Chai Lor1, Joseph D. Lopes2, Michelle K. Mattson-Hoss3, Jing Xu2* and Linda S. Hirst2*
1Biological Engineering and Small-scale Technologies Graduate Program, School of Engineering, University of California Merced, Merced, CA, USA
2Physics Department, School of Natural Science, University of California Merced, Merced, CA, USA
3Developmental and Cell Biology, School of Biological Sciences, University of California Irvine, Irvine, CA, USA
The presence of membrane tubules in living cells is essential to many biological processes. In cells, one mechanism to form nanosized lipid tubules is via molecular motor induced bilayer extraction. In this paper, we describe a simple experimental model to investigate the forces required for lipid tube formation using kinesin motors anchored to 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles. Previous related studies have used molecular motors actively pulling on the membrane to extract a nanotube. Here, we invert the system geometry; molecular motors are used as static anchors linking DOPC vesicles to a two-dimensional microtubule network and an external flow is introduced to generate nanotubes facilitated by the drag force. We found that a drag force of ≈7 pN was sufficient for tubule extraction for vesicles ranging from 1 to 2 μm in radius. By our method, we found that the force generated by a single molecular motor was sufficient for membrane tubule extraction from a spherical lipid vesicle.
Dr Chai Lor successfully defended his PhD thesis in the BEST (Bioengineering and small scale technologies) program on October 26th. Chai was a bioengineering undergraduate at UC Merced and a member of the first graduating class.
“Phase Behavior and Nanotube Formation in Lipid Membranes”
Biological cells are protected by a complex barrier called the lipid membrane. The lipid membrane is a soft material structure consisting of many lipid molecules held together by hydrophobic forces in an aqueous solution. Two simple experimental models were employed to investigate the role of specific lipid molecules in biological membranes. The first model investigated the phase behavior of the binary lipid mixture, 1-dipalmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (DHA-PE) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), using small-angle x-ray scattering (SAXS) and wide-angle x-ray scattering (WAXS). Our results shows that DHA-PE induces phase separation into a DHA rich liquid crystalline (Lα) phase and a DHA poor gel (Lβ’) phase at overall DHA-PE concentrations as low as 0.1mol%. In addition, we find that the structure of the Lβ’ phase, from which the DHA-PE molecules are largely excluded, is modified in the phase-separated state at low DHA-PE concentrations, with a decrease in bilayer thickness of 1.34nm for 0.1mol% at room temperature compared to pure DPPC bilayers. The second model investigated the formation of lipid nanotubes using an anchor system consisting of lipids, kinesin molecular motors, and microtubules in a flow cell. Lipid tubulation was conducted on two different membranes, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and DPPC. Lipid nanotubes were pulled from anchored giant unilamellar vesicles (GUVs) by drag force generated from the flow inside the channel. The results showed that DPPC membranes cannot generate lipid nanotubes while DOPC can, which was expected. We find that a drag force of approximately ≈7.9 pN is sufficient for tubule extraction and that it only requires 1-2 kinesin motor proteins for anchoring the GUV.
“Low concentrations of docosahexanoic acid significantly modify membrane structure and phase behavior” C. Lor and L.S. Hirst, MEMBRANES, 5(4), 857-874 Link (2015)
A dual-beam optical trap is used to trap and manipulate dielectric particles. When the refractive index of these particles is comparable to that of the surrounding medium, equilibrium trapping locations within the system shift from stable to unstable depending on fiber separation and particle size. This is due to to the relationship between gradient and scattering forces. We experimentally and computationally study the transitions between stable and unstable trapping of poly(methyl methacrylate) beads for a range of parameters relevant to experimental setups involving giant unilamellar vesicles. We present stability maps for various fiber separations and particle sizes, and find that careful attention to particle size and configuration is necessary to obtain reproducible quantitative results for soft matter stretching experiments.