Numerous studies provide evidence that the lipid bilayer of the plasma membrane contains lateral ... more Numerous studies provide evidence that the lipid bilayer of the plasma membrane contains lateral nanodomains, and that these are functionally important regulators of transmembrane cell signaling. Depending on their chemical composition and the biophysical mechanism bringing the lipids together, multiple types of nanodomains exist in the inner and the outer leaflet of the plasma membrane bilayer. In intact cells, these domains are smaller than the optical resolution limit of light microscopy and also highly dynamic. Recently, advanced fluorescence methods have provided data to characterize many biophysical and thermodynamic aspects of these nanodomains. In this review, we summarize the physicochemical determinants of nanodomain formation, stability and extent. Then, we detail how these nanodomains play a structural role by anchoring nucleation sites for the membrane cytoskeleton on the lipid bilayer. Further, we review the existing literature on mechanisms that modulate the nanodomain size and stability, both acute and chronic events. We conclude that regulation of the nanodomains distribution in the lipid bilayer of the plasma membrane is important for modulation of transmembrane signaling. However, only very few modulators of nanodomain stability and size have been quantified in cells, suggesting interesting directions for future studies.
The combination of multicomponent magnetic nanoparticles and a mechanosensitive ion channel has b... more The combination of multicomponent magnetic nanoparticles and a mechanosensitive ion channel has been shown to achieve fast magnetomechanical stimulation of neurons within the brain.
Many cell-membrane-associated processes require transient spatiotemporal separation of components... more Many cell-membrane-associated processes require transient spatiotemporal separation of components on scales ranging from a couple of molecules to micrometers in size. Understanding these processes mechanistically involves understanding how lipids and proteins self-organize and interact with the cell cortex. Here, we review recent advances in dissecting the mechanisms of cell membrane compartmentalization. We introduce the challenges in studying cell membrane organization, the current understanding of how complex membranes self-organize to form transient domains, and the role of protein scaffolds in membrane organization. We discuss the formation of signaling domains as an important example of transient membrane compartmentalization. We conclude by pointing to the current limitations of measuring membrane organization in living cells and the steps that are required to advance the field.
Establishing how neurocircuit activation causes particular behaviors requires modulating the acti... more Establishing how neurocircuit activation causes particular behaviors requires modulating the activity of specific neurons. Here, we demonstrate that magnetothermal genetic stimulation provides tetherless deep brain activation sufficient to evoke motor behavior in awake mice. The approach uses alternating magnetic fields to heat superparamagnetic nanoparticles on the neuronal membrane. Neurons heat-sensitized by expressing TRPV1 are activated with magnetic field application. Magnetothermal genetic stimulation in the motor cortex evoked ambulation, deep brain stimulation in the striatum caused rotation around the body-axis, and stimulation near the ridge between ventral and dorsal striatum caused freezing-of-gait. The duration of the behavior correlated tightly with field application. This approach provides genetically and spatially targetable, repeatable and temporarily precise activation of deep-brain circuits without need for surgical implantation of any device.
ABSTRACT We present the work on the synthesis of dye-doped monodisperse Fe/SiO2 core/shell nanopa... more ABSTRACT We present the work on the synthesis of dye-doped monodisperse Fe/SiO2 core/shell nanoparticles as bifunctional probes for bioapplications. Magnetic nanoparticles (NP) have been widely studied as nano-probes for bio-imaging, sensing as well as for cancer therapy. Among all the NPs, Fe NPs have been the focus because they have very high magnetization. However, Fe NPs are usually not stable in ambient due to the fast surface oxidation of the NPs. On the other hand, dye molecules have long been used as probes for bio-imaging. But they are sensitive to environmental conditions. It requires passivation for both so that they can be stable for applications. In this work, monodisperse Fe NPs with sizes ranging from 13-20 nm have been synthesized through the chemical thermal-decomposition in a solution. Silica shells were then coated on the Fe NPs by a two-phase oil-in-water method. Dye molecules were first bonded to a silica precursor and then encapsulated into the silica shell during the coating process. The silica shells protect both the Fe NPs and dye molecules, which makes them as robust probes. The dye doped Fe/SiO2 core/shell NPs remain both highly magnetic and highly fluorescent. The stable dye doped Fe/SiO2NPs have been used as a dual functional probe for both magnetic heating and local nanoscale temperature sending, and their performance will be reported.
ABSTRACT While the streptavidin-biotin interaction is the basis of many biotechnology and medical... more ABSTRACT While the streptavidin-biotin interaction is the basis of many biotechnology and medical applications, the large size of streptavidin tetramer and the multivalency of the molecule present obstacles in some situations. The focus of the current study is thus to engineer smaller streptavidin variants that have sufficient stability and affinity to make the technology accessible to other applications, including in vivo cell imaging. To this end, we adopted a rational design approach to engineer a monomer and a hairline dimer mutant with improved stability and affinity. We tested monomer mutants containing one or more engineered disulfides to stabilize the biotin-bound conformation. The best mutant was then engineered further by introducing surface mutations to create an extended stabilizing salt bridge network. The Tm = 47 C and Kd = 60 nM of the resulting monomer, which compare favorably with those of wt monomer (Tm = 32 C and Kd = 130 nM), allow the molecule to be used in a cell biology experiment performed at 37 C. It has long been suspected that hairline interaction across the dimer-dimer interface is important for high affinity biotin binding, but engineering such a dimer has been difficult due to limited interface between the subunits. To engineer a stable hairline dimer, we modeled the structure and introduced an interchain disulfide between two designed monomer subunits. We show that the purified dimer, which elutes as a single peak by gel filtration and has high monodispersity by dynamic light scattering, is more stable than the substituent monomer by ~ 6 C and has an 8 fold higher affinity compared to wt monomer, making it an ideal molecule for applications that require a stable labeling reagent. In addition to rational design of streptavidin protomers, we will present results from two other parallel works for improving the biophysical properties of streptavidin monomer. First, we have recently demonstrated that functional streptavidin monomer can be displayed on the yeast surface using biotin as a molecular chaperone. This allowed us to improve the stability of rationally designed molecule by directed evolution, which has not been possible until now due to the difficulties of folding streptavidin in yeast. Second, we used homology modeling of streptavidin and streptavidin-like molecules from other organisms to create a stable hybrid molecule that is roughly 20% of the mw of wt streptavidin. Toward improving the thermodynamic and binding properties of streptavidin monomer, we will present a comparative analysis of designed chimeras that reveal the challenges of engineering an optimized a monomer sequence.
... sensor in a spectrofluorimeter, epifluorescence intensity ratio imaging, donor fluorescence .... more ... sensor in a spectrofluorimeter, epifluorescence intensity ratio imaging, donor fluorescence ... 2. (Top) Commonly used single-molecule FΓΆrster (or fluorescence) resonance energy transfer (FRET) sensors ... Photobleaching of ECFP strongly shortens its fluorescence lifetime (2.59 vs. ...
The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactio... more The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-beta family. The Ran-importin-beta system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1 (refs 1, 4, 5). However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. Here we examine the Ran-importin-beta system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-beta by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vit...
Numerous studies provide evidence that the lipid bilayer of the plasma membrane contains lateral ... more Numerous studies provide evidence that the lipid bilayer of the plasma membrane contains lateral nanodomains, and that these are functionally important regulators of transmembrane cell signaling. Depending on their chemical composition and the biophysical mechanism bringing the lipids together, multiple types of nanodomains exist in the inner and the outer leaflet of the plasma membrane bilayer. In intact cells, these domains are smaller than the optical resolution limit of light microscopy and also highly dynamic. Recently, advanced fluorescence methods have provided data to characterize many biophysical and thermodynamic aspects of these nanodomains. In this review, we summarize the physicochemical determinants of nanodomain formation, stability and extent. Then, we detail how these nanodomains play a structural role by anchoring nucleation sites for the membrane cytoskeleton on the lipid bilayer. Further, we review the existing literature on mechanisms that modulate the nanodomain size and stability, both acute and chronic events. We conclude that regulation of the nanodomains distribution in the lipid bilayer of the plasma membrane is important for modulation of transmembrane signaling. However, only very few modulators of nanodomain stability and size have been quantified in cells, suggesting interesting directions for future studies.
The combination of multicomponent magnetic nanoparticles and a mechanosensitive ion channel has b... more The combination of multicomponent magnetic nanoparticles and a mechanosensitive ion channel has been shown to achieve fast magnetomechanical stimulation of neurons within the brain.
Many cell-membrane-associated processes require transient spatiotemporal separation of components... more Many cell-membrane-associated processes require transient spatiotemporal separation of components on scales ranging from a couple of molecules to micrometers in size. Understanding these processes mechanistically involves understanding how lipids and proteins self-organize and interact with the cell cortex. Here, we review recent advances in dissecting the mechanisms of cell membrane compartmentalization. We introduce the challenges in studying cell membrane organization, the current understanding of how complex membranes self-organize to form transient domains, and the role of protein scaffolds in membrane organization. We discuss the formation of signaling domains as an important example of transient membrane compartmentalization. We conclude by pointing to the current limitations of measuring membrane organization in living cells and the steps that are required to advance the field.
Establishing how neurocircuit activation causes particular behaviors requires modulating the acti... more Establishing how neurocircuit activation causes particular behaviors requires modulating the activity of specific neurons. Here, we demonstrate that magnetothermal genetic stimulation provides tetherless deep brain activation sufficient to evoke motor behavior in awake mice. The approach uses alternating magnetic fields to heat superparamagnetic nanoparticles on the neuronal membrane. Neurons heat-sensitized by expressing TRPV1 are activated with magnetic field application. Magnetothermal genetic stimulation in the motor cortex evoked ambulation, deep brain stimulation in the striatum caused rotation around the body-axis, and stimulation near the ridge between ventral and dorsal striatum caused freezing-of-gait. The duration of the behavior correlated tightly with field application. This approach provides genetically and spatially targetable, repeatable and temporarily precise activation of deep-brain circuits without need for surgical implantation of any device.
ABSTRACT We present the work on the synthesis of dye-doped monodisperse Fe/SiO2 core/shell nanopa... more ABSTRACT We present the work on the synthesis of dye-doped monodisperse Fe/SiO2 core/shell nanoparticles as bifunctional probes for bioapplications. Magnetic nanoparticles (NP) have been widely studied as nano-probes for bio-imaging, sensing as well as for cancer therapy. Among all the NPs, Fe NPs have been the focus because they have very high magnetization. However, Fe NPs are usually not stable in ambient due to the fast surface oxidation of the NPs. On the other hand, dye molecules have long been used as probes for bio-imaging. But they are sensitive to environmental conditions. It requires passivation for both so that they can be stable for applications. In this work, monodisperse Fe NPs with sizes ranging from 13-20 nm have been synthesized through the chemical thermal-decomposition in a solution. Silica shells were then coated on the Fe NPs by a two-phase oil-in-water method. Dye molecules were first bonded to a silica precursor and then encapsulated into the silica shell during the coating process. The silica shells protect both the Fe NPs and dye molecules, which makes them as robust probes. The dye doped Fe/SiO2 core/shell NPs remain both highly magnetic and highly fluorescent. The stable dye doped Fe/SiO2NPs have been used as a dual functional probe for both magnetic heating and local nanoscale temperature sending, and their performance will be reported.
ABSTRACT While the streptavidin-biotin interaction is the basis of many biotechnology and medical... more ABSTRACT While the streptavidin-biotin interaction is the basis of many biotechnology and medical applications, the large size of streptavidin tetramer and the multivalency of the molecule present obstacles in some situations. The focus of the current study is thus to engineer smaller streptavidin variants that have sufficient stability and affinity to make the technology accessible to other applications, including in vivo cell imaging. To this end, we adopted a rational design approach to engineer a monomer and a hairline dimer mutant with improved stability and affinity. We tested monomer mutants containing one or more engineered disulfides to stabilize the biotin-bound conformation. The best mutant was then engineered further by introducing surface mutations to create an extended stabilizing salt bridge network. The Tm = 47 C and Kd = 60 nM of the resulting monomer, which compare favorably with those of wt monomer (Tm = 32 C and Kd = 130 nM), allow the molecule to be used in a cell biology experiment performed at 37 C. It has long been suspected that hairline interaction across the dimer-dimer interface is important for high affinity biotin binding, but engineering such a dimer has been difficult due to limited interface between the subunits. To engineer a stable hairline dimer, we modeled the structure and introduced an interchain disulfide between two designed monomer subunits. We show that the purified dimer, which elutes as a single peak by gel filtration and has high monodispersity by dynamic light scattering, is more stable than the substituent monomer by ~ 6 C and has an 8 fold higher affinity compared to wt monomer, making it an ideal molecule for applications that require a stable labeling reagent. In addition to rational design of streptavidin protomers, we will present results from two other parallel works for improving the biophysical properties of streptavidin monomer. First, we have recently demonstrated that functional streptavidin monomer can be displayed on the yeast surface using biotin as a molecular chaperone. This allowed us to improve the stability of rationally designed molecule by directed evolution, which has not been possible until now due to the difficulties of folding streptavidin in yeast. Second, we used homology modeling of streptavidin and streptavidin-like molecules from other organisms to create a stable hybrid molecule that is roughly 20% of the mw of wt streptavidin. Toward improving the thermodynamic and binding properties of streptavidin monomer, we will present a comparative analysis of designed chimeras that reveal the challenges of engineering an optimized a monomer sequence.
... sensor in a spectrofluorimeter, epifluorescence intensity ratio imaging, donor fluorescence .... more ... sensor in a spectrofluorimeter, epifluorescence intensity ratio imaging, donor fluorescence ... 2. (Top) Commonly used single-molecule FΓΆrster (or fluorescence) resonance energy transfer (FRET) sensors ... Photobleaching of ECFP strongly shortens its fluorescence lifetime (2.59 vs. ...
The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactio... more The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-beta family. The Ran-importin-beta system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1 (refs 1, 4, 5). However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. Here we examine the Ran-importin-beta system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-beta by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vit...
Proteins in contrast to macroscopic machines are subject to thermal fluctuations in shape which p... more Proteins in contrast to macroscopic machines are subject to thermal fluctuations in shape which provide both opportunities and challenges. They have to be flexible enough to support turn-over rates up to hundreds per second, yet stable enough to maintain their three-dimensional structure over hours and days. As result of thermal excitation they fluctuate between structural conformations. We measured thermally excited
Proteins in contrast to macroscopic machines are subject to thermal fluctuations in shape which p... more Proteins in contrast to macroscopic machines are subject to thermal fluctuations in shape which provide both opportunities and challenges. They have to be flexible enough to support turn-over rates up to hundreds per second, yet stable enough to maintain their three-dimensional ...
ABSTRACT The cell membrane is thought to contain spatial domains, created by cholesterol-lipid cl... more ABSTRACT The cell membrane is thought to contain spatial domains, created by cholesterol-lipid clusters and by interactions with the membrane cytoskeleton. The influence of these domains on membrane protein mobility and cell signaling has clearly been demonstrate. Yet, due to their small size and transient nature, the cholesterol stabilized domains cannot be visualized directly. We show here that thermal noise imaging (TNI) which tracks the diffusion of a colloid labeled membrane protein with microsecond and nanometer precision, can visualize cholesterol stabilized domains, also know as lipid raft, in intact cells. Using TNI to confine a single membrane protein to diffuse for seconds in an area of 300nm x 300nm provides sufficient data for high resolutions maps of the local diffusion, local attraction potentials and membrane stiffness. Using a GPI-anchored GFP molecule to probe the membrane of PtK2 cells we detect domains of increased membrane stiffness, which also show increase viscosity and are the preferred location for the GPI-anchored protein. These domains are further stabilized by addition of ganglioside cross linking toxins and disappear after removal of the cholesterol.
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