Myosin-V Opposes Microtubule-Based Cargo Transport and Drives Directional Motility on Cortical Actin

SummaryIntracellular transport is driven by motor proteins that either use microtubules or actin filaments as their tracks [1], but the interplay between these transport pathways is poorly understood [2–4]. Whereas many microtubule-based motors are known to drive long-range transport, several actin-based motors have been proposed to function predominantly in cargo tethering [4–6]. How these opposing activities are integrated on cargoes that contain both types of motors is unknown. Here we use inducible intracellular transport assays to show that acute recruitment of myosin-V to kinesin-propelled cargo reduces their motility near the cell periphery and enhances their localization at the actin-rich cell cortex. Myosin-V arrests rapid microtubule-based transport without the need for regulated auto- or other inhibition of kinesin motors. In addition, myosin-V, despite being an ineffective long-range transporter, can drive slow, medium-range (1–5 μm), point-to-point transport in cortical cell regions. Altogether, these data support a model in which myosin-V establishes local cortical delivery of kinesin-bound cargos through a combination of tethering and active transport

Probing aggrephagy using chemically-induced protein aggregates

Selective types of autophagy mediate the clearance of specific cellular components and are essential to maintain cellular homeostasis. However, tools to directly induce and monitor such pathways are limited. Here we introduce the PIM (particles induced by multimerization) assay as a tool for the study of aggrephagy, the autophagic clearance of aggregates. The assay uses an inducible multimerization module to assemble protein clusters, which upon induction recruit ubiquitin, p62, and LC3 before being delivered to lysosomes. Moreover, use of a dual fluorescent tag allows for the direct observation of cluster delivery to the lysosome. Using flow cytometry and fluorescence microscopy, we show that delivery to the lysosome is partially dependent on p62 and ATG7. This assay will help in elucidating the spatiotemporal dynamics and control mechanisms underlying aggregate clearance by the autophagy-lysosomal system

Uptake, Transport, and Toxicity of Pristine and Weathered Micro- and Nanoplastics in Human Placenta Cells

BACKGROUND: The first evidence of micro- and nanoplastic (MNP) exposure in the human placenta is emerging. However, the toxicokinetics and toxicity of MNPs in the placenta, specifically environmentally relevant particles, remain unclear. OBJECTIVES: We examined the transport, uptake, and toxicity of pristine and experimentally weathered MNPs in nonsyncytialized and syncytialized BeWo b30 choriocarcinoma cells. METHODS: We performed untargeted chemical characterization of pristine and weathered MNPs using liquid chromatography high-resolution mass spectrometry to evaluate compositional differences following particle weathering. We investigated cellular internalization of pristine and weathered polystyrene (PS; 0.05 - 10 μ m ) and high-density polyethylene (HDPE; 0 - 80 μ m ) particles using high-resolution confocal imaging and three-dimensional rendering. We investigated the influence of particle coating with human plasma on the cellular transport of PS particles using a transwell setup and examined the influence of acute MNP exposure on cell viability, damage to the plasma membrane, and expression of genes involved in steroidogenesis. RESULTS: Chemical characterization of MNPs showed a significantly higher number of unique features in pristine particles in comparison with weathered particles. Size-dependent placental uptake of pristine and weathered MNPs was observed in both placental cell types after 24 h exposure. Cellular transport was limited and size-dependent and was not influenced by particle coating with human plasma. None of the MNPs affected cell viability. Damage to the plasma membrane was observed only for 0.05 μ m PS particles in the nonsyncytialized cells at the highest concentration tested ( 100 μ g / mL ). Modest down-regulation of hsd17b1 was observed in syncytialized cells exposed to pristine MNPs. DISCUSSION: Our results suggest that pristine and weathered MNPs are internalized and translocated in placental cells in vitro. Effects on gene expression observed upon pristine PS and HDPE particle exposure warrant further examination. More in-depth investigations are needed to better understand the potential health risks of MNP and chemicals associated with them under environmentally relevant exposure scenarios. https://doi.org/10.1289/EHP10873

Concerted action of kinesins kif5b and kif13b promotes efficient secretory vesicle transport to microtubule plus ends

Intracellular transport relies on multiple kinesins, but it is poorly understood which kinesins are present on particular cargos, what their contributions are and whether they act simultaneously on the same cargo. Here, we show that Rab6-positive secretory vesicles are transported from the Golgi apparatus to the cell periphery by kinesin-1 KIF5B and kinesin-3 KIF13B, which determine the location of secretion events. KIF5B plays a dominant role, whereas KIF13B helps Rab6 vesicles to reach freshly polymerized microtubule ends, to which KIF5B binds poorly, likely because its cofactors, MAP7-family proteins, are slow in populating these ends. Sub-pixel localization demonstrated that during microtubule plus-end directed transport, both kinesins localize to the vesicle front and can be engaged on the same vesicle. When vesicles reverse direction, KIF13B relocates to the middle of the vesicle, while KIF5B shifts to the back, suggesting that KIF5B but not KIF13B undergoes a tug-of-war with a minus-end directed motor.info:eu-repo/semantics/publishe

Тенденції розвитку національної інноваційної системи в Україні

Проаналізовано національну інноваційну систему України. Розглянуто галузі промисловості України за ознаками інноваційної активності та досліджено темпи зростання показників, враховуючи індекс інфляції. Встановлено, що спад темпів зростання динаміки реалізованої продукції призводить до зменшення витрат на інноваційну діяльність.Дан анализ национальной инновационной системы Украины. Рассмотрены отрасли промышленности Украины по признакам инновационной активности и исследованы темпы роста показателей, учитывая индекс инфляции. Установлено, что спад темпов роста динамики реализованной продукции приводит к уменьшению затрат на инновационную деятельность.This article analyses national innovation system of Ukraine. Examined the industry of Ukraine based on innovative activity and investigated the growth indicators, taking into account inflation-index. It is established that the slowdown in the dynamics realized production leads to a decrease in the cost of innovation

EGFR Dynamics Change during Activation in Native Membranes as Revealed by NMR

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase

EGFR Dynamics Change during Activation in Native Membranes as Revealed by NMR

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase

Article Optimization of Curvilinear Tracing Applied to Solar Physics and Biophysics

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Studying neuronal microtubule organization and microtubule-associated proteins using single molecule localization microscopy

The formation and maintenance of highly polarized neurons critically depends on the proper organization of the microtubule (MT) cytoskeleton. In axons, MTs are uniformly oriented with their plus-end pointing outward whereas in mature dendrites MTs have mixed orientations. MT organization and dynamics can be regulated by MT-associated proteins (MAPs). Plus-end tracking proteins are specialized MAPs that decorate plus-ends of growing MTs and regulate neuronal polarity, neurite extension, and dendritic spine morphology. Conventional fluorescence microscopy enables observation of specific cellular components through molecule-specific labeling but provides limited resolution (∼250 nm). Therefore, electron microscopy has until now provided most of our knowledge about the precise MT organization in neurons. In the past decade, super-resolution fluorescence microscopy techniques have emerged that circumvent the diffraction limit of light and enable high-resolution reconstruction of the MT network combined with selective protein labeling. However, preserving MT ultrastructure, MAP binding, high labeling density, and antibody specificity after fixation protocols is still quite challenging. In this chapter, we provide an optimized protocol for two-color direct stochastic optical reconstruction microscopy imaging of neuronal MTs together with their growing plus-ends to probe MT architecture and polarity

Control of endothelial cell polarity and sprouting angiogenesis by non-centrosomal microtubules

Microtubules control different aspects of cell polarization. In cells with a radial microtubule system, a pivotal role in setting up asymmetry is attributed to the relative positioning of the centrosome and the nucleus. Here, we show that centrosome loss had no effect on the ability of endothelial cells to polarize and move in 2D and 3D environments. In contrast, non-centrosomal microtubules stabilized by the microtubule minus-end-binding protein CAMSAP2 were required for directional migration on 2D substrates and for the establishment of polarized cell morphology in soft 3D matrices. CAMSAP2 was also important for persistent endothelial cell sprouting during in vivo zebrafish vessel development. In the absence of CAMSAP2, cell polarization in 3D could be partly rescued by centrosome depletion, indicating that in these conditions the centrosome inhibited cell polarity. We propose that CAMSAP2-protected non-centrosomal microtubules are needed for establishing cell asymmetry by enabling microtubule enrichment in a single-cell protrusion