Objectives/Hypothesis: In animal studies of vocal fold scarring and treatment, imaging-based eval... more Objectives/Hypothesis: In animal studies of vocal fold scarring and treatment, imaging-based evaluation is most often conducted by tissue slicing and histological staining. Given variation in anatomy, injury type, severity, and sacrifice timepoints, planar histological sections provide limited spatiotemporal details of tissue repair. Three-dimensional (3D) virtual histology may provide additional contextual spatial information, enhancing objective interpretation. The study's aim was to evaluate the suitability of magnetic resonance imaging (MRI), microscale computed tomography (CT), and nonlinear laser-scanning microscopy (NM) as virtual histology approaches for rabbit studies of vocal fold scarring. Methods: A unilateral injury was created using microcup forceps in the left vocal fold of three New Zealand White rabbits. Animals were sacrificed at 3, 10, and 39 days postinjury. ex vivo imaging of excised larynges was performed with MRI, CT, and NM modalities. Results: The MRI modality allowed visualization of injury location and morphological internal features with 100-μm spatial resolution. The CT modality provided a view of the injury defect surface with 12-μm spatial resolution. The NM modality with optical clearing resolved second-harmonic generation signal of collagen fibers and two-photon autofluorescence in vocal fold lamina propria, muscle, and surrounding cartilage structures at submicrometer spatial scales. Conclusions: Features of vocal fold injury and wound healing were observed with MRI, CT, and NM. The MRI and CT modalities provided contextual spatial information and dissection guidance, whereas NM resolved extracellular matrix structure. The results serve as a proof of concept to motivate incorporation of 3D virtual histology techniques in future vocal fold injury animal studies.
The spectrally distinct nature of these probes coupled with the fact that they can be readily fun... more The spectrally distinct nature of these probes coupled with the fact that they can be readily functionalized with various surface modifications suggests that they hold great promise for multi-color imaging applications.
bioRxiv (Cold Spring Harbor Laboratory), Jun 23, 2021
We present a fluorescence fluctuation image correlation analysis method that can rapidly and simu... more We present a fluorescence fluctuation image correlation analysis method that can rapidly and simultaneously measure the diffusion coefficient, photoblinking rates, and fraction of diffusing particles of fluorescent molecules in cells. Unlike other image correlation techniques, we demonstrated that our method could be applied irrespective of a non-uniformly distributed, immobile blinking fluorophore population. This allows us to measure blinking and transport dynamics in complex cell morphologies, a benefit for a range of super-resolution fluorescence imaging approaches that rely on probe emission blinking. Furthermore, we showed that our technique could be applied without directly accounting for photobleaching. We successfully employed our technique on several simulations with realistic EMCCD noise and photobleaching models, as well as on Dronpa-C12 labeled beta-actin in living NIH/3T3 and HeLa cells. We found that the diffusion coefficients measured using our method were consistent with previous literature values. We further found that photoblinking rates measured in the live HeLa cells varied as expected with changing excitation power.
The International Journal of Biochemistry & Cell Biology, 2021
Nonlinear microscopy is a technique that utilizes nonlinear interactions between light and matter... more Nonlinear microscopy is a technique that utilizes nonlinear interactions between light and matter to image fluorescence and scattering phenomena in biological tissues. Very high peak intensities from focused short pulsed lasers are required for nonlinear excitation due to the extremely low probability of the simultaneous arrival of multiple photons of lower energy to excite fluorophores or interact with selective structures for harmonic generation. Combined with reduced scattering from the utilization of longer wavelengths, the inherent spatial confinement associated with achieving simultaneous arrival of photons within the focal volume enables deep imaging with low out-of-focus background for nonlinear imaging. This review provides an introduction to the different contrast mechanisms available with nonlinear imaging and instrumentation commonly used in nonlinear microscopy. Furthermore, we discuss some recent advances in nonlinear microscopy to extend the imaging penetration depth, conduct histopathological investigations on fresh tissues and examine the molecular order and orientation of molecules using polarization nonlinear microscopy.
The formation of memory and the process of learning are believed to be regulated, at least in par... more The formation of memory and the process of learning are believed to be regulated, at least in part, by the expression, distribution, and redistribution of glutamate receptors. The expression of these receptors at synaptic sites has a major impact on the strength of synaptic connections, and the AMPA receptor subunit GluR1 appears to play a key role within this system. Increasing evidence suggests that previously silent synapses become activated through rapid AMPA receptor insertion upon appropriate stimulation, and thus the trafficking of this receptor subunit from cellular stores to the synapse is of prime interest.We are using a transgenic mouse expressing a GFP-tagged form of GluR1 (GluR1-GFP) in order to study the dynamic changes in GluR1 expression and distribution occurring during brain development and following induction of long-term potentiation (LTP). The fusion protein is transcribed under the control of the CaMKIIα promoter, which restricts the localization to forebrain n...
Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive ac... more Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive actin-rich protrusions, to generate forces, migrate, and patrol for foreign antigens. Individual podosomes probe their microenvironment through periodic protrusion and retraction cycles (height oscillations), while oscillations of multiple podosomes in a cluster are coordinated in a wave-like fashion. However, the mechanisms governing both the individual oscillations and the collective wave-like dynamics remain unclear. Here, by integrating actin polymerization, myosin contractility, actin diffusion, and mechanosensitive signaling, we develop a chemo-mechanical model for podosome dynamics in clusters. Our model reveals that podosomes show oscillatory growth when actin polymerization-driven protrusion and signalingassociated myosin contraction occur at similar rates, while the diffusion of actin monomers drives wave-like coordination of podosome oscillations. Our theoretical predictions are validated by different pharmacological treatments and the impact of microenvironment stiffness on chemo-mechanical waves. Our proposed framework can shed light on the role of podosomes in immune cell mechanosensing within the context of wound healing and cancer immunotherapy. Dendritic cells (DCs) and macrophages, act as gatekeepers of the immune system by surveilling peripheral tissues for foreign antigens. During this process, these cells form dozens of specialized actin-rich protrusions called podosomes, which are typically organized into large clusters. By utilizing podosomes, DCs and macrophages can control adhesions, degrade the surrounding extracellular matrix (ECM), and remodel the extracellular environment to facilitate their migration 1-3. Podosomes are characterized by an actin-based core surrounded by an adhesive ring consisting of integrins and adaptor proteins, such as vinculin and talin 4,5. Podosomes generate protrusive forces to penetrate the underlying ECMs at the cores, while applying tensile forces to pull the ECMs at the rings 6,7. Studies in DCs and macrophages have shown that individual podosomes exhibit periodic oscillations in the fluorescence intensity of core actin, ring components, and the protrusive forces exerted at their cores 7-9. Collectively, podosomes in a cluster show spatially correlated behaviors, where the oscillations of podosome components are coordinated in a wave-like manner within the cluster 10,11. However, the relationship which directs the core protrusive and ring tensile forces into oscillations of individual podosomes and subsequent spatiotemporal wave patterns in clusters remains unknown. Understanding these processes can provide insights on how podosomes in a cluster collectively probe and respond
Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invas... more Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invasion relies on invadosomes, a collective term for invadopodia and podosomes. An adequate structural framework for this process is still missing. Here, we reveal the modular actin nano-architecture that enables podosome protrusion and mechanosensing. The podosome protrusive core contains a central branched actin module encased by a linear actin module, each harboring specific actin interactors and actin isoforms. From the core, two actin modules radiate: ventral filaments bound by vinculin and connected to the plasma membrane and dorsal interpodosomal filaments crosslinked by myosin IIA. On stiff substrates, the actin modules mediate long-range substrate exploration, associated with degradative behavior. On compliant substrates, the vinculin-bound ventral actin filaments shorten, resulting in shortrange connectivity and a focally protrusive, non-degradative state. Our findings redefine podosome nanoscale architecture and reveal a paradigm for how actin modularity drives invadosome mechanosensing in cells that breach tissue boundaries.
acid interactions. Visualization is achieved through the application of two complementary procedu... more acid interactions. Visualization is achieved through the application of two complementary procedures. In one, a single DNA molecule is attached to a polystyrene bead, which is captured in an optical trap. The DNA is extended either by the force of solution flow in a micro-flowcell, or by capturing the opposite DNA end in a second optical trap. In the second procedure, DNA is attached by one end to a glass surface. The coiled DNA is elongated either by continuous solution flow or by subsequently tethering the opposite end to the surface. Proteins and DNA are visualized via fluorescent reporters. Individual molecules are imaged using either epifluorescence microscopy or total internal reflection fluorescence (TIRF) microscopy. Molecules are introduced and supramolecular complexes are built, one component at a time, using microfluidic flowcells. Using these approaches, we have watched proteins functioning in the repair, replication, and manipulation of DNA. We have imaged unwinding of DNA by helicases, translocation along DNA by motor proteins, self-assembly of protein filaments on DNA, the search for DNA sequence homology by protein-DNA filaments, real-time replication of DNA, and nucleosome remodeling. I will summarize how these experiments were done, and what we've learned about biology from the stochastic behavior of individual molecules.
We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation ... more We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) that relies on complete analysis of both the temporal and spatial correlation lags for intensity fluctuations from a laserscanning microscopy image series. This new approach allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) for fluorescently labeled membrane proteins in living cells through monitoring of the time evolution of the full space-time correlation function. By using filtering in Fourier space to remove frequencies associated with immobile components, we are able to measure the protein transport even in the presence of a large fraction (.90%) of immobile species. We present the background theory, computer simulations, and analysis of measurements on fluorescent microspheres to demonstrate proof of principle, capabilities, and limitations of the method. We demonstrate mapping of flow vectors for mixed samples containing fluorescent microspheres with different emission wavelengths using space time image cross-correlation. We also present results from two-photon laser-scanning microscopy studies of a-actinin/enhanced green fluorescent protein fusion constructs at the basal membrane of living CHO cells. Using space-time image correlation spectroscopy (STICS), we are able to measure protein fluxes with magnitudes of mm/min from retracting lamellar regions and protrusions for adherent cells. We also demonstrate the measurement of correlated directed flows (magnitudes of mm/min) and diffusion of interacting a5 integrin/enhanced cyan fluorescent protein and a-actinin/enhanced yellow fluorescent protein within living CHO cells. The STICS method permits us to generate complete transport maps of proteins within subregions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements.
Smart fluorophores'', such as reversibly switchable fluorescent proteins, are crucial for advance... more Smart fluorophores'', such as reversibly switchable fluorescent proteins, are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available, and many display reduced biological performance compared to more classical variants. We present the development of robustly photoswitchable variants of enhanced green fluorescent protein (EGFP), named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37 C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multimodal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of~70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the ''on''-and ''off''-conformation we were able to confirm the presence of a cis-trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded ''smart fluorophores'' can be readily optimized for biological performance and provides a practical strategy for developing maturation-and stability-enhanced photochromic fluorescent proteins.
There is evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) anion chann... more There is evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is highly expressed at the apical pole of ciliated cells in human bronchial epithelium (HBE), however recent studies have detected little CFTR mRNA in those cells. To understand this discrepancy we immunostained well differentiated primary HBE cells using CFTR antibodies. We confirmed apical immunofluorescence in ciliated cells and quantified the covariance of the fluorescence signals and that of an antibody against the ciliary marker centrin-2 using image cross-correlation spectroscopy (ICCS). Super-resolution stimulated emission depletion (STED) imaging localized the immunofluorescence in distinct clusters at the bases of the cilia. However, similar apical fluorescence was observed when the monoclonal CFTR antibodies 596, 528 and 769 were used to immunostain ciliated cells expressing F508del-CFTR, or cells lacking CFTR due to a Class I mutation. A BLAST search using the CFTR epitope identified a similar amino acid sequence in the ciliary protein rootletin X1. Its expression level correlated with the intensity of immunostaining by CFTR antibodies and it was detected by 596 antibody after transfection into CFBE cells. These results may explain the high apparent expression of CFTR in ciliated cells and reports of anomalous apical immunofluorescence in well differentiated cells that express F508del-CFTR. Human airway bronchial epithelium contains ciliated cells, secretory goblet and club cells, basal cells, and pulmonary neuroendocrine cells 1,2 , each with distinct roles in airway physiology and host defense. Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP regulated anion channel required for normal secretion of airway surface liquid 3-5. CFTR mutations lead to abnormal mucus and impaired mucociliary clearance of inhaled bacteria that are hallmarks of cystic fibrosis (CF) 6. CFTR is thought to be highly expressed in ciliated cells of the airway epithelium, however recent single cell mRNA sequencing (scRNAseq) reports detected low CFTR mRNA levels in ciliated cells from primary human bronchial epithelial (HBE) cultures and mouse lung tissue 7-9. It was suggested that most CFTR transcripts (> 45%) are in a rare (< 2% of total) epithelial cell type called pulmonary ionocytes 7,8 , although one scRNAseq study found most CFTR transcripts (80% of the total) in secretory and basal cells 9. Further analysis at the protein level is needed to assess CFTR immunofluorescence in ciliated cells and understand F508del-CFTR apical immunofluorescence reported in some studies of CF airways 10,11. Many CFTR mutations have been identified and classified as Class I-VI based on the predominant molecular defects they produce 12-14. Class I mutations include nonsense, frameshift and splicing mutations that prevent expression of the full-length protein. Class II mutations cause protein misfolding and impair trafficking to the plasma membrane. Class III mutations are those that inhibit channel gating or regulation, while class IV-VI mutations reduce pore conduction, protein expression and CFTR stability, respectively. F508del is by far the most frequent mutation, occurring on at least one chromosome in ~ 90% of the CF population. It is a Class II mutation that causes misfolding, retention in the endoplasmic reticulum, and premature degradation by the proteasome and other pathways 15-17. We used monoclonal antibodies to immunofluorescence label and localize CFTR in well-differentiated primary cultures of human bronchial epithelial cells from non-CF donors and F508del homozygotes. Importantly,
Objectives/Hypothesis: In animal studies of vocal fold scarring and treatment, imaging-based eval... more Objectives/Hypothesis: In animal studies of vocal fold scarring and treatment, imaging-based evaluation is most often conducted by tissue slicing and histological staining. Given variation in anatomy, injury type, severity, and sacrifice timepoints, planar histological sections provide limited spatiotemporal details of tissue repair. Three-dimensional (3D) virtual histology may provide additional contextual spatial information, enhancing objective interpretation. The study's aim was to evaluate the suitability of magnetic resonance imaging (MRI), microscale computed tomography (CT), and nonlinear laser-scanning microscopy (NM) as virtual histology approaches for rabbit studies of vocal fold scarring. Methods: A unilateral injury was created using microcup forceps in the left vocal fold of three New Zealand White rabbits. Animals were sacrificed at 3, 10, and 39 days postinjury. ex vivo imaging of excised larynges was performed with MRI, CT, and NM modalities. Results: The MRI modality allowed visualization of injury location and morphological internal features with 100-μm spatial resolution. The CT modality provided a view of the injury defect surface with 12-μm spatial resolution. The NM modality with optical clearing resolved second-harmonic generation signal of collagen fibers and two-photon autofluorescence in vocal fold lamina propria, muscle, and surrounding cartilage structures at submicrometer spatial scales. Conclusions: Features of vocal fold injury and wound healing were observed with MRI, CT, and NM. The MRI and CT modalities provided contextual spatial information and dissection guidance, whereas NM resolved extracellular matrix structure. The results serve as a proof of concept to motivate incorporation of 3D virtual histology techniques in future vocal fold injury animal studies.
The spectrally distinct nature of these probes coupled with the fact that they can be readily fun... more The spectrally distinct nature of these probes coupled with the fact that they can be readily functionalized with various surface modifications suggests that they hold great promise for multi-color imaging applications.
bioRxiv (Cold Spring Harbor Laboratory), Jun 23, 2021
We present a fluorescence fluctuation image correlation analysis method that can rapidly and simu... more We present a fluorescence fluctuation image correlation analysis method that can rapidly and simultaneously measure the diffusion coefficient, photoblinking rates, and fraction of diffusing particles of fluorescent molecules in cells. Unlike other image correlation techniques, we demonstrated that our method could be applied irrespective of a non-uniformly distributed, immobile blinking fluorophore population. This allows us to measure blinking and transport dynamics in complex cell morphologies, a benefit for a range of super-resolution fluorescence imaging approaches that rely on probe emission blinking. Furthermore, we showed that our technique could be applied without directly accounting for photobleaching. We successfully employed our technique on several simulations with realistic EMCCD noise and photobleaching models, as well as on Dronpa-C12 labeled beta-actin in living NIH/3T3 and HeLa cells. We found that the diffusion coefficients measured using our method were consistent with previous literature values. We further found that photoblinking rates measured in the live HeLa cells varied as expected with changing excitation power.
The International Journal of Biochemistry & Cell Biology, 2021
Nonlinear microscopy is a technique that utilizes nonlinear interactions between light and matter... more Nonlinear microscopy is a technique that utilizes nonlinear interactions between light and matter to image fluorescence and scattering phenomena in biological tissues. Very high peak intensities from focused short pulsed lasers are required for nonlinear excitation due to the extremely low probability of the simultaneous arrival of multiple photons of lower energy to excite fluorophores or interact with selective structures for harmonic generation. Combined with reduced scattering from the utilization of longer wavelengths, the inherent spatial confinement associated with achieving simultaneous arrival of photons within the focal volume enables deep imaging with low out-of-focus background for nonlinear imaging. This review provides an introduction to the different contrast mechanisms available with nonlinear imaging and instrumentation commonly used in nonlinear microscopy. Furthermore, we discuss some recent advances in nonlinear microscopy to extend the imaging penetration depth, conduct histopathological investigations on fresh tissues and examine the molecular order and orientation of molecules using polarization nonlinear microscopy.
The formation of memory and the process of learning are believed to be regulated, at least in par... more The formation of memory and the process of learning are believed to be regulated, at least in part, by the expression, distribution, and redistribution of glutamate receptors. The expression of these receptors at synaptic sites has a major impact on the strength of synaptic connections, and the AMPA receptor subunit GluR1 appears to play a key role within this system. Increasing evidence suggests that previously silent synapses become activated through rapid AMPA receptor insertion upon appropriate stimulation, and thus the trafficking of this receptor subunit from cellular stores to the synapse is of prime interest.We are using a transgenic mouse expressing a GFP-tagged form of GluR1 (GluR1-GFP) in order to study the dynamic changes in GluR1 expression and distribution occurring during brain development and following induction of long-term potentiation (LTP). The fusion protein is transcribed under the control of the CaMKIIα promoter, which restricts the localization to forebrain n...
Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive ac... more Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive actin-rich protrusions, to generate forces, migrate, and patrol for foreign antigens. Individual podosomes probe their microenvironment through periodic protrusion and retraction cycles (height oscillations), while oscillations of multiple podosomes in a cluster are coordinated in a wave-like fashion. However, the mechanisms governing both the individual oscillations and the collective wave-like dynamics remain unclear. Here, by integrating actin polymerization, myosin contractility, actin diffusion, and mechanosensitive signaling, we develop a chemo-mechanical model for podosome dynamics in clusters. Our model reveals that podosomes show oscillatory growth when actin polymerization-driven protrusion and signalingassociated myosin contraction occur at similar rates, while the diffusion of actin monomers drives wave-like coordination of podosome oscillations. Our theoretical predictions are validated by different pharmacological treatments and the impact of microenvironment stiffness on chemo-mechanical waves. Our proposed framework can shed light on the role of podosomes in immune cell mechanosensing within the context of wound healing and cancer immunotherapy. Dendritic cells (DCs) and macrophages, act as gatekeepers of the immune system by surveilling peripheral tissues for foreign antigens. During this process, these cells form dozens of specialized actin-rich protrusions called podosomes, which are typically organized into large clusters. By utilizing podosomes, DCs and macrophages can control adhesions, degrade the surrounding extracellular matrix (ECM), and remodel the extracellular environment to facilitate their migration 1-3. Podosomes are characterized by an actin-based core surrounded by an adhesive ring consisting of integrins and adaptor proteins, such as vinculin and talin 4,5. Podosomes generate protrusive forces to penetrate the underlying ECMs at the cores, while applying tensile forces to pull the ECMs at the rings 6,7. Studies in DCs and macrophages have shown that individual podosomes exhibit periodic oscillations in the fluorescence intensity of core actin, ring components, and the protrusive forces exerted at their cores 7-9. Collectively, podosomes in a cluster show spatially correlated behaviors, where the oscillations of podosome components are coordinated in a wave-like manner within the cluster 10,11. However, the relationship which directs the core protrusive and ring tensile forces into oscillations of individual podosomes and subsequent spatiotemporal wave patterns in clusters remains unknown. Understanding these processes can provide insights on how podosomes in a cluster collectively probe and respond
Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invas... more Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invasion relies on invadosomes, a collective term for invadopodia and podosomes. An adequate structural framework for this process is still missing. Here, we reveal the modular actin nano-architecture that enables podosome protrusion and mechanosensing. The podosome protrusive core contains a central branched actin module encased by a linear actin module, each harboring specific actin interactors and actin isoforms. From the core, two actin modules radiate: ventral filaments bound by vinculin and connected to the plasma membrane and dorsal interpodosomal filaments crosslinked by myosin IIA. On stiff substrates, the actin modules mediate long-range substrate exploration, associated with degradative behavior. On compliant substrates, the vinculin-bound ventral actin filaments shorten, resulting in shortrange connectivity and a focally protrusive, non-degradative state. Our findings redefine podosome nanoscale architecture and reveal a paradigm for how actin modularity drives invadosome mechanosensing in cells that breach tissue boundaries.
acid interactions. Visualization is achieved through the application of two complementary procedu... more acid interactions. Visualization is achieved through the application of two complementary procedures. In one, a single DNA molecule is attached to a polystyrene bead, which is captured in an optical trap. The DNA is extended either by the force of solution flow in a micro-flowcell, or by capturing the opposite DNA end in a second optical trap. In the second procedure, DNA is attached by one end to a glass surface. The coiled DNA is elongated either by continuous solution flow or by subsequently tethering the opposite end to the surface. Proteins and DNA are visualized via fluorescent reporters. Individual molecules are imaged using either epifluorescence microscopy or total internal reflection fluorescence (TIRF) microscopy. Molecules are introduced and supramolecular complexes are built, one component at a time, using microfluidic flowcells. Using these approaches, we have watched proteins functioning in the repair, replication, and manipulation of DNA. We have imaged unwinding of DNA by helicases, translocation along DNA by motor proteins, self-assembly of protein filaments on DNA, the search for DNA sequence homology by protein-DNA filaments, real-time replication of DNA, and nucleosome remodeling. I will summarize how these experiments were done, and what we've learned about biology from the stochastic behavior of individual molecules.
We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation ... more We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) that relies on complete analysis of both the temporal and spatial correlation lags for intensity fluctuations from a laserscanning microscopy image series. This new approach allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) for fluorescently labeled membrane proteins in living cells through monitoring of the time evolution of the full space-time correlation function. By using filtering in Fourier space to remove frequencies associated with immobile components, we are able to measure the protein transport even in the presence of a large fraction (.90%) of immobile species. We present the background theory, computer simulations, and analysis of measurements on fluorescent microspheres to demonstrate proof of principle, capabilities, and limitations of the method. We demonstrate mapping of flow vectors for mixed samples containing fluorescent microspheres with different emission wavelengths using space time image cross-correlation. We also present results from two-photon laser-scanning microscopy studies of a-actinin/enhanced green fluorescent protein fusion constructs at the basal membrane of living CHO cells. Using space-time image correlation spectroscopy (STICS), we are able to measure protein fluxes with magnitudes of mm/min from retracting lamellar regions and protrusions for adherent cells. We also demonstrate the measurement of correlated directed flows (magnitudes of mm/min) and diffusion of interacting a5 integrin/enhanced cyan fluorescent protein and a-actinin/enhanced yellow fluorescent protein within living CHO cells. The STICS method permits us to generate complete transport maps of proteins within subregions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements.
Smart fluorophores'', such as reversibly switchable fluorescent proteins, are crucial for advance... more Smart fluorophores'', such as reversibly switchable fluorescent proteins, are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available, and many display reduced biological performance compared to more classical variants. We present the development of robustly photoswitchable variants of enhanced green fluorescent protein (EGFP), named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37 C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multimodal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of~70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the ''on''-and ''off''-conformation we were able to confirm the presence of a cis-trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded ''smart fluorophores'' can be readily optimized for biological performance and provides a practical strategy for developing maturation-and stability-enhanced photochromic fluorescent proteins.
There is evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) anion chann... more There is evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is highly expressed at the apical pole of ciliated cells in human bronchial epithelium (HBE), however recent studies have detected little CFTR mRNA in those cells. To understand this discrepancy we immunostained well differentiated primary HBE cells using CFTR antibodies. We confirmed apical immunofluorescence in ciliated cells and quantified the covariance of the fluorescence signals and that of an antibody against the ciliary marker centrin-2 using image cross-correlation spectroscopy (ICCS). Super-resolution stimulated emission depletion (STED) imaging localized the immunofluorescence in distinct clusters at the bases of the cilia. However, similar apical fluorescence was observed when the monoclonal CFTR antibodies 596, 528 and 769 were used to immunostain ciliated cells expressing F508del-CFTR, or cells lacking CFTR due to a Class I mutation. A BLAST search using the CFTR epitope identified a similar amino acid sequence in the ciliary protein rootletin X1. Its expression level correlated with the intensity of immunostaining by CFTR antibodies and it was detected by 596 antibody after transfection into CFBE cells. These results may explain the high apparent expression of CFTR in ciliated cells and reports of anomalous apical immunofluorescence in well differentiated cells that express F508del-CFTR. Human airway bronchial epithelium contains ciliated cells, secretory goblet and club cells, basal cells, and pulmonary neuroendocrine cells 1,2 , each with distinct roles in airway physiology and host defense. Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP regulated anion channel required for normal secretion of airway surface liquid 3-5. CFTR mutations lead to abnormal mucus and impaired mucociliary clearance of inhaled bacteria that are hallmarks of cystic fibrosis (CF) 6. CFTR is thought to be highly expressed in ciliated cells of the airway epithelium, however recent single cell mRNA sequencing (scRNAseq) reports detected low CFTR mRNA levels in ciliated cells from primary human bronchial epithelial (HBE) cultures and mouse lung tissue 7-9. It was suggested that most CFTR transcripts (> 45%) are in a rare (< 2% of total) epithelial cell type called pulmonary ionocytes 7,8 , although one scRNAseq study found most CFTR transcripts (80% of the total) in secretory and basal cells 9. Further analysis at the protein level is needed to assess CFTR immunofluorescence in ciliated cells and understand F508del-CFTR apical immunofluorescence reported in some studies of CF airways 10,11. Many CFTR mutations have been identified and classified as Class I-VI based on the predominant molecular defects they produce 12-14. Class I mutations include nonsense, frameshift and splicing mutations that prevent expression of the full-length protein. Class II mutations cause protein misfolding and impair trafficking to the plasma membrane. Class III mutations are those that inhibit channel gating or regulation, while class IV-VI mutations reduce pore conduction, protein expression and CFTR stability, respectively. F508del is by far the most frequent mutation, occurring on at least one chromosome in ~ 90% of the CF population. It is a Class II mutation that causes misfolding, retention in the endoplasmic reticulum, and premature degradation by the proteasome and other pathways 15-17. We used monoclonal antibodies to immunofluorescence label and localize CFTR in well-differentiated primary cultures of human bronchial epithelial cells from non-CF donors and F508del homozygotes. Importantly,
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Papers by Paul Wiseman