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The gliding motility of microtubule filaments has been used to study the biophysical properties of kinesin motors, as well as being used in a variety of nanotechnological applications. While microtubules are generally stabilized in vitro with paclitaxel (Taxol®), osmolytes such as polyethylene glycol (PEG) and trimethylamine N-oxide (TMAO) are also able to inhibit depolymerization over extended periods of time. High concentrations of TMAO have also been reported to reversibly inhibit kinesin motility of paclitaxel-stabilized microtubules. Here, we examined the effects of the osmolytes PEG, TMAO, and glycerol on stabilizing microtubules during gliding motility on kinesin-coated substrates. As previously observed, microtubule depolymerization was inhibited in a concentration dependent manner by the addition of the different osmolytes. Kinesin-driven motility also exhibited concentration dependent effects with the addition of the osmolytes, specifically reducing the velocity, increasing rates of pinning, and altering trajectories of the microtubules. These data suggest that there is a delicate balance between the ability of osmolytes to stabilize microtubules without inhibiting motility. Overall, these findings provide a more comprehensive understanding of how osmolytes affect the dynamics of microtubules and kinesin motors, and their interactions in crowded environments.

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (

Most small molecule chemotherapeutics must cross one or more cellular membrane barriers to reach their biochemical targets. Owing to the relatively low solubility of chemotherapeutics in the lipid membrane environment, high doses are often required to achieve a therapeutic effect. The resulting systemic toxicity has motivated efforts to improve the efficiency of chemotherapeutic delivery to the cellular interior. Toward this end, liposomes containing lipids with cationic head groups have been shown to permeabilize cellular membranes, resulting in the more efficient release of encapsulated drugs into the cytoplasm. However, the high concentrations of cationic lipids required to achieve efficient delivery remain a key limitation, frequently resulting in toxicity. Toward overcoming this limitation, here, we investigate the ability of ternary lipid mixtures to enhance liposomal delivery. Specifically, we investigate the delivery of the chemotherapeutic, doxorubicin, using ternary liposomes that are homogeneous at physiological temperature but have the potential to undergo membrane phase separation upon contact with the cell surface. This approach, which relies upon the ability of membrane phase boundaries to promote drug release, provides a novel method for reducing the overall concentration of cationic lipids required for efficient delivery. Our results show that this approach improves the performance of doxorubicin by up to 5-fold in comparison to the delivery of the same drug by conventional liposomes. These data demonstrate that ternary lipid compositions and cationic lipids can be combined synergistically to substantially improve the efficiency of chemotherapeutic delivery in vitro.

The gliding motility of microtubule filaments has been used to study the biophysical properties of kinesin motors, as well as being used in a variety of nanotechnological applications. While microtubules are generally stabilized

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.

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.

From viruses to organelles, fusion of biological membranes is used by diverse biological systems to deliver macromolecules across membrane barriers. Membrane fusion is also a potentially efficient mechanism for the delivery of macromolecular therapeutics to the cellular cytoplasm. However, a key shortcoming of existing fusogenic liposomal systems is that they are inefficient, requiring a high concentration of fusion-promoting lipids in order to cross cellular membrane barriers. Toward addressing this limitation, our experiments explore the extent to which membrane fusion can be amplified by using the process of lipid membrane phase separation to concentrate fusion-promoting lipids within distinct regions of the membrane surface. We used confocal fluorescence microscopy to investigate the integration of fusion-promoting lipids into a ternary lipid membrane system that separated into liquid-ordered and liquid-disordered membrane phases. Additionally, we quantified the impact of membrane phase separation on the efficiency with which liposomes transferred lipids and encapsulated macromolecules to cells, using a combination of confocal fluorescence imaging and flow cytometry. Here we report that concentrating fusion-promoting lipids within phase-separated lipid domains on the surfaces of liposomes significantly increases the efficiency of liposome fusion with model membranes and cells. In particular, membrane phase separation enhanced the delivery of lipids and model macromolecules to the cytoplasm of tumor cells by at least four-fold in comparison to homogenous liposomes. Our findings demonstrate that phase separation can enhance membrane fusion by locally concentrating fusion-promoting lipids on the surface of liposomes. This work represents the first application of lipid membrane phase separation in the design of biomaterials-based delivery systems. Additionally, these results lay the ground work for developing fusogenic liposomes that are triggered by physical and molecular cues associated with target cells.

From viruses to organelles, fusion of biological membranes is used by diverse biological systems to deliver macromolecules across membrane barriers. Membrane fusion is also a potentially efficient mechanism for the delivery of macromolecular therapeutics to the cellular cytoplasm. However, a key shortcoming of existing fusogenic liposomal systems is that they are inefficient, requiring a high concentration of fusion-promoting lipids in order to cross cellular membrane barriers.Toward addressing this limitation, our experiments explore the extent to which membrane fusion can be amplified by using the process of lipid membrane phase separation to concentrate fusion-promoting lipids within distinct regions of the membrane surface.We used confocal fluorescence microscopy to investigate the integration of fusion-promoting lipids into a ternary lipid membrane system that separated into liquid-ordered and liquid-disordered membrane phases. Additionally, we quantified the impact of membrane phase separation on the efficiency with which liposomes transferred lipids and encapsulated macromolecules to cells, using a combination of confocal fluorescence imaging and flow cytometry.Here we report that concentrating fusion-promoting lipids within phase-separated lipid domains on the surfaces of liposomes significantly increases the efficiency of liposome fusion with model membranes and cells. In particular, membrane phase separation enhanced the delivery of lipids and model macromolecules to the cytoplasm of tumor cells by at least 4-fold in comparison to homogenous liposomes.Our findings demonstrate that phase separation can enhance membrane fusion by locally concentrating fusion-promoting lipids on the surface of liposomes. This work represents the first application of lipid membrane phase separation in the design of biomaterials-based delivery systems. Additionally, these results lay the ground work for developing fusogenic liposomes that are triggered by physical and molecular cues associated with target cells.

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.

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.

Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins that interact with rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers provides insights into the roles rafts play in cells. Multiple studies have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure at membrane surfaces. Furthermore, our recent work has shown that lateral steric pressure at membrane surfaces opposes the assembly of membrane domains. Building on these findings, here we report that membrane-bound polymers are potent suppressors of membrane phase separation, which can destabilize lipid domains with substantially greater efficiency than globular domains such as membrane-bound proteins. Specifically, we created giant vesicles with a ternary lipid composition, which separated into coexisting liquid ordered and disordered phases. Lipids with saturated tails and poly(ethylene glycol) (PEG) chains conjugated to their head groups were included at increasing molar concentrations. When these lipids were sparse on the membrane surface they partitioned to the liquid ordered phase. However, as they became more concentrated, the fraction of GUVs that were phase-separated decreased dramatically, ultimately yielding a population of homogeneous membrane vesicles. Experiments and physical modeling using compositions of increasing PEG molecular weight and lipid miscibility phase transition temperature demonstrate that longer polymers are the most efficient suppressors of membrane phase separation when the energetic barrier to lipid mixing is low. In contrast, as the miscibility transition temperature increases, longer polymers are more readily driven out of domains by the increased steric pressure. Therefore, the concentration of shorter polymers required to suppress phase separation decreases relative to longer polymers. Collectively, our results demonstrate that crowded, membrane-bound polymers are highly efficient suppressors of phase separation and suggest that the ability of lipid domains to resist steric pressure depends on both their lipid composition and the size and concentration of the membrane-bound polymers they incorporate.

Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins thought to congregate in rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers may provide insights into the roles rafts play in cells. Multiple studies of PEG-conjugated lipids have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure. Furthermore, our recent work has shown that steric pressure on membrane surfaces can oppose the assembly of lipid domains. Based on these findings, this work investigates whether steric pressure among membrane-bound polymeric domains could oppose membrane phase separation. To address this question we created GUVs with a ternary composition (DPPC:DOPC:Chol), which separated into coexisting liquid ordered and disordered domains. DPPE-PEG lipids (1, 2, or 5 kDa PEG) were included at increasing molar concentrations. As the distance between PEG molecules on membrane surfaces decreased, the fraction of GUVs that phase separated decreased, suggesting that membrane-bound PEG chains opposed phase separation. Interestingly, for lipid mixtures with a Tm just above room temperature, domains were destabilized when the spacing among PEG chains was greater than their hydrodynamic diameter, suggesting that relatively little pressure was required. In contrast, when we examined GUVs with a Tm significantly above room temperature, domains were not destabilized until the PEG spacing was much less than their hydrodynamic diameter, suggesting that the polymer chains had to pass through the high pressure brush transition to drive lipid mixing. These results demonstrate that steric pressure among membrane-bound polymers can drive mixing of lipid domains and suggest that the ability of cellular lipid rafts to resist this pressure and concentrate polymeric domains likely depends heavily on their lipid composition.