Spermiogenesis is a complex and tightly regulated process,consisting of acrosomal biogenesis,condensation of chromatin,flagellar assembly,and disposal of extra cytoplasm.Previous studies have reported that sperm flage...Spermiogenesis is a complex and tightly regulated process,consisting of acrosomal biogenesis,condensation of chromatin,flagellar assembly,and disposal of extra cytoplasm.Previous studies have reported that sperm flagellar 2(SPEF2)deficiency causes severe asthenoteratozoospermia owing to spermiogenesis failure,but the underlying molecular mechanism in humans remains unclear.Here,we performed proteomic analysis on spermatozoa from three SPEF2 mutant patients to study the functional role of SPEF2 during sperm tail development.A total of 1262 differentially expressed proteins were detected,including 486 upregulated and 776 downregulated.The constructed heat map of the differentially expressed proteins showed similar trends.Among these,the expression of proteins related to flagellar assembly,including SPEF2,sperm associated antigen 6(SPAG6),dynein light chain tctex-type 1(DYNLT1),radial spoke head component 1(RSPH1),translocase of outer mitochondrial membrane 20(TOM20),EF-hand domain containing 1(EFHC1),meiosis-specific nuclear structural 1(MNS1)and intraflagellar transport 20(IFT20),was verified by western blot.Functional clustering analysis indicated that these differentially expressed proteins were specifically enriched for terms such as spermatid development and flagellar assembly.Furthermore,we showed that SPEF2 interacts with radial spoke head component 9(RSPH9)and IFT20 in vitro,which are well-studied components of radial spokes or intra-flagellar transport and are essential for flagellar assembly.These results provide a rich resource for further investigation into the molecular mechanism underlying the role that SPEF2 plays in sperm tail development and could provide a theoretical basis for gene therapy in SPEF2 mutant patients in the future.展开更多
Flagellar biosynthesis and motility are subject to a four-tiered transcriptional regulatory circuit in Pseudomonas,and the master regulator FleQ appears to be the highest-level regulator in this hierarchical regulator...Flagellar biosynthesis and motility are subject to a four-tiered transcriptional regulatory circuit in Pseudomonas,and the master regulator FleQ appears to be the highest-level regulator in this hierarchical regulatory cascade.Pseudomonas stutzeri A1501 is motile by a polar flagellum;however,the motility and regulatory mechanisms involved in this process are unknown.Here,we searched the A1501 genome for flagella and motility genes and found that approximately 50 genes,which were distributed in three non-contiguous chromosomal regions,contribute to the formation,regulation and function of the flagella.The non-polar mutation of fleQ impaired flagellar biosynthesis,motility and root colonization but enhanced biofilm formation.FleQ positively regulates the expression of flagellar class Ⅱ-Ⅳ genes,suggesting a regulatory cascade that is coordinated similar to that of the well-known P.aeruginosa.Based on our results,we propose that flagellar genes in P.stutzeri A1501 are regulated in a cascade regulated by FleQ and that flagellum-driven motility properties may be necessary for competitive rhizosphere colonization.展开更多
We extend the 2D Landau phase transition theory to the bacterial flagellar filament which displays the phase transition between the left handed normal form and the right handed semi-coiled form. The bacterial flagella...We extend the 2D Landau phase transition theory to the bacterial flagellar filament which displays the phase transition between the left handed normal form and the right handed semi-coiled form. The bacterial flagellar filament is treated as an elastic thin rod based on the Kirchhoff’s thin rod theory. Mechanical analysis is performed on the periodical phase transition of the filament between the two helical structures of the opposite charity. The curvature and twist are chosen as the order parameters in constructing the phase transition model of the filament. The established model is applied to study the instability properties of the filament and to investigate the loading and deformation conditions of the phase transition. In addition, the curvature and twist gradient energy are considered to describe the interface properties of the two phases.展开更多
Bacterial flagellar filament can undergo a stress-induced polymorphic phase transition in both vitro and vivo environments.The filament has 12 different helical forms(phases) characterized by different pitch lengths a...Bacterial flagellar filament can undergo a stress-induced polymorphic phase transition in both vitro and vivo environments.The filament has 12 different helical forms(phases) characterized by different pitch lengths and helix radii.When subjected to the frictional force of flowing fluid,the filament changes between a left-handed normal phase and a right-handed semi-coiled phase via phase nucleation and growth.This paper develops non-local finite element method(FEM) to simulate the phase transition under a displacement-controlled loading condition(controlled helix-twist).The FEM formulation is based on the Ginzburg-Landau theory using a one-dimensional non-convex and non-local continuum model.To describe the processes of the phase nucleation and growth,viscosity-type kinetics is also used.The non-local FEM simulation captures the main features of the phase transition:two-phase coexistence with an interface of finite thickness,phase nucleation and phase growth with interface propagation.The non-local FEM model provides a tool to study the effects of the interfacial energy/thickness and loading conditions on the phase transition.展开更多
Bacterial flagellar filaments can undergo a polymorphic phase transition in both vitro and vivo environments. Each bacterial flagellar filament has 12 different helical forms which are macroscopically represented by d...Bacterial flagellar filaments can undergo a polymorphic phase transition in both vitro and vivo environments. Each bacterial flagellar filament has 12 different helical forms which are macroscopically represented by different pitch lengths and helix radii. For external mechanical force induced filament phase transitions, there is so far only one experiment performed by Hotani in 1982, who showed a very beautiful cyclic phase transition phenomenon in his experiment on isolated flagellar filaments. In the present paper, we give a detailed mechanical analysis on Hotani＇s experiments. Through theoretical computations, we obtained a phase transition rule based on the phase transition mechanism. The theoretical analysis provides a foundation facilitating the establishment of phase transition theory for bacterial flagellar filaments.展开更多
A new type of propeller that is optimized for low Reynolds numbers is required to propel a small object in a medium where the flow is dominated by viscous rather than inertial forces. A propeller in the shape of a bac...A new type of propeller that is optimized for low Reynolds numbers is required to propel a small object in a medium where the flow is dominated by viscous rather than inertial forces. A propeller in the shape of a bacterial flagellum seems an appro- priate choice for driving a small object. Accordingly, in this study, we visualized the velocity field induced by a spring-like propeller inspired by the Escherichia coli flagellum, using a macroscopic model and applying stereoscopic particle image velocimetry. We also experimentally evaluated the effect of pitch and rotational speed on the performance of this flagellar propeller. Silicone oil, which has a kinematic viscosity 100,000 times that of water, was used as the working fluid to generate a low Reynolds number for the macroscopic model. Thrust, torque, and velocity were measured as functions of pitch and rota- tional speed, and the efficiency of the propeller was calculated from the measured results. We found that the flagellar propeller reached a max!mum efficiency when the pitch angle was approximately 53°. Compared to pitch, rotational speed had a relatively small effect on the efficiency, and the pitch altered the flow pattern behind the rotating propeller.展开更多
Cilia and flagella are organelles of motility that enable cells to swim or move liquid over its surface. An exhaustive literature survey for the presence of the organelle in organisms across phyla showed that most ani...Cilia and flagella are organelles of motility that enable cells to swim or move liquid over its surface. An exhaustive literature survey for the presence of the organelle in organisms across phyla showed that most animal cells harbor cilia in contrast to very few fungal cells. While this was not unexpected, it was the position and arrangement of this organelle in each cell that intrigued our attention. Natural selection might have favored motility over chemotaxis;and it would have done so to evolve a stable structure that could have undergone an optimization process requiring a precise geometry in the shape of cells and the structure that would help cells to move. The positioning of such a structure would play a pre-dominant role in optimal motility. It is now known that the flagellar position of a cell is a genetically distinct trait, occasionally used in phylogeny of bacteria, distributed in distinguishing patterns over cellular surface, but basically are of two types, either polar (one flagellum arising from one pole per cell) or peritrichous (lateral flagella distributed over the entire cell surface). Irrespective of the cellular habitat, flagella origin, ultrastructure and proteome, the present investigation surveyed 26 sub-types of flagellar arrangements from as many species as possible. A peculiar pattern ensued-Prokaryotes harbored predominantly polar and peritrichous types;eukaryotes showed a mere change of the peritrichous one. These numbers when used to create a Similarity tree depicted a similarity distance of 14 between the Eubacteria and Archaebacteria forming the first neighborhood;Protozoans, Algae, Fungi, Plantae and Animalia formed a second neighborhood. We offer a working hypothesis for this pattern and the gradual shift in the flagellar arrangement from polar, peritrichous, sub-apical, and apical to lateral throughout evolution.展开更多
This paper presents a simple technique to fabricate new electrofluidic devices for the three-dimensional(3D)manipulation of microorganisms by hybrid subtractive and additive femtosecond(fs)laser microfabrication(fs la...This paper presents a simple technique to fabricate new electrofluidic devices for the three-dimensional(3D)manipulation of microorganisms by hybrid subtractive and additive femtosecond(fs)laser microfabrication(fs laser-assisted wet etching of glass followed by water-assisted fs laser modification combined with electroless metal plating).The technique enables the formation of patterned metal electrodes in arbitrary regions in closed glass microfluidic channels,which can spatially and temporally control the direction of electric fields in 3D microfluidic environments.The fabricated electrofluidic devices were applied to nanoaquariums to demonstrate the 3D electro-orientation of Euglena gracilis(an elongated unicellular microorganism)in microfluidics with high controllability and reliability.In particular,swimming Euglena cells can be oriented along the z-direction(perpendicular to the device surface)using electrodes with square outlines formed at the top and bottom of the channel,which is quite useful for observing the motions of cells parallel to their swimming directions.Specifically,z-directional electric field control ensured efficient observation of manipulated cells on the front side(45 cells were captured in a minute in an imaging area of~160×120μm),resulting in a reduction of the average time required to capture the images of five Euglena cells swimming continuously along the z-direction by a factor of~43 compared with the case of no electric field.In addition,the combination of the electrofluidic devices and dynamic imaging enabled observation of the flagella of Euglena cells,revealing that the swimming direction of each Euglena cell under the electric field application was determined by the initial body angle.展开更多
Clostridium acetobutylicum has been extensively exploited to produce biofuels and solvents and its biofilm could dramatically improve the productivities.However,genetic control of C.acetobutylicum biofilm has not been...Clostridium acetobutylicum has been extensively exploited to produce biofuels and solvents and its biofilm could dramatically improve the productivities.However,genetic control of C.acetobutylicum biofilm has not been dissected so far.Here,to identify potential genes controlling C.acetobutylicum biofilm formation,over 40 gene candidates associated with extracellular matrix,cell surface,cell signaling or gene transcription,were tried to be disrupted to examine their individual impact.A total of 25 disruptants were finally obtained over years of attempts,for which biofilm and relevant phenotypes were characterized.Most of these disruptants formed robust biofilm still,or suffered both growth and biofilm defect.Only a strain with a disrupted histidine kinase gene(CA_C2730,designated bfcK in this study)abolished biofilm formation without impairing cell growth or solvent production.Further analysis revealed that bfcK could control flagellar biogenesis and cell motility at protein levels.The bfcK also appeared to repress the phosphorylation of a serine/threonine protein kinase(encoded by CA_C0404)that might negatively regulate biofilm formation.Based on these findings,possible bfcK-mediated mechanisms for biofilm formation were proposed.This is a big step toward understanding the biofilm formation in C.acetobutylicum and will help further engineering of its biofilm-based industrial processes.展开更多
We report a high-throughput and label-free computational imaging technique that simultaneously measures in three-dimensional(3D)space the locomotion and angular spin of the freely moving heads of microswimmers and the...We report a high-throughput and label-free computational imaging technique that simultaneously measures in three-dimensional(3D)space the locomotion and angular spin of the freely moving heads of microswimmers and the beating patterns of their flagella over a sample volume more than two orders-of-magnitude larger compared to existing optical modalities.Using this platform,we quantified the 3D locomotion of 2133 bovine sperms and determined the spin axis and the angular velocity of the sperm head,providing the perspective of an observer seated at the moving and spinning sperm head.In this constantly transforming perspective,flagellum-beating patterns are decoupled from both the 3D translation and spin of the head,which provides the opportunity to truly investigate the 3D spatio-temporal kinematics of the flagellum.In addition to providing unprecedented information on the 3D locomotion of microswimmers,this computational imaging technique could also be instrumental for micro-robotics and sensing research,enabling the high-throughput quantification of the impact of various stimuli and chemicals on the 3D swimming patterns of sperms,motile bacteria and other micro-organisms,generating new insights into taxis behaviors and the underlying biophysics.展开更多
Trypanosoma brucei is a protozoan flagellate that causes African sleeping sickness.Flagellar function in this organism is critical for life cycle progression and pathogenesis,however the regulation of flagellar motili...Trypanosoma brucei is a protozoan flagellate that causes African sleeping sickness.Flagellar function in this organism is critical for life cycle progression and pathogenesis,however the regulation of flagellar motility is not well understood.The flagellar axoneme produces a complex beat through the pre-cisely coordinated firing of many proteins,including multiple dynein motors.These motors are found in the inner arm and outer arm complexes.We are studying one of the inner arm dynein motors in the T.brucei flagellum:dynein-f.RNAi knockdown of genes for two components of dynein-f:DNAH10,theαheavy chain,and IC138,an intermediate chain,cause severe motility defects including immotility.To determine if motility defects result from structural disruption of the axoneme,we used two different flagellar preparations to carefully examine axoneme structure in these strains using transmission electron microscopy(TEM).Our analysis showed that inner arm dynein size,axoneme structural integrity and fixed central pair orientation are not significantly different in either knockdown culture when compared to control cultures.These results support the idea that immotility in knockdowns affecting DNAH10 or IC138 results from loss of dynein-f function rather than from obvious structural defects in the axoneme.展开更多
基金support of the National Key Research&Developmental Program of China(2018YFC1004900 to YQT)the National Natural Science Foundation of China(81971447 to YQT)the Key Grant of Prevention and Treatment of Birth Defect from Hunan province(2019SK1012 to YQT),and the research grant ofCITIC-Xiangya(YNXM-202004,YNXM-202006).
文摘Spermiogenesis is a complex and tightly regulated process,consisting of acrosomal biogenesis,condensation of chromatin,flagellar assembly,and disposal of extra cytoplasm.Previous studies have reported that sperm flagellar 2(SPEF2)deficiency causes severe asthenoteratozoospermia owing to spermiogenesis failure,but the underlying molecular mechanism in humans remains unclear.Here,we performed proteomic analysis on spermatozoa from three SPEF2 mutant patients to study the functional role of SPEF2 during sperm tail development.A total of 1262 differentially expressed proteins were detected,including 486 upregulated and 776 downregulated.The constructed heat map of the differentially expressed proteins showed similar trends.Among these,the expression of proteins related to flagellar assembly,including SPEF2,sperm associated antigen 6(SPAG6),dynein light chain tctex-type 1(DYNLT1),radial spoke head component 1(RSPH1),translocase of outer mitochondrial membrane 20(TOM20),EF-hand domain containing 1(EFHC1),meiosis-specific nuclear structural 1(MNS1)and intraflagellar transport 20(IFT20),was verified by western blot.Functional clustering analysis indicated that these differentially expressed proteins were specifically enriched for terms such as spermatid development and flagellar assembly.Furthermore,we showed that SPEF2 interacts with radial spoke head component 9(RSPH9)and IFT20 in vitro,which are well-studied components of radial spokes or intra-flagellar transport and are essential for flagellar assembly.These results provide a rich resource for further investigation into the molecular mechanism underlying the role that SPEF2 plays in sperm tail development and could provide a theoretical basis for gene therapy in SPEF2 mutant patients in the future.
基金supported by grants from the National Basic Research(973) Program of China(2015CB755700)the National High-Tech R&D(863) Program of China (2012AA02A703)+2 种基金the National Natural Science Foundation of China(31170081)the Special Fund for Agro-scientific Research in the Public Interest,China(201103007)the Guangdong Innovative and Entrepreneurial Research Team Program,China(2013S033).
文摘Flagellar biosynthesis and motility are subject to a four-tiered transcriptional regulatory circuit in Pseudomonas,and the master regulator FleQ appears to be the highest-level regulator in this hierarchical regulatory cascade.Pseudomonas stutzeri A1501 is motile by a polar flagellum;however,the motility and regulatory mechanisms involved in this process are unknown.Here,we searched the A1501 genome for flagella and motility genes and found that approximately 50 genes,which were distributed in three non-contiguous chromosomal regions,contribute to the formation,regulation and function of the flagella.The non-polar mutation of fleQ impaired flagellar biosynthesis,motility and root colonization but enhanced biofilm formation.FleQ positively regulates the expression of flagellar class Ⅱ-Ⅳ genes,suggesting a regulatory cascade that is coordinated similar to that of the well-known P.aeruginosa.Based on our results,we propose that flagellar genes in P.stutzeri A1501 are regulated in a cascade regulated by FleQ and that flagellum-driven motility properties may be necessary for competitive rhizosphere colonization.
基金supported by the Hong Kong University of Science & Technology, and the National Natural Science Foundation of China (No. 10902013)
文摘We extend the 2D Landau phase transition theory to the bacterial flagellar filament which displays the phase transition between the left handed normal form and the right handed semi-coiled form. The bacterial flagellar filament is treated as an elastic thin rod based on the Kirchhoff’s thin rod theory. Mechanical analysis is performed on the periodical phase transition of the filament between the two helical structures of the opposite charity. The curvature and twist are chosen as the order parameters in constructing the phase transition model of the filament. The established model is applied to study the instability properties of the filament and to investigate the loading and deformation conditions of the phase transition. In addition, the curvature and twist gradient energy are considered to describe the interface properties of the two phases.
基金supported by the Hong Kong University of Science and Technology and the National Natural Science Foundation of China (10902013)
文摘Bacterial flagellar filament can undergo a stress-induced polymorphic phase transition in both vitro and vivo environments.The filament has 12 different helical forms(phases) characterized by different pitch lengths and helix radii.When subjected to the frictional force of flowing fluid,the filament changes between a left-handed normal phase and a right-handed semi-coiled phase via phase nucleation and growth.This paper develops non-local finite element method(FEM) to simulate the phase transition under a displacement-controlled loading condition(controlled helix-twist).The FEM formulation is based on the Ginzburg-Landau theory using a one-dimensional non-convex and non-local continuum model.To describe the processes of the phase nucleation and growth,viscosity-type kinetics is also used.The non-local FEM simulation captures the main features of the phase transition:two-phase coexistence with an interface of finite thickness,phase nucleation and phase growth with interface propagation.The non-local FEM model provides a tool to study the effects of the interfacial energy/thickness and loading conditions on the phase transition.
基金supported by the Hong Kong University of Science and Technology and the National Natural Science Foundation of China (10902013)
文摘Bacterial flagellar filaments can undergo a polymorphic phase transition in both vitro and vivo environments. Each bacterial flagellar filament has 12 different helical forms which are macroscopically represented by different pitch lengths and helix radii. For external mechanical force induced filament phase transitions, there is so far only one experiment performed by Hotani in 1982, who showed a very beautiful cyclic phase transition phenomenon in his experiment on isolated flagellar filaments. In the present paper, we give a detailed mechanical analysis on Hotani＇s experiments. Through theoretical computations, we obtained a phase transition rule based on the phase transition mechanism. The theoretical analysis provides a foundation facilitating the establishment of phase transition theory for bacterial flagellar filaments.
文摘A new type of propeller that is optimized for low Reynolds numbers is required to propel a small object in a medium where the flow is dominated by viscous rather than inertial forces. A propeller in the shape of a bacterial flagellum seems an appro- priate choice for driving a small object. Accordingly, in this study, we visualized the velocity field induced by a spring-like propeller inspired by the Escherichia coli flagellum, using a macroscopic model and applying stereoscopic particle image velocimetry. We also experimentally evaluated the effect of pitch and rotational speed on the performance of this flagellar propeller. Silicone oil, which has a kinematic viscosity 100,000 times that of water, was used as the working fluid to generate a low Reynolds number for the macroscopic model. Thrust, torque, and velocity were measured as functions of pitch and rota- tional speed, and the efficiency of the propeller was calculated from the measured results. We found that the flagellar propeller reached a max!mum efficiency when the pitch angle was approximately 53°. Compared to pitch, rotational speed had a relatively small effect on the efficiency, and the pitch altered the flow pattern behind the rotating propeller.
文摘Cilia and flagella are organelles of motility that enable cells to swim or move liquid over its surface. An exhaustive literature survey for the presence of the organelle in organisms across phyla showed that most animal cells harbor cilia in contrast to very few fungal cells. While this was not unexpected, it was the position and arrangement of this organelle in each cell that intrigued our attention. Natural selection might have favored motility over chemotaxis;and it would have done so to evolve a stable structure that could have undergone an optimization process requiring a precise geometry in the shape of cells and the structure that would help cells to move. The positioning of such a structure would play a pre-dominant role in optimal motility. It is now known that the flagellar position of a cell is a genetically distinct trait, occasionally used in phylogeny of bacteria, distributed in distinguishing patterns over cellular surface, but basically are of two types, either polar (one flagellum arising from one pole per cell) or peritrichous (lateral flagella distributed over the entire cell surface). Irrespective of the cellular habitat, flagella origin, ultrastructure and proteome, the present investigation surveyed 26 sub-types of flagellar arrangements from as many species as possible. A peculiar pattern ensued-Prokaryotes harbored predominantly polar and peritrichous types;eukaryotes showed a mere change of the peritrichous one. These numbers when used to create a Similarity tree depicted a similarity distance of 14 between the Eubacteria and Archaebacteria forming the first neighborhood;Protozoans, Algae, Fungi, Plantae and Animalia formed a second neighborhood. We offer a working hypothesis for this pattern and the gradual shift in the flagellar arrangement from polar, peritrichous, sub-apical, and apical to lateral throughout evolution.
文摘This paper presents a simple technique to fabricate new electrofluidic devices for the three-dimensional(3D)manipulation of microorganisms by hybrid subtractive and additive femtosecond(fs)laser microfabrication(fs laser-assisted wet etching of glass followed by water-assisted fs laser modification combined with electroless metal plating).The technique enables the formation of patterned metal electrodes in arbitrary regions in closed glass microfluidic channels,which can spatially and temporally control the direction of electric fields in 3D microfluidic environments.The fabricated electrofluidic devices were applied to nanoaquariums to demonstrate the 3D electro-orientation of Euglena gracilis(an elongated unicellular microorganism)in microfluidics with high controllability and reliability.In particular,swimming Euglena cells can be oriented along the z-direction(perpendicular to the device surface)using electrodes with square outlines formed at the top and bottom of the channel,which is quite useful for observing the motions of cells parallel to their swimming directions.Specifically,z-directional electric field control ensured efficient observation of manipulated cells on the front side(45 cells were captured in a minute in an imaging area of~160×120μm),resulting in a reduction of the average time required to capture the images of five Euglena cells swimming continuously along the z-direction by a factor of~43 compared with the case of no electric field.In addition,the combination of the electrofluidic devices and dynamic imaging enabled observation of the flagella of Euglena cells,revealing that the swimming direction of each Euglena cell under the electric field application was determined by the initial body angle.
基金This work was supported by the Key Program of the National Natural Science Foundation of China(Grant No.21636003)the Outstanding Youth Foundation of Jiangsu(Grant No.SBK2017010373)+1 种基金the National Key Research and Development Program of China(Grant No.2019YFD1101204)the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture.Dong Liu is supported by the Jiangsu Qinglan Talent Program.
文摘Clostridium acetobutylicum has been extensively exploited to produce biofuels and solvents and its biofilm could dramatically improve the productivities.However,genetic control of C.acetobutylicum biofilm has not been dissected so far.Here,to identify potential genes controlling C.acetobutylicum biofilm formation,over 40 gene candidates associated with extracellular matrix,cell surface,cell signaling or gene transcription,were tried to be disrupted to examine their individual impact.A total of 25 disruptants were finally obtained over years of attempts,for which biofilm and relevant phenotypes were characterized.Most of these disruptants formed robust biofilm still,or suffered both growth and biofilm defect.Only a strain with a disrupted histidine kinase gene(CA_C2730,designated bfcK in this study)abolished biofilm formation without impairing cell growth or solvent production.Further analysis revealed that bfcK could control flagellar biogenesis and cell motility at protein levels.The bfcK also appeared to repress the phosphorylation of a serine/threonine protein kinase(encoded by CA_C0404)that might negatively regulate biofilm formation.Based on these findings,possible bfcK-mediated mechanisms for biofilm formation were proposed.This is a big step toward understanding the biofilm formation in C.acetobutylicum and will help further engineering of its biofilm-based industrial processes.
基金the support of the Presidential Early Career Award for Scientists and Engineers(PECASE)the Army Research Office(ARO,W911NF-13-1-0419 and W911NF-13-1-0197)+11 种基金the ARO Life Sciences Divisionthe National Science Foundation(NSF)CBET Division Biophotonics Programthe NSF Emerging Frontiers in Research and Innovation(EFRI)Awardthe NSF EAGER Award,NSF INSPIRE Award,NSF Partnerships for Innovation:Building Innovation Capacity(PFI:BIC)ProgramOffice of Naval Research(ONR)the National Institutes of Health(NIH)the Howard Hughes Medical Institute(HHMI)Vodafone Americas Foundationthe Mary Kay FoundationSteven&Alexandra Cohen Foundation,and KAUSTbased upon research performed in a laboratory renovated by the National Science Foundation under Grant No.0963183award funded under the American Recovery and Reinvestment Act of 2009(ARRA).
文摘We report a high-throughput and label-free computational imaging technique that simultaneously measures in three-dimensional(3D)space the locomotion and angular spin of the freely moving heads of microswimmers and the beating patterns of their flagella over a sample volume more than two orders-of-magnitude larger compared to existing optical modalities.Using this platform,we quantified the 3D locomotion of 2133 bovine sperms and determined the spin axis and the angular velocity of the sperm head,providing the perspective of an observer seated at the moving and spinning sperm head.In this constantly transforming perspective,flagellum-beating patterns are decoupled from both the 3D translation and spin of the head,which provides the opportunity to truly investigate the 3D spatio-temporal kinematics of the flagellum.In addition to providing unprecedented information on the 3D locomotion of microswimmers,this computational imaging technique could also be instrumental for micro-robotics and sensing research,enabling the high-throughput quantification of the impact of various stimuli and chemicals on the 3D swimming patterns of sperms,motile bacteria and other micro-organisms,generating new insights into taxis behaviors and the underlying biophysics.
文摘Trypanosoma brucei is a protozoan flagellate that causes African sleeping sickness.Flagellar function in this organism is critical for life cycle progression and pathogenesis,however the regulation of flagellar motility is not well understood.The flagellar axoneme produces a complex beat through the pre-cisely coordinated firing of many proteins,including multiple dynein motors.These motors are found in the inner arm and outer arm complexes.We are studying one of the inner arm dynein motors in the T.brucei flagellum:dynein-f.RNAi knockdown of genes for two components of dynein-f:DNAH10,theαheavy chain,and IC138,an intermediate chain,cause severe motility defects including immotility.To determine if motility defects result from structural disruption of the axoneme,we used two different flagellar preparations to carefully examine axoneme structure in these strains using transmission electron microscopy(TEM).Our analysis showed that inner arm dynein size,axoneme structural integrity and fixed central pair orientation are not significantly different in either knockdown culture when compared to control cultures.These results support the idea that immotility in knockdowns affecting DNAH10 or IC138 results from loss of dynein-f function rather than from obvious structural defects in the axoneme.
