TIRF microscopy has provided a means to view mobile granules within 100 nm in size in two dimensions.However quantitative analysis of the position and motion of those granules requires an appropriate tracking method.I...TIRF microscopy has provided a means to view mobile granules within 100 nm in size in two dimensions.However quantitative analysis of the position and motion of those granules requires an appropriate tracking method.In this paper,we present a new tracking algorithm combined with the unique features of TIRF.Firstly a fluorescence correction procedure was processed to solve the problem of fluorescence bleaching over time.Mobile granules were then segmented from a time-lapse image stack by an adaptive background subtraction method.Kalman filter was introduced to estimate and track the granules that allowed reducing searching range and hence greater reliability in tracking process.After the tracked granules were located in x-y plane,the z-position was indirectly inferred from the changes in their intensities.In the experiments the algorithm was applied in tracking GLUT4 vesicles in living adipose cells.The results indicate that the algorithm has achieved robust estimation and tracking of the vesicles in three dimensions.展开更多
As studies on life sciences progress toward the single-molecule level,new experiments have put forward more requirements for simultaneously displaying the mechanical properties and conformational changes of biomolecul...As studies on life sciences progress toward the single-molecule level,new experiments have put forward more requirements for simultaneously displaying the mechanical properties and conformational changes of biomolecules.Optical tweezers and fluorescence microscopy have been combined to solve this problem.The combination of instruments forms a new generation of hybrid single-molecule technology that breaks through the limitations of traditional biochemical analysis.Powerfulmanipulation and fluorescence visualization have beenwidely used,and these techniques provide new possibilities for studying complex biochemical reactions at the singlemolecule level.This paper explains the features of this combined technique,including the application characteristics of single-trap and dual-traps,the anti-bleaching method,and optical tweezers combined with epifluorescence,confocal fluorescence,total internal reflection fluorescence,and other fluorescence methods.Using typical experiments,we analyze technical solutions and explain the factors and principles that instrument designers should consider.This review aims to give an introduction to this novel fusion technology process and describe important biological results.展开更多
We have developed and tested transparent microelectrode arrays capable of simultaneous amperometric measurement of oxidizable molecules and fluorescence imaging through the electrodes. Surface patterned microelectrode...We have developed and tested transparent microelectrode arrays capable of simultaneous amperometric measurement of oxidizable molecules and fluorescence imaging through the electrodes. Surface patterned microelectrodes were fabricated from three different conducting materials: Indium-tin-oxide (ITO), nitrogen-doped diamond-like carbon (DLC) deposited on top of ITO, or very thin (12 - 17 nm) gold films on glass substrates. Chromaffin cells loaded with lysotracker green or acridine orange dye were placed atop the electrodes and vesicle fluorescence imaged with total internal reflection fluorescence (TIRF) microscopy while catecholamine release from single vesicles was measured as amperometric spikes with the surface patterned electrodes. Electrodes fabricated from all three materials were capable of detecting amperometric signals with high resolution. Unexpectedly, amperometric spikes recorded with ITO electrodes had only about half the amplitude and about half as much charge as those detected with DLC or gold electrodes, indicating that the ITO electrodes are not as sensitive as gold or DLC electrodes for measurement of quantal catecholamine release. The lower sensitivity of ITO electrodes was confirmed by chronoamperometry measurements comparing the currents in the presence of different analytes with the different electrode materials.展开更多
The complex signaling mechanisms in red blood cells(RBCs)enable them to adapt to physiological stresses such as exposure to low O_(2)levels,metabolic demands,oxidative stress,and shear stress.Since Ca^(2+)is a crucial...The complex signaling mechanisms in red blood cells(RBCs)enable them to adapt to physiological stresses such as exposure to low O_(2)levels,metabolic demands,oxidative stress,and shear stress.Since Ca^(2+)is a crucial determinant of RBC fate,various ion channels,pumps,and exchangers regulate the delicate balance of Ca^(2+)influx and efflux in RBCs.Elevated intracellular Ca^(2+)can activate processes such as membrane phospholipid scrambling and alter RBC deformability,which is essential for effective capillary transit.However,the dynamic information about Ca^(2+)regulation in RBCs is limited.Although static mapping and bioanalytical methods have been utilized,the absence of a nucleus and the presence of hemoglobin create challenges for real-time probing of RBC signaling,necessitating innovative approaches.This work introduces a synthetic chemistry−recombinant protein-based strategy to assemble sensors at genetically intact healthy human RBC surfaces for measuring dynamic signaling.Using this approach,we measured autocrine regulation of RBC Ca^(2+)influx in response to low O_(2)tension-induced ATP release.The study also explores the utilization of synthetic glycosylphosphatidylinositol(GPI)anchor mimics and sortagging for targeting sensors to the surfaces of primary as well as immortalized cells.This demonstrated the wide applicability of this approach to probe dynamic signaling in intact cells.展开更多
The ribosome,a 2.6 megadalton biomolecule measuring approximately 20 nm in diameter,coordinates numerous ligands,factors,and regulators to translate proteins with high fidelity and speed.Understanding its complex func...The ribosome,a 2.6 megadalton biomolecule measuring approximately 20 nm in diameter,coordinates numerous ligands,factors,and regulators to translate proteins with high fidelity and speed.Understanding its complex functions necessitates multiperspective observations.We developed a dualFRET single-molecule Förste Resonance Energy Transfer method(dual-smFRET),allowing simultaneous observation and correlation of tRNA dynamics and Elongation Factor G(EF-G)conformations in the same complex,in a 10 s time window.By synchronizing laser shutters and motorized filter sets,two FRET signals are captured in consecutive 5 s intervals with a time gap of 50-100 ms.We observed distinct fluorescent emissions from single-,double-,and quadruple-labeled ribosome complexes.Through comprehensive spectrum analysis and correction,we distinguish and correlate conformational changes in two parts of the ribosome,offering additional perspectives on its coordination and timing during translocation.Our setup’s versatility,accommodating up to six FRET pairs,suggests broader applications in studying large biomolecules and various biological systems.展开更多
Formins are conserved regulators of actin cytoskeletal organization and dynamics that have been impli- cated to be important for cell division and cell polarity. The mechanism by which diverse formins regulate actin d...Formins are conserved regulators of actin cytoskeletal organization and dynamics that have been impli- cated to be important for cell division and cell polarity. The mechanism by which diverse formins regulate actin dynamics in plants is still not well understood. Using in vitro single-molecule imaging technology, we directly observed that the FH1-FH2 domain of an Arabidopsis thaliana formin, AtFH14, processively at- taches to the barbed end of actin filaments as a dimer and slows their elongation rate by 90%. The attach- ment persistence of FH1-FH2 is concentration dependent. Furthermore, by use of the triple-color total internal reflection fluorescence microscopy, we found that ABP29, a barbed-end capping protein, com- petes with FH1-FH2 at the filament barbed end, where its binding is mutually exclusive with AtFH14. In the presence of different plant profilin isoforms, FH1-FH2 enhances filament elongation rates from about 10 to 42 times. Filaments buckle when FH1-FH2 is anchored specifically to cover slides, further indicating that AtFH 14 moves processively on the elongating barbed end. At high concentration, AtFH 14 bundles actin filaments randomly into antiparallel or parallel spindle-like structures; however, the FH1-FH2-mediated bundles become thinner and longer in the presence of plant profilins. This is the direct demonstration of a processive formin from plants. Our results also illuminate the molecular mechanism of AtFH14 in regulating actin dynamics via association with profilin.展开更多
Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion,growth,proliferation and differentiation.Recently,covalent and selective functionalization of glass and titanium surfaces with an...Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion,growth,proliferation and differentiation.Recently,covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide(HVP)mapped on[351e359]sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays,and to promote osseointegration in in vivo studies.For the first time to our knowledge,in this study we investigated the resistance of adhesion sequences to proteolytic digestion:HVP was completely cleaved after 5 h.In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence.A retro-inverted peptide D-2HVP,composed of D amino acids,was completely stable in serum-containing medium.In addition,glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium.Interestingly,D-2HVP increased expression of IBSP,VTN and SPP1 genes as compared to HVP functionalized surfaces.Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces.Therefore,the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.展开更多
Microtubules consisting of a/b-tubulin dimers play critical roles in cells.More than seven genes encode a-tubulin in vertebrates.However,the property of microtubules composed of different a-tubulin isotypes is largely...Microtubules consisting of a/b-tubulin dimers play critical roles in cells.More than seven genes encode a-tubulin in vertebrates.However,the property of microtubules composed of different a-tubulin isotypes is largely unknown.Here,we purified recombinant tubulin heterodimers of mouse a-tubulin isotypes including a1A and a1C with b-tubulin isotype b2A.In vitro microtubule reconstitution assay detected that a1C/b2A microtubules grew faster and underwent catastrophe less frequently than a1A/b2A microtubules.Generation of chimeric tail-swapped and point-mutation tubulins revealed that the carboxyl-terminal(C-terminal)tails of a-tubulin isotypes largely accounted for the differences in polymerization dynamics of a1A/b2A and a1C/b2A microtubules.Kinetics analysis showed that in comparison to a1A/b2A microtubules,a1C/b2A microtubules displayed higher on-rate,lower off-rate,and similar GTP hydrolysis rate at the plus-end,suggesting a contribution of higher plus-end affinity to faster growth and less frequent catastrophe of a1C/b2A microtubules.Furthermore,EB1 had a higher binding ability to a1C/b2A microtubules than to a1A/b2A ones,which could also be attributed to the difference in the C-terminal tails of these two a-tubulin isotypes.Thus,a-tubulin isotypes diversify microtubule properties,which,to a great extent,could be accounted by their C-terminal tails.展开更多
基金Project supported by the National Natural Science Foundation ofChina (No. 30770596)the Key Laboratory for Biomedical En-gineering of Ministry of Education of China
文摘TIRF microscopy has provided a means to view mobile granules within 100 nm in size in two dimensions.However quantitative analysis of the position and motion of those granules requires an appropriate tracking method.In this paper,we present a new tracking algorithm combined with the unique features of TIRF.Firstly a fluorescence correction procedure was processed to solve the problem of fluorescence bleaching over time.Mobile granules were then segmented from a time-lapse image stack by an adaptive background subtraction method.Kalman filter was introduced to estimate and track the granules that allowed reducing searching range and hence greater reliability in tracking process.After the tracked granules were located in x-y plane,the z-position was indirectly inferred from the changes in their intensities.In the experiments the algorithm was applied in tracking GLUT4 vesicles in living adipose cells.The results indicate that the algorithm has achieved robust estimation and tracking of the vesicles in three dimensions.
基金supported by the National Key Research and Development Program of China [grant numbers 2016YFB1102203, 2017YFF0107003]
文摘As studies on life sciences progress toward the single-molecule level,new experiments have put forward more requirements for simultaneously displaying the mechanical properties and conformational changes of biomolecules.Optical tweezers and fluorescence microscopy have been combined to solve this problem.The combination of instruments forms a new generation of hybrid single-molecule technology that breaks through the limitations of traditional biochemical analysis.Powerfulmanipulation and fluorescence visualization have beenwidely used,and these techniques provide new possibilities for studying complex biochemical reactions at the singlemolecule level.This paper explains the features of this combined technique,including the application characteristics of single-trap and dual-traps,the anti-bleaching method,and optical tweezers combined with epifluorescence,confocal fluorescence,total internal reflection fluorescence,and other fluorescence methods.Using typical experiments,we analyze technical solutions and explain the factors and principles that instrument designers should consider.This review aims to give an introduction to this novel fusion technology process and describe important biological results.
文摘We have developed and tested transparent microelectrode arrays capable of simultaneous amperometric measurement of oxidizable molecules and fluorescence imaging through the electrodes. Surface patterned microelectrodes were fabricated from three different conducting materials: Indium-tin-oxide (ITO), nitrogen-doped diamond-like carbon (DLC) deposited on top of ITO, or very thin (12 - 17 nm) gold films on glass substrates. Chromaffin cells loaded with lysotracker green or acridine orange dye were placed atop the electrodes and vesicle fluorescence imaged with total internal reflection fluorescence (TIRF) microscopy while catecholamine release from single vesicles was measured as amperometric spikes with the surface patterned electrodes. Electrodes fabricated from all three materials were capable of detecting amperometric signals with high resolution. Unexpectedly, amperometric spikes recorded with ITO electrodes had only about half the amplitude and about half as much charge as those detected with DLC or gold electrodes, indicating that the ITO electrodes are not as sensitive as gold or DLC electrodes for measurement of quantal catecholamine release. The lower sensitivity of ITO electrodes was confirmed by chronoamperometry measurements comparing the currents in the presence of different analytes with the different electrode materials.
文摘The complex signaling mechanisms in red blood cells(RBCs)enable them to adapt to physiological stresses such as exposure to low O_(2)levels,metabolic demands,oxidative stress,and shear stress.Since Ca^(2+)is a crucial determinant of RBC fate,various ion channels,pumps,and exchangers regulate the delicate balance of Ca^(2+)influx and efflux in RBCs.Elevated intracellular Ca^(2+)can activate processes such as membrane phospholipid scrambling and alter RBC deformability,which is essential for effective capillary transit.However,the dynamic information about Ca^(2+)regulation in RBCs is limited.Although static mapping and bioanalytical methods have been utilized,the absence of a nucleus and the presence of hemoglobin create challenges for real-time probing of RBC signaling,necessitating innovative approaches.This work introduces a synthetic chemistry−recombinant protein-based strategy to assemble sensors at genetically intact healthy human RBC surfaces for measuring dynamic signaling.Using this approach,we measured autocrine regulation of RBC Ca^(2+)influx in response to low O_(2)tension-induced ATP release.The study also explores the utilization of synthetic glycosylphosphatidylinositol(GPI)anchor mimics and sortagging for targeting sensors to the surfaces of primary as well as immortalized cells.This demonstrated the wide applicability of this approach to probe dynamic signaling in intact cells.
基金supported by NIGMS grant:R01GM111452,NSF:2130427the Welch Foundation:E-1721 to Y.Wang。
文摘The ribosome,a 2.6 megadalton biomolecule measuring approximately 20 nm in diameter,coordinates numerous ligands,factors,and regulators to translate proteins with high fidelity and speed.Understanding its complex functions necessitates multiperspective observations.We developed a dualFRET single-molecule Förste Resonance Energy Transfer method(dual-smFRET),allowing simultaneous observation and correlation of tRNA dynamics and Elongation Factor G(EF-G)conformations in the same complex,in a 10 s time window.By synchronizing laser shutters and motorized filter sets,two FRET signals are captured in consecutive 5 s intervals with a time gap of 50-100 ms.We observed distinct fluorescent emissions from single-,double-,and quadruple-labeled ribosome complexes.Through comprehensive spectrum analysis and correction,we distinguish and correlate conformational changes in two parts of the ribosome,offering additional perspectives on its coordination and timing during translocation.Our setup’s versatility,accommodating up to six FRET pairs,suggests broader applications in studying large biomolecules and various biological systems.
文摘Formins are conserved regulators of actin cytoskeletal organization and dynamics that have been impli- cated to be important for cell division and cell polarity. The mechanism by which diverse formins regulate actin dynamics in plants is still not well understood. Using in vitro single-molecule imaging technology, we directly observed that the FH1-FH2 domain of an Arabidopsis thaliana formin, AtFH14, processively at- taches to the barbed end of actin filaments as a dimer and slows their elongation rate by 90%. The attach- ment persistence of FH1-FH2 is concentration dependent. Furthermore, by use of the triple-color total internal reflection fluorescence microscopy, we found that ABP29, a barbed-end capping protein, com- petes with FH1-FH2 at the filament barbed end, where its binding is mutually exclusive with AtFH14. In the presence of different plant profilin isoforms, FH1-FH2 enhances filament elongation rates from about 10 to 42 times. Filaments buckle when FH1-FH2 is anchored specifically to cover slides, further indicating that AtFH 14 moves processively on the elongating barbed end. At high concentration, AtFH 14 bundles actin filaments randomly into antiparallel or parallel spindle-like structures; however, the FH1-FH2-mediated bundles become thinner and longer in the presence of plant profilins. This is the direct demonstration of a processive formin from plants. Our results also illuminate the molecular mechanism of AtFH14 in regulating actin dynamics via association with profilin.
文摘Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion,growth,proliferation and differentiation.Recently,covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide(HVP)mapped on[351e359]sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays,and to promote osseointegration in in vivo studies.For the first time to our knowledge,in this study we investigated the resistance of adhesion sequences to proteolytic digestion:HVP was completely cleaved after 5 h.In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence.A retro-inverted peptide D-2HVP,composed of D amino acids,was completely stable in serum-containing medium.In addition,glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium.Interestingly,D-2HVP increased expression of IBSP,VTN and SPP1 genes as compared to HVP functionalized surfaces.Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces.Therefore,the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.
基金This work was supported by grants from the National Natural Science Foundation of China(31991194,31330046,and 31922018)the Strategic Priority Research Program of Chinese Academy of Sciences(XDB19000000).
文摘Microtubules consisting of a/b-tubulin dimers play critical roles in cells.More than seven genes encode a-tubulin in vertebrates.However,the property of microtubules composed of different a-tubulin isotypes is largely unknown.Here,we purified recombinant tubulin heterodimers of mouse a-tubulin isotypes including a1A and a1C with b-tubulin isotype b2A.In vitro microtubule reconstitution assay detected that a1C/b2A microtubules grew faster and underwent catastrophe less frequently than a1A/b2A microtubules.Generation of chimeric tail-swapped and point-mutation tubulins revealed that the carboxyl-terminal(C-terminal)tails of a-tubulin isotypes largely accounted for the differences in polymerization dynamics of a1A/b2A and a1C/b2A microtubules.Kinetics analysis showed that in comparison to a1A/b2A microtubules,a1C/b2A microtubules displayed higher on-rate,lower off-rate,and similar GTP hydrolysis rate at the plus-end,suggesting a contribution of higher plus-end affinity to faster growth and less frequent catastrophe of a1C/b2A microtubules.Furthermore,EB1 had a higher binding ability to a1C/b2A microtubules than to a1A/b2A ones,which could also be attributed to the difference in the C-terminal tails of these two a-tubulin isotypes.Thus,a-tubulin isotypes diversify microtubule properties,which,to a great extent,could be accounted by their C-terminal tails.
