The Role Of Kinesin-2 In Navigating Microtubule Obstacles: Implications For The Regulation Of Axonal Transport

Neurons are specialized cells that transmit information through electrical and chemical signals using structural processes known as dendrites and axons. Dendrites receive information for the cell to interpret while the exceedingly long axon transmits the processed information to its target destination. To ensure the neuron properly carries out its extracellular functions, the orchestration of intracellular cargo (e.g. mitochondria) is critical. This is especially true in the axon, which can be up to a meter in length. There are many challenges involved in the spatial and temporal regulation of cargo over such vast cellular distances. In order to accomplish cargo transport between the cell body and axon terminus the neuron has developed an efficient process to overcome this challenge called axonal transport. Axonal transport utilizes a system of molecular motors coupled to cargo, creating a multi-motor complex, which walks along a set of tracks to position the cargo at the right time and place. One class of molecular motors, called kinesin, are used to traffic cargo away from the cell body and walk along microtubule tracks. These motors work in teams to navigate a complex microtubule landscape that is rich in microtubule-associated proteins (MAPs). One MAP abundantly found within the axon is called Tau and is implicated in a variety of neurodegenerative disorders (e.g. Alzheimer\u27s disease). Much attention has been focused on the kinesin-1 motor while investigating the axonal transport process. However, kinesin-2 plays an equally important role and is not as well characterized as kinesin-1. Previously, it has been demonstrated, in vitro, that Tau disrupts kinesin-1 transport, even below physiological concentrations, however, in vivo evidence suggests the contrary. Given this discrepancy, there are likely other cellular systems in place to provide the necessary navigation of Tau obstacles. One solution may involve multi-motor complexes using two kinesin family members attached to cargo, as both kinesin-1 and kinesin-2 have been observed coupled to cargo. In order to peel away the complex layers of kinesin-1 and kinesin-2 coupled cargo inside the axon, single-molecule imaging techniques were employed to observe the individual behavior of both kinesin-1 and kinesin-2, in vitro. Further, using a combination of genetic engineering, single-molecule analysis and mathematical modeling has helped elucidate differences between these two motors. Kinesin-2 was found to be insensitive to Tau obstacles, unlike kinesin-1, which is in part due to a longer region of the motor called the neck-linker. This region connects the motor domain, which interfaces with the microtubule track, to the coiled-coil stock, which interfaces with the cargo. When the neck-linker lengths were swapped between the motors their behavior in the presence of Tau also switched, and kinesin-2 became sensitive to Tau. To understand kinesin-2\u27s mechanism of navigating Tau obstacles, we looked at the lateral stepping characteristics of both motors. We observed kinesin-2\u27s lateral stepping frequency to be 2-4 fold higher than kinesin-1 and independent of the microtubule obstacle concentration. Thus, kinein-2\u27s longer neck-linker allows a more agile walk along the microtubule surface to navigate obstacles more efficiently than kinesin-1. In a multi-motor complex containing both motors, kinesin-2 is more efficient at maneuvering around MAPs while kinesin-1, which has previously been demonstrated to sustain a higher stall force, is more efficient at towing cargo. This work demonstrates how teams of directionally similar motors may work together to position cargo during axonal transport

Near Real-Time Measurement of Forces Applied by An Optical Trap to A Rigid Cylindrical Object

An automated data acquisition and processing system is established to measure the force applied by an optical trap to an object of unknown composition in real time. Optical traps have been in use for the past 40 years to manipulate microscopic particles, but the magnitude of applied force is often unknown and requires extensive instrument characterization. Measuring or calculating the force applied by an optical trap to nonspherical particles presents additional difficulties which are also overcome with our system. Extensive experiments and measurements using well-characterized objects were performed to verify the system performance

Near Real-Time Measurement of Forces Applied by An Optical Trap to A Rigid Cylindrical Object

An automated data acquisition and processing system is established to measure the force applied by an optical trap to an object of unknown composition in real time. Optical traps have been in use for the past 40 years to manipulate microscopic particles, but the magnitude of applied force is often unknown and requires extensive instrument characterization. Measuring or calculating the force applied by an optical trap to nonspherical particles presents additional difficulties which are also overcome with our system. Extensive experiments and measurements using well-characterized objects were performed to verify the system performance

EB1 directly regulates APC-mediated actin nucleation

EB1 was discovered 25 years ago as a binding partner of the tumor suppressor Adenomatous Polyposis Coli (APC) [1]; however, the significance of EB1-APC interactions has remained poorly understood. EB1 functions at the center of a network of microtubule end-tracking proteins (+TIPs) [2–5], and APC binding to EB1 promotes EB1 association with microtubule ends and microtubule stabilization [6, 7]. Whether or not EB1 interactions govern functions of APC beyond microtubule regulation has not been explored. The C-terminal Basic domain of APC (APC-B) directly nucleates actin assembly, and this activity is required in vivo for directed cell migration and for maintaining normal levels of F-actin [8–10]. Here, we show that EB1 binds APC-B and inhibits its actin nucleation function by blocking actin monomer recruitment. Consistent with these biochemical observations, knocking down EB1 increases F-actin levels in cells, and this can be rescued by disrupting APC-mediated actin nucleation. Conversely, overexpressing EB1 decreases F-actin levels and impairs directed cell migration, without altering microtubule organization and independent of its direct binding interactions with microtubules. Overall, our results define a new function for EB1 in negatively regulating APC-mediated actin assembly. Combining these findings with other recent studies showing that APC interactions regulate EB1-dependent effects on microtubule dynamics [7], we propose that EB1-APC interactions govern bidirectional cytoskeletal crosstalk by coordinating microtubule and actin dynamics

Amphotericin B induced interdigitation of apolipoprotein stabilized nanodisk bilayers

Amphotericin B nanodisks (AMB-ND) are ternary complexes of AMB, phospholipid (PL) and apolipoprotein organized as discrete nanometer scale disk-shaped bilayers. In gel filtration chromatography experiments, empty ND lacking AMB elute as a single population of particles with a molecular weight in the range of 200 kDa. AMB-ND formulated at a 4:1 PL:AMB weight ratio, separated into two peaks. Peak 1 eluted at the position of control ND lacking AMB while the second peak, containing all of the AMB present in the original sample, eluted in the void volume. When ND prepared with increased AMB (1:1 phospholipid:AMB molar ratio) were subjected to gel filtration chromatography, an increased proportion of phospholipid and apolipoprotein were recovered in the void volume with the AMB. Prior to gel filtration the AMB-ND sample could be passed through a 0.22 {micro}m filter without loss of AMB while the voided material was lost. Native gel electrophoresis studies corroborated the gel permeation chromatography data. Far UV circular dichroism analyses revealed that apoA-I associated with AMB-ND denatures at a lower guanidine HCl concentration than apoA-I associated with ND lacking AMB. Atomic force microscopy revealed that AMB induces compression of the ND bilayer thickness consistent with bilayer interdigitation, a phenomenon that is likely related to the ability of AMB to induce pore formation in susceptible membranes

Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles

AbstractTo better understand the incorporation of membrane proteins into discoidal nanolipoprotein particles (NLPs) we have used atomic force microscopy (AFM) to image and analyze NLPs assembled in the presence of bacteriorhodopsin (bR), lipoprotein E4 n-terminal 22k fragment scaffold and DMPC lipid. The self-assembly process produced two distinct NLP populations: those containing inserted bR (bR-NLPs) and those that did not (empty-NLPs). The bR-NLPs were distinguishable from empty-NLPs by an average increase in height of 1.0 nm as measured by AFM. Streptavidin binding to biotinylated bR confirmed that the original 1.0 nm height increase corresponds to br-NLP incorporation. AFM and ion mobility spectrometry (IMS) measurements suggest that NLP size did not vary around a single mean but instead there were several subpopulations, which were separated by discrete diameters. Interestingly, when bR was present during assembly the diameter distribution was shifted to larger particles and the larger particles had a greater likelihood of containing bR than smaller particles, suggesting that membrane proteins alter the mechanism of NLP assembly

Reproducing Musical Instrument Components from Manufacturers’ Technical Drawings using 3D Printing: Boosey & Hawkes as a Case Study

This paper demonstrates how component musical instrument parts can be recreated from manufacturers’ technical drawings using 3D printing. While the application of this technology is not new, previous approaches have usually involved Computer Tomography scanning of extant models. Instead we used surviving technical drawings of clarinet mouthpieces originally manufactured by Boosey & Hawkes. We demonstrated that this non-invasive technique can be used to 3D print musical component parts if the original plans survive, even when extant parts do not. We also demonstrated through qualitative feedback from professional musicians that hand finishing of 3D printed mouthpieces remains essential for skilled performers

Characterization and Reconstruction of Nanolipoprotein Particles (Nlps) by Cryo-EM and Image Reconstruction

Nanolipoprotein particles (NLPs) are small 10-20 nm diameter assemblies of apolipoproteins and lipids. At Lawrence Livermore National Laboratory (LLNL), they have constructed multiple variants of these assemblies. NLPs have been generated from a variety of lipoproteins, including apolipoprotein Al, apolipophorin III, apolipoprotein E4 22K, and MSP1T2 (nanodisc, Inc.). Lipids used included DMPC (bulk of the bilayer material), DMPE (in various amounts), and DPPC. NLPs were made in either the absence or presence of the detergent cholate. They have collected electron microscopy data as a part of the characterization component of this research. Although purified by size exclusion chromatography (SEC), samples are somewhat heterogeneous when analyzed at the nanoscale by negative stained cryo-EM. Images reveal a broad range of shape heterogeneity, suggesting variability in conformational flexibility, in fact, modeling studies point to dynamics of inter-helical loop regions within apolipoproteins as being a possible source for observed variation in NLP size. Initial attempts at three-dimensional reconstructions have proven to be challenging due to this size and shape disparity. They are pursuing a strategy of computational size exclusion to group particles into subpopulations based on average particle diameter. They show here results from their ongoing efforts at statistically and computationally subdividing NLP populations to realize greater homogeneity and then generate 3D reconstructions