Genetically encoded biosensors based on Förster resonance energy transfer (FRET) can visualize re... more Genetically encoded biosensors based on Förster resonance energy transfer (FRET) can visualize responses of individual cells in real time. Here, we evaluated whether FRET-based biosensors provide sufficient contrast and specificity to measure activity of G-protein-coupled receptors. The four histamine receptor subtypes (H 1 R, H 2 R, H 3 R, and H 4 R) respond to the ligand histamine by activating three canonical heterotrimeric G-protein-mediated signaling pathways with a reported high degree of specificity. Using FRET-based biosensors, we demonstrate that H 1 R activates Gaq. We also observed that H 1 R activates Gai, albeit at a 10-fold lower potency. In addition to increasing cAMP levels, most likely via Gas, we found that the H 2 R induces Gaq-mediated calcium release. The H 3 R and H 4 R activated Gai with high specificity and a high potency. We demonstrate that a number of FRET sensors provide sufficient contrast to: 1) analyze the specificity of the histamine receptor subtypes for different heterotrimeric G-protein families with single-cell resolution, 2) probe for antagonist specificity, and 3) allow the measurement of single-cell concentration-response curves.
Background: Rho guanine exchange factors (RhoGEFs) control cellular processes such as migration, ... more Background: Rho guanine exchange factors (RhoGEFs) control cellular processes such as migration, adhesion and proliferation. Alternative splicing of the RhoGEF Trio produces TGAT. The RhoGEF TGAT is an oncoprotein with constitutive RhoGEF activity. We investigated whether the subcellular location of TGAT is critical for its RhoGEF activity. Methods: Since plasma membrane associated RhoGEFs are particularly effective at activating RhoA, plasma membrane localization of TGAT was examined. To this end, we developed a highly sensitive image analysis method to quantitatively measure plasma membrane association. The method requires a cytoplasmic marker and a plasma membrane marker, which are co-imaged with the tagged protein of interest. Linear unmixing is performed to determine the plasma membrane and cytoplasmic component in the fluorescence signal of protein of interest. Results: The analysis revealed that wild-type TGAT is partially co-localized with the plasma membrane. Strikingly, cysteine TGAT-mutants lacking one or more putative palmitoylation sites in the C-tail, still showed membrane association. In contrast, a truncated variant, lacking the last 15 amino acids, TGAT Δ15 , lost membrane association. We show that membrane localization of TGAT was responsible for high RhoGEF activity by using a RhoA FRET-sensor and by determining F-actin levels. Mutants of TGAT that still maintained membrane association showed similar activity as wild-type TGAT. In contrast, the activity was abrogated for the cytoplasmic TGAT Δ15 variant. Synthetic recruitment of TGAT Δ15 to membranes confirmed that TGAT effectively activates RhoA at the plasma membrane. Conclusion: Together, these results show that membrane association of TGAT is critical for its activity.
Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed t... more Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.
Chemotaxis is the result of a refined interplay among various intracellular molecules that proces... more Chemotaxis is the result of a refined interplay among various intracellular molecules that process spatial and temporal information. Here we present a modular scheme of the complex interactions between the front and the back of cells that allows them to navigate. First, at the front of the cell, activated Rho-type GTPases induce actin polymerization and pseudopod formation. Second, phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P 3) is produced in a patch at the leading edge, where it binds pleckstrinhomology-domain-containing proteins, which enhance actin polymerization and translocation of the pseudopod. Third, in Dictyostelium amoebae, a cyclic-GMP-signalling cascade has been identified that regulates myosin filament formation in the posterior of the cell, thereby inhibiting the formation of lateral pseudopodia that could misdirect the cell.
Background: In fly photoreceptors, light is focused onto a photosensitive waveguide, the rhabdome... more Background: In fly photoreceptors, light is focused onto a photosensitive waveguide, the rhabdomere, consisting of tens of thousands of microvilli. Each microvillus is capable of generating elementary responses, quantum bumps, in response to single photons using a stochastically operating phototransduction cascade. Whereas much is known about the cascade reactions, less is known about how the concerted action of the microvilli population encodes light changes into neural information and how the ultrastructure and biochemical machinery of photoreceptors of flies and other insects evolved in relation to the information sampling and processing they perform. Results: We generated biophysically realistic fly photoreceptor models, which accurately simulate the encoding of visual information. By comparing stochastic simulations with single cell recordings from Drosophila photoreceptors, we show how adaptive sampling by 30,000 microvilli captures the temporal structure of natural contrast changes. Following each bump, individual microvilli are rendered briefly (w100-200 ms) refractory, thereby reducing quantum efficiency with increasing intensity. The refractory period opposes saturation, dynamically and stochastically adjusting availability of microvilli (bump production rate: sample rate), whereas intracellular calcium and voltage adapt bump amplitude and waveform (sample size). These adapting sampling principles result in robust encoding of natural light changes, which both approximates perceptual contrast constancy and enhances novel events under different light conditions, and predict information processing across a range of species with different visual ecologies. Conclusions: These results clarify why fly photoreceptors are structured the way they are and function as they do, linking sensory information to sensory evolution and revealing benefits of stochasticity for neural information processing.
It remains unclear how visual information is co-processed by different layers of neurons in the r... more It remains unclear how visual information is co-processed by different layers of neurons in the retina. In particular, relatively little is known how retina translates vast environmental light changes into neural responses of limited range. We began examining this question in a bottom-up way in a relatively simple °y eye. To gain understanding of how complex bio-molecular interactions govern the conversion of light input into voltage output (phototransduction), we are building a biophysical model of the Drosophila R1-R6 photoreceptor. Our model, which relates molecular dynamics of the underlying biochemical reactions to external light input, attempts to capture the molecular dynamics of phototransduction gain control in a quantitative way
Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed t... more Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.
Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed t... more Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocy...
Background: Rho guanine exchange factors (RhoGEFs) control cellular processes such as migration, ... more Background: Rho guanine exchange factors (RhoGEFs) control cellular processes such as migration, adhesion and proliferation. Alternative splicing of the RhoGEF Trio produces TGAT. The RhoGEF TGAT is an oncoprotein with constitutive RhoGEF activity. We investigated whether the subcellular location of TGAT is critical for its RhoGEF activity. Methods: Since plasma membrane associated RhoGEFs are particularly effective at activating RhoA, plasma membrane localization of TGAT was examined. To this end, we developed a highly sensitive image analysis method to quantitatively measure plasma membrane association. The method requires a cytoplasmic marker and a plasma membrane marker, which are co-imaged with the tagged protein of interest. Linear unmixing is performed to determine the plasma membrane and cytoplasmic component in the fluorescence signal of protein of interest. Results: The analysis revealed that wild-type TGAT is partially co-localized with the plasma membrane. Strikingly, cysteine TGAT-mutants lacking one or more putative palmitoylation sites in the C-tail, still showed membrane association. In contrast, a truncated variant, lacking the last 15 amino acids, TGAT Δ15 , lost membrane association. We show that membrane localization of TGAT was responsible for high RhoGEF activity by using a RhoA FRET-sensor and by determining F-actin levels. Mutants of TGAT that still maintained membrane association showed similar activity as wild-type TGAT. In contrast, the activity was abrogated for the cytoplasmic TGAT Δ15 variant. Synthetic recruitment of TGAT Δ15 to membranes confirmed that TGAT effectively activates RhoA at the plasma membrane. Conclusion: Together, these results show that membrane association of TGAT is critical for its activity.
The performance of Förster Resonance Energy Transfer (FRET) biosensors depends on brightness and ... more The performance of Förster Resonance Energy Transfer (FRET) biosensors depends on brightness and photostability, which are dependent on the characteristics of the fluorescent proteins that are employed. Yellow fluorescent protein (YFP) is often used as an acceptor but YFP is prone to photobleaching and pH changes. In this study, we evaluated the properties of a diverse set of acceptor fluorescent proteins in combination with the optimized CFP variant mTurquoise2 as the donor. To determine the theoretical performance of acceptors, the Förster radius was determined. The practical performance was determined by measuring FRET efficiency and photostability of tandem fusion proteins in mammalian cells. Our results show that mNeonGreen is the most efficient acceptor for mTurquoise2 and that the photostability is better than SYFP2. The non-fluorescent YFP variant sREACh is an efficient acceptor, which is useful in lifetime-based FRET experiments. Among the orange and red fluorescent protein...
We report the engineering of mscarlet, a truly monomeric red fluorescent protein with record brig... more We report the engineering of mscarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mscarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mscarlet's crystal structure reveals a planar and rigidified chromophore. mscarlet outperforms existing red fluorescent proteins as a fusion tag, and it is especially useful as a förster resonance energy transfer (fret) acceptor in ratiometric imaging. Fluorescent proteins (FPs) have become indispensable in biological research 1. After the cloning of GFP from the jellyfish Aequorea victoria 2 , several GFP spectral variants were developed, including blue, cyan and yellow FPs 3. The palette of FPs was greatly expanded after the cloning of red FP (RFP) homologs from corals and other Anthozoa species 4,5. However, all Anthozoa RFPs form obligate tetramers, which can seriously interfere with localization and functioning of RFP-fusion proteins. Monomerization of tetrameric RFPs was accompanied by a serious deterioration of the brightness and incomplete and/or partial green maturation of the resulting monomer 6. After the development of the first monomeric RFP (mRFP), mRFP1 (ref. 6), several improved mRFPs have been reported: mCherry 7 , mApple 8 , TagRFP(-T) 8,9 , mKate2 (ref. 10), mRuby2 (ref. 11), mRuby3 (ref. 12) and FusionRed 13 (reviewed in ref. 14). But all these mRFPs are dimmer than their tetrameric ancestors; they have quantum yields below 50%, and several still harbor additional problems due to incomplete or partial green maturation and a residual tendency to dimerize 15. Spectral variants of FPs can be applied in FRET-based biosensors to probe molecular interactions, conformational changes and metabolite concentrations within living cells 16. While good FP-based FRET pairs are available with cyan FPs (CFPs) as donors
Rho guanine exchange factors (RhoGEFs) control many aspects of the cellular cytoskeleton, and the... more Rho guanine exchange factors (RhoGEFs) control many aspects of the cellular cytoskeleton, and thereby regulate and control processes such as cell migration, cell adhesion and proliferation. TGAT is a splice variant of the RhoGEF Trio, with oncogenic potential. Whether the subcellular location of TGAT is critical for its activity is unknown. Confocal microscopy of fluorescent protein tagged TGAT revealed co-localization with a Golgi marker. Because plasma membrane localized RhoGEFs are particularly effective at activating RhoA, plasma membrane localization of TGAT was studied. In order to quantitatively measure plasma membrane association we developed a novel, highly sensitive image analysis method. The method requires a cytoplasmic marker and a plasma membrane marker, which are co-imaged with the tagged protein of interest. Linear unmixing is performed to determine the plasma membrane and cytoplasmic component in the fluorescence signal of protein of interest. The analysis revealed ...
Genetically encoded biosensors based on Forster resonance energy transfer (FRET) can visualize re... more Genetically encoded biosensors based on Forster resonance energy transfer (FRET) can visualize responses of individual cells in real time. Here, we evaluated whether FRET based biosensors provide sufficient contrast and specificity to measure activity of G protein coupled receptors. The four histamine receptor subtypes (H1R, H2R, H3R and H4R) respond to the ligand histamine by activating three canonical heterotrimeric G-protein mediated signaling pathways with a reported high degree of specificity. Using FRET based biosensors we demonstrate that H1R activates Gαq. We also observed that H1R activates Gαi, albeit at a 10-fold lower potency. In addition to increasing cAMP levels, most likely via Gαs, we found that the H2R induces Gαq mediated calcium release. The H3R and H4R activated Gαi with high specificity and a high potency. We demonstrate that a number of FRET sensors provide sufficient contrast to (i) analyze the specificity of the histamine receptor subtypes for different heter...
The small GTPase RhoA is involved in cell morphology and migration. RhoA activity is tightly regu... more The small GTPase RhoA is involved in cell morphology and migration. RhoA activity is tightly regulated in time and space and depends on guanine exchange factors (GEFs). However, the kinetics and subcellular localization of GEF activity towards RhoA are poorly defined. To study the mechanism underlying the spatiotemporal control of RhoA activity by GEFs, we performed single cell imaging with an improved FRET sensor reporting on the nucleotide loading state of RhoA. By employing the FRET sensor we show that a plasma membrane located RhoGEF, p63RhoGEF, can rapidly activate RhoA through endogenous GPCRs and that localized RhoA activity at the cell periphery correlates with actin polymerization. Moreover, synthetic recruitment of the catalytic domain derived from p63RhoGEF to the plasma membrane, but not to the Golgi apparatus, is sufficient to activate RhoA. The synthetic system enables local activation of endogenous RhoA and effectively induces actin polymerization and changes in cellu...
SNAIL transcriptional factors are key regulators during development and disease. They arose early... more SNAIL transcriptional factors are key regulators during development and disease. They arose early during evolution and in cnidarians such as Nematostella vectensis, NvSNAILA/B are detected in invaginating tissues during gastrulation. The function of SNAIL proteins is well established in bilaterians but their roles in cnidarians remain unknown. The structure of NvSNAILA and B is similar to the human SNAIL1 and 2, including SNAG and zinc-finger domains. Here, we performed a molecular analysis on localization and mobility of NvSNAILA/B using mammalian cells and Nematostella embryos. NvSNAILA/B display nuclear localization and mobility similar to HsSNAIL1/2. Strikingly, NvSNAILA is highly enriched in the nucleoli and shuttles between the nucleoli and the nucleoplasm. Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility. Truncation of the C-terminal zinc-fingers, i...
Null mutations in thetransient receptor potential(trp) gene eliminate the major, Ca2+-selective c... more Null mutations in thetransient receptor potential(trp) gene eliminate the major, Ca2+-selective component of the light-sensitive conductance inDrosophilaphotoreceptors. Although it is the prototypical member of the TRP ion channel superfamily, conclusive evidence that TRP is a pore-forming channel subunitin vivois lacking. We show here that mutating a specific acidic residue (Asp621) in the putative pore virtually eliminated Ca2+permeationin vivoand altered other biophysical properties of the native TRP conductance. The results identify Asp621as a critical residue of the TRP Ca2+selectivity filter, provide the first rigorous demonstration that a TRP protein is a pore-forming subunit in any native system, and point to the likely location of the pore in mammalian canonical TRP channels. The specific elimination of Ca2+permeation in TRP also provided a unique opportunity to address the roles of Ca2+influxin vivo. We found that Asp621mutations profoundly affected several key aspects of ...
Pflügers Archiv - European Journal of Physiology, 2005
Neuronal depolarization causes larger intracellular pH (pH i) shifts in axonal and dendritic regi... more Neuronal depolarization causes larger intracellular pH (pH i) shifts in axonal and dendritic regions than in the cell body. In this paper, we present evidence relating the time for collapse of these gradients to neuronal morphology. We have used ratiometric pH i measurements using 8hydroxypyrene-1,3,6-trisulfonic acid (HPTS) in whole-cell patch-clamped snail neurons to study the collapse of longitudinal pH gradients. Using depolarization to open voltage-gated proton channels, we produced alkaline pH i microdomains. In the absence of added mobile buffers, facilitated H + diffusion down the length of the axon plays a critical role in determining pH i microdomain lifetime, with axons of ∼100 μm allowing pH differences to be maintained for >60 s. An application of mobile, membranepermeant pH buffers accelerated the collapse of the alkaline-pH gradients but, even at 30 mM, was unable to abolish them. Modeling of the pH i dynamics showed that both the relatively weak effect of the weak acid/base on the peak size of the pH gradient and the accelerated collapse of the pH gradient could be due to the time taken for equilibration of the weak acid and base across the cell. We propose that appropriate weak acid/base mixes may provide a simple method for studying the role of local pH i signals without perturbing steady-state pH i. Furthermore, an extrapolation of our in vitro data to longer and thinner neuronal structures found in the mammalian nervous system suggests that dendritic and axonal pH i are likely to be dominated by local pH i-regulating mechanisms rather than simply following the soma pH i .
Phototransduction in flies is the fastest known G protein-coupled signaling cascade, but how this... more Phototransduction in flies is the fastest known G protein-coupled signaling cascade, but how this performance is achieved remains unclear. Here, we investigate the mechanism and role of rhodopsin inactivation. We determined the lifetime of activated rhodopsin (metarhodopsin = M*) in whole-cell recordings from Drosophila photoreceptors by measuring the time window within which inactivating M* by photoreisomerization to rhodopsin could suppress responses to prior illumination. M* was inactivated rapidly (t $20 ms) under control conditions, but $10-fold more slowly in Ca 2+-free solutions. This pronounced Ca 2+ dependence of M* inactivation was unaffected by mutations affecting phosphorylation of rhodopsin or arrestin but was abolished in mutants of calmodulin (CaM) or the CaM-binding myosin III, NINAC. This suggests a mechanism whereby Ca 2+ influx acting via CaM and NINAC accelerates the binding of arrestin to M*. Our results indicate that this strategy promotes quantum efficiency, temporal resolution, and fidelity of visual signaling.
Genetically encoded biosensors based on Förster resonance energy transfer (FRET) can visualize re... more Genetically encoded biosensors based on Förster resonance energy transfer (FRET) can visualize responses of individual cells in real time. Here, we evaluated whether FRET-based biosensors provide sufficient contrast and specificity to measure activity of G-protein-coupled receptors. The four histamine receptor subtypes (H 1 R, H 2 R, H 3 R, and H 4 R) respond to the ligand histamine by activating three canonical heterotrimeric G-protein-mediated signaling pathways with a reported high degree of specificity. Using FRET-based biosensors, we demonstrate that H 1 R activates Gaq. We also observed that H 1 R activates Gai, albeit at a 10-fold lower potency. In addition to increasing cAMP levels, most likely via Gas, we found that the H 2 R induces Gaq-mediated calcium release. The H 3 R and H 4 R activated Gai with high specificity and a high potency. We demonstrate that a number of FRET sensors provide sufficient contrast to: 1) analyze the specificity of the histamine receptor subtypes for different heterotrimeric G-protein families with single-cell resolution, 2) probe for antagonist specificity, and 3) allow the measurement of single-cell concentration-response curves.
Background: Rho guanine exchange factors (RhoGEFs) control cellular processes such as migration, ... more Background: Rho guanine exchange factors (RhoGEFs) control cellular processes such as migration, adhesion and proliferation. Alternative splicing of the RhoGEF Trio produces TGAT. The RhoGEF TGAT is an oncoprotein with constitutive RhoGEF activity. We investigated whether the subcellular location of TGAT is critical for its RhoGEF activity. Methods: Since plasma membrane associated RhoGEFs are particularly effective at activating RhoA, plasma membrane localization of TGAT was examined. To this end, we developed a highly sensitive image analysis method to quantitatively measure plasma membrane association. The method requires a cytoplasmic marker and a plasma membrane marker, which are co-imaged with the tagged protein of interest. Linear unmixing is performed to determine the plasma membrane and cytoplasmic component in the fluorescence signal of protein of interest. Results: The analysis revealed that wild-type TGAT is partially co-localized with the plasma membrane. Strikingly, cysteine TGAT-mutants lacking one or more putative palmitoylation sites in the C-tail, still showed membrane association. In contrast, a truncated variant, lacking the last 15 amino acids, TGAT Δ15 , lost membrane association. We show that membrane localization of TGAT was responsible for high RhoGEF activity by using a RhoA FRET-sensor and by determining F-actin levels. Mutants of TGAT that still maintained membrane association showed similar activity as wild-type TGAT. In contrast, the activity was abrogated for the cytoplasmic TGAT Δ15 variant. Synthetic recruitment of TGAT Δ15 to membranes confirmed that TGAT effectively activates RhoA at the plasma membrane. Conclusion: Together, these results show that membrane association of TGAT is critical for its activity.
Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed t... more Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.
Chemotaxis is the result of a refined interplay among various intracellular molecules that proces... more Chemotaxis is the result of a refined interplay among various intracellular molecules that process spatial and temporal information. Here we present a modular scheme of the complex interactions between the front and the back of cells that allows them to navigate. First, at the front of the cell, activated Rho-type GTPases induce actin polymerization and pseudopod formation. Second, phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P 3) is produced in a patch at the leading edge, where it binds pleckstrinhomology-domain-containing proteins, which enhance actin polymerization and translocation of the pseudopod. Third, in Dictyostelium amoebae, a cyclic-GMP-signalling cascade has been identified that regulates myosin filament formation in the posterior of the cell, thereby inhibiting the formation of lateral pseudopodia that could misdirect the cell.
Background: In fly photoreceptors, light is focused onto a photosensitive waveguide, the rhabdome... more Background: In fly photoreceptors, light is focused onto a photosensitive waveguide, the rhabdomere, consisting of tens of thousands of microvilli. Each microvillus is capable of generating elementary responses, quantum bumps, in response to single photons using a stochastically operating phototransduction cascade. Whereas much is known about the cascade reactions, less is known about how the concerted action of the microvilli population encodes light changes into neural information and how the ultrastructure and biochemical machinery of photoreceptors of flies and other insects evolved in relation to the information sampling and processing they perform. Results: We generated biophysically realistic fly photoreceptor models, which accurately simulate the encoding of visual information. By comparing stochastic simulations with single cell recordings from Drosophila photoreceptors, we show how adaptive sampling by 30,000 microvilli captures the temporal structure of natural contrast changes. Following each bump, individual microvilli are rendered briefly (w100-200 ms) refractory, thereby reducing quantum efficiency with increasing intensity. The refractory period opposes saturation, dynamically and stochastically adjusting availability of microvilli (bump production rate: sample rate), whereas intracellular calcium and voltage adapt bump amplitude and waveform (sample size). These adapting sampling principles result in robust encoding of natural light changes, which both approximates perceptual contrast constancy and enhances novel events under different light conditions, and predict information processing across a range of species with different visual ecologies. Conclusions: These results clarify why fly photoreceptors are structured the way they are and function as they do, linking sensory information to sensory evolution and revealing benefits of stochasticity for neural information processing.
It remains unclear how visual information is co-processed by different layers of neurons in the r... more It remains unclear how visual information is co-processed by different layers of neurons in the retina. In particular, relatively little is known how retina translates vast environmental light changes into neural responses of limited range. We began examining this question in a bottom-up way in a relatively simple °y eye. To gain understanding of how complex bio-molecular interactions govern the conversion of light input into voltage output (phototransduction), we are building a biophysical model of the Drosophila R1-R6 photoreceptor. Our model, which relates molecular dynamics of the underlying biochemical reactions to external light input, attempts to capture the molecular dynamics of phototransduction gain control in a quantitative way
Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed t... more Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.
Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed t... more Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocy...
Background: Rho guanine exchange factors (RhoGEFs) control cellular processes such as migration, ... more Background: Rho guanine exchange factors (RhoGEFs) control cellular processes such as migration, adhesion and proliferation. Alternative splicing of the RhoGEF Trio produces TGAT. The RhoGEF TGAT is an oncoprotein with constitutive RhoGEF activity. We investigated whether the subcellular location of TGAT is critical for its RhoGEF activity. Methods: Since plasma membrane associated RhoGEFs are particularly effective at activating RhoA, plasma membrane localization of TGAT was examined. To this end, we developed a highly sensitive image analysis method to quantitatively measure plasma membrane association. The method requires a cytoplasmic marker and a plasma membrane marker, which are co-imaged with the tagged protein of interest. Linear unmixing is performed to determine the plasma membrane and cytoplasmic component in the fluorescence signal of protein of interest. Results: The analysis revealed that wild-type TGAT is partially co-localized with the plasma membrane. Strikingly, cysteine TGAT-mutants lacking one or more putative palmitoylation sites in the C-tail, still showed membrane association. In contrast, a truncated variant, lacking the last 15 amino acids, TGAT Δ15 , lost membrane association. We show that membrane localization of TGAT was responsible for high RhoGEF activity by using a RhoA FRET-sensor and by determining F-actin levels. Mutants of TGAT that still maintained membrane association showed similar activity as wild-type TGAT. In contrast, the activity was abrogated for the cytoplasmic TGAT Δ15 variant. Synthetic recruitment of TGAT Δ15 to membranes confirmed that TGAT effectively activates RhoA at the plasma membrane. Conclusion: Together, these results show that membrane association of TGAT is critical for its activity.
The performance of Förster Resonance Energy Transfer (FRET) biosensors depends on brightness and ... more The performance of Förster Resonance Energy Transfer (FRET) biosensors depends on brightness and photostability, which are dependent on the characteristics of the fluorescent proteins that are employed. Yellow fluorescent protein (YFP) is often used as an acceptor but YFP is prone to photobleaching and pH changes. In this study, we evaluated the properties of a diverse set of acceptor fluorescent proteins in combination with the optimized CFP variant mTurquoise2 as the donor. To determine the theoretical performance of acceptors, the Förster radius was determined. The practical performance was determined by measuring FRET efficiency and photostability of tandem fusion proteins in mammalian cells. Our results show that mNeonGreen is the most efficient acceptor for mTurquoise2 and that the photostability is better than SYFP2. The non-fluorescent YFP variant sREACh is an efficient acceptor, which is useful in lifetime-based FRET experiments. Among the orange and red fluorescent protein...
We report the engineering of mscarlet, a truly monomeric red fluorescent protein with record brig... more We report the engineering of mscarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mscarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mscarlet's crystal structure reveals a planar and rigidified chromophore. mscarlet outperforms existing red fluorescent proteins as a fusion tag, and it is especially useful as a förster resonance energy transfer (fret) acceptor in ratiometric imaging. Fluorescent proteins (FPs) have become indispensable in biological research 1. After the cloning of GFP from the jellyfish Aequorea victoria 2 , several GFP spectral variants were developed, including blue, cyan and yellow FPs 3. The palette of FPs was greatly expanded after the cloning of red FP (RFP) homologs from corals and other Anthozoa species 4,5. However, all Anthozoa RFPs form obligate tetramers, which can seriously interfere with localization and functioning of RFP-fusion proteins. Monomerization of tetrameric RFPs was accompanied by a serious deterioration of the brightness and incomplete and/or partial green maturation of the resulting monomer 6. After the development of the first monomeric RFP (mRFP), mRFP1 (ref. 6), several improved mRFPs have been reported: mCherry 7 , mApple 8 , TagRFP(-T) 8,9 , mKate2 (ref. 10), mRuby2 (ref. 11), mRuby3 (ref. 12) and FusionRed 13 (reviewed in ref. 14). But all these mRFPs are dimmer than their tetrameric ancestors; they have quantum yields below 50%, and several still harbor additional problems due to incomplete or partial green maturation and a residual tendency to dimerize 15. Spectral variants of FPs can be applied in FRET-based biosensors to probe molecular interactions, conformational changes and metabolite concentrations within living cells 16. While good FP-based FRET pairs are available with cyan FPs (CFPs) as donors
Rho guanine exchange factors (RhoGEFs) control many aspects of the cellular cytoskeleton, and the... more Rho guanine exchange factors (RhoGEFs) control many aspects of the cellular cytoskeleton, and thereby regulate and control processes such as cell migration, cell adhesion and proliferation. TGAT is a splice variant of the RhoGEF Trio, with oncogenic potential. Whether the subcellular location of TGAT is critical for its activity is unknown. Confocal microscopy of fluorescent protein tagged TGAT revealed co-localization with a Golgi marker. Because plasma membrane localized RhoGEFs are particularly effective at activating RhoA, plasma membrane localization of TGAT was studied. In order to quantitatively measure plasma membrane association we developed a novel, highly sensitive image analysis method. The method requires a cytoplasmic marker and a plasma membrane marker, which are co-imaged with the tagged protein of interest. Linear unmixing is performed to determine the plasma membrane and cytoplasmic component in the fluorescence signal of protein of interest. The analysis revealed ...
Genetically encoded biosensors based on Forster resonance energy transfer (FRET) can visualize re... more Genetically encoded biosensors based on Forster resonance energy transfer (FRET) can visualize responses of individual cells in real time. Here, we evaluated whether FRET based biosensors provide sufficient contrast and specificity to measure activity of G protein coupled receptors. The four histamine receptor subtypes (H1R, H2R, H3R and H4R) respond to the ligand histamine by activating three canonical heterotrimeric G-protein mediated signaling pathways with a reported high degree of specificity. Using FRET based biosensors we demonstrate that H1R activates Gαq. We also observed that H1R activates Gαi, albeit at a 10-fold lower potency. In addition to increasing cAMP levels, most likely via Gαs, we found that the H2R induces Gαq mediated calcium release. The H3R and H4R activated Gαi with high specificity and a high potency. We demonstrate that a number of FRET sensors provide sufficient contrast to (i) analyze the specificity of the histamine receptor subtypes for different heter...
The small GTPase RhoA is involved in cell morphology and migration. RhoA activity is tightly regu... more The small GTPase RhoA is involved in cell morphology and migration. RhoA activity is tightly regulated in time and space and depends on guanine exchange factors (GEFs). However, the kinetics and subcellular localization of GEF activity towards RhoA are poorly defined. To study the mechanism underlying the spatiotemporal control of RhoA activity by GEFs, we performed single cell imaging with an improved FRET sensor reporting on the nucleotide loading state of RhoA. By employing the FRET sensor we show that a plasma membrane located RhoGEF, p63RhoGEF, can rapidly activate RhoA through endogenous GPCRs and that localized RhoA activity at the cell periphery correlates with actin polymerization. Moreover, synthetic recruitment of the catalytic domain derived from p63RhoGEF to the plasma membrane, but not to the Golgi apparatus, is sufficient to activate RhoA. The synthetic system enables local activation of endogenous RhoA and effectively induces actin polymerization and changes in cellu...
SNAIL transcriptional factors are key regulators during development and disease. They arose early... more SNAIL transcriptional factors are key regulators during development and disease. They arose early during evolution and in cnidarians such as Nematostella vectensis, NvSNAILA/B are detected in invaginating tissues during gastrulation. The function of SNAIL proteins is well established in bilaterians but their roles in cnidarians remain unknown. The structure of NvSNAILA and B is similar to the human SNAIL1 and 2, including SNAG and zinc-finger domains. Here, we performed a molecular analysis on localization and mobility of NvSNAILA/B using mammalian cells and Nematostella embryos. NvSNAILA/B display nuclear localization and mobility similar to HsSNAIL1/2. Strikingly, NvSNAILA is highly enriched in the nucleoli and shuttles between the nucleoli and the nucleoplasm. Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility. Truncation of the C-terminal zinc-fingers, i...
Null mutations in thetransient receptor potential(trp) gene eliminate the major, Ca2+-selective c... more Null mutations in thetransient receptor potential(trp) gene eliminate the major, Ca2+-selective component of the light-sensitive conductance inDrosophilaphotoreceptors. Although it is the prototypical member of the TRP ion channel superfamily, conclusive evidence that TRP is a pore-forming channel subunitin vivois lacking. We show here that mutating a specific acidic residue (Asp621) in the putative pore virtually eliminated Ca2+permeationin vivoand altered other biophysical properties of the native TRP conductance. The results identify Asp621as a critical residue of the TRP Ca2+selectivity filter, provide the first rigorous demonstration that a TRP protein is a pore-forming subunit in any native system, and point to the likely location of the pore in mammalian canonical TRP channels. The specific elimination of Ca2+permeation in TRP also provided a unique opportunity to address the roles of Ca2+influxin vivo. We found that Asp621mutations profoundly affected several key aspects of ...
Pflügers Archiv - European Journal of Physiology, 2005
Neuronal depolarization causes larger intracellular pH (pH i) shifts in axonal and dendritic regi... more Neuronal depolarization causes larger intracellular pH (pH i) shifts in axonal and dendritic regions than in the cell body. In this paper, we present evidence relating the time for collapse of these gradients to neuronal morphology. We have used ratiometric pH i measurements using 8hydroxypyrene-1,3,6-trisulfonic acid (HPTS) in whole-cell patch-clamped snail neurons to study the collapse of longitudinal pH gradients. Using depolarization to open voltage-gated proton channels, we produced alkaline pH i microdomains. In the absence of added mobile buffers, facilitated H + diffusion down the length of the axon plays a critical role in determining pH i microdomain lifetime, with axons of ∼100 μm allowing pH differences to be maintained for >60 s. An application of mobile, membranepermeant pH buffers accelerated the collapse of the alkaline-pH gradients but, even at 30 mM, was unable to abolish them. Modeling of the pH i dynamics showed that both the relatively weak effect of the weak acid/base on the peak size of the pH gradient and the accelerated collapse of the pH gradient could be due to the time taken for equilibration of the weak acid and base across the cell. We propose that appropriate weak acid/base mixes may provide a simple method for studying the role of local pH i signals without perturbing steady-state pH i. Furthermore, an extrapolation of our in vitro data to longer and thinner neuronal structures found in the mammalian nervous system suggests that dendritic and axonal pH i are likely to be dominated by local pH i-regulating mechanisms rather than simply following the soma pH i .
Phototransduction in flies is the fastest known G protein-coupled signaling cascade, but how this... more Phototransduction in flies is the fastest known G protein-coupled signaling cascade, but how this performance is achieved remains unclear. Here, we investigate the mechanism and role of rhodopsin inactivation. We determined the lifetime of activated rhodopsin (metarhodopsin = M*) in whole-cell recordings from Drosophila photoreceptors by measuring the time window within which inactivating M* by photoreisomerization to rhodopsin could suppress responses to prior illumination. M* was inactivated rapidly (t $20 ms) under control conditions, but $10-fold more slowly in Ca 2+-free solutions. This pronounced Ca 2+ dependence of M* inactivation was unaffected by mutations affecting phosphorylation of rhodopsin or arrestin but was abolished in mutants of calmodulin (CaM) or the CaM-binding myosin III, NINAC. This suggests a mechanism whereby Ca 2+ influx acting via CaM and NINAC accelerates the binding of arrestin to M*. Our results indicate that this strategy promotes quantum efficiency, temporal resolution, and fidelity of visual signaling.
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Papers by Marten Postma