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Howard E Buhse - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Vorticella Convallaria calcium‐binding centrin proteins
Journal of Eukaryotic Microbiology, 2005Co-Authors: Katarzyna Konior, Suzanne M Mccutcheon, Howard E BuhseAbstract:The stalked ciliate, Vorticella Convallaria, is a good model system to study mechanochemical motility because its contractile organelles (spasmoneme and myonemes) use a mode of contraction that differs from most other eukaryotic motile systems. Since calcium triggers this contraction, we have undertaken the molecular characterization of the calcium-binding proteins associated with these organelles. We have isolated and identified seven unique centrin-like cDNAs from V. Convallaria. Each encodes an acidic protein of approximately 20-kDa, containing a unique N-terminus and four potential calcium-binding domains. We predict that each centrin has a distinct function within the cell. To define these functions, we have initiated immunofluorescence localization studies utilizing various anti-centrin antibodies. Western analysis indicates that each antibody recognizes a distinct protein or subset of proteins in Vorticella. Using these antibodies, we have localized centrin to various structures within the cell; myonemes, spasmoneme, and the oral apparatus. Because each of these antibodies recognizes a different protein on Westerern analysis, we conclude that a number of calcium-binding proteins are associated with the contractile organelles. To further characterize this gene family, we have initiated immunolocalization at the ultrastructural level. This will permit subcellular localization of all Vorticella centrins and enable us to dissect the function of this multi-gene family.
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characterization of vorticella Convallaria calcium binding centrin proteins
Journal of Eukaryotic Microbiology, 2005Co-Authors: Katarzyna Konior, Suzanne M Mccutcheon, Howard E BuhseAbstract:The stalked ciliate, Vorticella Convallaria, is a good model system to study mechanochemical motility because its contractile organelles (spasmoneme and myonemes) use a mode of contraction that differs from most other eukaryotic motile systems. Since calcium triggers this contraction, we have undertaken the molecular characterization of the calcium-binding proteins associated with these organelles. We have isolated and identified seven unique centrin-like cDNAs from V. Convallaria. Each encodes an acidic protein of approximately 20-kDa, containing a unique N-terminus and four potential calcium-binding domains. We predict that each centrin has a distinct function within the cell. To define these functions, we have initiated immunofluorescence localization studies utilizing various anti-centrin antibodies. Western analysis indicates that each antibody recognizes a distinct protein or subset of proteins in Vorticella. Using these antibodies, we have localized centrin to various structures within the cell; myonemes, spasmoneme, and the oral apparatus. Because each of these antibodies recognizes a different protein on Westerern analysis, we conclude that a number of calcium-binding proteins are associated with the contractile organelles. To further characterize this gene family, we have initiated immunolocalization at the ultrastructural level. This will permit subcellular localization of all Vorticella centrins and enable us to dissect the function of this multi-gene family.
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cloning and expression of a cdna encoding a vorticella Convallaria spasmin an ef hand calcium binding protein
Journal of Eukaryotic Microbiology, 1999Co-Authors: John J Maciejewski, Emanuel J Vacchiano, Suzanne M Mccutcheon, Howard E BuhseAbstract:The stalked, ciliated protozoan Vorticella Convallaria possesses a highly contractile cytoskeleton consisting of spasmonemes and myonemes. The major component of these contractile organelles is the calcium-binding protein(s) called spasmin. Cloning and characterization of spasmin would help elucidate this contractile system. Therefore, enriched spasmoneme protein preparations from these contractile stalks were used to produce a monoclonal antibody to spasmin. A monoclonal antibody, 1F5, was obtained that immunolocalized specifically to the spasmonemes and the myonemes and recognized a 20-kD calcium-binding protein in spasmoneme protein preparations. A putative spasmin cDNA was obtained from a V. Convallaria cDNA library and the derived amino acid sequence of this cDNA revealed an acidic, 20-kD protein with calcium-binding helix-loop-helix domains. The physical properties of the putative spasmin were assessed by characterization of a recombinantly-produced spasmin protein. The recombinant spasmin protein was shown to bind calcium using calcium gel-shift assays and was recognized by the anti-spasmin antibody. Therefore, a V. Convallaria spasmin was cloned and shown to be a member of the EF-hand superfamily of calcium-binding proteins.
Paul Matsudaira - One of the best experts on this subject based on the ideXlab platform.
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Fluid dynamic estimation of the effective spring constant of the relaxing stalk of Vorticella Convallaria
JMST Advances, 2020Co-Authors: Paul MatsudairaAbstract:The stalk of Vorticella Convallaria , a sessile ciliated protozoan, contracts in a few milliseconds at a maximum speed of ~ 10 mm/s and generates a contractile force of ~ 10 nN. After powerful contraction, the stalk slowly returns to its extended state, and this relaxation process completes and resets the contraction cycle. The stalk relaxation needs to be better characterized because it is indispensable to the contraction-relaxation cycle of V. Convallaria . In contrast to the spasmoneme-based contraction force, the driving force for the stalk relaxation is thought to be the elastic restoring force of the coiled stalk. In this study, relaxing V. Convallaria was modeled as the damped spring system to estimate the effective spring constant of the relaxing stalk in different viscous media. In the order of 0.1 pN/μm, the effective spring constant was found to increase with the medium viscosity, which suggests that the stalk relaxation is affected by the final status of the contraction phase. Graphical Abstract
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direct measurement of vorticella contraction force by micropipette deflection
FEBS Letters, 2017Co-Authors: Jonathan Tejada, Paul MatsudairaAbstract:: The ciliated protozoan Vorticella Convallaria is noted for its exceptionally fast adenosine triphosphate-independent cellular contraction, but direct measurements of contractile force have proven difficult given the length scale, speed, and forces involved. We used high-speed video microscopy to image live Vorticella stalled in midcontraction by deflection of an attached micropipette. Stall forces correlate with both distance contracted and the resting stalk length. Estimated isometric forces range from 95 to 177 nanonewtons (nN), or 1.12 nN·μm-1 of the stalk. Maximum velocity and work are also proportional to distance contracted. These parameters constrain proposed biochemical/physical models of the contractile mechanism.
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maximal force characteristics of the ca2 powered actuator of vorticella Convallaria
Biophysical Journal, 2012Co-Authors: Matthew J Lang, Paul MatsudairaAbstract:The millisecond stalk contraction of the sessile ciliate Vorticella Convallaria is powered by energy from Ca2+ binding to generate contractile forces of ∼10 nN. Its contractile organelle, the spasmoneme, generates higher contractile force under increased stall resistances. By applying viscous drag force to contracting V. Convallaria in a microfluidic channel, we observed that the mechanical force and work of the spasmoneme depended on the stalk length, i.e., the maximum tension (150–350 nN) and work linearly depended on the stalk length (∼2.5 nN and ∼30 fJ per 1 μm of the stalk). This stalk-length dependency suggests that motor units of the spasmoneme may be organized in such a way that the mechanical force and work of each unit cumulate in series along the spasmoneme.
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unsteady motion finite reynolds numbers and wall effect on vorticella Convallaria contribute contraction force greater than the stokes drag
Biophysical Journal, 2010Co-Authors: Paul MatsudairaAbstract:Contraction of Vorticella Convallaria, a sessile ciliated protozoan, is completed within a few milliseconds and results in a retraction of its cell body toward the substratum by coiling its stalk. Previous studies have modeled the cell body as a sphere and assumed a drag force that satisfies Stokes' law. However, the contraction-induced flow of the medium is transient and bounded by the substrate, and the maximum Reynolds number is larger than unity. Thus, calculations of contractile force from the drag force are incomplete. In this study, we analyzed fluid flow during contraction by the particle tracking velocimetry and computational fluid dynamics simulations to estimate the contractile force. Particle paths show that the induced flow is limited by the substrate. Simulation-based force estimates suggest that the combined effect of the flow unsteadiness, the finite Reynolds number, and the substrate comprises 35% of the total force. The work done in the early stage of contraction and the maximum power output are similar regardless of the medium viscosity. These results suggest that, during the initial development of force, V. Convallaria uses a common mechanism for performing mechanical work irrespective of viscous loading conditions.
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Chemical control of Vorticella bioactuator using microfluidics
Lab on a Chip, 2010Co-Authors: Moeto Nagai, Todd Thorsen, Paul Matsudaira, Hiroyuki FujitaAbstract:In this report, we demonstrate a microfluidic platform to control the stalk contraction and extension of Vorticella Convallaria by changing concentration of Ca2+ with pneumatically-actuated elastomeric microvalves. Habitation, extraction and control of V. Convallaria were carried out in a PDMS-based microfluidic device. By treating the cells with the permeant saponin, external actuation of cell-anchoring stalk between an extended and contracted state was achieved by cyclic exposure of the cells to a Ca2+ buffer (10−6 M) and a rinse buffer containing EGTA as a chelation agent. When solutions were switched, the stalk contracted and extended responding to the ambient Ca2+ concentration change. The length of the stalk changed between 20 and 60 μm, resulting in a working distance of about 40 μm.
Hiroshi Asai - One of the best experts on this subject based on the ideXlab platform.
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Contraction and extension of Vorticella and its mechanical characterization under flow loading
Biomicrofluidics, 2010Co-Authors: Moeto Nagai, Hiroshi Asai, Hiroyuki FujitaAbstract:We have studied the contraction and extension of Vorticella Convallaria and its mechanical properties with a microfluidic loading system. Cells of V. Convallaria were injected to a microfluidic channel (500 μm in width and 100 μm in height) and loaded by flow up to ∼350 mm s−1. The flow produced a drag force on the order of nanonewton on a typical vorticellid cell body. We gradually increased the loading force on the same V. Convallaria specimen and examined its mechanical property and stalk motion of V. Convallaria. With greater drag forces, the contraction distance linearly decreased; the contracted length was close to around 90% of the stretched length. We estimated the drag force on Vorticella in the channel by calculating the force on a sphere in a linear shear flow.
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phylogenetic relationships between vorticella Convallaria and other species inferred from small subunit rrna gene sequences
Zoological Science, 2002Co-Authors: Takeshi Itabashi, Kazuyuki Mikami, Jie Fang, Hiroshi AsaiAbstract:Abstract Vorticellid ciliates generally dwell in freshwater. In nature, the species have up until now been identified by comparison with previous descriptions. It is difficult to identify between species of the genus Vorticella, because the morphological markers of vorticellid ciliates described in reports are limited and variable. Unfortunately, culturing them has only succeeded with certain species such as Vorticella Convallaria, but many others have been impossible to culture. To find out whether the sequence of a small subunit rRNA gene was an appropriate marker to identify vorticellid ciliates, the gene was aligned and compared. Finding a new convenient method will contribute to research on vorticellid ciliates. In strains of V. Convallaria, classified morphologically, some varieties of the SSrRNA gene sequences were recognized, but there were large variations within the same species. According to the phylogenetic tree, these strains are closely related. However, the difference was not as big as betw...
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high speed video cinematographic demonstration of stalk and zooid contraction of vorticella Convallaria
Biophysical Journal, 1998Co-Authors: Yasushige Moriyama, Shigeo Hiyama, Hiroshi AsaiAbstract:Stalk contraction and zooid contraction of living Vorticella Convallaria were studied by high-speed video cinematography. Contraction was monitored at a speed of 9000 frames per second to study the contractile process in detail. Complete stalk contraction required approximately 9 ms. The maximal contraction velocity, 8.8 cm/s, was observed 2 ms after the start of contraction. We found that a twist appeared in the zooid during contraction. As this twist unwound, the zooid began to rotate like a right-handed screw. The subsequent stalk contraction steps, the behavior of which was similar to that of a damped harmonic oscillator, were analyzed by means of the equation of motion. From the beginning of stalk contraction, the Hookean force constant increased, and reached an upper limit of 2.23 x 10(-4) N/m 2-3 ms after the start of contraction. Thus, within 2 ms, the contraction signal spread to the entire stalk, allowing the stalk to generate the full force of contraction. The tension of an extended stalk was estimated to be 5.58 x 10(-8) N from the Hookean force constant of a stalk. This value coincides with that of the isometric tension of a glycerol-treated V. Convallaria, confirming that the contractile system of V. Convallaria is well preserved despite glycerol treatment.
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ca2 induced tension development in the stalks of glycerinated vorticella Convallaria
Cytoskeleton, 1996Co-Authors: Yasushige Moriyama, Kenji Yasuda, Shinichi Ishiwata, Hiroshi AsaiAbstract:: We have developed a method of measuring the isometric tension in glycerinated stalks of Vorticella Convallaria. Using this method, we measured tension vs. pCa relations in glycerinated V. Convallaria stalks. The maximum isometric tension was 4 x 10(-8) N on average. The Hill's parameter, n, which is the number of calcium ions bound simultaneously and cooperatively to a contractile element (a force generating element), is approximately 3.2 when the Ca2+ concentration is increased and 2.5 when it is decreased. In order to estimate the efficiency of the energy conversion of Ca2+ binding to mechanical work, we measured the Ca(2+)-induced Carnot cycle in the Vorticella stalk. The energy efficiency was tentatively estimated to be about 7%. With this method, we have also succeeded in measuring the isometric tension of isolated spasmoneme, the rubber-like contractile fibrous organelle in the stalk. The maximum tension of spasmoneme was approximately one tenth that of the glycerinated stalk. We speculate that the isolated spasmoneme was only partially functional due to damage sustained when it was pulled out of the stalk.
Suzanne M Mccutcheon - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Vorticella Convallaria calcium‐binding centrin proteins
Journal of Eukaryotic Microbiology, 2005Co-Authors: Katarzyna Konior, Suzanne M Mccutcheon, Howard E BuhseAbstract:The stalked ciliate, Vorticella Convallaria, is a good model system to study mechanochemical motility because its contractile organelles (spasmoneme and myonemes) use a mode of contraction that differs from most other eukaryotic motile systems. Since calcium triggers this contraction, we have undertaken the molecular characterization of the calcium-binding proteins associated with these organelles. We have isolated and identified seven unique centrin-like cDNAs from V. Convallaria. Each encodes an acidic protein of approximately 20-kDa, containing a unique N-terminus and four potential calcium-binding domains. We predict that each centrin has a distinct function within the cell. To define these functions, we have initiated immunofluorescence localization studies utilizing various anti-centrin antibodies. Western analysis indicates that each antibody recognizes a distinct protein or subset of proteins in Vorticella. Using these antibodies, we have localized centrin to various structures within the cell; myonemes, spasmoneme, and the oral apparatus. Because each of these antibodies recognizes a different protein on Westerern analysis, we conclude that a number of calcium-binding proteins are associated with the contractile organelles. To further characterize this gene family, we have initiated immunolocalization at the ultrastructural level. This will permit subcellular localization of all Vorticella centrins and enable us to dissect the function of this multi-gene family.
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characterization of vorticella Convallaria calcium binding centrin proteins
Journal of Eukaryotic Microbiology, 2005Co-Authors: Katarzyna Konior, Suzanne M Mccutcheon, Howard E BuhseAbstract:The stalked ciliate, Vorticella Convallaria, is a good model system to study mechanochemical motility because its contractile organelles (spasmoneme and myonemes) use a mode of contraction that differs from most other eukaryotic motile systems. Since calcium triggers this contraction, we have undertaken the molecular characterization of the calcium-binding proteins associated with these organelles. We have isolated and identified seven unique centrin-like cDNAs from V. Convallaria. Each encodes an acidic protein of approximately 20-kDa, containing a unique N-terminus and four potential calcium-binding domains. We predict that each centrin has a distinct function within the cell. To define these functions, we have initiated immunofluorescence localization studies utilizing various anti-centrin antibodies. Western analysis indicates that each antibody recognizes a distinct protein or subset of proteins in Vorticella. Using these antibodies, we have localized centrin to various structures within the cell; myonemes, spasmoneme, and the oral apparatus. Because each of these antibodies recognizes a different protein on Westerern analysis, we conclude that a number of calcium-binding proteins are associated with the contractile organelles. To further characterize this gene family, we have initiated immunolocalization at the ultrastructural level. This will permit subcellular localization of all Vorticella centrins and enable us to dissect the function of this multi-gene family.
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cloning and expression of a cdna encoding a vorticella Convallaria spasmin an ef hand calcium binding protein
Journal of Eukaryotic Microbiology, 1999Co-Authors: John J Maciejewski, Emanuel J Vacchiano, Suzanne M Mccutcheon, Howard E BuhseAbstract:The stalked, ciliated protozoan Vorticella Convallaria possesses a highly contractile cytoskeleton consisting of spasmonemes and myonemes. The major component of these contractile organelles is the calcium-binding protein(s) called spasmin. Cloning and characterization of spasmin would help elucidate this contractile system. Therefore, enriched spasmoneme protein preparations from these contractile stalks were used to produce a monoclonal antibody to spasmin. A monoclonal antibody, 1F5, was obtained that immunolocalized specifically to the spasmonemes and the myonemes and recognized a 20-kD calcium-binding protein in spasmoneme protein preparations. A putative spasmin cDNA was obtained from a V. Convallaria cDNA library and the derived amino acid sequence of this cDNA revealed an acidic, 20-kD protein with calcium-binding helix-loop-helix domains. The physical properties of the putative spasmin were assessed by characterization of a recombinantly-produced spasmin protein. The recombinant spasmin protein was shown to bind calcium using calcium gel-shift assays and was recognized by the anti-spasmin antibody. Therefore, a V. Convallaria spasmin was cloned and shown to be a member of the EF-hand superfamily of calcium-binding proteins.
Viviana A Alder - One of the best experts on this subject based on the ideXlab platform.
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Microplanktonic distributional patterns west of the Antarctic Peninsula, with special emphasis on the Tintinnids
Polar Biology, 1991Co-Authors: Viviana A Alder, Demetrio BoltovskoyAbstract:Microplankton was sampled with a centrifugal suction pump in the surface layer (approx. 9 m) of the Bellingshausen Sea and the Bransfield Strait in March 1987, and concentrated with a 26 μ m-mesh net. Bulk microplanktonic settling volumes were assessed, silicoflagellates and large thecate dinoflagellates were counted, and tintinnids were counted and identified to species. Average (and maximum) values for the entire area surveyed were as follows, settling volume: 6.7 (43.3) ml/m^3; silicoflagellates: 674 (7777) ind./l, 0.57 (6.54) mg C/m^3; dinoflagellates: 109 (1321) ind./l, 1.40 (16.98) mg C/m^3; tintinnids: 52 (589) ind./l, 1.15 (9.87) mg C/m^3. The three geographic zones defined objectively on the basis of tintinnid specific assemblages also differed sharply in their surface salinity, overall microplanktonic abundance and bulk settling volume. The Bransfield Strait, with lowest settling volume values (2.1 ml/m^3) and cell concentrations, was characterized by the dominance of Cymatocylis affinis/Convallaria. In waters around the tip of the Antarctic Peninsula microplanktonic settling volumes averaged 4.6 ml/m^3, cell concentrations were intermediate, and 79% of the tintinnids were represented by Codonellopsis balechi . The Bellingshausen Sea was characterized by the lowest salinities and the highest settling volumes (8.7 ml/m^3) and cell counts; Laackmanniella spp. and Cymatocylis drygalskii , f. typica dominated this area. Almost all biological variables were significantly intercorrelated, and showed strong and mostly significant negative correlations with surface salinity, yet relationships between enhanced standing stock and ice meltwater were not obvious; rather, highest microplanktonic concentrations seemed to be due to ice-associated growth. Extremely high spatial correlations were found between the tintinnids and the dinoflagellates (r^2: 0.941), suggesting the existence of close links between these two groups. Tintinnid species-specific assemblages show a coherent distributional pattern and well defined environment-related trends; most clearly differentiated preferences are exhibited by Laackmanniella prolongata (closely associated with ice-covered areas), Cymatocylis affinis/Convallaria (oligotrophic open-ocean waters), and Codonellopsis balechi (coastal regions).
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Intraspecific variability in Antarctic tintinnids: the Cymatocylis affinis/Convallaria species group
Journal of Plankton Research, 1990Co-Authors: Demetrio Boltovskoy, Estela O Dinofrio, Viviana A AlderAbstract:Analyses of the morphometry of 630 loricae belonging to the described species Cymatocylis affinis and Cymatocylis Convallaria retrieved from 21 samples collected in the Weddel Sea (6O-73°S) strongly suggest that both taxa, including the four formae into which C.affinis was originally divided, are members of a single species. The only feature which varies significantly and consistently among the samples surveyed is the total length of the lorica, lowest average lengths being positively correlated with lowest absolute abundances of this tintinnid in the waters sampled. This relationship points to some unfavorable factor(s) which affects both tintinnid reproduction/ survival and growth; the data analysed suggest that drops in the availability of food might be responsible for the size (and abundance) variations observed.
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intraspecific variability in antarctic tintinnids the cymatocylis affinis Convallaria species group
Journal of Plankton Research, 1990Co-Authors: Demetrio Boltovskoy, Estela O Dinofrio, Viviana A AlderAbstract:Analyses of the morphometry of 630 loricae belonging to the described species Cymatocylis affinis and Cymatocylis Convallaria retrieved from 21 samples collected in the Weddel Sea (6O-73°S) strongly suggest that both taxa, including the four formae into which C.affinis was originally divided, are members of a single species. The only feature which varies significantly and consistently among the samples surveyed is the total length of the lorica, lowest average lengths being positively correlated with lowest absolute abundances of this tintinnid in the waters sampled. This relationship points to some unfavorable factor(s) which affects both tintinnid reproduction/ survival and growth; the data analysed suggest that drops in the availability of food might be responsible for the size (and abundance) variations observed.