Localization and diffusion of tracer particles in viscoelastic media with active force dipoles

Optical tracking in vivo experiments reveal that diffusion of particles in biological cells is strongly enhanced in the presence of ATP and the experimental data for animal cells could previously be reproduced within a phenomenological model of a gel with myosin motors acting within it [EPL 110, 48005 (2015)]. Here, the two-fluid model of a gel is considered where active macromolecules, described as force dipoles, cyclically operate both in the elastic and the fluid components. Through coarse-graining, effective equations of motions for tracer particles displaying local deformations and local fluid flows are derived. The equation for deformation tracers coincides with the earlier phenomenological model and thus confirms it. For flow tracers, diffusion enhancement caused by active force dipoles in the fluid component, and thus due to metabolic activity, is found. The latter effect may explain why ATP-dependent diffusion enhancement could also be observed in bacteria that lack molecular motors in their skeleton or when the activity of myosin motors was chemically inhibited

A Neural Circuit Arbitrates between Persistence and Withdrawal in Hungry Drosophila

In pursuit of food, hungry animals mobilize significant energy resources and overcome exhaustion and fear. How need and motivation control the decision to continue or change behavior is not understood. Using a single fly treadmill, we show that hungry flies persistently track a food odor and increase their effort over repeated trials in the absence of reward suggesting that need dominates negative experience. We further show that odor tracking is regulated by two mushroom body output neurons (MBONs) connecting the MB to the lateral horn. These MBONs, together with dopaminergic neurons and Dop1R2 signaling, control behavioral persistence. Conversely, an octopaminergic neuron, VPM4, which directly innervates one of the MBONs, acts as a brake on odor tracking by connecting feeding and olfaction. Together, our data suggest a function for the MB in internal state-dependent expression of behavior that can be suppressed by external inputs conveying a competing behavioral drive

[89Zr]Oxinate4 for long-term in vivo cell tracking by positron emission tomography

Purpose 111In (typically as [111In]oxinate3) is a gold standard radiolabel for cell tracking in humans by scintigraphy. A long half-life positron-emitting radiolabel to serve the same purpose using positron emission tomography (PET) has long been sought. We aimed to develop an 89Zr PET tracer for cell labelling and compare it with [111In]oxinate3 single photon emission computed tomography (SPECT). Methods [89Zr]Oxinate4 was synthesised and its uptake and efflux were measured in vitro in three cell lines and in human leukocytes. The in vivo biodistribution of eGFP-5T33 murine myeloma cells labelled using [89Zr]oxinate4 or [111In]oxinate3 was monitored for up to 14 days. 89Zr retention by living radiolabelled eGFP-positive cells in vivo was monitored by FACS sorting of liver, spleen and bone marrow cells followed by gamma counting. Results Zr labelling was effective in all cell types with yields comparable with 111In labelling. Retention of 89Zr in cells in vitro after 24 h was significantly better (range 71 to >90 %) than 111In (43–52 %). eGFP-5T33 cells in vivo showed the same early biodistribution whether labelled with 111In or 89Zr (initial pulmonary accumulation followed by migration to liver, spleen and bone marrow), but later translocation of radioactivity to kidneys was much greater for 111In. In liver, spleen and bone marrow at least 92 % of 89Zr remained associated with eGFP-positive cells after 7 days in vivo. Conclusion [89Zr]Oxinate4 offers a potential solution to the emerging need for a long half-life PET tracer for cell tracking in vivo and deserves further evaluation of its effects on survival and behaviour of different cell types

Active mechanics reveal molecular-scale force kinetics in living oocytes

Active diffusion of intracellular components is emerging as an important process in cell biology. This process is mediated by complex assemblies of molecular motors and cytoskeletal filaments that drive force generation in the cytoplasm and facilitate enhanced motion. The kinetics of molecular motors have been precisely characterized in-vitro by single molecule approaches, however, their in-vivo behavior remains elusive. Here, we study the active diffusion of vesicles in mouse oocytes, where this process plays a key role in nuclear positioning during development, and combine an experimental and theoretical framework to extract molecular-scale force kinetics (force, power-stroke, and velocity) of the in-vivo active process. Assuming a single dominant process, we find that the nonequilibrium activity induces rapid kicks of duration $\tau \sim$ 300 $\mu$s resulting in an average force of $F \sim$ 0.4 pN on vesicles in in-vivo oocytes, remarkably similar to the kinetics of in-vitro myosin-V. Our results reveal that measuring in-vivo active fluctuations allows extraction of the molecular-scale activity in agreement with single-molecule studies and demonstrates a mesoscopic framework to access force kinetics.Comment: 20 pages, 4 figures, see ancillary files for Supplementary Materials, * equally contributing author

Lysosomal Delivery of Bioactive Proteins to Living Human Cells via Engineered Exosomes

Exosomes are naturally secreted nanovesicles derived from mammalian cells that are used for intercellular communication in vivo. As a result, they can potentially be used for intracellular delivery of therapeutics for disease treatment. We have developed an exosome pseudotyping approach using vesicular stomatitis virus glycoprotein (VSVG) to produce protein chimeras that optimize production of modified exosomes containing protein therapeutics and facilitate effective delivery to their target cells. To the VSVG transmembrane scaffold, we have fused both fluorescent and luminescent reporters for exosome tracking/visualization and quantification of activity respectively. Through our design, we have shown the biogenesis of VSVG modified exosomes from transfected producer cells through fluorescence imaging and the production of a VSVG-based stable cell line. In addition, we have characterized isolated engineered exosomes and shown that they exhibited the correct size, distribution, and molecular markers, while retaining the bioactivity of their protein cargo. Furthermore, we show that our engineered exosomes and their protein cargo are internalized by multiple cell lines into the endosomal and lysosomal compartments of those cells. Lastly, these modified exosomes can confer their bioactive cargo, either a luminescent reporter or puromycin resistance into these target cells. In summary, this study presents a novel approach to exosome engineering to enhance therapeutic protein loading and delivery, and more importantly, shows the delivery of modified exosomes to intracellular lysosomal compartments. This aspect leads to the assumption that in future studies, these engineered exosomes can be used as a vehicle for delivery of therapeutic proteins for treatment of lysosomal storage diseases

Behavioral Comorbidities and Drug Treatments in a Zebrafish scn1lab Model of Dravet Syndrome.

Loss-of-function mutations in SCN1A cause Dravet syndrome (DS), a catastrophic childhood epilepsy in which patients experience comorbid behavioral conditions, including movement disorders, sleep abnormalities, anxiety, and intellectual disability. To study the functional consequences of voltage-gated sodium channel mutations, we use zebrafish with a loss-of-function mutation in scn1lab, a zebrafish homolog of human SCN1A. Homozygous scn1labs552/s552 mutants exhibit early-life seizures, metabolic deficits, and early death. Here, we developed in vivo assays using scn1labs552 mutants between 3 and 6 d postfertilization (dpf). To evaluate sleep disturbances, we monitored larvae for 24 h with locomotion tracking software. Locomotor activity during dark (night phase) was significantly higher in mutants than in controls. Among anticonvulsant drugs, clemizole and diazepam, but not trazodone or valproic acid, decreased distance moved at night for scn1labs552 mutant larvae. To monitor exploratory behavior in an open field, we tracked larvae in a novel arena. Mutant larvae exhibited impaired exploratory behavior, with increased time spent near the edge of the arena and decreased mobility, suggesting greater anxiety. Both clemizole and diazepam, but not trazodone or valproic acid, decreased distance moved and increased time spent in the center of the arena. Counting inhibitory neurons in vivo revealed no differences between scn1labs552 mutants and siblings. Taken together, our results demonstrate conserved features of sleep, anxiety, and movement disorders in scn1lab mutant zebrafish, and provide evidence that a zebrafish model allows effective tests of treatments for behavioral comorbidities associated with DS

MRI Tracking of Macrophages Labeled with Glucan Particles Entrapping a Water Insoluble Paramagnetic Gd-Based Agent.

PURPOSE: This study is aimed at demonstrating the in vivo potential of Gd(III)-loaded glucan particles (Gd-GPs) as magnetic resonance imaging (MRI)-positive agents for labeling and tracking phagocytic cells. PROCEDURE: GPs were obtained from Saccharomyces cerevisae and loaded with the water-insoluble complex Gd-DOTAMA(C18)2. The uptake kinetics of Gd-GPs by murine macrophages was studied in vitro and the internalization mechanism was assessed by competition assays. The in vivo performance of Gd-GPs was tested at 7.05 T on a mouse model of acute liver inflammation. RESULTS: The minimum number of Gd-GPs-labeled J774.A1 macrophages detected in vitro by MRI was ca. 300 cells/μl of agar, which is the lowest number ever reported for cells labeled with a positive T1 agent. Intravenous injection of macrophages labeled with Gd-GPs in a mouse model of liver inflammation enabled the MRI visualization of the cellular infiltration in the diseased area. CONCLUSIONS: Gd-GPs represent a promising platform for tracking macrophages by MRI as a T1 alternative to the golden standard T2-based iron oxide particles

Experimental and computational analyses reveal that environmental restrictions shape HIV-1 spread in 3D cultures

Here, using an integrative experimental and computational approach, Imle et al. show how cell motility and density affect HIV cell-associated transmission in a three-dimensional tissue-like culture system of CD4+ T cells and collagen, and how different collagen matrices restrict infection by cell-free virions