fabricating-multi-component-lipid-nanotube-networks-using-gliding

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...

Sandia National Laboratories

Kinesin motors and their associated filaments, microtubules, are essential to many biological processes. The motor and filament system can be reconstituted in vitro with the surface-adhered motors transporting the filaments along the surface. In this format, the system has been used to study active self-assembly and to power microdevices or perform analyte detection. However, fundamental properties of the system, such as the spacing of the kinesin motors bound to the microtubule and the dynamics of binding, remain poorly understood. We show that Fluorescence Interference Contrast (FLIC) microscopy can illuminate the exact height of the microtubule, which for a sufficiently low surface density of kinesin, reveals the locations of the bound motors. We examine the spacing of the kinesin motors on the microtubules at various kinesin surface densities and compare the results with theory. FLIC reveals that the system is highly dynamic, with kinesin binding and unbinding along the length of the microtubule as it is transported along the surface.

Kinesin motor density and dynamics in gliding microtubule motility.

Structural defects can determine and influence various properties of materials, and many technologies rely on the manipulation of defects (e.g., semiconductor industries). In biological systems, management of defects/errors (e.g. DNA repair) is critical to an organism's survival, which has inspired the design of artificial nanomachines that mimic nature's ability to detect defects and repair damage. Biological motors have captured considerable attention in developing such capabilities due to their ability to convert energy into directed motion in response to environmental stimuli, which maximizes their ability for detection and repair. The objective of the present study was to develop an understanding of how the presence of non-bonding domains, here considered as a "defect", in microtubule (MT) building blocks affect the kinesin-driven, active assembly of MT spools. The assembly/joining of micron-scale bonding (i.e., biotin-containing) and non-bonding (i.e., no biotin) MTs resulted in segmented MT building blocks consisting of alternating bonding and non-bonding domains. Here, the introduction of these MT building blocks into a kinesin gliding motility assay along with streptavidin-coated quantum dots resulted in the active assembly of spools with altered morphology but retained functionality. Moreover, it was noted that non-bonding domains were autonomously and preferentially released from the spools over time, representing a mechanism by which defects may be removed from these structures. Overall, our findings demonstrate that this active assembly system has an intrinsic ability for quality control, which can be potentially expanded to a wide range of applications such as self-regulation and healing of active materials.

How non-bonding domains affect the active assembly of microtubule spools.

Cytoskeletal filaments and motor proteins are critical components in the transport and reorganization of membrane-based organelles in eukaryotic cells. Previous studies have recapitulated the microtubule-kinesin transport system in vitro to dynamically assemble large-scale nanotube networks from multilamellar liposomes and polymersomes. Moving toward more biologically relevant systems, the present work examines whether lipid nanotube (LNT) networks can be generated from giant unilamellar vesicles (GUVs) and subsequently characterizes how the lipid composition may be tuned to alter the dynamics, structure, and fluidity of networks. Here, we describe a two-step process in which microtubule motility (i) drives the transport and aggregation of GUVs to form structures with a decreased energy barrier for LNT formation and (ii) extrudes LNTs without destroying parent GUVs, allowing for the formation of large LNT networks. We further show that the lipid composition of the GUV influences formation and morphology of the extruded LNTs and associated networks. For example, LNTs formed from phase-separated GUVs (e.g., liquid-solid phase-separated and coexisting liquid-ordered and liquid-disordered phase-separated) display morphologies related to the specific phase behavior reflective of the parent GUVs. Overall, the ability to form nanotubes from compositionally complex vesicles opens the door to generating lipid networks that more closely mimic the structure and function of those found in cellular systems.

Multicomponent and Multiphase Lipid Nanotubes Formed by Gliding Microtubule-Kinesin Motility and Phase-Separated Giant Unilamellar Vesicles.

Dynamic instability of microtubules is characterized by stochastically alternating phases of growth and shrinkage and is hypothesized to be controlled by the conformation and nucleotide state of tubulin dimers within the microtubule lattice. Specifically, conformation changes (compression) in the tubulin dimer following the hydrolysis of GTP have been suggested to generate stress and drive depolymerization. In the present study, molecular dynamics simulations were used in tandem with in vitro experiments to investigate changes in depolymerization based on the presence of islands of uncompressed (GMPCPP) dimers in the microtubule lattice. Both methods revealed an exponential decay in the kinetic rate of depolymerization corresponding to the relative level of uncompressed (GMPCPP) dimers, beginning at approximately 20% incorporation. This slowdown was accompanied by a distinct morphological change from unpeeling "ram's horns" to blunt-ended dissociation at the microtubule end. Collectively these data demonstrated that islands of uncompressed dimers can alter the mechanism and kinetics of depolymerization in a manner consistent with promoting rescue events.

Zachary I. Imam

Center for Integrated Nanotechnologies

Sandia National Laboratories

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Zachary I. Imam

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Sandia National Laboratories

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Center for Integrated Nanotechnologies