single-molecule-analysis-sf9-purified-superprocessive-kinesin-3

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Indian Institute of Technology Gandhinagar

Access to safe drinking water is still a distant dream to millions of people around the world. Especially, people from the low-income group in the developing countries remain deprived of this fundamental right and causes millions of death. There is an urgent need to develop affordable and easy to handle water filter which can provide desired drinking water quality without any electricity. In the present work, a simple and low-cost surface engineered particle (SEP) based filter is developed via alkali treatment of soda-lime-silica particle. The SEP based filter can be used as a portable, non-electric, gravity-driven Point-of-Use (POU) water disinfection system. The developed SEP-based filter is capable to arrest the 99.48% (~2 to 2.5 log reduction) of gram-negative bacteria Escherichia coli (E. coli OP50) on its surface from the water containing 3 × 10 cells/ml. No bacterial regrowth is observed in the purified water for 12 h. The performance of SEP bed filter is implicated to the nano-scale surface roughness, its distribution along with the surface charge and surface hydrophobicity which are favorable to attract and adhere the bacteria in the flowing water. The observation is consistent over multiple filtration cycles indicating the suitability of SEP based bed filter for POU water disinfection. The SEP surface with 0.05 mM Ag loading (SEP) completely inactivated (>99.99999%) bacteria and protects any bacteria recontamination in the purified water for its long term usage. The strong and effective silver binding property of SEP surface enables very minimal silver loading and eliminates any health hazard due to low silver leaching (~50 ppb) which is well below the drinking water equivalent level (DWEL ≤ 100 ppb). In rural and urban slum areas of developing countries where no water purification system exists prior to consumption, the easy-to-implement and affordable SEP-based gravity-driven non-electric point-of-use water purifier (materials cost ~ 0.25 USD) can be used to protect millions of lives from water borne diseases.

A Non-electric and Affordable Surface Engineered Particle (SEP) based Point-of-Use (POU) Water Disinfection System.

Investigating the dynamics of different biomolecules in the cellular milieu through microscopic imaging has gained paramount importance in the last decade. Continuous developments in the field of microscopy are paralleled by the design and synthesis of fluorophores that target specific compartments within a cell. In this study, we have synthesized four fluorescent styrene derivatives, a neutral styrylpridine, three cationic styrylpyridinium probes with and without cholesterol tether, and investigated their absorption, emission, and cellular imaging properties. The fluorophores show solvatochromic emission attributed to intramolecular charge transfer from donor to acceptor with an emission range of 500-600 nm. The fluorescent cholesterol conjugate labels plasma membrane effectively while the fluorophores devoid of the cholesterol tether label mitochondria. Cholesterol conjugate also shows strong interaction with liposome membrane. Furthermore, the fluorophores alsotrack the mitochondria in live cells with high specificity. Cell viability assay showed overall non-toxic nature of the probes even at higher fluorophore concentrations. Through sidearm modifications, keeping the fluorescent core intact, we successfully targeted specific subcellular compartments of neuronal (N2a) and non-neuronal (HeLa) mammalian cell lines. This strategy of using a single molecular scaffold with subtle substitutions could be ideal in generating a variety of fluorophores targeting other subcellular compartments.

Imaging mitochondria and plasma membrane in live cells using solvatochromic styrylpyridines.

Lipid droplets (LDs), the lipid-rich intracellular organelles, serve to regulate many physiological processes and therefore attention has been attracted towards their selective detection. We report positively solvatochromic lipophilic dyes, based on the push-pull framework containing coumarin-pyridine heterocycles for selective live-cell imaging of lipid droplets (LDs) in Cos-7 and McA-RH7777 cells at ultralow concentrations (200 nM). The fluorescent probes show a remarkable increase in fluorescence intensity with time with the hydrophobic core of the lipid droplets contributing to the observed intensity enhancement. The simple structural framework, red emission, strong Stokes shift (>80 nm), and excellent biocompatibility highlight their significance as a versatile imaging tool for studying lipid droplets (LDs).

Live-cell imaging of lipid droplets using solvatochromic coumarin derivatives.

Endocytic recycling is a complex itinerary, critical for many cellular processes. Membrane tubulation is a hallmark of recycling endosomes (REs), mediated by KIF13A, a kinesin-3 family motor. Understanding the regulatory mechanism of KIF13A in RE tubulation and cargo recycling is of fundamental importance but is overlooked. Here, we report a unique mechanism of KIF13A dimerization modulated by Rab22A, a small guanosine triphosphatase, during RE tubulation. A conserved proline between neck coil-coiled-coil (NC-CC1) domains of KIF13A creates steric hindrance, rendering the motors as inactive monomers. Rab22A plays an unusual role by binding to NC-CC1 domains of KIF13A, relieving proline-mediated inhibition and facilitating motor dimerization. As a result, KIF13A motors produce balanced motility and force against multiple dyneins in a molecular tug-of-war to regulate RE tubulation and homeostasis. Together, our findings demonstrate that KIF13A motors are tuned at a single-molecule level to function as weak dimers on the cellular cargo.

KIF13A motors are regulated by Rab22A to function as weak dimers inside the cell.

Visualization of sub-cellular organelles allows the determination of various cellular processes and the underlying mechanisms. Herein, we report a fluorescent probe, bearing push-pull substituents emitting at 600 nm and its application in cellular imaging. The probe shows dual imaging of mitochondria and nucleoli and maps mitochondrial viscosity in live cells under various physiological variations and show minimum cytotoxicity. Nucleolar staining is confirmed by RNAase digestion.

Live-cell imaging of the nucleolus and mapping mitochondrial viscosity with a dual function fluorescent probe.

The 'powerhouses' of cell, mitochondria have seen an upsurge of interest in investigations pertaining to the imaging and mapping of physiological processes. By utilizing sterol-modified rhodamine, we have performed the live-cell imaging of mitochondria without dependence on a membrane potential. The sterol probes are highly biocompatible, and they can track the mitochondrial live-cell dynamics in a background-free manner with improved brightness and impressive contrast. This is the first attempt to study the stress response using a direct fluorescence readout with bio-conjugates of rhodamine inside mitochondria. The results pave the way for developing different sterol markers for understanding cellular responses and function.

Stress-responsive rhodamine bioconjugates for membrane-potential-independent mitochondrial live-cell imaging and tracking.

We use two-state ratchet models containing single and coupled Brownian motors to understand the role of motor-microtubule binding, ATPase reaction rate and dimerisation on the translational velocities of Kinesin motors. We use model parameters derived from the experimental measurements on KIF1A, KIF13A, KIF13B, and KIF16B motors to compute velocities in μm/s. We observe that both the models show the same trend in velocities (KIF1A > KIF13A > KIF13B > KIF16B) as the experimental results. However, the models significantly underpredict the velocities when compared with the experiments. The predictions of the coupled-motor model are closer to the experiments than those of the single-motor model. Our results indicate that the variation of ATPase reaction rate governs the trend in velocities for the above four motors. The variation of motor-microtubule binding affinity and the coupling strength between the motor domains may only have a secondary effect. More rigorous models that incorporate the power-stroke mechanism are necessary for better quantitative compliance with the experiments.

Effect of Binding-Affinity and ATPase Activity on the Velocities of Kinesins Using Ratchet Models.

Metal Organic Frameworks (MOFs) are extensively used for a wide range of applications due to their exceptionally high surface area. MOF particles are conventionally in micron size, but the nanosized MOFs show good transportation/mobility due to their small size, and when combined with the high surface area of MOFs, it makes MOF nanoparticles an ideal candidate to study for environmental remediation. Therefore, it is important to study the ecotoxicological impact of these MOFs. In this study, we developed rhodamine labelled nanoparticles of zinc imidazolate metal organic framework (ZIF-8 MOFs) as a means of  tracing the MOF translocation in . Rhodamine B isothiocyanate functionalized ZIF-8 MOFs nanoparticles (RBITC@ZIF-8 MOF nanoparticles; size 44 ± 7 nm) were fed to the worms naturally within a concentration range of 0.16-16.4 μg mg. Fluorescence was detected in the pharyngeal and gut lumen regions of the worms after 4 h of treatment, for exposure concentrations >0.163 μg mg. A higher intensity of fluorescence was observed at the end of 24 h for all exposure concentrations. Worms treated with RBITC@ZIF-8 MOF concentrations of ≥1.63 μg mg for 24 h showed a bright stable fluorescence signal at the tail region. The uptake of RBITC@ZIF-8 MOF for an exposure concentration of 0.163, 1.63, and 8.2 μg mg was found to be 52.1, 11.4 and 28.6%, respectively. Through this study, we showed that RBITC@ZIF-8 MOFs can be exposed to  and imaged at low concentrations of ∼0.16 μg mg.

Article	Year
A Non-electric and Affordable Surface Engineered Particle (SEP) based Point-of-Use (POU) Water Disinfection System. Scientific reports\| PubMed ID: 31796814	2019
Imaging mitochondria and plasma membrane in live cells using solvatochromic styrylpyridines. Journal of photochemistry and photobiology. B, Biology\| PubMed ID: 31864089	2020
Live-cell imaging of lipid droplets using solvatochromic coumarin derivatives. Organic & biomolecular chemistry\| PubMed ID: 32648565	2020
KIF13A motors are regulated by Rab22A to function as weak dimers inside the cell. Science advances\| PubMed ID: 33536208	2021
Live-cell imaging of the nucleolus and mapping mitochondrial viscosity with a dual function fluorescent probe. Organic & biomolecular chemistry\| PubMed ID: 33555275	2021
Stress-responsive rhodamine bioconjugates for membrane-potential-independent mitochondrial live-cell imaging and tracking. Organic & biomolecular chemistry\| PubMed ID: 34610076	2021
Effect of Binding-Affinity and ATPase Activity on the Velocities of Kinesins Using Ratchet Models. Cell biochemistry and biophysics\| PubMed ID: 35088234	2022
Toxicological Impact and Tracing of Rhodamine Functionalised ZIF-8 Nanoparticles. Frontiers in toxicology\| PubMed ID: 35846435	2022

Virupakshi Soppina

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