CYK4 relaxes the bias in the off-axis motion by MKLP1 kinesin-6

Centralspindlin, a complex of the MKLP1 kinesin-6 and CYK4 GAP subunits, plays key roles in metazoan cytokinesis. CYK4-binding to the long neck region of MKLP1 restricts the configuration of the two MKLP1 motor domains in the centralspindlin. However, it is unclear how the CYK4-binding modulates the interaction of MKLP1 with a microtubule. Here, we performed three-dimensional nanometry of a microbead coated with multiple MKLP1 molecules on a freely suspended microtubule. We found that beads driven by dimeric MKLP1 exhibited persistently left-handed helical trajectories around the microtubule axis, indicating torque generation. By contrast, centralspindlin, like monomeric MKLP1, showed similarly left-handed but less persistent helical movement with occasional rightward movements. Analysis of the fluctuating helical movement indicated that the MKLP1 stochastically makes off-axis motions biased towards the protofilament on the left. CYK4-binding to the neck domains in MKLP1 enables more flexible off-axis motion of centralspindlin, which would help to avoid obstacles along crowded spindle microtubules

Multichannel Simultaneous Measurements of Single-Molecule Translocation in α-Hemolysin Nanopore Array

We present a microarray system that enables simultaneous monitoring of multiple ionic currents through transmembrane α-hemolysin nanopores arrayed at bilayer lipid membranes. We applied the self-assembling ability of lipid molecules interfaced between an aqueous solution and organic solvent to induce bilayer membrane formation at a microfluidic device; the device consists of a hydrophobic polymer film that serves to suspend the lipid-containing solvent at micrometer-sized apertures as well as to separate the aqueous solution into two chambers. In this study, we confirmed that expeditious and reproducible bilayer formation is realized by control of the composition of the solvent, a mixture of n-decane and 1-hexanol, which permits simultaneous incorporation of the α-hemolysin nanopores to the membrane array. Monitoring the eight wells on the array at once, we obtained a maximum of four relevant, synchronous signals of translocating ionic current through the nanopores. The system was also able to detect translocation events of nucleic acid molecules through the pore via the profile of a blocked current, promising its potential for high-throughput applications

Open-source and low-cost miniature microscope for on-site fluorescence detection

The development of a compact and affordable fluorescence microscope can be a formidable challenge for growing needs in on-site testing and detection of fluorescent labeled biological systems, especially for those who specialize in biology rather than in engineering. In response to such a situation, we present an open-source miniature fluorescence microscope using Raspberry Pi. Our fluorescence microscope, with dimensions of 19.2 × 13.6 × 8.2 cm3 (including the display, computer, light-blocking case, and other operational requirements), not only offers cost-effectiveness (costing less than $500) but is also highly customizable to meet specific application needs. The 12.3-megapixel Raspberry Pi HQ Camera captures high-resolution imagery, while the equipped wide-angle lens provides a field of view measuring 21 × 15 mm2. The integrated wireless LAN in the Raspberry Pi, along with software-controllable high-powered fluorescence LEDs, holds potential for a wide range of applications. This open-source fluorescence microscope offers biohybrid sensor developers a versatile tool to streamline unfamiliar mechanical design tasks and open new opportunities for on-site fluorescence detections

Molecular resolution of a dioleoyl-Sn-glycero-phosphocholine lipid bilayer in liquid by phase modulation atomic force microscopy

International audienceWe present a molecular resolved image of a supported lipid bilayer using atomic force microscopy in liquid conditions. Due to the well-known molecular diffusion processes with velocities of a few nm/s occurring in free-standing membranes, this type of mapping is difficult to achieve. We have obtained a sub-nanometer scale resolution of a dioleoyl-sn-glycero-phosphocholine membrane because of the high sensitivity of our phase modulated microscope and the slowed down diffusion due to the interaction with the substrate. A pair function analysis has revealed local disordered and gel phases with an order range limited to the first neighbour

Efficient Lipid Bilayer Formation by Dipping Lipid-Loaded Microperforated Sheet in Aqueous Solution

This paper describes a method for a bilayer lipid membrane (BLM) formation using a perforated sheet along with an open chamber. Microscopic observation of the formed membrane showed a typical droplet interface bilayer. We proved that the formed membrane was a BLM based on electrical measurements of the membrane protein α-hemolysin, which produces nanopores in BLMs. Unlike the conventional approach for BLM formation based on the droplet contact method, this method provides aqueous surfaces with no organic solvent coating layer. Hence, this method is suitable for producing BLMs that facilitate the direct addition of chemicals into the aqueous phase