Intracellular transport by kinesin motors moving along their associated cytoskeletal filaments, microtubules, is essential to many biological processes. This active transport system can be reconstituted in vitro with the surface-adhered motors transporting the microtubules across a planar surface. In this geometry, the kinesin-microtubule system has been used to study active self-assembly, to power microdevices, and to perform analyte detection. Fundamental to these applications is the ability to characterize the interactions between the surface tethered motors and microtubules. Fluorescence Interference Contrast (FLIC) microscopy can illuminate the height of the microtubule above a surface, which, at sufficiently low surface densities of kinesin, also reveals the number, locations, and dynamics of the bound motors.
Nuclear pore complexes (NPCs) regulate all cargo traffic across the nuclear envelope. The transport conduit of NPCs is highly enriched in disordered phenylalanine/glycine-rich nucleoporins (FG-Nups), which form a permeability barrier of still elusive and highly debated molecular structure. Here we present a microfluidic device that triggered liquid-to-liquid phase separation of FG-Nups, which yielded droplets that showed typical properties of a liquid state. On the microfluidic chip, droplets were perfused with different transport-competent or -incompetent cargo complexes, and then the permeability barrier properties of the droplets were optically interrogated. We show that the liquid state mimics permeability barrier properties of the physiological nuclear transport pathway in intact NPCs in cells: that is, inert cargoes ranging from small proteins to large capsids were excluded from liquid FG-Nup droplets, but functional import complexes underwent facilitated import into droplets. Collectively, these data provide an experimental model of how NPCs can facilitate fast passage of cargoes across an order of magnitude in cargo size.
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.
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.
Microtubule dynamics play a critical role in the normal physiology of eukaryotic cells as well as a number of cancers and neurodegenerative disorders. The polymerization/depolymerization of microtubules is regulated by a variety of stabilizing and destabilizing factors, including microtubule-associated proteins and therapeutic agents (e.g., paclitaxel, nocodazole). Here we describe the ability of the osmolytes polyethylene glycol (PEG) and trimethylamine-N-oxide (TMAO) to inhibit the depolymerization of individual microtubule filaments for extended periods of time (up to 30 days). We further show that PEG stabilizes microtubules against both temperature- and calcium-induced depolymerization. Our results collectively suggest that the observed inhibition may be related to combination of the kosmotropic behavior and excluded volume/osmotic pressure effects associated with PEG and TMAO. Taken together with prior studies, our data suggest that the physiochemical properties of the local environment can regulate microtubule depolymerization and may potentially play an important role in in vivo microtubule dynamics.
The fundamental biophysics of gliding microtubule (MT) motility by surface-tethered kinesin-1 motor proteins has been widely studied, as well as applied to capture and transport analytes in bioanalytical microdevices. In these systems, phenomena such as molecular wear and fracture into shorter MTs have been reported due the mechanical forces applied on the MT during transport. In the present work, we show that MTs can be split longitudinally into protofilament bundles (PFBs) by the work performed by surface-bound kinesin motors. We examine the properties of these PFBs using several techniques (e.g., fluorescence microscopy, SEM, AFM), and show that the PFBs continue to be mobile on the surface and display very high curvature compared to MT. Further, higher surface density of kinesin motors and shorter kinesin-surface tethers promote PFB formation, whereas modifying MT with GMPCPP or higher paclitaxel concentrations did not affect PFB formation.
Polymersomes are being widely explored as synthetic analogs of lipid vesicles based on their enhanced stability and potential uses in a wide variety of applications in (e.g., drug delivery, cell analogs, etc.). Controlled formation of giant polymersomes for use in membrane studies and cell mimetic systems, however, is currently limited by low-yield production methodologies. Here, we describe for the first time, how the size distribution of giant poly(ethylene glycol)-poly(butadiene) (PEO-PBD) polymersomes formed by gel-assisted rehydration may be controlled based on membrane fluidization. We first show that the average diameter and size distribution of PEO-PBD polymersomes may be readily increased by increasing the temperature of the rehydration solution. Further, we describe a correlative relationship between polymersome size and membrane fluidization through the addition of sucrose during rehydration, enabling the formation of PEO-PBD polymersomes with a range of diameters, including giant-sized vesicles (>100 μm). This correlative relationship suggests that sucrose may function as a small molecule fluidizer during rehydration, enhancing polymer diffusivity during formation and increasing polymersome size. Overall the ability to easily regulate the size of PEO-PBD polymersomes based on membrane fluidity, either through temperature or fluidizers, has broadly applicability in areas including targeted therapeutic delivery and synthetic biology.
Active self-assembly offers a powerful route for the creation of dynamic multiscale structures that are presently inaccessible with standard microfabrication techniques. One such system uses the translation of microtubule filaments by surface-tethered kinesin to actively assemble nanocomposites with bundle, ring, and spool morphologies. Attempts to observe mechanisms involved in this active assembly system have been hampered by experimental difficulties with performing observation during buffer exchange and photodamage from fluorescent excitation. In the present work, we used a custom microfluidic device to remove these limitations and directly study ring/spool formation, including the earliest events (nucleation) that drive subsequent nanocomposite assembly. Three distinct formation events were observed: pinning, collisions, and induced curvature. Of these three, collisions accounted for the majority of event leading to ring/spool formation, while the rate of pinning was shown to be dependent on the amount of photodamage in the system. We further showed that formation mechanism directly affects the diameter and rotation direction of the resultant rings and spools. Overall, the fundamental understanding described in this work provides a foundation by which the properties of motor-driven, actively assembled nanocomposites may be tailored toward specific applications.
Polycarbonate is a desirable material for many applications due to its favorable mechanical and optical properties. Here, we report a simple, safe, environmentally friendly aqueous method that uses diamines to functionalize a polycarbonate surface with amino groups. The use of water as the solvent for the functionalization ensures that solvent induced swelling does not affect the optical or mechanical properties of the polycarbonate. We characterize the efficacy of the surface amination using X-ray photo spectroscopy, Fourier transform infrared spectroscopy (FT-IR), atomic force microscopy (AFM), and contact angle measurements. Furthermore, we demonstrate the ability of this facile method to serve as a foundation upon which other functionalities may be attached, including antifouling coatings and oriented membrane proteins. (Chemical Presented).