Unfolded proteins may contain a native or nonnative residual structure, which has important impli... more Unfolded proteins may contain a native or nonnative residual structure, which has important implications for the thermodynamics and kinetics of folding, as well as for misfolding and aggregation diseases. However, it has been universally accepted that residual structure should not affect the global size scaling of the denatured chain, which obeys the statistics of random coil polymers. Here we use a single-molecule optical technique— fluorescence correlation spectroscopy—to probe the denatured state of a set of repeat proteins containing an increasing number of identical domains, from 2 to 20. The availability of this set allows us to obtain the scaling law for the unfolded state of these proteins, which turns out to be unusually compact, strongly deviating from random coil statistics. The origin of this unexpected behavior is traced to the presence of an extensive nonnative polyproline II helical structure, which we localize to specific segments of the polypeptide chain. We show th...
Tight junctions (TJs) between blood-brain barrier (BBB) endothelial cells construct a robust phys... more Tight junctions (TJs) between blood-brain barrier (BBB) endothelial cells construct a robust physical barrier, whose damage underlies BBB dysfunctions related to several neurodegenerative diseases. What makes these highly specialized BBB-TJs extremely restrictive remains unknown. Here, we use super-resolution microscopy (dSTORM) to uncover new structural and functional properties of BBB TJs. Focusing on three major components, Nano-scale resolution revealed sparse (occludin) vs. clustered (ZO1/claudin-5) molecular architecture. Developmentally, permeable TJs become first restrictive to large molecules, and only later to small molecules, with claudin-5 proteins arrangement compacting during this maturation process. Mechanistically, we reveal that ZO1 clustering is independent of claudin-5 in-vivo. In contrast to accepted knowledge, we found that in the developmental context, total levels of claudin-5 inversely correlate with TJ functionality. Our super-resolution studies provide a un...
Detecting targets using Infra-Red (IR) sensor is a common field of investigation. The main proble... more Detecting targets using Infra-Red (IR) sensor is a common field of investigation. The main problematic issue is that the target appears in front of a background that causes false alarms. Increasing the detection threshold decreases the false alarms but also decreases the probability of detection. Knowing the relation between the background's common correlation distance and the target's displacement between sequential samplings is an apriori information that may be used to improve the detection abilities. This apriori information may be estimated from the scene. In this paper we derive a model relating the movement of the target with the statistics of the background so that lower probability of false alarm may be obtained for similar probability of detection or on the other hand higher detection probability for equal probability of false alarm. The obtained improvement is due to the fact that instead of placing a global threshold chosen according to the total spatial and temp...
Cell-cell interfaces convey mechanical and chemical information in multicellular systems. Microsc... more Cell-cell interfaces convey mechanical and chemical information in multicellular systems. Microscopy has revealed intricate structure of such interfaces, yet typically with limited resolution due to diffraction and unfavourable orthogonal orientation of the interface to the coverslip. We present a simple and robust way to align cell-cell interfaces in parallel to the coverslip by adhering the interacting cells to two opposing coverslips. We demonstrate high-quality diffraction-limited and super-resolution imaging of interfaces (immune-synapses) between fixed and live CD8+ T-cells and either antigen presenting cells or melanoma cells. Imaging methods include bright-field, confocal, STED, dSTORM, SOFI, SRRF and large-scale tiled images. The low background, lack of aberrations and enhanced spatial stability of our method relative to existing cell-trapping techniques allow use of these methods. We expect that the simplicity and wide-compatibility of our approach will allow its wide diss...
Intracellular dynamics is highly complex, and includes diffusion of poly-dispersed objects in a n... more Intracellular dynamics is highly complex, and includes diffusion of poly-dispersed objects in a non-homogeneous, out-of-equilibrium medium. Assuming non-equilibrium steady-state, we developed a framework that relates non-equilibrium fluctuations to diffusion, and generalized entropy in cells. We employed imaging of live Jurkat T cells, and showed that active cells have higher diffusion parameters (Kα and α) and entropy relative to the same cells after ATP depletion. Kα and α were related in ATP-depleted cells while this relation was not apparent in untreated cells, probably due to non-equilibrium applied work. Next we evaluated the effect of intracellular diffusion and entropy on the cell content homogeneity, which was displayed by the extent of its liquid–liquid phase separation (LLPS). Correlations between intracellular diffusion parameters, entropy and cell homogeneity could be demonstrated only in active cells while these correlations disappeared after ATP depletion. We conclude...
Organization of intracellular content is affected by multiple simultaneous processes, including d... more Organization of intracellular content is affected by multiple simultaneous processes, including diffusion in a viscoelastic and structured environment, intracellular mechanical work and vibrations. The combined effects of these processes on intracellular organization are complex and remain poorly understood. Here, we studied the organization and dynamics of a free Ca++ probe as a small and mobile tracer in live T cells. Ca++, highlighted by Fluo-4, is localized in intracellular organelles. Inhibiting intracellular mechanical work by myosin II through blebbistatin treatment increased cellular dis-homogeneity of Ca++-rich features in length scale < 1.1 μm. We detected a similar effect in cells imaged by label-free bright-field (BF) microscopy, in mitochondria-highlighted cells and in ATP-depleted cells. Blebbistatin treatment also reduced the dynamics of the Ca++-rich features and generated prominent negative temporal correlations in their signals. Following Guggenberger et al. and...
Single-molecule-localization-microscopy (SMLM) enables superresolution imaging of biological samp... more Single-molecule-localization-microscopy (SMLM) enables superresolution imaging of biological samples down to ~ 10–20 nm and in single molecule detail. However, common SMLM reconstruction largely disregards information embedded in the entire intensity trajectories of individual emitters. Here, we develop and demonstrate an approach, termed time-correlated-SMLM (tcSMLM), that uses such information for enhancing SMLM reconstruction. Specifically, tcSMLM is shown to increase the spatial resolution and fidelity of SMLM reconstruction of both simulated and experimental data; esp. upon acquisition under stringent conditions of low SNR, high acquisition rate and high density of emitters. We further provide detailed guidelines and optimization procedures for effectively applying tcSMLM to data of choice. Importantly, our approach can be readily added in tandem to multiple SMLM and related superresolution reconstruction algorithms. Thus, we expect that our approach will become an effective an...
Intracellular mechanical work facilitates multiple cell functions, such as material transport, ce... more Intracellular mechanical work facilitates multiple cell functions, such as material transport, cell motility, etc., and is indicative of the cell’s physiological condition. Still, the characterization of intracellular mechanical work and resultant dynamics remain hard to determine in intact label-free cells. For that, we imaged live T cells via bright-field microscopy and studied fluctuations in the homogeneity of their intracellular medium. Specifically, we characterized medium homogeneity and dynamics by using the information entropy of its related intensity gray levels (termed Gray Level Information Entropy (GLIE)) and spectral analysis of GLIE fluctuations, respectively. First, we provide simple examples of particle motion, to demonstrate the utility of our approach. Using this approach, we could further study and distinguish mitochondrial dysfunction and ATP depletion state in live Jurkat cells. The relation of our results to intracellular dynamics was confirmed by comparison t...
Unfolded proteins may contain a native or nonnative residual structure, which has important impli... more Unfolded proteins may contain a native or nonnative residual structure, which has important implications for the thermodynamics and kinetics of folding, as well as for misfolding and aggregation diseases. However, it has been universally accepted that residual structure should not affect the global size scaling of the denatured chain, which obeys the statistics of random coil polymers. Here we use a single-molecule optical technique— fluorescence correlation spectroscopy—to probe the denatured state of a set of repeat proteins containing an increasing number of identical domains, from 2 to 20. The availability of this set allows us to obtain the scaling law for the unfolded state of these proteins, which turns out to be unusually compact, strongly deviating from random coil statistics. The origin of this unexpected behavior is traced to the presence of an extensive nonnative polyproline II helical structure, which we localize to specific segments of the polypeptide chain. We show th...
Tight junctions (TJs) between blood-brain barrier (BBB) endothelial cells construct a robust phys... more Tight junctions (TJs) between blood-brain barrier (BBB) endothelial cells construct a robust physical barrier, whose damage underlies BBB dysfunctions related to several neurodegenerative diseases. What makes these highly specialized BBB-TJs extremely restrictive remains unknown. Here, we use super-resolution microscopy (dSTORM) to uncover new structural and functional properties of BBB TJs. Focusing on three major components, Nano-scale resolution revealed sparse (occludin) vs. clustered (ZO1/claudin-5) molecular architecture. Developmentally, permeable TJs become first restrictive to large molecules, and only later to small molecules, with claudin-5 proteins arrangement compacting during this maturation process. Mechanistically, we reveal that ZO1 clustering is independent of claudin-5 in-vivo. In contrast to accepted knowledge, we found that in the developmental context, total levels of claudin-5 inversely correlate with TJ functionality. Our super-resolution studies provide a un...
Detecting targets using Infra-Red (IR) sensor is a common field of investigation. The main proble... more Detecting targets using Infra-Red (IR) sensor is a common field of investigation. The main problematic issue is that the target appears in front of a background that causes false alarms. Increasing the detection threshold decreases the false alarms but also decreases the probability of detection. Knowing the relation between the background's common correlation distance and the target's displacement between sequential samplings is an apriori information that may be used to improve the detection abilities. This apriori information may be estimated from the scene. In this paper we derive a model relating the movement of the target with the statistics of the background so that lower probability of false alarm may be obtained for similar probability of detection or on the other hand higher detection probability for equal probability of false alarm. The obtained improvement is due to the fact that instead of placing a global threshold chosen according to the total spatial and temp...
Cell-cell interfaces convey mechanical and chemical information in multicellular systems. Microsc... more Cell-cell interfaces convey mechanical and chemical information in multicellular systems. Microscopy has revealed intricate structure of such interfaces, yet typically with limited resolution due to diffraction and unfavourable orthogonal orientation of the interface to the coverslip. We present a simple and robust way to align cell-cell interfaces in parallel to the coverslip by adhering the interacting cells to two opposing coverslips. We demonstrate high-quality diffraction-limited and super-resolution imaging of interfaces (immune-synapses) between fixed and live CD8+ T-cells and either antigen presenting cells or melanoma cells. Imaging methods include bright-field, confocal, STED, dSTORM, SOFI, SRRF and large-scale tiled images. The low background, lack of aberrations and enhanced spatial stability of our method relative to existing cell-trapping techniques allow use of these methods. We expect that the simplicity and wide-compatibility of our approach will allow its wide diss...
Intracellular dynamics is highly complex, and includes diffusion of poly-dispersed objects in a n... more Intracellular dynamics is highly complex, and includes diffusion of poly-dispersed objects in a non-homogeneous, out-of-equilibrium medium. Assuming non-equilibrium steady-state, we developed a framework that relates non-equilibrium fluctuations to diffusion, and generalized entropy in cells. We employed imaging of live Jurkat T cells, and showed that active cells have higher diffusion parameters (Kα and α) and entropy relative to the same cells after ATP depletion. Kα and α were related in ATP-depleted cells while this relation was not apparent in untreated cells, probably due to non-equilibrium applied work. Next we evaluated the effect of intracellular diffusion and entropy on the cell content homogeneity, which was displayed by the extent of its liquid–liquid phase separation (LLPS). Correlations between intracellular diffusion parameters, entropy and cell homogeneity could be demonstrated only in active cells while these correlations disappeared after ATP depletion. We conclude...
Organization of intracellular content is affected by multiple simultaneous processes, including d... more Organization of intracellular content is affected by multiple simultaneous processes, including diffusion in a viscoelastic and structured environment, intracellular mechanical work and vibrations. The combined effects of these processes on intracellular organization are complex and remain poorly understood. Here, we studied the organization and dynamics of a free Ca++ probe as a small and mobile tracer in live T cells. Ca++, highlighted by Fluo-4, is localized in intracellular organelles. Inhibiting intracellular mechanical work by myosin II through blebbistatin treatment increased cellular dis-homogeneity of Ca++-rich features in length scale < 1.1 μm. We detected a similar effect in cells imaged by label-free bright-field (BF) microscopy, in mitochondria-highlighted cells and in ATP-depleted cells. Blebbistatin treatment also reduced the dynamics of the Ca++-rich features and generated prominent negative temporal correlations in their signals. Following Guggenberger et al. and...
Single-molecule-localization-microscopy (SMLM) enables superresolution imaging of biological samp... more Single-molecule-localization-microscopy (SMLM) enables superresolution imaging of biological samples down to ~ 10–20 nm and in single molecule detail. However, common SMLM reconstruction largely disregards information embedded in the entire intensity trajectories of individual emitters. Here, we develop and demonstrate an approach, termed time-correlated-SMLM (tcSMLM), that uses such information for enhancing SMLM reconstruction. Specifically, tcSMLM is shown to increase the spatial resolution and fidelity of SMLM reconstruction of both simulated and experimental data; esp. upon acquisition under stringent conditions of low SNR, high acquisition rate and high density of emitters. We further provide detailed guidelines and optimization procedures for effectively applying tcSMLM to data of choice. Importantly, our approach can be readily added in tandem to multiple SMLM and related superresolution reconstruction algorithms. Thus, we expect that our approach will become an effective an...
Intracellular mechanical work facilitates multiple cell functions, such as material transport, ce... more Intracellular mechanical work facilitates multiple cell functions, such as material transport, cell motility, etc., and is indicative of the cell’s physiological condition. Still, the characterization of intracellular mechanical work and resultant dynamics remain hard to determine in intact label-free cells. For that, we imaged live T cells via bright-field microscopy and studied fluctuations in the homogeneity of their intracellular medium. Specifically, we characterized medium homogeneity and dynamics by using the information entropy of its related intensity gray levels (termed Gray Level Information Entropy (GLIE)) and spectral analysis of GLIE fluctuations, respectively. First, we provide simple examples of particle motion, to demonstrate the utility of our approach. Using this approach, we could further study and distinguish mitochondrial dysfunction and ATP depletion state in live Jurkat cells. The relation of our results to intracellular dynamics was confirmed by comparison t...
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