1.1. Overview of Vorticella
Among various microorganisms, protozoa have been widely studied as model systems for biology, physics, engineering and biomimetics. They form a diverse group of unicellular eukaryotic organisms including ciliates, flagellates and amoeboids. Among such protists, Vorticella has drawn the attention of scientists since Anthony van Leeuwenhoek first described its unique motility in his letter dated 9 October 1676. In a recent study, Vorticella was discovered in a fossil that is more than 200 million year old, bolstering the fossil record of soft-bodied organisms [3]. While we primarily describe biomechanical and biomimetic aspects of Vorticella, uncovered since the 1950s, it is worth noting that Vorticella has played a long and ongoing role in many fields of biological, physical and engineering study.
Vorticella is a suspension-feeding ciliate that lives in two forms: free swimming telotroch and sessile stalked trophont [4]. A sessile Vorticella consists of the zooid (inverted-bell-shaped cell body; usually about 30–40 μm in diameter when contracted) and the stalk (3–4 μm in diameter and about 100 μm in length). The zooid has two bands of cilia around the peristome, the mouth-like part of the zooid, which are used for suspension feeding. The cilia of the inner band generate water flow to draw food particles toward the zooid (Figure 1B), and these particles are filtered by the cilia of the outer band. An example of such a feeding current observed in the laboratory shows a vortex (Figure 1B), and it was observed that this flow could move micro-diameter particles at least 450 μm away from the peristome with the maximum flow velocity of 360 μm/s [5,6].
In the sessile form, the vertex or scopula of the zooid is connected to the proximal end of the stalk while the distal end is rooted on a habitat surface (Figure 1A). Therefore, the stalk tethers the zooid to the habitat. The stalk also coils very quickly and moves the zooid toward the surface through the action of its contractile organelle, the spasmoneme (Figure 1C). Afterwards, the stalk slowly relaxes and moves the zooid away from the surface over a few seconds [10,11,12]. Although the stalk contraction-relaxation cycle of Vorticella has been hypothesized as a way either to avoid dangers, or to mix the surrounding fluid, no explanation is completely satisfactory.
During stalk contraction, Vorticella stops ciliary beating and contracts the zooid to a nearly spherical shape, and the stalk shortens to 20%–40% of its extended length in less than 10 ms. Therefore, Vorticella contracts at the average rate of 10–20 mm/s, and its zooid reaches a maximum speed of 60–90 mm/s. In terms of specific velocity (body length/s), this maximum speed corresponds to ~1200 body length/s, making Vorticella among the fastest living creatures using this metric [12]. It has also been estimated that in terms of specific power (power dissipation per mass) the stalk of Vorticella performs better than both automotive engines and striated muscle [15]. Recent measurements showed that live spasmonemes could generate a peak contractile force of ~30 nN with maximum power of ~1.6 nW during normal contraction, and isometric tension of 150–350 nN.
In addition to its remarkable motility, the energy source for the stalk contraction makes Vorticella unique. While most biological contractile systems depend on ATP (adenosine triphosphate) for their biochemical fuel, the Vorticella spasmoneme is powered by calcium ions [17]. In the absence of ATP, extracted Vorticella stalks can repeat the contraction–relaxation cycle and generate contractile force, driven only by external calcium ions diffusing into the spasmoneme . Because of its surpassing contractility and unique energy source, therefore, the Vorticella stalk is regarded as a candidate for Ca2+-powered cell motility and biomimetic actuating materials
1.2. Similar Microorganisms
There are various other microorganisms which contract using Ca2+, such as Stentor and Paramecium. Among them, Zoothamnium and Carchesium are close to Vorticella in that these species also have contractile stalks. These larger ciliates have one main stalk with many branches ending in zooids, which is a major difference from Vorticella, which has only a single stalk and zooid. Upon stimulation, Zoothamnium contracts the entire colony into one large globule and then folds the main stalk. In contrast, Carchesium contracts each zooid and its stalk separately. This difference is due to a structural difference of the spasmoneme between the two species: the Zoothamniumspasmoneme is continuous among zooids in the colony, whereas the Carchesium spasmoneme is discontinuous between zooids. Because Zoothamnium and Carchesium have bigger spasmonemes than Vorticella does (the spasmoneme of Zoothamnium is about 30 μm in diameter and 1 mm long [30]), they have also been employed for studying the spasmonemal contraction
In this review, we focus on Vorticella in the sessile form and summarize previous studies about its beating cilia and contractile stalk. Because the most recent reviews on Vorticella focus on its biological aspects and stalk contraction, our review is complementary to them, providing an in-depth review of Vorticella from cellular mechanics and bio-inspired engineering perspectives. Furthermore, we intend to introduce Vorticella as a promising biological model system for bio-inspired engineering and biomimetics.
This review consists of two main parts. In the first part, we introduce the common features of cilia and review previous studies on the flow-generating-capability of the Vorticella cilia. Then, we discuss possibilities of using the Vorticella cilia for engineering applications. In the second section, we focus on the stalk of Vorticella and its Ca2+-powered contraction, including relevant studies on Zoothamnium and Carchesium. We also discuss how Vorticella with the contractile stalk can be used in microscale systems as an actuator and as a model for bio-inspired engineering.
2. Cilia and Cilia-Generated Flow of Vorticella
2.1. Structure of Cilia Rows
In his first description of Vorticella, Leeuwenhoek thought that Vorticella had two horns moving like horse ears near the oral part [1,2]. What he actually observed were beating oral cilia generating water flow. These cilia have a structure common to eukaryotic cells: each cilium includes a bundle of microtubules extending continuously for the length of the cilium with the so called “9 + 2” pattern. As the stiffest cellular filament, a microtubule is a hollow tube-like polymer of a globular protein tubulin. In this 9 + 2 arrangement, a pair of singlet microtubules is surrounded by nine doublet microtubules.
Ciliary beating is characterized by a series of bends of the cilium, which depends on the sliding of adjacent doublet microtubules caused by dynein, one of the microtubule motor proteins [63]. Arms of dynein molecules are attached periodically along the length of the microtubule, and these arms of one doublet walk along the adjacent doublet powered by hydrolysis of ATP [62]. While each cilium beats periodically, their synchronized motion forms metachronal waves along the periphery of the oral part which causes a circulating current in the surrounding water. This ciliary metachronal wave is a common motility mechanism among microorganisms.
2.2. Ciliary Performance
Using Vorticella as a model protozoan for microscale fluid control requires evaluating its ciliary performance and understanding the flow caused by the cilia. Such studies experimentally measured the field and strength of the ciliary flow of Vorticella by tracing particles in the flow. Fluid dynamics modeling has also been employed to model the ciliary flow based on the fact that the flow is dominated by viscosity. Characterizations made with both experiments and theoretical models are discussed here.
2.2.1. Flow Measurement
Sleigh and Barlow first examined the feeding current of V. convallaria by tracing micron-sized particles in the vicinity of the zooid using high-speed cinematography [5]. They observed that 17 μm-long cilia beat at about 50 Hz with the tip speed of 4 mm/s while particles moved at 2.5 mm/s near ciliary tips. This ciliary beating enabled V. convallaria to draw particles 450 μm away from the oral part and to collect particles in a volume of about 0.3 mm3.
Later, Vopel et al. measured the velocity of the feeding current of a marine Vorticella using a flow microsensor [69]. The measured flow velocity was about 18 mm/s and 2.6 mm/s at a horizontal distance of 50 μm and 350 μm from the oral part, respectively, and 0.8 mm/s at the stalk base. Having observed that the Vorticella moved the surrounding seawater at least 400 μm from the zooid, which agrees with Sleigh and Barlow’s observation, Vopel et al. suggested that the ciliate could generate sufficient feeding flow by raising the zooid above the substrate by the stalk.
Recently, Nagai et al. rigorously measured the two-dimensional (2D) velocity components of the feeding current of V. picta using the confocal micro-particle image velocimetry (μ-PIV) [6]. Their reconstructed flow velocity field showed the same double-vortex structure seen by Sleigh and Barlow [5]. Although Sleigh and Barlow extrapolated an axisymmetric toroidal vortex structure from these twin vortices, truly three-dimensional flow measurement is still required to validate the seemingly toroidal feeding current of Vorticella.
Nagai et al. observed the maximum flow speed of about 360 μm/s occurring in front of the peristome, and evaluated the volume flow rate toward the oral part to be about 3 × 10−4 mm3/s. This maximum flow speed is about one order of magnitude lower than that of the previous two studies. This is because flow speed measurements depend on the region of the measurement in the vicinity of cilia and the resolution of imaging. It is noticeable that 1/3 of this inflow was carried from fresh water whereas 2/3 was recirculated. Therefore, V. picta appeared to clear water at a rate of 0.36 mm3/h and to collect particles from a fluid volume of 1.7 × 10−3 mm3.
Pepper et al. also measured the feeding current of V. convallaria using particle tracking velocimetry (PTV). Individual V. covallaria cells were anchored to the thin edge of a coverslip, confined between two closely-spaced surfaces. Having observed that increasing the spacing between the confining surfaces increased the size of the observed vortices, Pepper et al. suggested that the feeding flow in nature without confining boundaries nearby might differ significantly from the dual-vortex structure seen for Vorticella sandwiched between surfaces. A full 3D measurement of the feeding current around a Vorticella without nearby boundaries has not yet been made.
2.2.2. Fluid Dynamic Model
Since Vorticellae are small and their feeding current is relatively slow, they feed in the regime of low Reynolds number flow (i.e., Stokes flow) [70]. The Reynolds number is the ratio of inertial to viscous forces in a fluid flow, and is defined as Re = UL/ν. Here, U and L are a characteristic velocity and length of the flow, and ν is the kinematic viscosity of the fluid. For Vorticella in water (zooid diameter L ≈ 40 μm and feeding flow speed U ≈ 100 μm/s), a typical Reynolds number of the feeding current is 4 × 10−3. Thus, viscous forces dominate over inertia forces in the feeding current of Vorticella.
The feeding current of microscopic suspension feeders including Vorticella is often modeled as a single stokeslet, a point force in Stokes flow. A stokeslet in free space does not have the characteristic toroidal flow pattern seen in, whereas several different models of Vorticella feeding between two closely-spaced parallel boundaries result in such recirculating flow with tight, circular streamlines. This theoretical observation suggests that the circular vortices are due to the two boundaries sandwiching the organism, which are glass surfaces present in most experimental observations of Vorticella feeding flow. Indeed, the feeding flow field produced by Vorticella is likely to be determined mostly by the geometry of nearby boundaries.
A more realistic model for feeding Vorticellae is likely a stokeslet forcing fluid towards a single plane which represents the habitat substrate. Having modeled Vorticella as such a stokeslet, Blake and Otto found that the feeding current consists of a toroidal vortex around the organism, but not with the characteristic circular shape found in experiments and with a stokeslet between two boundaries [72].
Recently, Pepper et al. showed that the single stokeslet model with nearby boundaries agrees well with experimentally-measured feeding flow fields around Vorticella [8]. They also confirmed that the size of these eddies is determined by the distance between the two boundaries, which indicates that the eddies would not be present if no boundaries were nearby, as shown by. Moreover, they estimated that the total force exerted by the cilia of V. convallaria on the surrounding water is 0.05–0.5 nN. It has also been shown that when only one boundary is nearby, the fluid flow changes dramatically depending on the angle of Vorticella to the surface [84]. Therefore, effects of nearby boundaries need to be considered when utilizing the ciliary flow of Vorticella and other microorganisms.
2.3 Engineering Applications
It has been suggested that the biological motors of microorganisms can be used or mimicked in microsystems to reduce the overall size of devices, and cilia beating has been envisaged as a potential strategy for inducing fluid flow and mixing in microsystems. Cilia-mimicking devices such as artificial cilia have succeeded in generating pumping and mixing, but they require external power sources for operation, which enlarges the system. In contrast, cellular cilia do not require such external sources because they convert in situ biochemical energy to mechanical work [97], which seems ideal for components of microsystems. For instance, live carpets of flagellated bacteria have been used to produce directional flows and thus to transport fluid.
Similarly, the cilia of live Vorticella can function as a driving source for fluid motion in microfluidic devices. Recently, Nagai et al. used the oral cilia motion of live V. convallaria in a microchannel for mixing enhancement. Several V. convallaria cells on a channel surface caused active mixing between two streams in a Y-channel based on fluid transport generated by their cilia. Another possible application could be fluid pumping, for which multiple Vorticellae generate directional flow and thus transport target objects in the flow direction. For better performance, Vorticellae can be patterned in microsystems using a guiding flow into the system. However, drawbacks exist in using live Vorticellae in the microsystem, such as providing nutrients and removing waste for sustainable culture of the microorganisms, controlling their location, and packaging fluidic systems for controlling microenvironment.
3. Stalk and Ultrafast Contraction of Vorticella
3.1. Structure and Coiling of the Stalk
Arguably, the vast majority of the attention paid to sessile Vorticella has focused on the contractility of its stalk and thus the stalk structure. Although the coiling stalk of Vorticella was witnessed centuries ago under the first microscope [1,2], it has taken more modern investigations at various length scales to reveal the structural elements that enact Vorticella’s rapid movement.
The Vorticella stalk consists of a fibrous contractile organelle (the spasmoneme), a relatively robust sheath, a matrix of tiny fibers (the fibrillar matrix), and rod-like bundles of filaments (the bâtonnets) stiffening the sheath. The spasmoneme runs through the entire length of the stalk as a left-handed helix [21], whereas the bâtonnets run as a right-handed helix, always located on the opposite side of the sheath from the spasmoneme. The spasmoneme is intracellular, connected with the zooid whereas the sheath material is extracellular and primarily considered structural in nature.
Coiling of the stalk involves the mechanical interplay of these elements. As the stalk coils, the spasmoneme takes a shorter path through the coil while the bâtonnets remain on the outside of the coil indicating the least deformable part of the sheath and stalk. During the course of rapid contraction, the spasmoneme twists itself, and the stored strain is relaxed by the rotation of the zooid at the end of contraction [12]. Re-extension or relaxation of the stalk is also a mechanical interplay of these elements. The roles of each element have not been precisely defined, although the elastic restoring force of the deformed sheath was suggested to be responsible for the stalk re-extension.
The fibrous structure of the spasmoneme was first shown in transmission electron microscope (TEM) images in the early 1970s. These early observations revealed hundreds of filaments, each roughly 2–4 nm in diameter, in the contracted spasmoneme. These fibrils are interspersed with tubules which are roughly 50 nm in diameter with ~250 nm spacing. These round membranated structures have been proposed as mitochondria and/or calcium storage sites.
As previously mentioned, calcium ions induce and power the spasmonemal contraction [17]. In the absence of ATP, the permeabilized stalk can coil if the free Ca2+ concentration of the medium ([Ca2+]free) is higher than 10−6 M. This is because external calcium ions can diffuse into the stalk and trigger the spasmoneme. The permeabilized stalk remains extended when [Ca2+]free < 10−8 M. The stalk can repeat the contraction-relaxation cycle and generate tension upon changes in [Ca2+]free, although it loses contractility over repeated cycles.
Should the membranous tubules seen by TEM be proven to contain high concentrations of calcium, they could feasibly serve as energy reservoirs for the spasmoneme. As a contraction is triggered, calcium is thought to be released from the tubules. Relaxation of the stalk then requires that calcium be pumped back to its storage organelles by ATP-dependent pumps. These two processes occur on vastly different time scales: milliseconds for contraction and seconds for relaxation.
3.2. Contraction Mechanism
When considered together, the stalk elements point towards Vorticella stalk coiling being carried out by a fibrous, ordered substructure of the spasmoneme actuated by calcium ions. Two prominent models were proposed that relied heavily on the filamentous structure of the spasmoneme. In the electrostatic model , the nanofibrils of the spasmoneme are negatively charged, so they are aligned in parallel due to electrostatic repulsive force among the filaments. Binding of calcium ions neutralizes the charged filaments, which results in the collapse of the filaments and thus contraction of the spasmoneme. However, the electrostatic model does not consider the Ca2+-specificity of the spasmoneme. The spasmonemal contraction can be induced by a divalent cation of around the same size as Ca2+ whereas electrostatic screening by monovalent salt ions cannot do so [106]. The second model assumes the spasmoneme to be a rubber-like material, wherein calcium binding induces the intramolecular folding of contractile elements or peptides. A common drawback of the above models is that they do not consider the biochemistry of the spasmoneme components.
Routledge et al. first identified the major protein component of the Vorticella spasmoneme, a 20 kilodalton (kDa) Ca2+-binding protein termed spasmin [40]. Spasmin is negatively charged and becomes more hydrophobic upon calcium binding. Because of its abundance in the spasmoneme and affinity for calcium, spasmin is widely considered to be the actuating protein of the spasmoneme. Spasmin appears to have a binding partner in the spasmoneme, a 50 kDa spasmin-receptor protein named spaconnectin. This protein pair is also found in the spasmoneme of Carchesium and Zoothamnium [110]. However, the roles of spasmin and spaconnectin in Vorticella contractility have yet to be identified.
Centrin, a homolog of spasmin, provides clues to elucidate the contraction mechanism of the spasmoneme. As a eukaryotic signature protein, centrin is a highly conserved Ca2+-binding protein found contractile organelles of some eukaryotic cells . Because centrin does not readily form nanofibers on its own, its binding partners have been identified which have multiple repeated conserved sites for centrin binding and higher fibril-forming potentia. For instance, the contractile infraciliary lattice (ICL) of Paramecium contains centrin and a centrin-binding protein named PtCenBP. It has been suggested that the Ca2+-responding nanofilaments of the ICL consist of PtCenBP1 molecules containing repeated centrin-binding motifs, so multiple centrin molecules bind to each extended PtCenBP1 molecule at low [Ca2+]free. Upon influx of Ca2+, centrin molecules undergo conformational changes, which causes shortening of the filaments and thus the contraction of the ICL [107].
Recently, a similar two-component model has been proposed for the spasmoneme based on spasmin and spaconnectin. This model assumes that the spasmonemal nanofibrils consist of an α-helix of spaconnectin joining a pair of spasmin molecules. Upon increase in [Ca2+]free, spaconnectin molecules become random coils, so the filaments shorten. This two-component model leaves room to accommodate several key aspects from the experimental evidence: specificity for calcium and hydrophobic interactions that become available when spasmin binds calcium. Identifying the contraction mechanism of the spasmoneme based on the roles of its protein components is crucial for exploiting the Ca2+-induced contraction of the spasmoneme in bio-inspired engineering.
3.3. Contractile Performance
Studies on the stalk contractility of Vorticella not only give us insights into how the spasmoneme operates, but also frame the motor capabilities of the spasmoneme as a model for bioinspired actuator. Such studies measured key parameters including contraction speed, coiling propagation speed, contractile force (or tension), mechanical work, and power, using both live and extracted Vorticella cells. High-speed imaging techniques have enabled observations of the millisecond contraction sequence of live Vorticella. In contrast, extracted cells have enabled easier manipulations and observations of the spasmonemal contraction as shown and if stored frozen in glycerin, remain contractile for up to several month. Observations made with both live and extracted cells are discussed here.
3.3.1. Dynamics of Stalk Contraction
Two types of speed measurements have been taken for Vorticella: the contracting speed of the whole stalk, which equals the moving speed of the zooid during stalk contraction, and the propagation speed of the contraction onset, which proceeds from the zooid to the distal end of the stalk. The former is defined by the capabilities of the contractile machinery as a whole, while the latter is believed to be dictated by the molecular signaling mechanism that drives calcium release within the spasmoneme, thereby instigating contraction.
Various imaging methods have been employed for time-resolved investigations of live Vorticella’s contraction. To the best knowledge of the authors, Ueda made the first measurements of the contraction speed of Carchesium polypinum, which was 17.8–24.7 cm/s, using a photographic method for which a detailed description was not given. Later, Sugi projected the image of the contracting stalk of C. polypinum, with carbon granules attached, through a narrow slit onto a rotating photographic paper roll, and measured that the contraction propagation speed was 20–50 cm/s, which is higher than the contraction speed [33]. A similar approach was employed for Vorticella by Katoh and Naitoh, and the measured contraction speed (6.7 cm/s) was lower than that of C. polypinum. These measurements are summarized along with the latent period (the time between observed cell body contraction and the onset of stalk contraction.
ΑΝΑΔΗΜΟΣΙΕΥΣΗ ΑΠΟ ΠΗΓΗ ΤΟΥ ΙΝΤΕΡΝΕΤ 1/9/2019Hyoung Jin Cho, Academic Editor and Sung Kwon Cho, Academic Editor
Δεν υπάρχουν σχόλια:
Δημοσίευση σχολίου