Tuning axial and lateral localization precision in 3D super-resolution microscopy with variable astigmatism.

Astigmatism imaging is a three-dimensional (3D) single molecule fluorescence microscopy approach that yields super-resolved spatial information on a rapid time scale from a single image. It is ideally suited for resolving structures on a sub-micrometer scale and temporal behavior in the millisecond regime. While traditional astigmatism imaging utilizes a cylindrical lens, adaptive optics enables the astigmatism to be tuned for the experiment. We demonstrate here how the precisions in x, y, and z are inter-linked and vary with the astigmatism, z-position, and photon level. This experimentally driven and verified approach provides a guide for astigmatism selection in biological imaging strategies.

Calmodulin regulates MGRN1-GP78 interaction mediated ubiquitin proteasomal degradation system.

The mechanism by which the endoplasmic reticulum (ER) ubiquitin ligases sense stress to potentiate their activity is poorly understood. GP78, an ER E3 ligase, is best known for its role in ER-associated protein degradation, although its activity is also linked to mitophagy, ER-mitochondria junctions, and MAPK signaling, thus highlighting the importance of understanding its regulation. In healthy cells, Mahogunin really interesting new gene (RING) finger 1 (MGRN1) interacts with GP78 and proteasomally degrades it to alleviate mitophagy. Here, we identify calmodulin (CaM) as the adapter protein that senses fluctuating cytosolic Ca2+ levels and modulates the Ca2+-dependent MGRN1-GP78 interactions. When stress elevates cytosolic Ca2+ levels in cultured and primary neuronal cells, CaM binds to both E3 ligases and inhibits their interaction. Molecular docking, simulation, and biophysical studies show that CaM interacts with both proteins with different affinities and binding modes. The physiological impact of this interaction switch manifests in the regulation of ER-associated protein degradation, ER-mitochondria junctions, and relative distribution of smooth ER and rough ER.-Mukherjee, R., Bhattacharya, A., Sau, A., Basu, S., Chakrabarti, S., Chakrabarti, O. Calmodulin regulates MGRN1-GP78 interaction mediated ubiquitin proteasomal degradation system.

Constrained Photophysics of 5,7-dimethoxy-2,3,4,9-tetrahydro-1H-carbazol-1-one in the Bioenvironment of Serum Albumins: A Spectroscopic Endeavour Supported by Molecular Docking Analysis.

This paper vividly indicates that steady state as well as time-resolved fluorescence techniques can serve as highly sensitive monitors to explore the interactions of 5,7-dimethoxy-2,3,4,9-tetrahydro-1H-carbazol-1-one with model transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA). Besides these, we have used fluorescence anisotropy study to assess the degree of restrictions imparted by the micro-environments of serum albumins. Again, to speculate the triplet excited state interaction between such fluorophore and albumin proteins (BSA& HSA), laser flash-photolysis experiments have been carried out. Molecular docking experiments have also been performed to support the conclusions obtained from steady state experiments.

Monitoring the Competence of a New Keto-tetrahydrocarbazole Based Fluorosensor Under Homogeneous, Micro-Heterogeneous and Serum Albumin Environments.

We present here a detailed photophysical study of a recently synthesised fluorophore 8-methyl-8,9-dihydro-5H-[1,3]dioxolo[4,5-b]carbazol-6(7H)-one. This is a synthetic precursor of bio-active carbazole skeleton Clausenalene. Spectroscopic investigation of the fluorophore has been carried out in different protic and aprotic solvents, as well as in binary solvent mixtures, using absorption, steady-state and time-resolved fluorescence techniques. This fluorophore is particularly responsive to the hydrogen bonding nature as well as polarity of the solvent molecules. When considered in micelles and ß-cyclodextrin, this behaves as a reporter of its immediate microenvironment. Steady state and time resolved fluorometric and circular dichroism techniques have been used to explore the binding interaction of the fluorophore with transport proteins, bovine serum albumin and human serum albumin. The probable binding sites of the fluorophore in the proteinous environments have been evaluated from fluorescence resonance energy transfer study. Laser flash photolysis experiments also have been performed to observe the triplet excited state interaction between the fluorophore and albumin proteins.

A series of spontaneously blinking dyes for super-resolution microscopy.

Spontaneously blinking fluorophores permit the detection and localization of individual molecules without reducing buffers or caging groups, thus simplifying single-molecule localization microscopy (SMLM). The intrinsic blinking properties of such dyes are dictated by molecular structure and modulated by environment, which can limit utility. We report a series of tuned spontaneously blinking dyes with duty cycles that span two orders of magnitude, allowing facile SMLM in cells and dense biomolecular structures.

Super-resolved 3D tracking of cargo transport through nuclear pore complexes.

Nuclear pore complexes (NPCs) embedded within the nuclear envelope mediate rapid, selective and bidirectional traffic between the cytoplasm and the nucleoplasm. Deciphering the mechanism and dynamics of this process is challenged by the need for high spatial and temporal resolution. We report here a multicolour imaging approach that enables direct three-dimensional visualization of cargo transport trajectories relative to a super-resolved octagonal double-ring structure of the NPC scaffold. The success of this approach is enabled by the high positional stability of NPCs within permeabilized cells, as verified by a combined experimental and simulation analysis. Hourglass-shaped translocation conduits for two cargo complexes representing different nuclear transport receptor pathways indicate rapid migration through the permeability barrier on or near the NPC scaffold. Binding sites for cargo complexes extend more than 100 nm from the pore openings, which is consistent with a wide distribution of the phenylalanine-glycine polypeptides that bind nuclear transport receptors.

Metal nanoparticle alters adenine induced charge transfer kinetics of vitamin K3 in magnetic field.

In this article, we highlight the alterations in the photoinduced electron transfer (ET) and hydrogen atom transfer (HAT) pathways between an anti-tumor drug vitamin-K3 (MQ) and a nucleobase adenine (ADN) in the presence of gold (Au) and iron (Fe) nanoparticles (NPs). Inside the confined micellar media, with laser flash photolysis corroborated with an external magnetic field (MF), we have detected the transient geminate radicals of MQ and ADN, photo-generated through ET and HAT. We observe that the presence of AuNP on the MQ-ADN complex (AuMQ-ADN) assists HAT by limiting the ET channel, on the other hand, FeNP on the MQ-ADN complex (FeMQ-ADN) mostly favors a facile PET. We hypothesize that through selective interactions of the ADN molecules with AuNP and MQ molecules with FeNP, a preferential HAT and PET process is eased. The enhanced HAT and PET have been confirmed by the escape yields of radical intermediates by time-resolved transient absorption spectroscopy in the presence of MF.

Preferential photochemical interaction of Ru (III) doped carbon nano dots with bovine serum albumin over human serum albumin.

The excitation wavelength dependent emission of carbon nano dots (CNDs) restricts their use in photophysical studies. However, instead of bare CNDs, the amine coated Ru (III) doped CNDs (Ru:CNDEDAs) are quite eligible to generate excitation wavelength independent fluorescence with high quantum yield. Herein, we report a detailed study on the photochemical interaction between two different serum albumins, bovine serum albumin (BSA) and human serum albumin (HSA), with Ru:CNDEDAs synthesized in our laboratory, using steady-state and time-resolved spectroscopic techniques. Absorption study reveals the formation of ground state complex between Ru:CNDEDAs and BSA/HSA while the circular dichroism study implies that Ru:CNDEDAs perturbs the secondary structure of the albumin proteins. Steady-state fluorescence study helps in understanding energy transfer from tryptophan, the fluorophore moiety of BSA and HSA, to Ru:CNDEDAs. Time-resolved studies within nanosecond time domain clarify the phenomenon of energy transfer from BSA/HSA to Ru:CNDEDAs with varied efficiency. Molecular dynamic simulation ascertains that the efficiency of energy transfer is highly dependent on the stability of protein-nanoparticle complex. This study provides a qualitative description regarding the structural rigidity of transport protein, BSA compared to HSA, which determines the transport ability of CNDs to deliver the desired drug molecule to the targeted cells.

DNA Damage and Apoptosis Induction in Cancer Cells by Chemically Engineered Thiolated Riboflavin Gold Nanoassembly.

Herein we have engineered a smart nuclear targeting thiol-modified riboflavin-gold nano assembly, RfS@AuNPs, which accumulates selectively in the nucleus without any nuclear-targeting peptides (NLS/RGD) and shows photophysically in vitro DNA intercalation. A theoretical model using Molecular Dynamics has been developed to probe the mechanism of formation and stability as well as dynamics of the RfS@AuNPs in aqueous solution and within the DNA microenvironment. The RfS@AuNPs facilitate the binucleated cell formation that is reflected in the significant increase of DNA damage marker, γ-H2AX as well as the arrest of most of the HeLa cells at the pre-G1 phase indicating cell death. Moreover, a significant upregulation of apoptotic markers confirms that the cell death occurs through the apoptotic pathway. Analyses of the microarray gene expression of RfS@AuNPs treated HeLa cells show significant alterations in vital biological processes necessary for cell survival. Taken together, our study reports a unique nuclear targeting mechanism through targeting the riboflavin receptors, which are upregulated in cancer cells and induce apoptosis in the targeted cells.

Micellar control over tautomerization and photo-induced electron transfer of Lumichrome in the presence of aliphatic and aromatic amines: a transient absorption study.

Lumichrome (Lc), a molecule consisting of a trinuclear alloxazine moiety is our present subject of interest. This molecule is subjected to tautomerization in the presence of pyridine, acetic acid, etc, through the formation of an eight-membered ring. In our present contribution, we have attempted to analyze the influence of the presence of an aliphatic amine, triethylamine (TEA) and an aromatic amine, N,N-dimethylaniline (DMA) in the double proton transfer step of the tautomerization as well as the photo-induced electron transfer (PET) from those amines to Lc. We have studied these phenomena within micelles, anionic and neutral, to observe the effect of confinement. Through our experiments, it could be stated that along with tautomerization and proton transfer, there is also evidence of PET in triplet excited state.

Development of a Triplet-Triplet Absorption Ruler: DNA- and Chromatin-Mediated Drug Molecule Release from a Nanosurface.

Triplet-triplet (T-T) absorption spectroscopy has been used successfully as a molecular ruler to understand the actual release process of sanguinarine as a drug molecule from a gold nanoparticle surface in the presence of cell components, that is, DNA and chromatin. The obtained results have been verified by fluorescence and surface-enhanced Raman spectroscopy (SERS), and a plausible explanation has been put forward to describe the underestimation and overestimation of the percentage (%) of the release of drug molecules measured by fluorescence- and SERS-based techniques, respectively, over the highlighted T-T absorption spectroscopy. Because of the intrinsic nature of absorption, the reported T-T absorption spectroscopic assay overpowers fluorescence- and SERS-based assays, which are limited by the long-range interaction and nonlinear dependence of the concentration of analytes, respectively.