Imaging of mitochondria/lysosomes in live cells and C. elegans.

Two rhodamine-phenothiazine conjugates, RP1 and RP2, were synthesized, and their photophysical properties, subcellular localization, and photocytotoxicity were investigated. We observed robust localization of RP1 in mitochondria and dual localization in mitochondria and lysosomes with RP2 in live cells. Live cell imaging with these probes allowed us to track the dynamics of mitochondria and lysosomes during ROS-induced mitochondrial damage and the subsequent lysosomal digestion of the damaged mitochondria. The fluorophores also demonstrated preferential accumulation in cancer cells compared to normal cells and had strong photo-cytotoxicity. However, no cytotoxicity was observed in the dark. The mitochondrial staining and light-induced ROS production were not limited to mammalian cell lines, but were also observed in the animal model C. elegans. The study demonstrated the potential applications of these probes in visualizing the mitochondria-lysosome cross-talk after ROS production and for photodynamic therapy.

Imaging of lipid droplets using coumarin fluorophores in live cells and C. elegans.

Fluorescent probes offer incredibly effective tools for visualizing the dynamic morphology of lipid droplets (LDs) and investigating their physiological interactions. In this work, we have utilized solvatochromic coumarin probes bearing nitrile and ester substituents for live-cell imaging. The fluorescence probes are characterized by a donor (diethylamino) and acceptor (nitrile and/or ester) substituents and a rotatable double bond. The designed architecture allows investigation of environmental sensitivity apart from providing excellent ability to target sub-cellular organelles. The synthesized fluorophores showed low cytotoxicity and excellent localization within the lipid droplets. Further, the fluorophores were also utilized to study viscosity changes within the LDs induced by Nystatin. More importantly, we also demonstrate imaging of LDs in multi-cellular animal models such as C. elegans.

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 single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

Kinesin-3 motors are fine-tuned at the molecular level to endow distinct mechanical outputs.

BACKGROUND: Kinesin-3 family motors drive diverse cellular processes and have significant clinical importance. The ATPase cycle is integral to the processive motility of kinesin motors to drive long-distance intracellular transport. Our previous work has demonstrated that kinesin-3 motors are fast and superprocessive with high microtubule affinity. However, chemomechanics of these motors remain poorly understood. RESULTS: We purified kinesin-3 motors using the Sf9-baculovirus expression system and demonstrated that their motility properties are on par with the motors expressed in mammalian cells. Using biochemical analysis, we show for the first time that kinesin-3 motors exhibited high ATP turnover rates, which is 1.3- to threefold higher compared to the well-studied kinesin-1 motor. Remarkably, these ATPase rates correlate to their stepping rate, suggesting a tight coupling between chemical and mechanical cycles. Intriguingly, kinesin-3 velocities (KIF1A > KIF13A > KIF13B > KIF16B) show an inverse correlation with their microtubule-binding affinities (KIF1A < KIF13A < KIF13B < KIF16B). We demonstrate that this differential microtubule-binding affinity is largely contributed by the positively charged residues in loop8 of the kinesin-3 motor domain. Furthermore, microtubule gliding and cellular expression studies displayed significant microtubule bending that is influenced by the positively charged insert in the motor domain, K-loop, a hallmark of kinesin-3 family. CONCLUSIONS: Together, we propose that a fine balance between the rate of ATP hydrolysis and microtubule affinity endows kinesin-3 motors with distinct mechanical outputs. The K-loop, a positively charged insert in the loop12 of the kinesin-3 motor domain promotes microtubule bending, an interesting phenomenon often observed in cells, which requires further investigation to understand its cellular and physiological significance.

Toxicological Impact and in Vivo Tracing of Rhodamine Functionalised ZIF-8 Nanoparticles.

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 in vivo tracing the MOF translocation in C. elegans. 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-1. 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-1. 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-1 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-1 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 C. elegans and imaged at low concentrations of ∼0.16 µg mg-1.

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

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.

Novel dual labelled nanoprobes for nanosafety studies: Quantification and imaging experiment of CuO nanoparticles in C. elegans.

Metal oxide nanoparticles have been extensively studied for their toxicological impacts. However, accurate tracing/quantification of the nanomaterials and their biological responses are difficult to measure at low concentrations. To overcome the challenge, we developed a dual-labelling technique of CuO nanoparticles with a stable isotope of 65Cu, and with rhodamine dye. In vivo experiments on C. elegans were performed using natural feeding of Rhodamine B isothiocyanate-(3 aminopropyl) triethoxysilane functionalized 65CuO nanoprobes (RBITC-APTES@65CuO) (size = 7.41 ± 1 nm) within the range of Predicted Environmental Concentration (PEC) of CuO nanoparticles in soil and sediments. Fluorescence emission (570 nm) was detected in the lumen of the intestine and the pharynx of C. elegans with no impact of nanoparticle exposure on the brood size and life span of worms. The ingested fluorescent labelled RBITC-APTES@65CuO nanoprobes did not enter the reproductive system and were distributed in the alimentary canal of C. elegans. Strong fluorescent signals from the ingested RBITC-APTES@65CuO nanoprobes were achieved even after 24 h of exposure demonstrating the high stability of these nanoprobes in vivo. The net accumulation measured of 65Cu in C. elegans after background subtraction was 0.001 µg mg-1 (3.52 %), 0.005 µg mg-1 (1.76 %) and 0.024 µg mg-1 (1.69 %) for an exposure concentration of 0.0284 µg mg-1, 0.284 µg mg-1, and 1.42 µg mg-1 of 65Cu, respectively. Using C. elegans as a model organism, we demonstrated that RBITC-APTES tagged 65CuO nanoparticles acted as novel nanoprobes for measuring the uptake, accumulation, and biodistribution through quantification and imaging the nanoprobes at a very low exposure concentration (65CuO concentration: 0.033 µg mg-1).

Regulation of longevity by depolarization-induced activation of PLC-ß-IP3R signaling in neurons.

Mitochondrial ATP production is a well-known regulator of neuronal excitability. The reciprocal influence of plasma-membrane potential on ATP production, however, remains poorly understood. Here, we describe a mechanism by which depolarized neurons elevate the somatic ATP/ADP ratio in Drosophila glutamatergic neurons. We show that depolarization increased phospholipase-Cß (PLC-ß) activity by promoting the association of the enzyme with its phosphoinositide substrate. Augmented PLC-ß activity led to greater release of endoplasmic reticulum Ca2+ via the inositol trisphosphate receptor (IP3R), increased mitochondrial Ca2+ uptake, and promoted ATP synthesis. Perturbations that decoupled membrane potential from this mode of ATP synthesis led to untrammeled PLC-ß-IP3R activation and a dramatic shortening of Drosophila lifespan. Upon investigating the underlying mechanisms, we found that increased sequestration of Ca2+ into endolysosomes was an intermediary in the regulation of lifespan by IP3Rs. Manipulations that either lowered PLC-ß/IP3R abundance or attenuated endolysosomal Ca2+ overload restored animal longevity. Collectively, our findings demonstrate that depolarization-dependent regulation of PLC-ß-IP3R signaling is required for modulation of the ATP/ADP ratio in healthy glutamatergic neurons, whereas hyperactivation of this axis in chronically depolarized glutamatergic neurons shortens animal lifespan by promoting endolysosomal Ca2+ overload.

Restraint of presynaptic protein levels by Wnd/DLK signaling mediates synaptic defects associated with the kinesin-3 motor Unc-104.

The kinesin-3 family member Unc-104/KIF1A is required for axonal transport of many presynaptic components to synapses, and mutation of this gene results in synaptic dysfunction in mice, flies and worms. Our studies at the Drosophila neuromuscular junction indicate that many synaptic defects in unc-104-null mutants are mediated independently of Unc-104's transport function, via the Wallenda (Wnd)/DLK MAP kinase axonal damage signaling pathway. Wnd signaling becomes activated when Unc-104's function is disrupted, and leads to impairment of synaptic structure and function by restraining the expression level of active zone (AZ) and synaptic vesicle (SV) components. This action concomitantly suppresses the buildup of synaptic proteins in neuronal cell bodies, hence may play an adaptive role to stresses that impair axonal transport. Wnd signaling also becomes activated when pre-synaptic proteins are over-expressed, suggesting the existence of a feedback circuit to match synaptic protein levels to the transport capacity of the axon.

Engineered kinesin motor proteins amenable to small-molecule inhibition.

The human genome encodes 45 kinesin motor proteins that drive cell division, cell motility, intracellular trafficking and ciliary function. Determining the cellular function of each kinesin would benefit from specific small-molecule inhibitors. However, screens have yielded only a few specific inhibitors. Here we present a novel chemical-genetic approach to engineer kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-permeable molecules. Using kinesin-1 as a prototype, we develop two independent strategies to generate inhibitable motors, and characterize the resulting inhibition in single-molecule assays and in cells. We further apply these two strategies to create analogously inhibitable kinesin-3 motors. These inhibitable motors will be of great utility to study the functions of specific kinesins in a dynamic manner in cells and animals. Furthermore, these strategies can be used to generate inhibitable versions of any motor protein of interest.

The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein.

Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury.