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DNA intercalators are widely used as fluorescent probes to visualize DNA and DNA transactions in vivo and in vitro . It is well known that they perturb DNA structure and stability, which can in turn influence DNA-processing by proteins. Here we elucidate this perturbation by combining single-dye fluorescence microscopy with force spectroscopy and measuring the kinetics of DNA intercalation by the mono- and bis-intercalating cyanine dyes SYTOX Orange, SYTOX Green, SYBR Gold, YO-PRO-1, YOYO-1 and POPO-3. We show that their DNA-binding affinity is mainly governed by a strongly tension-dependent dissociation rate. These rates can be tuned over a range of seven orders of magnitude by changing DNA tension, intercalating species and ionic strength. We show that optimizing these rates minimizes the impact of intercalators on strand separation and enzymatic activity. These new insights provide handles for the improved use of intercalators as DNA probes with minimal perturbation and maximal efficacy. DNA intercalators, a type of fluorescent probes widely used to visualize DNA, can perturb DNA structure and stability. Here, the authors show how DNA-binding affinity can be tuned using DNA tension, ionic strength and dye species, and how this can be used to minimize DNA structural perturbations.

Kymographs are graphical representations of spatial position over time, which are often used in biology to visualise the motion of fluorescent particles, molecules, vesicles, or organelles moving along a predictable path. Although in kymographs tracks of individual particles are qualitatively easily distinguished, their automated quantitative analysis is much more challenging. Kymographs often exhibit low signal-to-noise-ratios (SNRs), and available tools that automate their analysis usually require manual supervision. Here we developed KymoButler, a Deep Learning-based software to automatically track dynamic processes in kymographs. We demonstrate that KymoButler performs as well as expert manual data analysis on kymographs with complex particle trajectories from a variety of different biological systems. The software was packaged in a web-based ‘one-click’ application for use by the wider scientific community ( https://deepmirror.ai/kymobutler ). Our approach significantly speeds up data analysis, avoids unconscious bias, and represents another step towards the widespread adaptation of Machine Learning techniques in biological data analysis. Many molecules and structures within cells have to move about to do their job. Studying these movements is important to understand many biological processes, including the development of the brain or the spread of viruses. Kymographs are images that represent the movement of particles in time and space. Unfortunately, tracing the lines that represent movement in kymographs of biological particles is hard to do automatically, so currently this analysis is done by hand. Manually annotating kymographs is tedious, time-consuming and prone to the researcher’s unconscious bias. In an effort to simplify the analysis of kymographs, Jakobs et al. have developed KymoButler, a software tool that can do it automatically. KymoButler uses artificial intelligence to trace the lines in a kymograph and extract the information about particle movement. It speeds up analysis of kymographs by between 50 and 250 times, and comparisons show that it is as reliable as manual analysis. KymoButler is also significantly more effective than any previously existing automatic kymograph analysis programme. To make KymoButler accessible, Jakobs et al. have also created a website with a drag-and-drop facility that allows researchers to easily use the tool. KymoButler has been tested in many areas of biological research, from quantifying the movement of molecules in neurons to analysing the dynamics of the scaffolds that help cells keep their shape. This variety of applications showcases KymoButler’s versatility, and its potential applications. Jakobs et al. are further contributing to the field of machine learning in biology with ‘deepmirror.ai’, an online hub with the goal of accelerating the adoption of artificial intelligence in biology.

Cytoplasmic dynein and kinesin-1 are microtubule-based motors with opposite polarity that transport a wide variety of cargo in eukaryotic cells. Many cellular cargos demonstrate bidirectional movement due to the presence of ensembles of dynein and kinesin, but are ultimately sorted with spatial and temporal precision. To investigate the mechanisms that coordinate motor ensemble behavior, we built a programmable synthetic cargo using three-dimensional DNA origami to which varying numbers of DNA oligonucleotide-linked motors could be attached, allowing for control of motor type, number, spacing, and orientation in vitro. In ensembles of one to seven identical-polarity motors, motor number had minimal affect on directional velocity, whereas ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one motor species.

ALS is a fatal neurodegenerative disease characterized by selective motor neuron death resulting in muscle paralysis. Mutations in superoxide dismutase 1 (SOD1) are responsible for a subset of familial cases of ALS. Although evidence from transgenic mice expressing human mutant SOD1 G93A suggests that axonal transport defects may contribute to ALS pathogenesis, our understanding of how these relate to disease progression remains unclear. Using an in vivo assay that allows the characterization of axonal transport in single axons in the intact sciatic nerve, we have identified clear axonal transport deficits in presymptomatic mutant mice. An impairment of axonal retrograde transport may therefore represent one of the earliest axonal pathologies in SOD1 G93A mice, which worsens at an early symptomatic stage. A deficit in axonal transport may therefore be a key pathogenic event in ALS and an early disease indicator of motor neuron degeneration.

Purpose The study aimed to assess the relevance of objective vibratory parameters derived from high-speed videolaryngoscopy (HSV) as a supporting tool, to assist clinicians in establishing the initial diagnosis of benign and malignant glottal organic lesions. Methods The HSV examinations were conducted in 175 subjects: 50 normophonic, 85 subjects with benign vocal fold lesions, and 40 with early glottic cancer; organic lesions were confirmed by histopathologic examination. The parameters, derived from HSV kymography: amplitude, symmetry, and glottal dynamic characteristics, were compared statistically between the groups with the following ROC analysis. Results Among 14 calculated parameters, 10 differed significantly between the groups. Four of them, the average resultant amplitude of the involved vocal fold (AmpInvolvedAvg), average amplitude asymmetry for the whole glottis and its middle third part (AmplAsymAvg; AmplAsymAvg_2/3), and absolute average phase difference (AbsPhaseDiffAvg), showed significant differences between benign and malignant lesions. Amplitude values were decreasing, while asymmetry and phase difference values were increasing with the risk of malignancy. In ROC analysis, the highest AUC was observed for AmpAsymAvg (0.719; p  < 0.0001), and next in order was AmpInvolvedAvg (0.70; p  = 0.0002). Conclusion The golden standard in the diagnosis of organic lesions of glottis remains clinical examination with videolaryngoscopy, confirmed by histopathological examination. Our results showed that measurements of amplitude, asymmetry, and phase of vibrations in malignant vocal fold masses deteriorate significantly in comparison to benign vocal lesions. High-speed videolaryngoscopy could aid their preliminary differentiation noninvasively before histopathological examination; however, further research on larger groups is needed.

Mammalian oocyte maturation involves two asymmetric meiotic divisions that require the positioning of the meiotic spindle near the cortical area from which the extrusion of the polar bodies occurs. Li and colleagues show that the nucleating activity of the Arp2/3 complex, localized at the cortical actin cap, induces actin-filament flow away from the complex, creating a cytoplasmic streaming that pushes the spindle towards the cortex. Mature mammalian oocytes are poised for completing meiosis II (MII) on fertilization by positioning the spindle close to an actomyosin-rich cortical cap 1 , 2 , 3 . Here, we show that the Arp2/3 complex localizes to the cortical cap in a Ran-GTPase-dependent manner and nucleates actin filaments in the cortical cap and a cytoplasmic actin network. Inhibition of Arp2/3 activity leads to rapid dissociation of the spindle from the cortex. Live-cell imaging and spatiotemporal image correlation spectroscopy analysis reveal that actin filaments flow continuously away from the Arp2/3-rich cortex, driving a cytoplasmic streaming expected to exert a net pushing force on the spindle towards the cortex. Arp2/3 inhibition not only diminishes this actin flow and cytoplasmic streaming but also enables a reverse streaming driven by myosin-II-based cortical contraction, moving the spindle away from the cortex. Thus, the asymmetric MII spindle position is dynamically maintained as a result of balanced forces governed by the Arp2/3 complex.

MAIN CONCLUSION : Heat stress changes isoform content and distribution of cytoskeletal subunits in pollen tubes affecting accumulation of secretory vesicles and distribution of sucrose synthase, an enzyme involved in cell wall synthesis. Plants are sessile organisms and are therefore exposed to damages caused by the predictable increase in temperature. We have analyzed the effects of temperatures on the development of pollen tubes by focusing on the cytoskeleton and related processes, such as vesicular transport and cell wall synthesis. First, we show that heat stress affects pollen germination and, to a lesser extent, pollen tube growth. Both, microtubules and actin filaments, are damaged by heat treatment and changes of actin and tubulin isoforms were observed in both cases. Damages to actin filaments mainly concern the actin array present in the subapex, a region critical for determining organelle and vesicle content in the pollen tube apex. In support of this, green fluorescent protein-labeled vesicles are arranged differently between heat-stressed and control samples. In addition, newly secreted cell wall material (labeled by propidium iodide) shows an altered distribution. Damage induced by heat stress also extends to proteins that bind actin and participate in cell wall synthesis, such as sucrose synthase. Ultimately, heat stress affects the cytoskeleton thereby causing alterations in the process of vesicular transport and cell wall deposition.

High-Speed Videoendoscopy (HSV) is becoming a robust tool for the assessment of vocal fold vibration in laboratory investigation and clinical practice. We describe the first successful application of flexible High Speed Videoendoscopy with innovative laser light source conducted in clinical settings. The acquired image and simultaneously recorded audio data are compared to the results obtained by means of a rigid endoscope. We demonstrated that the HSV recordings with fiber-optic laryngoscope have enabled obtaining consistently bright, color images suitable for parametrization of vocal fold oscillation similarly as in the case of the HSV data obtained from a rigid laryngoscope. The comparison of period and amplitude perturbation parameters calculated on the basis of image and audio data acquired from flexible and rigid HSV recording objectively confirm that flexible High-Speed Videoendoscopy is a more suitable method for examination of natural phonation. The HSV-based measures generated from this kymographic analysis are arguably a superior representation of the vocal fold vibrations than the acoustic analysis because their quantification is independent of the vocal tract influences. This experimental study has several implications for further research in the field of HSV application in clinical assessment of glottal pathologies nature and its effect on vocal folds vibrations.

Protein-directed intracellular transport has not been observed in bacteria despite the existence of dynamic protein localization and a complex cytoskeleton. However, protein trafficking has clear potential uses for important cellular processes such as growth, development, chromosome segregation, and motility. Conflicting models have been proposed to explain Myxococcus xanthus motility on solid surfaces, some favoring secretion engines at the rear of cells and others evoking an unknown class of molecular motors distributed along the cell body. Through a combination of fluorescence imaging, force microscopy, and genetic manipulation, we show that membrane-bound cytoplasmic complexes consisting of motor and regulatory proteins are directionally transported down the axis of a cell at constant velocity. This intracellular motion is transmitted to the exterior of the cell and converted to traction forces on the substrate. Thus, this study demonstrates the existence of a conserved class of processive intracellular motors in bacteria and shows how these motors have been adapted to produce cell motility.

In many cell types, bidirectional long-range endosome transport is mediated by the opposing motor proteins dynein and kinesin-3. Here we use a fungal model system to investigate how both motors cooperate in early endosome (EE) motility. It was previously reported that Kin3, a member of the kinesin-3 family, and cytoplasmic dynein mediate bidirectional motility of EEs in the fungus Ustilago maydis. We fused the green fluorescent protein to the endogenous dynein heavy chain and the kin3 gene and visualized both motors and their cargo in the living cells. Whereas kinesin-3 was found on anterograde and retrograde EEs, dynein motors localize only to retrograde organelles. Live cell imaging shows that binding of retrograde moving dynein to anterograde moving endosomes changes the transport direction of the organelles. When dynein is leaving the EEs, the organelles switch back to anterograde kinesin-3-based motility. Quantitative photobleaching and comparison with nuclear pores as an internal calibration standard show that single dynein motors and four to five kinesin-3 motors bind to the organelles. These data suggest that dynein controls kinesin-3 activity on the EEs and thereby determines the long-range motility behavior of the organelles.