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Apparent Flexural Rigidity of Single Flagella Depends on Inter-Doublet Shear Stiffness
Monday, February 13, 2017 1 Mechanical Engineering and Materials Science, Washington University in Saint Louis, Saint Louis, MO, USA, 2Genetics, Washington University in Saint Louis, Saint Louis, MO, USA. The oscillatory, wavelike motion of cilia and flagella remains mysterious, although the basic structure of the 9þ2 axoneme and the properties of dynein motors are well known. It is widely believed that feedback from mechanical deformation of the axoneme (microtubule doublet curvature, sliding or spacing, e.g.) to dynein activity leads to dynamic instability and sustained oscillations. However, a prior analysis of the partial differential equations (PDEs) of a simplified, 2-doublet axoneme model suggests that dynamic dynein regulation is not necessary to produce oscillatory behavior. In this model, if dynein arms pull steadily above a critical threshold level, the resulting internal stresses, combined with viscous resistance, lead to a dynamic instability (‘‘viscoelastic flutter’’) and wavelike oscillations. In the current work, a finite element (FE) model of the 9-doublet flagellar axoneme was constructed using commercial software (ABAQUS, Dassault Systemes) to investigate the possible role of viscoelastic instability in a more realistic model. The model consisted of 1890 beam elements representing 9 microtubule doublets surrounding a single central axis, connected by radial spokes and nexin-dynein regulatory complexes (N-DRCs), all subject to viscous resistance. Steady, equal, and opposing axial loads, representing dynein activity, were applied between doublet pairs on opposing sides of the axoneme. No feedback was included between deformation and dynein activity. The effects of numerical parameters such as mesh size and time step were checked to verify that the models are robust to these choices. Consistent with the prior stability analysis, the FE model exhibited wavelike oscillations (flutter instability) above a critical load. This study provides evidence for the viscoelastic flutter hypothesis - that steady dynein activity is sufficient to produce propagating, oscillatory, flagellar waveforms, and dynein switching or dynamic regulation is not necessary for flagella beating.
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response to mechanical stimuli arising from contact with the substrate. However, it is unclear how the substrate properties affect motor-remodeling and the subsequent adaptation in motility. We will discuss our recent measurements of remodeling and motor functions in various mutants of E. coli under substrate conditions that are swarm-conducive. We will present estimated bounds on the magnitude of viscous drag that is responsible for remodeling and possibly, in the initiation of swarming. These results provide important insights into the dynamics of swarmers at a single-cell level and might likely provide tools in the future to combat antibiotic-resistance and infections. 1319-Pos Board B387 Quantification of Flagella-Driven Cellular Motility under Various Viscous Resistances Kara M. Clark, Daniel Fijalka, Gang Xu. Engineering and Physics, University of Central Oklahoma, Edmond, OK, USA. Motile cilia and flagella are hair-like microscopic structures in the human body that move cells or materials in airways, brain ventricles, and the oviduct. As a result, these cilia and flagella need to grow and bend actively in various physical environments with a wide range of viscous resistance. In order to improve our understanding on how cilia and flagella may adapt to the physical environment by altering their structure and mechanics, we studied the biflagellate green algae Chlamydomonas reinhardtii and quantified their flagelladriven cellular motility. Cells were grown in mediums of various viscosities that were controlled by different concentrations of methylcellulose. We demonstrated that while these cells and flagella grew relatively normally in physically-stressed conditions (mediums with higher viscosities), the flagelladriven cellular motility (manifested by the average cell speed) was significantly reduced under higher viscous resistances. When these cells were ‘rescued’ by returning to the normal viscous medium, their motility recovered to about the normal level, suggesting the maintenance of regular flagellar function. Further investigation is underway to correlate the biophysical properties with potential changes in flagella-related gene expression.
1317-Pos Board B385 Resurrection of Flagellar Bending Movements in Chlamydomonas Paralyzed Mutants at High Pressure Toshiki Yagi1, Masayoshi Nishiyama2. 1 Department of Life Science, Prefectural University of Hiroshima, Hiroshima, Japan, 2The HAKUBI Center, Kyoto University, Kyoto, Japan. Application of hydrostatic pressure modulates the activities of organisms [1, 2]. To examine the effect of the pressure on ciliary and flagellar motility, here we observed the flagellar movements of chlamydomonas under high-pressure conditions. Wild type cell movements were inhibited by the application of pressure; the swimming velocity and the percentage of moving cells decreased with the pressure increased, and the movements stopped under the pressure of >80 MPa at 25 C. Surprising, however, paralyzed mutants lacking the central structures of the flagellum (central-pair microtubules: CP, or radial spokes: RS) displayed vigorous beating under the pressure of 40-80 MPa, but the movements stopped under the pressure of >80 MPa at 25 C. Axonemes (isolated and demembranated flagella) of the mutants did not display beating in the presence of 1 mM ATP, but they displayed beating under the pressure of 40-60 MPa. This suggests that pressure directly modulate some components in axoneme. Cilia and flagella have two kinds of dyneins, inner- and outer-arm dyneins, in general. The motility analysis of the double mutants lacking both of CP/ RS and dyneins showed that outer-arm dynein is indispensable for the movements. Previous studies showed that the microtubule-sliding movements powered by dynein were reduced in the axonemes of the CP/RS-deficient mutants [3], suggesting that the application of pressure directly or indirectly activates the outer arm dynein and increases the sliding movements, which induces bending movements in the paralyzed mutants. [1] Nishiyama M. and Y. Sowa. 2012. Biophys J. 102:1872-1880. doi: 10.1016/ j.bpj.2012.03.033. [2] Nishiyama M. 2015. Subcell Biochem. 72:593-611. doi: 10.1007/978-94017-9918-8_27. [3] Kamiya R. and Yagi T. 2014. Zool. Sci. 31: 633-644. doi: 10.2108/ zs140066.
1320-Pos Board B388 Apparent Flexural Rigidity of Single Flagella Depends on Inter-Doublet Shear Stiffness Gang Xu1, Kate S. Wilson2, Ruth J. Okamoto2, Jin-Yu Shao3, Susan K. Dutcher4, Philip V. Bayly2. 1 Engineering and Physics, University of Central Oklahoma, Edmond, OK, USA, 2Mechanical Engineering and Materials Science, Washington University, St. Louis, MO, USA, 3Biomedical Engineering, Washington University, St. Louis, MO, USA, 4Genetics, Washington University, St. Louis, MO, USA. Motile cilia and flagella are whip-like subcellular organelles that bend actively to propel cells or move fluids. Normal motile functions of cilia and flagella play a critical role in many developmental and physiological processes, while ciliary dysfunction is associated with a number of ciliopathies. Efficient bending deformation of cilia and flagella depends on coordinated interactions between active forces from an array of motor proteins and passive mechanical resistance from the complex cytoskeletal structure (the axoneme). However, details of this coordination, especially axonemal mechanics, remain unclear. We investigated two major biophysical parameters, flexural rigidity and inter-doublet shear stiffness, of single flagella in the unicellular alga Chlamydomonas reinhardtii. Combining theoretical analysis with optical tweezers and counterbend experiments, we demonstrated that the apparent flexural rigidity of the axoneme depends on both the intrinsic flexural rigidity and the elastic inter-doublet shear stiffness. By comparing wild-type flagella with specific structural mutants, we found that the lack of nexin-dynein regulatory complexes (N-DRC) or dynein arms significantly reduces inter-doublet shear stiffness. The quantitative understanding of axonemal mechanics will ultimately lead to the development of novel diagnostic and therapeutic methods for cilia-related disorders.
1318-Pos Board B386 Surface Drag and Swarming Bacteria Katie M. Ford, Pushkar L. Lele. Chemical Engineering, Texas A&M University, College Station, TX, USA. Motile bacteria sense higher viscous drags near substrates and initiate swarming – a two-dimensional group motility that is markedly different from motility in the bulk. Our recent work suggests that flagellar motors act as sensors for increased viscous loads and undergo remodeling in
1321-Pos Board B389 Mechanical Properties of Trypanosoma Cruzi Flagellum Nancy E. Ruiz Uribe1, John Mario Gonzalez2, Antonio Manu Forero Shelton1. 1 Physics, Universidad de los Andes, Bogota´, Colombia, 2Medicine, Universidad de los Andes, Bogota´, Colombia. Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected tropical disease and a public health problem in Latin America. The
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response to mechanical stimuli arising from contact with the substrate. However, it is unclear how the substrate properties affect motor-remodeling and the subsequent adaptation in motility. We will discuss our recent measurements of remodeling and motor functions in various mutants of E. coli under substrate conditions that are swarm-conducive. We will present estimated bounds on the magnitude of viscous drag that is responsible for remodeling and possibly, in the initiation of swarming. These results provide important insights into the dynamics of swarmers at a single-cell level and might likely provide tools in the future to combat antibiotic-resistance and infections. 1319-Pos Board B387 Quantification of Flagella-Driven Cellular Motility under Various Viscous Resistances Kara M. Clark, Daniel Fijalka, Gang Xu. Engineering and Physics, University of Central Oklahoma, Edmond, OK, USA. Motile cilia and flagella are hair-like microscopic structures in the human body that move cells or materials in airways, brain ventricles, and the oviduct. As a result, these cilia and flagella need to grow and bend actively in various physical environments with a wide range of viscous resistance. In order to improve our understanding on how cilia and flagella may adapt to the physical environment by altering their structure and mechanics, we studied the biflagellate green algae Chlamydomonas reinhardtii and quantified their flagelladriven cellular motility. Cells were grown in mediums of various viscosities that were controlled by different concentrations of methylcellulose. We demonstrated that while these cells and flagella grew relatively normally in physically-stressed conditions (mediums with higher viscosities), the flagelladriven cellular motility (manifested by the average cell speed) was significantly reduced under higher viscous resistances. When these cells were ‘rescued’ by returning to the normal viscous medium, their motility recovered to about the normal level, suggesting the maintenance of regular flagellar function. Further investigation is underway to correlate the biophysical properties with potential changes in flagella-related gene expression.
1317-Pos Board B385 Resurrection of Flagellar Bending Movements in Chlamydomonas Paralyzed Mutants at High Pressure Toshiki Yagi1, Masayoshi Nishiyama2. 1 Department of Life Science, Prefectural University of Hiroshima, Hiroshima, Japan, 2The HAKUBI Center, Kyoto University, Kyoto, Japan. Application of hydrostatic pressure modulates the activities of organisms [1, 2]. To examine the effect of the pressure on ciliary and flagellar motility, here we observed the flagellar movements of chlamydomonas under high-pressure conditions. Wild type cell movements were inhibited by the application of pressure; the swimming velocity and the percentage of moving cells decreased with the pressure increased, and the movements stopped under the pressure of >80 MPa at 25 C. Surprising, however, paralyzed mutants lacking the central structures of the flagellum (central-pair microtubules: CP, or radial spokes: RS) displayed vigorous beating under the pressure of 40-80 MPa, but the movements stopped under the pressure of >80 MPa at 25 C. Axonemes (isolated and demembranated flagella) of the mutants did not display beating in the presence of 1 mM ATP, but they displayed beating under the pressure of 40-60 MPa. This suggests that pressure directly modulate some components in axoneme. Cilia and flagella have two kinds of dyneins, inner- and outer-arm dyneins, in general. The motility analysis of the double mutants lacking both of CP/ RS and dyneins showed that outer-arm dynein is indispensable for the movements. Previous studies showed that the microtubule-sliding movements powered by dynein were reduced in the axonemes of the CP/RS-deficient mutants [3], suggesting that the application of pressure directly or indirectly activates the outer arm dynein and increases the sliding movements, which induces bending movements in the paralyzed mutants. [1] Nishiyama M. and Y. Sowa. 2012. Biophys J. 102:1872-1880. doi: 10.1016/ j.bpj.2012.03.033. [2] Nishiyama M. 2015. Subcell Biochem. 72:593-611. doi: 10.1007/978-94017-9918-8_27. [3] Kamiya R. and Yagi T. 2014. Zool. Sci. 31: 633-644. doi: 10.2108/ zs140066.
1320-Pos Board B388 Apparent Flexural Rigidity of Single Flagella Depends on Inter-Doublet Shear Stiffness Gang Xu1, Kate S. Wilson2, Ruth J. Okamoto2, Jin-Yu Shao3, Susan K. Dutcher4, Philip V. Bayly2. 1 Engineering and Physics, University of Central Oklahoma, Edmond, OK, USA, 2Mechanical Engineering and Materials Science, Washington University, St. Louis, MO, USA, 3Biomedical Engineering, Washington University, St. Louis, MO, USA, 4Genetics, Washington University, St. Louis, MO, USA. Motile cilia and flagella are whip-like subcellular organelles that bend actively to propel cells or move fluids. Normal motile functions of cilia and flagella play a critical role in many developmental and physiological processes, while ciliary dysfunction is associated with a number of ciliopathies. Efficient bending deformation of cilia and flagella depends on coordinated interactions between active forces from an array of motor proteins and passive mechanical resistance from the complex cytoskeletal structure (the axoneme). However, details of this coordination, especially axonemal mechanics, remain unclear. We investigated two major biophysical parameters, flexural rigidity and inter-doublet shear stiffness, of single flagella in the unicellular alga Chlamydomonas reinhardtii. Combining theoretical analysis with optical tweezers and counterbend experiments, we demonstrated that the apparent flexural rigidity of the axoneme depends on both the intrinsic flexural rigidity and the elastic inter-doublet shear stiffness. By comparing wild-type flagella with specific structural mutants, we found that the lack of nexin-dynein regulatory complexes (N-DRC) or dynein arms significantly reduces inter-doublet shear stiffness. The quantitative understanding of axonemal mechanics will ultimately lead to the development of novel diagnostic and therapeutic methods for cilia-related disorders.
1318-Pos Board B386 Surface Drag and Swarming Bacteria Katie M. Ford, Pushkar L. Lele. Chemical Engineering, Texas A&M University, College Station, TX, USA. Motile bacteria sense higher viscous drags near substrates and initiate swarming – a two-dimensional group motility that is markedly different from motility in the bulk. Our recent work suggests that flagellar motors act as sensors for increased viscous loads and undergo remodeling in
1321-Pos Board B389 Mechanical Properties of Trypanosoma Cruzi Flagellum Nancy E. Ruiz Uribe1, John Mario Gonzalez2, Antonio Manu Forero Shelton1. 1 Physics, Universidad de los Andes, Bogota´, Colombia, 2Medicine, Universidad de los Andes, Bogota´, Colombia. Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected tropical disease and a public health problem in Latin America. The