Barun Kumar Maity - Academia.edu (original) (raw)

Papers by Barun Kumar Maity

Nanoscale, 2022

A high-throughput molecular force sensor that uses single-stranded DNA as a force sensing molecul... more A high-throughput molecular force sensor that uses single-stranded DNA as a force sensing molecule. It can measure pico-Newton level forces of multiple kinesin motors transporting a microtubule.

Cell reports methods, Feb 1, 2023

bioRxiv (Cold Spring Harbor Laboratory), Jan 13, 2021

A cargo encounters many obstacles during its transport by molecular motors as it moves throughout... more A cargo encounters many obstacles during its transport by molecular motors as it moves throughout the cell. Multiple motors on the cargo exert forces to steer the cargo to its destination. Measuring these forces is essential for understanding intracellular transport. Using kinesin as an example, we measured the force exerted by multiple stationary kinesins in vitro, driving a common microtubule. We find that individual kinesins generally exert less than a piconewton (pN) of force, even while bypassing obstacles, whether these are artificially placed 20-100 nm particles or tau, a Microtubule Associated Protein. We demonstrate that when a kinesin encounters an obstacle, the kinesin either becomes dislodged and then re-engages or switches protofilaments while the other kinesins continue to apply their (sub-)pN forces. By designing a high-throughput assay involving nanometer-resolved multicolor-fluorescence and a force-sensor able to measure picoNewtons of force, our technique is expected to be generally useful for many different types of molecular motors.

A cargo encounters many obstacles during its transport by molecular motors as it moves throughout... more A cargo encounters many obstacles during its transport by molecular motors as it moves throughout the cell. Multiple motors on the cargo exert forces to steer the cargo to its destination. Measuring these forces is essential for understanding intracellular transport. Using kinesin as an example, we measured the force exerted by multiple stationary kinesinsin vitro, driving a common microtubule. We find that individual kinesins generally exert less than a piconewton (pN) of force, even while bypassing obstacles, whether these are artificially placed 20-100 nm particles or tau, a Microtubule Associated Protein. We demonstrate that when a kinesin encounters an obstacle, the kinesin either becomes dislodged and then re-engages or switches protofilaments while the other kinesins continue to apply their (sub-)pN forces. By designing a high-throughput assay involving nanometer-resolved multicolor-fluorescence and a force-sensor able to measure picoNewtons of force, our technique is expecte...

Nanoscale

A high-throughput molecular force sensor that uses single-stranded DNA as a force sensing molecul... more A high-throughput molecular force sensor that uses single-stranded DNA as a force sensing molecule. It can measure pico-Newton level forces of multiple kinesin motors transporting a microtubule.

Biophysical Journal, 2018

calorimetry provided insights into the nature of interactions and mechanism of inhibition. We fou... more calorimetry provided insights into the nature of interactions and mechanism of inhibition. We found inhibition efficiency in the order, micellar SDS > 45 mM DCF > monomeric SDS > 5 mM DCF. The energetics of interaction, correlated with the molecular structure of inhibitors provide guidelines for effective synthesis and design of inhibitors. ITC results have imparted important relationship between inhibition efficiency and exothermicity of interactions and have demonstrated the significance of polar interactions in fibril prevention by these inhibitors. Interestingly it was found that the micellar SDS not only inhibits the process but also effectively disintegrates the formed fibrils.

Biophysical Journal, 2021

ATP hydrolysis. These data support a model of mechanotransduction that accounts for two proton tr... more ATP hydrolysis. These data support a model of mechanotransduction that accounts for two proton transfer reactions. The first proton transfer, between two Zundel cation waters to allow for nucleophilic attack of the g-phosphate of ATP, has a tunneling component. Experimental determination of a proton quantum mechanical event, by which a particle transfers through a reaction barrier, offers an alternate explanation to classical transition-state for acceleration of biological catalysis. The second transfer measured is likely between the proton from the separated water molecules and the salt bridge. We conclude that proton transfer reactions in motor proteins can involve quantum tunneling, solvent control of the reaction coordinate, and strong anharmonic couplings to many degrees of freedom in mechanotransduction.

ABSTRACTPoint accumulation for imaging in nanoscale topography (PAINT) is a single-molecule techn... more ABSTRACTPoint accumulation for imaging in nanoscale topography (PAINT) is a single-molecule technique for super-resolution microscopy, achieving ∼5-25 nanometer resolution. Here we show that by transfecting the protein-of-interest with a docker-coil, rather than by adding the docker externally—as is the norm when using DNA tethers or antibodies as dockers—we can achieve similar localization, ∼10 nm. However, using a transfected docker has several experimental advances and simplifications. Most importantly, it allows Peptide-PAINT to be applied to transfected live cells, including surface proteins in mammalian cells and neurons under physiological conditions. The enhance resolution of Peptide-PAINT is also shown for organelles in fixed cells to unravel structural details including ≈40-nm and ≈60-nm axial repeats in vimentin filaments in the cytoplasm, and fiber shapes of sub-100-nm histone-rich regions in the nucleus.

Journal of Optics

Knowledge of the excitation profile in a confocal or multiphoton microscope can improve the image... more Knowledge of the excitation profile in a confocal or multiphoton microscope can improve the image resolution, e.g. by using deconvolution, pixel reassignment or adaptive optics strategies. Here we demonstrate a method by which the scanning beam can be used to place a stationary, virtual ‘guide star’ at any chosen location in the sample, during imaging. This can then be used to directly image the excitation profile. The major advantage of our easy-to-install method, compared to competing methods, is that it can work for non-descanned multiphoton microscopy, the method of choice for deep tissue or ultraviolet imaging. Our experimental results reproduce diffraction theory based calculations in a minimally-scattering sample, and provide detailed information about the aberrated excitation profile in a highly scattering sample.

Chemistry: A European Journal, 2021

Biophysical Journal, 2017

Biophysical Journal, 2016

also needed to induce hemolysis and to kill bacteria, and this minimum length was different for d... more also needed to induce hemolysis and to kill bacteria, and this minimum length was different for different bacterial strains, so that the series of peptides can be used as a ''molecular ruler'' to determine the thickness of erythrocyte or bacterial membranes in vivo. Using solid-state NMR we found that the orientation depends strongly on the lipid spontaneous curvature. The KIA peptides are always staying flat on the membrane surface in lipids with a negative spontaneous curvature. On the other hand, in lipids with a positive spontaneous curvature, like lyso-lipids, they can go deeper into the membrane and have a tilted or even inserted orientation. In the inserted state, the tilt of the peptides is found to be mismatch dependent. This is the first time a mismatch effect has been found for amphipathic peptides, and shows that the effect is more generally valid than has been appreciated until now.

Multiphoton Microscopy in the Biomedical Sciences XX

Deconvolution, pixel reassignment or adaptive optics-based strategies utilize information about t... more Deconvolution, pixel reassignment or adaptive optics-based strategies utilize information about the detection profile in improving the resolution of optical microscopy. Here, we show a novel method which allows us to obtain the single-photon detection volume of a laser scanning confocal microscope at any desired location of the object. It can create a stationary, virtual ‘guide star’ at the chosen location while the excitation beam is scanning the sample, by using an optical fiber placed in the non-descanned path of the microscope. Our experimental results are verified by diffraction theory-based calculations. The major advantages of our method are that it is alignment free, affordable, sensitive and applicable to many different modes of confocal imaging.

Chemistry – A European Journal

Serotonin is a neurotransmitter as well as a somatic signaling molecule, and the serotonergic sys... more Serotonin is a neurotransmitter as well as a somatic signaling molecule, and the serotonergic system is a major target for psychotropic drugs. Serotonin, together with a few related neurotransmitters, has recently been found to exhibit an unexpectedly high lipid membrane affinity1–3. It has been conjectured that extrasynaptic serotonin can diffuse in the lipid membrane to efficiently reach remote receptors (and receptors with buried ligand-binding sites)4, providing a mechanism for the diffuse ‘volume’ neurotransmission that serotonin is capable of5–10. Here we show that membrane binding by serotonin can directly modulate membrane properties and cellular function, independent of its receptor-mediated actions. Atomic force microscopy shows that serotonin binding makes artificial lipid bilayers softer. It induces nucleation of liquid disordered domains inside the raft-like liquid-ordered domains in a ternary bilayer displaying phase separation. Solid-state NMR spectroscopy corroborate...

Proceedings of the National Academy of Sciences, India Section A: Physical Sciences

Surface enhanced Raman spectroscopy (SERS) has great potential for investigating the secondary st... more Surface enhanced Raman spectroscopy (SERS) has great potential for investigating the secondary structure of biomolecules. While enhancements of many orders of magnitude are possible in SERS, the excitation powers required for adequate signal to noise may still be damaging for sensitive protein molecules. Here, we describe the design and construction of a line-confocal Raman micro-spectrometer, which allows investigations at much lower powers compared to a commercial Raman microscope, which uses a point-confocal design. Our design combines a commercial Raman spectrometer and a fluorescence microscope, and achieves simultaneous spectrometry over 400 pixels. This can be used for multiplexed probing of a homogeneous sample, using much lower power for each pixel at comparable signal-to-noise ratios.

ACS Chemical Neuroscience

While the roles of intrinsically disordered protein domains in driving interprotein interactions ... more While the roles of intrinsically disordered protein domains in driving interprotein interactions are increasingly well-appreciated, the mechanism of toxicity of disease-causing disordered proteins remains poorly understood. A prime example is Alzheimer's disease (AD) associated amyloid beta (Aβ). Aβ oligomers are highly toxic partially structured peptide assemblies with a distinct ordered region (residues ∼10-40) and a shorter disordered region (residues ∼1-9). Here, we investigate the role of this disordered domain and its relation to the ordered domain in the manifestation of toxicity through a set of Aβ fragments and stereoisomers designed for this purpose. We measure their effects on lipid membranes and cultured neurons, probing their toxicity, intracellular distributions, and specific molecular interactions using the techniques of confocal imaging, lattice light sheet imaging, fluorescence lifetime imaging, and fluorescence correlation spectroscopy. Remarkably, we find that neither part-Aβ10-40 or Aβ1-9, is toxic by itself. The ordered part (Aβ10-40) is the major determinant of how Aβ attaches to lipid bilayers, enters neuronal cells, and localizes primarily in the late endosomal compartments. However, once Aβ enters the cell, it is the disordered part (only when it is connected to the rest of the peptide) that has a strong and stereospecific interaction with an unknown cellular component, as demonstrated by distinct changes in the fluorescence lifetime of a fluorophore attached to the N-terminal. This interaction appears to commit Aβ to the toxic pathway. Our findings correlate well with Aβ sites of familial AD mutations, a significant fraction of which cluster in the disordered region. We conclude that, while the ordered region dictates attachment and cellular entry, the key to toxicity lies in the ordered part presenting the disordered part for a specific cellular interaction.

ACS chemical neuroscience, Jan 21, 2018

Monoamine neurotransmission is key to neuromodulation, but imaging monoamines in live neurons has... more Monoamine neurotransmission is key to neuromodulation, but imaging monoamines in live neurons has remained a challenge. Here we show that externally added ortho-phthalaldehyde (OPA) can permeate live cells and form bright fluorogenic adducts with intracellular monoamines (e.g., serotonin, dopamine, and norepinephrine) and with L-DOPA, which can be imaged sensitively using conventional single-photon excitation in a fluorescence microscope. The peak excitation and emission wavelengths (λ = 401 nm and λ = 490 nm for serotonin; λ = 446 nm and λ = 557 nm for dopamine; and λ = 446 nm and λ = 544 nm for norepinephrine, respectively) are accessible to most modern confocal imaging instruments. The identity of monoamine containing structures (possibly neurotransmitter vesicles) in serotonergic RN46A cells is established by quasi-simultaneous imaging of serotonin using three-photon excitation microscopy. Mass spectrometry of cell extracts and of in vitro solutions helps us identify the chemica...

Nanoscale, 2022

A high-throughput molecular force sensor that uses single-stranded DNA as a force sensing molecul... more A high-throughput molecular force sensor that uses single-stranded DNA as a force sensing molecule. It can measure pico-Newton level forces of multiple kinesin motors transporting a microtubule.

Cell reports methods, Feb 1, 2023

bioRxiv (Cold Spring Harbor Laboratory), Jan 13, 2021

A cargo encounters many obstacles during its transport by molecular motors as it moves throughout... more A cargo encounters many obstacles during its transport by molecular motors as it moves throughout the cell. Multiple motors on the cargo exert forces to steer the cargo to its destination. Measuring these forces is essential for understanding intracellular transport. Using kinesin as an example, we measured the force exerted by multiple stationary kinesins in vitro, driving a common microtubule. We find that individual kinesins generally exert less than a piconewton (pN) of force, even while bypassing obstacles, whether these are artificially placed 20-100 nm particles or tau, a Microtubule Associated Protein. We demonstrate that when a kinesin encounters an obstacle, the kinesin either becomes dislodged and then re-engages or switches protofilaments while the other kinesins continue to apply their (sub-)pN forces. By designing a high-throughput assay involving nanometer-resolved multicolor-fluorescence and a force-sensor able to measure picoNewtons of force, our technique is expected to be generally useful for many different types of molecular motors.

A cargo encounters many obstacles during its transport by molecular motors as it moves throughout... more A cargo encounters many obstacles during its transport by molecular motors as it moves throughout the cell. Multiple motors on the cargo exert forces to steer the cargo to its destination. Measuring these forces is essential for understanding intracellular transport. Using kinesin as an example, we measured the force exerted by multiple stationary kinesinsin vitro, driving a common microtubule. We find that individual kinesins generally exert less than a piconewton (pN) of force, even while bypassing obstacles, whether these are artificially placed 20-100 nm particles or tau, a Microtubule Associated Protein. We demonstrate that when a kinesin encounters an obstacle, the kinesin either becomes dislodged and then re-engages or switches protofilaments while the other kinesins continue to apply their (sub-)pN forces. By designing a high-throughput assay involving nanometer-resolved multicolor-fluorescence and a force-sensor able to measure picoNewtons of force, our technique is expecte...

Nanoscale

A high-throughput molecular force sensor that uses single-stranded DNA as a force sensing molecul... more A high-throughput molecular force sensor that uses single-stranded DNA as a force sensing molecule. It can measure pico-Newton level forces of multiple kinesin motors transporting a microtubule.

Biophysical Journal, 2018

calorimetry provided insights into the nature of interactions and mechanism of inhibition. We fou... more calorimetry provided insights into the nature of interactions and mechanism of inhibition. We found inhibition efficiency in the order, micellar SDS > 45 mM DCF > monomeric SDS > 5 mM DCF. The energetics of interaction, correlated with the molecular structure of inhibitors provide guidelines for effective synthesis and design of inhibitors. ITC results have imparted important relationship between inhibition efficiency and exothermicity of interactions and have demonstrated the significance of polar interactions in fibril prevention by these inhibitors. Interestingly it was found that the micellar SDS not only inhibits the process but also effectively disintegrates the formed fibrils.

Biophysical Journal, 2021

ATP hydrolysis. These data support a model of mechanotransduction that accounts for two proton tr... more ATP hydrolysis. These data support a model of mechanotransduction that accounts for two proton transfer reactions. The first proton transfer, between two Zundel cation waters to allow for nucleophilic attack of the g-phosphate of ATP, has a tunneling component. Experimental determination of a proton quantum mechanical event, by which a particle transfers through a reaction barrier, offers an alternate explanation to classical transition-state for acceleration of biological catalysis. The second transfer measured is likely between the proton from the separated water molecules and the salt bridge. We conclude that proton transfer reactions in motor proteins can involve quantum tunneling, solvent control of the reaction coordinate, and strong anharmonic couplings to many degrees of freedom in mechanotransduction.

ABSTRACTPoint accumulation for imaging in nanoscale topography (PAINT) is a single-molecule techn... more ABSTRACTPoint accumulation for imaging in nanoscale topography (PAINT) is a single-molecule technique for super-resolution microscopy, achieving ∼5-25 nanometer resolution. Here we show that by transfecting the protein-of-interest with a docker-coil, rather than by adding the docker externally—as is the norm when using DNA tethers or antibodies as dockers—we can achieve similar localization, ∼10 nm. However, using a transfected docker has several experimental advances and simplifications. Most importantly, it allows Peptide-PAINT to be applied to transfected live cells, including surface proteins in mammalian cells and neurons under physiological conditions. The enhance resolution of Peptide-PAINT is also shown for organelles in fixed cells to unravel structural details including ≈40-nm and ≈60-nm axial repeats in vimentin filaments in the cytoplasm, and fiber shapes of sub-100-nm histone-rich regions in the nucleus.

Journal of Optics

Knowledge of the excitation profile in a confocal or multiphoton microscope can improve the image... more Knowledge of the excitation profile in a confocal or multiphoton microscope can improve the image resolution, e.g. by using deconvolution, pixel reassignment or adaptive optics strategies. Here we demonstrate a method by which the scanning beam can be used to place a stationary, virtual ‘guide star’ at any chosen location in the sample, during imaging. This can then be used to directly image the excitation profile. The major advantage of our easy-to-install method, compared to competing methods, is that it can work for non-descanned multiphoton microscopy, the method of choice for deep tissue or ultraviolet imaging. Our experimental results reproduce diffraction theory based calculations in a minimally-scattering sample, and provide detailed information about the aberrated excitation profile in a highly scattering sample.

Chemistry: A European Journal, 2021

Biophysical Journal, 2017

Biophysical Journal, 2016

also needed to induce hemolysis and to kill bacteria, and this minimum length was different for d... more also needed to induce hemolysis and to kill bacteria, and this minimum length was different for different bacterial strains, so that the series of peptides can be used as a ''molecular ruler'' to determine the thickness of erythrocyte or bacterial membranes in vivo. Using solid-state NMR we found that the orientation depends strongly on the lipid spontaneous curvature. The KIA peptides are always staying flat on the membrane surface in lipids with a negative spontaneous curvature. On the other hand, in lipids with a positive spontaneous curvature, like lyso-lipids, they can go deeper into the membrane and have a tilted or even inserted orientation. In the inserted state, the tilt of the peptides is found to be mismatch dependent. This is the first time a mismatch effect has been found for amphipathic peptides, and shows that the effect is more generally valid than has been appreciated until now.

Multiphoton Microscopy in the Biomedical Sciences XX

Deconvolution, pixel reassignment or adaptive optics-based strategies utilize information about t... more Deconvolution, pixel reassignment or adaptive optics-based strategies utilize information about the detection profile in improving the resolution of optical microscopy. Here, we show a novel method which allows us to obtain the single-photon detection volume of a laser scanning confocal microscope at any desired location of the object. It can create a stationary, virtual ‘guide star’ at the chosen location while the excitation beam is scanning the sample, by using an optical fiber placed in the non-descanned path of the microscope. Our experimental results are verified by diffraction theory-based calculations. The major advantages of our method are that it is alignment free, affordable, sensitive and applicable to many different modes of confocal imaging.

Chemistry – A European Journal

Serotonin is a neurotransmitter as well as a somatic signaling molecule, and the serotonergic sys... more Serotonin is a neurotransmitter as well as a somatic signaling molecule, and the serotonergic system is a major target for psychotropic drugs. Serotonin, together with a few related neurotransmitters, has recently been found to exhibit an unexpectedly high lipid membrane affinity1–3. It has been conjectured that extrasynaptic serotonin can diffuse in the lipid membrane to efficiently reach remote receptors (and receptors with buried ligand-binding sites)4, providing a mechanism for the diffuse ‘volume’ neurotransmission that serotonin is capable of5–10. Here we show that membrane binding by serotonin can directly modulate membrane properties and cellular function, independent of its receptor-mediated actions. Atomic force microscopy shows that serotonin binding makes artificial lipid bilayers softer. It induces nucleation of liquid disordered domains inside the raft-like liquid-ordered domains in a ternary bilayer displaying phase separation. Solid-state NMR spectroscopy corroborate...

Proceedings of the National Academy of Sciences, India Section A: Physical Sciences

Surface enhanced Raman spectroscopy (SERS) has great potential for investigating the secondary st... more Surface enhanced Raman spectroscopy (SERS) has great potential for investigating the secondary structure of biomolecules. While enhancements of many orders of magnitude are possible in SERS, the excitation powers required for adequate signal to noise may still be damaging for sensitive protein molecules. Here, we describe the design and construction of a line-confocal Raman micro-spectrometer, which allows investigations at much lower powers compared to a commercial Raman microscope, which uses a point-confocal design. Our design combines a commercial Raman spectrometer and a fluorescence microscope, and achieves simultaneous spectrometry over 400 pixels. This can be used for multiplexed probing of a homogeneous sample, using much lower power for each pixel at comparable signal-to-noise ratios.

ACS Chemical Neuroscience

While the roles of intrinsically disordered protein domains in driving interprotein interactions ... more While the roles of intrinsically disordered protein domains in driving interprotein interactions are increasingly well-appreciated, the mechanism of toxicity of disease-causing disordered proteins remains poorly understood. A prime example is Alzheimer's disease (AD) associated amyloid beta (Aβ). Aβ oligomers are highly toxic partially structured peptide assemblies with a distinct ordered region (residues ∼10-40) and a shorter disordered region (residues ∼1-9). Here, we investigate the role of this disordered domain and its relation to the ordered domain in the manifestation of toxicity through a set of Aβ fragments and stereoisomers designed for this purpose. We measure their effects on lipid membranes and cultured neurons, probing their toxicity, intracellular distributions, and specific molecular interactions using the techniques of confocal imaging, lattice light sheet imaging, fluorescence lifetime imaging, and fluorescence correlation spectroscopy. Remarkably, we find that neither part-Aβ10-40 or Aβ1-9, is toxic by itself. The ordered part (Aβ10-40) is the major determinant of how Aβ attaches to lipid bilayers, enters neuronal cells, and localizes primarily in the late endosomal compartments. However, once Aβ enters the cell, it is the disordered part (only when it is connected to the rest of the peptide) that has a strong and stereospecific interaction with an unknown cellular component, as demonstrated by distinct changes in the fluorescence lifetime of a fluorophore attached to the N-terminal. This interaction appears to commit Aβ to the toxic pathway. Our findings correlate well with Aβ sites of familial AD mutations, a significant fraction of which cluster in the disordered region. We conclude that, while the ordered region dictates attachment and cellular entry, the key to toxicity lies in the ordered part presenting the disordered part for a specific cellular interaction.

ACS chemical neuroscience, Jan 21, 2018

Monoamine neurotransmission is key to neuromodulation, but imaging monoamines in live neurons has... more Monoamine neurotransmission is key to neuromodulation, but imaging monoamines in live neurons has remained a challenge. Here we show that externally added ortho-phthalaldehyde (OPA) can permeate live cells and form bright fluorogenic adducts with intracellular monoamines (e.g., serotonin, dopamine, and norepinephrine) and with L-DOPA, which can be imaged sensitively using conventional single-photon excitation in a fluorescence microscope. The peak excitation and emission wavelengths (λ = 401 nm and λ = 490 nm for serotonin; λ = 446 nm and λ = 557 nm for dopamine; and λ = 446 nm and λ = 544 nm for norepinephrine, respectively) are accessible to most modern confocal imaging instruments. The identity of monoamine containing structures (possibly neurotransmitter vesicles) in serotonergic RN46A cells is established by quasi-simultaneous imaging of serotonin using three-photon excitation microscopy. Mass spectrometry of cell extracts and of in vitro solutions helps us identify the chemica...