In vivo pair correlation microscopy reveals dengue virus capsid protein nucleocytoplasmic bidirectional movement in mammalian infected cells

Flaviviruses are major human disease-causing pathogens, including dengue virus (DENV), Zika virus, yellow fever virus and others. DENV infects hundreds of millions of people per year around the world, causing a tremendous social and economic burden. DENV capsid (C) protein plays an essential role during genome encapsidation and viral particle formation. It has been previously shown that DENV C enters the nucleus in infected cells. However, whether DENV C protein exhibits nuclear export remains unclear. By spatially cross-correlating different regions of the cell, we investigated DENV C movement across the nuclear envelope during the infection cycle. We observed that transport takes place in both directions and with similar translocation times (in the ms time scale) suggesting a bidirectional movement of both C protein import and export.Furthermore, from the pair cross-correlation functions in cytoplasmic or nuclear regions we found two populations of C molecules in each compartment with fast and slow mobilities. While in the cytoplasm the correlation times were in the 2–6 and 40–110 ms range for the fast and slow mobility populations respectively, in the cell nucleus they were 1–10 and 25–140 ms range, respectively. The fast mobility of DENV C in cytoplasmic and nuclear regions agreed with the diffusion coefficients from Brownian motion previously reported from correlation analysis. These studies provide the first evidence of DENV C shuttling from and to the nucleus in infected cells, opening new venues for antiviral interventions.

Transient accumulation and bidirectional movement of KIF13B in primary cilia

Primary cilia are microtubule-based sensory organelles whose assembly and function rely on the conserved bidirectional intraflagellar transport (IFT) system, which is powered by anterograde kinesin-2 and retrograde cytoplasmic dynein 2 motors. Nematodes additionally employ a male-specific kinesin-3 motor, KLP-6, which regulates ciliary content and function by promoting release of bioactive extracellular vesicles (EVs) from cilia. Here we show by live cell imaging that a KLP-6 homolog, KIF13B, undergoes bursts of bidirectional movement within primary cilia of cultured mammalian cells at 0.64 +/- 0.07 μm/s in the anterograde direction and at 0.39 +/- 0.06 μm/s in the retrograde direction, reminiscent of conventional IFT. In addition, we found that KIF13B undergoes EV-like release from the ciliary tip whereas a ciliary membrane marker, SMO-tRFP, remains stably associated with cilia during such EV release. Our results suggest that KIF13B, similar to KLP-6, regulates ciliary membrane content by promoting ciliary EV release, possibly in coordination with conventional IFT.

Stable tug-of-war between kinesin-1 and cytoplasmic dynein upon different ATP and roadblock concentrations

ABSTRACTThe maintenance of intracellular processes, like organelle transport and cell division, depend on bidirectional movement along microtubules. These processes typically require kinesin and dynein motor proteins, which move with opposite directionality. Because both types of motors are often simultaneously bound to the cargo, regulatory mechanisms are required to ensure controlled directional transport. Recently, it has been shown that parameters like mechanical motor activation, ATP concentration and roadblocks on the microtubule surface differentially influence the activity of kinesin and dynein motors in distinct manners. However, how these parameters affect bidirectional transport systems has not been studied. Here, we investigate the regulatory influence of these three parameters using in vitro gliding motility assays and stochastic simulations. We find that the number of active kinesin and dynein motors determines the transport direction and velocity, but that variations in ATP concentration and roadblock density have no significant effect. Thus, factors influencing the force balance between opposite motors appear to be important, whereas the detailed stepping kinetics and bypassing capabilities of the motors only have a small effect.

Anterograde trafficking of ciliary MAP kinase–like ICK/CILK1 by the intraflagellar transport machinery is required for intraciliary retrograde protein trafficking

ICK (also known as CILK1) is a mitogen-activated protein kinase–like kinase localized at the ciliary tip. Its deficiency is known to result in the elongation of cilia and causes ciliopathies in humans. However, little is known about how ICK is transported to the ciliary tip. We here show that the C-terminal noncatalytic region of ICK interacts with the intraflagellar transport (IFT)–B complex of the IFT machinery and participates in its transport to the ciliary tip. Furthermore, total internal reflection fluorescence microscopy demonstrated that ICK undergoes bidirectional movement within cilia, similarly to IFT particles. Analysis of ICK knockout cells demonstrated that ICK deficiency severely impairs the retrograde trafficking of IFT particles and ciliary G protein–coupled receptors. In addition, we found that in ICK knockout cells, ciliary proteins are accumulated at the bulged ciliary tip, which appeared to be torn off and released into the environment as an extracellular vesicle. The exogenous expression of various ICK constructs in ICK knockout cells indicated that the IFT-dependent transport of ICK, as well as its kinase activity and phosphorylation at the canonical TDY motif, is essential for ICK function. Thus, we unequivocally show that ICK transported to the ciliary tip is required for retrograde ciliary protein trafficking and consequently for normal ciliary function.

Stable tug-of-war between kinesin-1 and cytoplasmic dynein upon different ATP and roadblock concentrations

ABSTRACTThe maintenance of intracellular processes like organelle transport and cell division depend on bidirectional movement along microtubules. These processes typically require kinesin and dynein motor proteins which move with opposite directionality. Because both types of motors are often simultaneously bound to the cargo, regulatory mechanisms are required to ensure controlled directional transport. Recently, it has been shown that parameters like mechanical motor activation, ATP concentration and roadblocks on the microtubule surface differentially influence the activity of kinesin and dynein motors in distinct manners. However, how these parameters affect bidirectional transport systems has not been studied. Here, we investigate the regulatory influence of these three parameter using in vitro gliding motility assays and stochastic simulations. We find that the number of active kinesin and dynein motors determines the transport direction and velocity, but that variations in ATP concentration and roadblock density have no significant effect. Thus, factors influencing the force balance between opposite motors appear to be important, whereas the detailed stepping kinetics and bypassing capabilities of the motors have only little effect.

Development of Hand Exoskeleton for Recuperation

About half a million people across the globe suffer from different types of hand injuries and hand impairments. These impairments reduce the quality of living. The present system demands a physician to perform the physiotherapy on the mutilated hand of the patient. Nowadays it has become difficult for the physiotherapists to cater to all their patients given time constraints and increasing numbers of patients. A Hand Exoskeleton was developed for the rehabilitation of the mutilated hand which enables the patient to conduct physiotherapy himself without having to go to a rehabilitation centre. The exoskeleton provides a convenient and cost-effective mechanism for physiotherapy since the patient can do the physiotherapy himself at a time of his convenience and any number of times he wants to. With the exoskeleton, the movement of the good hand is replicated in the mutilated hand of the patient. This exoskeleton can supervise the bidirectional movement of the mutilated fingers. That way, the injured fingers are provided with some movement by the help of the servo motors. The gyroscope attached to the stroke hand determines the angular deflection. This exoskeleton can be used for repetitive, intensive and continuous physiotherapy exercises. It can treat many patients daily with minimum supervision. In the long term, the patient would be rewarded physically, emotionally and economically. The exoskeleton is lightweight and economically affordable for everyone. In this manner, patients are no more dependent on physiotherapists for the exercise. No external help is required by the patients for their daily exercise.

Time-dependent three-dimensional Oldroyd-B nanofluid flow due to bidirectional movement of surface with zero mass flux

Unsteady three-dimensional flow of an incompressible Oldroyd-B nanomaterial is reported in this article. The origin of flow is time-dependent surface spreading in lateral directions transversely taking nanoparticles with zero mass flux. The formulated partial differential system is reframed by similarity variables into ordinary differential system. The obtained system is solved by the process of homotopy analysis for dimensional temperature and concentration of nanoparticles. Physical parameter behavior on temperature and concentrations of nanoparticles is examined using graph and tabular data. The surface temperature is also measured and evaluated, and it is found that the temperature is reduced for greater unsteadiness parameter values. We found that the higher [Formula: see text] enhances the curves of nanoparticle concentration and temperature while these curves retard for the incrementing values of [Formula: see text] The increasing nature of Brownian movement [Formula: see text] and Lewis number Le corresponds to lower profiles of nanoparticles concentration.