [20] J. J. Paulin and J. Bussey, “Oral Regeneration in the ciliate Stentor coeruleus: A Scanning and Transmission and Electron Optical and Study,” Journal of protozoology, vol. 18, pp. 201-213, 1971. [21] A. Lin, T. Makushok, U. Diaz and W. F. Marshall, “Methods for the Study of Regeneration in Stentor,” Journal of Visualized Experiments, vol 5, e57759, 2018. [22] W. Thielicke and E. J. Stamhuis, “PIVlab - Towards User-friendly, Affordable and Accurate Digital Particle Image Velocimetry in MATLAB,” Journal of Open Research Software, vol. 2, 2014. [23] M. A. Sleigh and Barlow, “Collection of Food by Vorticella,” Transactions of the American Microscopic Society, vol. 95, pp. 482-486, 1976. [24] R. E. Pepper, M. Roper, S. Ryu, P. Matsudaira and H. A. Stone, “Nearby boundaries create eddies near microscopic filter feeders,” Journal of The Royal Society Interface, vol. 7, pp. 851-862, 2010. [25] M. A. Sleigh, “Further Observations on Co-ordination and the Determination of Frequency in the Peristomial Cilia of Stentor,” J. Exp. Biol., vol. 34, p. 106, 1957. bioRxiv preprint doi: https://doi.org/10.1101/681908; this version posted June 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

[26] D. R. Brumley, K. Y. Wan, M. Polin and R. E. Goldstein, “Flagellar synchronization through direct hydrodynamic interactions,” elife, vol. 3, p. e02750, 2014. [27] K. Y. Wan and R. E. Goldstein, “Time Irreversibility and Criticality in the Motility of a Flagellate Microorganism,” Physical Review Letters, vol. 121, p. 058103, 2018. [28] K. Y. Wan and R. E. Goldstein, “Coordinated beating of algal flagella is mediated by basal coupling,” Proceedings of the National Academy of Sciences USA, vol. 113, pp. E2784--E2793, 2016. [29] L. H. Bannister and E. C. Tatchell, “Contractility and the fibre systems of Stentor coeruleus,” Journal of Cell Science, vol. 3, pp. 295-308, 1968. [30] M. A. Sleigh, “Metachronism And Frequency Of Beat In The Peristomial Cilia Of Stentor,” Journal of Experimental Biology, vol. 33, pp. 15-28, 1956. [31] L. G. Worley, “Ciliary metachronism and reversal in Paramecium, Spirostomum and Stentor,” J. Cell. Comp. Physiol., vol. 5, pp. 53-72, 2019. [32] J. C. Nawroth, H. Guo, E. Koch, E. A. C. Heath-Heckman, J. C. Hermanson, E. G. Ruby, J. O. Dabiri, E. Kanso and M. McFall-Ngai, “Motile cilia create fluid-mechanical microhabitats for the active recruitment of the host microbiome,” Proceedings of the National Academy of Sciences USA, vol. 114, pp. 9510-9516, 2017. [33] B. Nasouri and G. J. Elfring, “Hydrodynamic interactions of cilia on a spherical body,” Physical Review E, vol. 93, p. 033111, 2016. [34] T. Niedermayer, B. Eckhardt and P. Lenz, “Synchronization, phase locking, and metachronal wave formation in ciliary chains,” Chaos, vol. 18, 037128, 2008. [35] J. Han and C. S. Peskin, “Spontaneous oscillation and fluid-structure interaction of cilia,” Proceedings of the National Academy of Sciences USA, vol. 115, p. 4417, 2018. [36] J. Elgeti and G. Gompper, “Emergence of metachronal waves in cilia arrays,” Proceedings of the National Academy of Sciences USA, vol. 110, pp. 4470-4475, 2013. [37] D. R. Brumley, M. Polin, T. J. Pedley and R. E. Goldstein, “Metachronal waves in the flagellar beating of Volvox and their hydrodynamic origin,” Journal of the Royal Society Interface, vol. 12, p. 20141358, 2015. [38] L. Gheber and Z. Priel, “Synchronization between beating cilia,” Biophysical Journal, vol. 55, pp. 183-191, 1989. [39] L. Feriani, M. Juenet, C. J. Fowler, N. Bruot, M. Chioccioli, S. M. Holland, C. E. Bryant and P. Cicuta, “Assessing the Collective Dynamics of Motile Cilia in Cultures of Human Airway Cells by Multiscale DDM,” Biophysical Journal, vol. 113, pp. 109-119, 2017. [40] W. Marshall, H. Qin, M. Rodrigo Brenni and J. Rosenbaum, “Flagellar length control system: testing a simple model based on intraflagellar transport and turnover,” Molecular Biology of the Cell, vol. 16, pp. 20-278, 2005. [41] Y. Shigenaka, T. Yamaoka, Y. Ito and M. Kaneda, “An Electron and Microscopical Study on Ciliary and Detachment and and Reformation in a Heterotrichous Ciliate and Stentor coeruleus,” J. Electron Microscopy, vol. 28, pp. 73-82, 1979. [42] V. Tartar, “Regeneration in Situ of Membranellar Cilia in Stentor Coeruleus,” Transactions of the American Microscopical Society, vol. 87, pp. 297-306, 1968. [43] N. M. Stevens, “Notes on Regeneration in Stentor coeruleus,” vol. 16, pp 461-475, 1903. [44] J. Bakowska, E. Marlo Nerlon. and J. Frankel., “Development of the Ciliary and Pattern of the Oral and Apparatus of Tetrahymena thermophila”, J. Protozoology, vol. 29, pp 366-382, 1982. [45] W. Gilpin, V. N. Prakash and M. Prakash, “Vortex arrays and ciliary tangles underlie the feeding and swimming trade-off in starfish larvae,” Nature Physics, vol. 13, pp. 380-386, 2016. [46] C. Veraszto, N. Ueda, L. A. Bezares-Calderon, A. Panzera, E. A. Williams, R. Shahidi and G. Jekely, “Ciliomotor circuitry underlying whole-body coordination of ciliary activity in the Platynereis larva,” eLife, vol. 6, e26000, 2017. [47] H. Onsbring, M. Jamy and T. J. G. Ettema, “RNA Sequencing of Stentor Cell Fragments Reveals Transcriptional Changes during Cellular Regeneration,” Current Biology, vol. 28, pp. 1281-1288, 2018.