The shuttling of transcription factors and transcriptional regulators in and out of the nucleus i... more The shuttling of transcription factors and transcriptional regulators in and out of the nucleus is central to the regulation of many biological processes. Here we describe a new method for studying the rates of nuclear entry and exit of transcriptional regulators. A photo-responsive AsLOV (Avena sativa Light Oxygen Voltage) domain is used to sequester fluorescently-labelled transcriptional regulators YAP1 and TAZ/WWTR1 on the surface of mitochondria and reversibly release them upon blue light illumination. After dissociation, fluorescent signals from mitochondria, cytoplasm and nucleus are extracted with a bespoke app and used to generate rates of nuclear entry and exit. Using this method, we demonstrate that phosphorylation of YAP1 on canonical sites enhances its rate of nuclear export. Moreover, we provide evidences that, despite high intercellular variability, YAP1 import and export rates correlated within the same cell. By simultaneously releasing YAP1 and TAZ from sequestration...
Control of protein activity in living cells can reveal the role of spatio-temporal dynamics in si... more Control of protein activity in living cells can reveal the role of spatio-temporal dynamics in signaling circuits. Protein analogs with engineered allosteric responses can be particularly effective, as they can replace endogenous proteins with minimum perturbation of native interactions. However, it has been a challenge to identify allosteric sites in target proteins where insertion of responsive domains produces activation changes comparable to native proteins. Here we describe a detailed protocol to generate genetically-encoded analogs of proteins that can be allosterically controlled by either rapamycin or blue light, using computational methods to identify effective insertion sites. Rapamycin added to the medium activates the protein essentially irreversibly, while light-controlled allosteric switches provide reversible inactivation with higher spatio-temporal resolution. We discuss the computational framework to identify the insertion sites, and experimental procedures to produce and test the engineered proteins in vitro and in mammalian cell lines. This method has been successfully applied to catalytic domains of protein kinases, Rho family GTPase and guanine exchange factors, as well as binding domain of a guanine exchange factor Vav2. Computational tasks can be completed within a few hours, followed by 1-2 weeks of experimental validation. We provide protocols for computational design, cloning, and experimental testing of the engineered proteins, using Src tyrosine kinase, guanine exchange factor Vav2, and Rho GTPase Rac1 as examples.
<p>(A) Simplified schematic of the role of LIN-1 in vulval fate specification. (B) Top: Sch... more <p>(A) Simplified schematic of the role of LIN-1 in vulval fate specification. (B) Top: Schematic of the wild type LIN-1 protein. Bottom: Schematic of the LIN-1::LANS protein produced after modification of the native <i>lin-1</i> locus using Cas9-triggered homologous recombination. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#pone.0128443.s004" target="_blank">S4 Fig</a>. (C) DIC Images of the developing vulvae in mid-L4 larvae from the indicted strains and conditions. Top panel: Black arrow indicates the normal, symmetric vulval invagination. Middle panel: Black arrow indicates the main vulval invagination, and green arrowhead indicates an extra vulval invagination. Bottom panel: Orange arrowheads indicate small invaginations produced by the secondary vulval precursors, and black arrow indicates the plug of tissue derived from the failed primary cell. Scale bars represent 20 μm. (D) Quantification of phenotypes in the indicated strains and conditions. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#sec011" target="_blank">Methods</a> for detailed definitions of each phenotype. Numbers at the top of each bar indicate the total number of animals scored in this experiment. These data are from a single experiment; the experiment was repeated three times, using two independently isolated <i>lin-1</i>::<i>lans</i> alleles, with similar results.</p
<p>(A) Schematic of the mKate2::LANS construct that was expressed in <i>C</i>. ... more <p>(A) Schematic of the mKate2::LANS construct that was expressed in <i>C</i>. <i>elegans</i> embryos (B) Confocal images of an embryo expressing mKate2::LANS ubiquitously and subjected to photoactivation with blue light. Scale bars represent 10 μm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#pone.0128443.s008" target="_blank">S4 Movie</a>. (C) Left: Confocal images of four mKate2::LANS expressing MS lineage cells on the ventral surface of a late gastrulation-stage embryo. The blue box in the center image indicates the region that was photoactivated with blue light. Brightness and contrast were adjusted to compensate for photobleaching. Scale bar represents 5 μm. Right: Sketches summarizing the observed localization. Numbers correspond to the cell numbers in (D). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#pone.0128443.s009" target="_blank">S5 Movie</a>. (D) Quantification of nuclear and cytoplasmic fluorescence intensities as a function of time for the two cells labelled in (C). Cell 1 was illuminated with blue light, and Cell 2 is a neighboring cell. These measurements were corrected for photobleaching (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#sec011" target="_blank">materials and methods</a>).</p
On p. 483, an error appears in the subheading of the middle column. The correct subheading should... more On p. 483, an error appears in the subheading of the middle column. The correct subheading should read as follows.
Podosomes are actin-enriched adhesion structures important for multiple cellular processes, inclu... more Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterize the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy, a super-resolution microscopy technique capable of 15–20 nm resolution, together with structured illumination microscopy and localization-based super-resolution microscopy. Phagocytic podosomes are observed during frustrated phagocytosis, a model in which cells attempt to engulf micropatterned IgG antibodies. For circular patterns, this results in regular arrays of podosomes with well-defined geometry. Using persistent homology, we develop a pipeline for semi-automatic identification and measurement of podosome features. These studies reveal an hourglass shape of the podosome actin core, a protruding knob at the bottom of the core, and two actin networks extending from the core. Additionally, the dist...
Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been r... more Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrin αIIbβ3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbβ3 activation, but it activates αIIbβ3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbβ3 is tightly regulated by the topology of β3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the em...
Light-activatable proteins allow precise spatial and temporal control of biological processes in ... more Light-activatable proteins allow precise spatial and temporal control of biological processes in living cells and animals. Several approaches have been developed for controlling protein localization with light, including the conditional inhibition of a nuclear localization signal (NLS) with the Light Oxygen Voltage (AsLOV2) domain of phototropin 1 from Avena sativa. In the dark, the switch adopts a closed conformation that sterically blocks the NLS motif. Upon activation with blue light the C-terminus of the protein unfolds, freeing the NLS to direct the protein to the nucleus. A previous study showed that this approach can be used to control the localization and activity of proteins in mammalian tissue culture cells. Here, we extend this result by characterizing the binding properties of a LOV/NLS switch and demonstrating that it can be used to control gene transcription in yeast. Additionally, we show that the switch, referred to as LANS (light-activated nuclear shuttle), function...
Highlights d Generalizable biosensors based on changes in exposure of small peptides d A bright s... more Highlights d Generalizable biosensors based on changes in exposure of small peptides d A bright signal enables detection of single molecule conformations in living cells d Conformation is revealed by FRET or simply by localization of a fluorophore d Quantitation of Src dynamics and activation in clusters and at adhesions
A long-standing question in neurodevelopment is how neurons develop a single axon and multiple de... more A long-standing question in neurodevelopment is how neurons develop a single axon and multiple dendrites from common immature neurites. Long-range inhibitory signaling from the growing axon is hypothesized to prevent outgrowth of other immature neurites and to differentiate them into dendrites, but the existence and nature of this inhibitory signaling remains unknown. Here, we demonstrate that axonal growth triggered by neurotrophin-3 remotely inhibits neurite outgrowth through long-range Ca(2+) waves, which are delivered from the growing axon to the cell body. These Ca(2+) waves increase RhoA activity in the cell body through calcium/calmodulin-dependent protein kinase I. Optogenetic control of Rho-kinase combined with computational modeling reveals that active Rho-kinase diffuses to growing other immature neurites and inhibits their outgrowth. Mechanistically, calmodulin-dependent protein kinase I phosphorylates a RhoA-specific GEF, GEF-H1, whose phosphorylation enhances its GEF a...
Advances in live cell fluorescence tagging, automated microscopy and computing enable a vision of... more Advances in live cell fluorescence tagging, automated microscopy and computing enable a vision of fully automated high-resolution imaging of the detailed intracellular molecular dynamics, functionally linked with cellular behaviors. Given the heterogeneity of all cell populations, statistically relevant studies of molecular-cellular dynamics would be an important result of improved automation. Here, we explore fully automating computerized, microscope-based data extraction and analyses that monitor spatiotemporal localizations of cellular protein activities through ratiometric, two-channel fluorescent biosensors. Novel image processing methods include: K-means clustering segmentation preprocessing before modified discrete, normalized cross-correlational alignment of 2-color images; ratiometric processing of the two colors for fluorescence resonance energy transfer (FRET) measurements; and intracellular spatial distribution measurements of RhoA GTPase activity. These advances lay the foundation for extraction and correlation of features characterizing the functional relationships of spatial location and degree of protein activation with cell polarization, protrusion and retraction, and direction of movement.
The small GTPases Rac1 and Rap1 can fulfill multiple cellular functions because their activation ... more The small GTPases Rac1 and Rap1 can fulfill multiple cellular functions because their activation kinetics and localization are precisely controlled. To probe the role of their spatio-temporal dynamics, we generated optogenetic tools that activate or inhibit endogenous Rac and Rap1 in living cells. An improved version of the light-induced dimerization (iLID) system was used to control plasma membrane localization of protein domains that specifically activate or inactivate Rap1 and Rac (Tiam1 and Chimerin for Rac, RasGRP2 and Rap1GAP for Rap1). Irradiation yielded a 50-230% increase in the concentration of these domains at the membrane, leading to effects on cell morphodynamics consistent with the known roles of Rac1 and Rap1.
The accuracy of biosensor ratio imaging is limited by signal/noise. Signals can be weak when bios... more The accuracy of biosensor ratio imaging is limited by signal/noise. Signals can be weak when biosensor concentrations must be limited to avoid cell perturbation. This can be especially problematic in imaging of low volume regions, e.g., along the cell edge. The cell edge is an important imaging target in studies of cell motility. We show how the division of fluorescence intensities with low signal-to-noise at the cell edge creates specific artifacts due to background subtraction and division by small numbers, and that simply improving the accuracy of background subtraction cannot address these issues. We propose a new approach where, rather than simply subtracting background from the numerator and denominator, we subtract a noise correction factor (NCF) from the numerator only. This NCF can be derived from the analysis of noise distribution in the background near the cell edge or from ratio measurements in the cell regions where signal-to-noise is high. We test the performance of th...
The shuttling of transcription factors and transcriptional regulators in and out of the nucleus i... more The shuttling of transcription factors and transcriptional regulators in and out of the nucleus is central to the regulation of many biological processes. Here we describe a new method for studying the rates of nuclear entry and exit of transcriptional regulators. A photo-responsive AsLOV (Avena sativa Light Oxygen Voltage) domain is used to sequester fluorescently-labelled transcriptional regulators YAP1 and TAZ/WWTR1 on the surface of mitochondria and reversibly release them upon blue light illumination. After dissociation, fluorescent signals from mitochondria, cytoplasm and nucleus are extracted with a bespoke app and used to generate rates of nuclear entry and exit. Using this method, we demonstrate that phosphorylation of YAP1 on canonical sites enhances its rate of nuclear export. Moreover, we provide evidences that, despite high intercellular variability, YAP1 import and export rates correlated within the same cell. By simultaneously releasing YAP1 and TAZ from sequestration...
Control of protein activity in living cells can reveal the role of spatio-temporal dynamics in si... more Control of protein activity in living cells can reveal the role of spatio-temporal dynamics in signaling circuits. Protein analogs with engineered allosteric responses can be particularly effective, as they can replace endogenous proteins with minimum perturbation of native interactions. However, it has been a challenge to identify allosteric sites in target proteins where insertion of responsive domains produces activation changes comparable to native proteins. Here we describe a detailed protocol to generate genetically-encoded analogs of proteins that can be allosterically controlled by either rapamycin or blue light, using computational methods to identify effective insertion sites. Rapamycin added to the medium activates the protein essentially irreversibly, while light-controlled allosteric switches provide reversible inactivation with higher spatio-temporal resolution. We discuss the computational framework to identify the insertion sites, and experimental procedures to produce and test the engineered proteins in vitro and in mammalian cell lines. This method has been successfully applied to catalytic domains of protein kinases, Rho family GTPase and guanine exchange factors, as well as binding domain of a guanine exchange factor Vav2. Computational tasks can be completed within a few hours, followed by 1-2 weeks of experimental validation. We provide protocols for computational design, cloning, and experimental testing of the engineered proteins, using Src tyrosine kinase, guanine exchange factor Vav2, and Rho GTPase Rac1 as examples.
<p>(A) Simplified schematic of the role of LIN-1 in vulval fate specification. (B) Top: Sch... more <p>(A) Simplified schematic of the role of LIN-1 in vulval fate specification. (B) Top: Schematic of the wild type LIN-1 protein. Bottom: Schematic of the LIN-1::LANS protein produced after modification of the native <i>lin-1</i> locus using Cas9-triggered homologous recombination. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#pone.0128443.s004" target="_blank">S4 Fig</a>. (C) DIC Images of the developing vulvae in mid-L4 larvae from the indicted strains and conditions. Top panel: Black arrow indicates the normal, symmetric vulval invagination. Middle panel: Black arrow indicates the main vulval invagination, and green arrowhead indicates an extra vulval invagination. Bottom panel: Orange arrowheads indicate small invaginations produced by the secondary vulval precursors, and black arrow indicates the plug of tissue derived from the failed primary cell. Scale bars represent 20 μm. (D) Quantification of phenotypes in the indicated strains and conditions. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#sec011" target="_blank">Methods</a> for detailed definitions of each phenotype. Numbers at the top of each bar indicate the total number of animals scored in this experiment. These data are from a single experiment; the experiment was repeated three times, using two independently isolated <i>lin-1</i>::<i>lans</i> alleles, with similar results.</p
<p>(A) Schematic of the mKate2::LANS construct that was expressed in <i>C</i>. ... more <p>(A) Schematic of the mKate2::LANS construct that was expressed in <i>C</i>. <i>elegans</i> embryos (B) Confocal images of an embryo expressing mKate2::LANS ubiquitously and subjected to photoactivation with blue light. Scale bars represent 10 μm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#pone.0128443.s008" target="_blank">S4 Movie</a>. (C) Left: Confocal images of four mKate2::LANS expressing MS lineage cells on the ventral surface of a late gastrulation-stage embryo. The blue box in the center image indicates the region that was photoactivated with blue light. Brightness and contrast were adjusted to compensate for photobleaching. Scale bar represents 5 μm. Right: Sketches summarizing the observed localization. Numbers correspond to the cell numbers in (D). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#pone.0128443.s009" target="_blank">S5 Movie</a>. (D) Quantification of nuclear and cytoplasmic fluorescence intensities as a function of time for the two cells labelled in (C). Cell 1 was illuminated with blue light, and Cell 2 is a neighboring cell. These measurements were corrected for photobleaching (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#sec011" target="_blank">materials and methods</a>).</p
On p. 483, an error appears in the subheading of the middle column. The correct subheading should... more On p. 483, an error appears in the subheading of the middle column. The correct subheading should read as follows.
Podosomes are actin-enriched adhesion structures important for multiple cellular processes, inclu... more Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterize the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy, a super-resolution microscopy technique capable of 15–20 nm resolution, together with structured illumination microscopy and localization-based super-resolution microscopy. Phagocytic podosomes are observed during frustrated phagocytosis, a model in which cells attempt to engulf micropatterned IgG antibodies. For circular patterns, this results in regular arrays of podosomes with well-defined geometry. Using persistent homology, we develop a pipeline for semi-automatic identification and measurement of podosome features. These studies reveal an hourglass shape of the podosome actin core, a protruding knob at the bottom of the core, and two actin networks extending from the core. Additionally, the dist...
Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been r... more Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrin αIIbβ3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbβ3 activation, but it activates αIIbβ3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbβ3 is tightly regulated by the topology of β3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the em...
Light-activatable proteins allow precise spatial and temporal control of biological processes in ... more Light-activatable proteins allow precise spatial and temporal control of biological processes in living cells and animals. Several approaches have been developed for controlling protein localization with light, including the conditional inhibition of a nuclear localization signal (NLS) with the Light Oxygen Voltage (AsLOV2) domain of phototropin 1 from Avena sativa. In the dark, the switch adopts a closed conformation that sterically blocks the NLS motif. Upon activation with blue light the C-terminus of the protein unfolds, freeing the NLS to direct the protein to the nucleus. A previous study showed that this approach can be used to control the localization and activity of proteins in mammalian tissue culture cells. Here, we extend this result by characterizing the binding properties of a LOV/NLS switch and demonstrating that it can be used to control gene transcription in yeast. Additionally, we show that the switch, referred to as LANS (light-activated nuclear shuttle), function...
Highlights d Generalizable biosensors based on changes in exposure of small peptides d A bright s... more Highlights d Generalizable biosensors based on changes in exposure of small peptides d A bright signal enables detection of single molecule conformations in living cells d Conformation is revealed by FRET or simply by localization of a fluorophore d Quantitation of Src dynamics and activation in clusters and at adhesions
A long-standing question in neurodevelopment is how neurons develop a single axon and multiple de... more A long-standing question in neurodevelopment is how neurons develop a single axon and multiple dendrites from common immature neurites. Long-range inhibitory signaling from the growing axon is hypothesized to prevent outgrowth of other immature neurites and to differentiate them into dendrites, but the existence and nature of this inhibitory signaling remains unknown. Here, we demonstrate that axonal growth triggered by neurotrophin-3 remotely inhibits neurite outgrowth through long-range Ca(2+) waves, which are delivered from the growing axon to the cell body. These Ca(2+) waves increase RhoA activity in the cell body through calcium/calmodulin-dependent protein kinase I. Optogenetic control of Rho-kinase combined with computational modeling reveals that active Rho-kinase diffuses to growing other immature neurites and inhibits their outgrowth. Mechanistically, calmodulin-dependent protein kinase I phosphorylates a RhoA-specific GEF, GEF-H1, whose phosphorylation enhances its GEF a...
Advances in live cell fluorescence tagging, automated microscopy and computing enable a vision of... more Advances in live cell fluorescence tagging, automated microscopy and computing enable a vision of fully automated high-resolution imaging of the detailed intracellular molecular dynamics, functionally linked with cellular behaviors. Given the heterogeneity of all cell populations, statistically relevant studies of molecular-cellular dynamics would be an important result of improved automation. Here, we explore fully automating computerized, microscope-based data extraction and analyses that monitor spatiotemporal localizations of cellular protein activities through ratiometric, two-channel fluorescent biosensors. Novel image processing methods include: K-means clustering segmentation preprocessing before modified discrete, normalized cross-correlational alignment of 2-color images; ratiometric processing of the two colors for fluorescence resonance energy transfer (FRET) measurements; and intracellular spatial distribution measurements of RhoA GTPase activity. These advances lay the foundation for extraction and correlation of features characterizing the functional relationships of spatial location and degree of protein activation with cell polarization, protrusion and retraction, and direction of movement.
The small GTPases Rac1 and Rap1 can fulfill multiple cellular functions because their activation ... more The small GTPases Rac1 and Rap1 can fulfill multiple cellular functions because their activation kinetics and localization are precisely controlled. To probe the role of their spatio-temporal dynamics, we generated optogenetic tools that activate or inhibit endogenous Rac and Rap1 in living cells. An improved version of the light-induced dimerization (iLID) system was used to control plasma membrane localization of protein domains that specifically activate or inactivate Rap1 and Rac (Tiam1 and Chimerin for Rac, RasGRP2 and Rap1GAP for Rap1). Irradiation yielded a 50-230% increase in the concentration of these domains at the membrane, leading to effects on cell morphodynamics consistent with the known roles of Rac1 and Rap1.
The accuracy of biosensor ratio imaging is limited by signal/noise. Signals can be weak when bios... more The accuracy of biosensor ratio imaging is limited by signal/noise. Signals can be weak when biosensor concentrations must be limited to avoid cell perturbation. This can be especially problematic in imaging of low volume regions, e.g., along the cell edge. The cell edge is an important imaging target in studies of cell motility. We show how the division of fluorescence intensities with low signal-to-noise at the cell edge creates specific artifacts due to background subtraction and division by small numbers, and that simply improving the accuracy of background subtraction cannot address these issues. We propose a new approach where, rather than simply subtracting background from the numerator and denominator, we subtract a noise correction factor (NCF) from the numerator only. This NCF can be derived from the analysis of noise distribution in the background near the cell edge or from ratio measurements in the cell regions where signal-to-noise is high. We test the performance of th...
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Papers by Klaus M Hahn