Kinesin Moving through the Spotlight: Single-Motor Fluorescence Microscopy with Submillisecond Time Resolution

AbstractKinesin-1 is one of the motor proteins that drive intracellular transport in eukaryotes. This motor makes hundreds of 8-nm steps along a microtubule before releasing. Kinesin-1 can move at velocities of up to ∼800nm/s, which means that one turnover on average takes 10ms. Important details, however, concerning the coordination between the two motor domains have not been determined due to limitations of the techniques used. In this study, we present an approach that allows the observation of fluorescence intensity changes on individual kinesins with a time resolution far better than the duration of a single step. In our approach, the laser focus of a confocal fluorescence microscope is pointed at a microtubule and the photons emitted by fluorescently labeled kinesin motors walking through the spot are detected with submicrosecond accuracy. We show that the autocorrelation of a fluorescence time trace of an individual kinesin motor contains information at time lags down to 0.1ms. The quality and time resolution of the autocorrelation is primarily determined by the amount of signal photons used. By adding the autocorrelations of several tens of kinesins, fluorescence intensity changes can be observed at a timescale below 100μs

Axial accuracy in localization microscopy with 3D point spread function engineering

Single-molecule localization microscopy has developed into a widely used technique to overcome the diffraction limit and enables 3D localization of single-emitters with nanometer precision. A widely used method to enable 3D encoding is to use a cylindrical lens or a phase mask to engineer the point spread function (PSF). The performance of these PSFs is often assessed by comparing the precision they achieve, ignoring accuracy. Nonetheless, accurate localization is required in many applications, such as multi-plane imaging, measuring and modelling of physical processes based on volumetric data, and 3D particle averaging. However, there are PSF model mismatches in the localization schemes due to how reference PSFs are obtained, look-up-tables are created, or spots are fitted. Currently there is little insight in how these model mismatches give rise to systematic axial localization errors, how large these errors are, and how to mitigate them. In this theoretical and simulation work we use a vector PSF model, which incorporates super-critical angle fluorescence (SAF) and the appropriate aplanatic correction factor, to analyze the errors in z-localization. We introduce theory for defining the focal plane in SAF conditions and analyze the predicted axial errors for an astigmatic PSF, double-helix PSF, and saddle-point PSF. These simulations indicate that the absolute axial biases can be as large as 140 nm, 250 nm, and 120 nm for the astigmatic, saddle-point, and double-helix PSF respectively, with relative errors of more than 50%. Finally, we discuss potential experimental methods to verify these findings and propose a workflow to mitigate these effects

Non-genomic steroid signaling through the mineralocorticoid receptor:Involvement of a membrane-associated receptor?

Corticosteroid receptors in the mammalian brain mediate genomic as well as non-genomic actions. Although receptors mediating genomic actions were already cloned 35 years ago, it remains unclear whether the same molecules are responsible for the non-genomic actions or that the latter involve a separate class of receptors. Here we focus on one type of corticosteroid receptors, i.e. the mineralocorticoid receptor (MR). We summarize some of the known properties and the current insight in the localization of the MR in peripheral cells and neurons, especially in relation to non-genomic signaling. Previous studies from our own and other labs provided evidence that MRs mediating non-genomic actions are identical to the ones involved in genomic signaling, but may be translocated to the plasma cell membrane instead of the nucleus. With fixed cell imaging and live cell imaging techniques we tried to visualize these presumed membrane-associated MRs, using antibodies or overexpression of MR-GFP in COS7 and hippocampal cultured neurons. Despite the physiological evidence for MR location in or close to the cell membrane, we could not convincingly visualize membrane localization of endogenous MRs or GFP-MR molecules. However, we did find punctae of labeled antibodies intracellularly, which might indicate transactivating spots of MR near the membrane. We also found some evidence for trafficking of MR via beta-arrestins. In beta-arrestin knockout mice, we didn't observe metaplasticity in the basolateral amygdala anymore, indicating that internalization of MRs could play a role during corticosterone activation. Furthermore, we speculate that membrane-associated MRs could act indirectly via activating other membrane located structures like e.g. GPER and/or receptor tyrosine kinases

К вопросу об устойчивости сопряжений капитальных выработок глубоких шахт

Наведений аналіз стану сполучень протяжних виробок. Розглянуті умови підтримання похилих виробок та сполучень на шахті ім. В.М. Бажанова. Визначені розрахункові показники параметрів сполучень виробок. Наведені результати шахтних досліджень за станом сполучень капітальних похилих виробок шахти ім. В.М. Бажанова.The analysis of the state of pairings of the extended workings is resulted. The terms of maintenance of the sloping workings and pairings are considered on a mine the name of V.M. Bazhanova. The calculation indexes of parameters of pairings of workings are certain. The results of the mine researches are resulted after the state of pairings of the capital sloping workings of mine the name of V.M. Bazhanova

Microtubule cross-linking triggers the directional motility of kinesin-5

Although assembly of the mitotic spindle is known to be a precisely controlled process, regulation of the key motor proteins involved remains poorly understood. In eukaryotes, homotetrameric kinesin-5 motors are required for bipolar spindle formation. Eg5, the vertebrate kinesin-5, has two modes of motion: an adenosine triphosphate (ATP)–dependent directional mode and a diffusive mode that does not require ATP hydrolysis. We use single-molecule experiments to examine how the switching between these modes is controlled. We find that Eg5 diffuses along individual microtubules without detectable directional bias at close to physiological ionic strength. Eg5's motility becomes directional when bound between two microtubules. Such activation through binding cargo, which, for Eg5, is a second microtubule, is analogous to known mechanisms for other kinesins. In the spindle, this might allow Eg5 to diffuse on single microtubules without hydrolyzing ATP until the motor is activated by binding to another microtubule. This mechanism would increase energy and filament cross-linking efficiency

Local changes in microtubule network mobility instruct neuronal polarization and axon specification

The polarization of neurons into axons and dendrites depends on extracellular cues, intracellular signaling, cytoskeletal rearrangements, and polarized transport, but the interplay between these processes during polarization remains unresolved. Here, we show that axon specification is determined by differences in microtubule network mobility between neurites, regulated by Rho guanosine triphosphatases (GTPases) and extracellular cues. In developing neurons, retrograde microtubule flow prevents the entry of the axon-selective motor protein Kinesin-1 into most neurites. Using inducible assays to control microtubule network flow, we demonstrate that local inhibition of microtubule mobility is sufficient to guide Kinesin-1 into a specific neurite, whereas long-term global inhibition induces the formation of multiple axons. We furthermore show that extracellular mechanical cues and intracellular Rho GTPase signaling control the local differences in microtubule network flow. These results reveal a novel cytoskeletal mechanism for neuronal polarization

Opto-katanin, an optogenetic tool for localized, microtubule disassembly

Microtubules are cytoskeletal polymers that separate chromosomes during mitosis and serve as rails for intracellular transport and organelle positioning. Manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubules are currently lacking. Here, we describe a light-activated system for localized recruitment of the microtubule-severing enzyme katanin. This system, named opto-katanin, uses targeted illumination with blue light to induce rapid, localized, and reversible microtubule depolymerization. This tool allows precise clearing of a subcellular region of microtubules while preserving the rest of the microtubule network, demonstrating that regulation of katanin recruitment to microtubules is sufficient to control its severing activity. The tool is not toxic in the absence of blue light and can be used to disassemble both dynamic and stable microtubules in primary neurons as well as in dividing cells. We show that opto-katanin can be used to locally block vesicle transport and to clarify the dependence of organelle morphology and dynamics on microtubules. Specifically, our data indicate that microtubules are not required for the maintenance of the Golgi stacks or the tubules of the endoplasmic reticulum but are needed for the formation of new membrane tubules. Finally, we demonstrate that this tool can be applied to study the contribution of microtubules to cell mechanics by showing that microtubule bundles can exert forces constricting the nucleus

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

Extending the bandwidth of optical-tweezers interferometry

The extension of the bandwidth of optical-tweezers interferometry was discussed. It was found that the detection bandwidth was extended to at least 100 KHz, either by using wavelengths below 850 nm or by using different detectors at longer wavelengths. The power spectral density of the Brownian motion of micron-sized beads in optical tweezers was also measured

A kinesin-based approach for inducing chromosome-specific mis-segregation in human cells

Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve.</p