Results and Discussion
Originally, we experimented with using 1 μm streptavidin beads, which were labeled with fluorescent green dye. In order to link the beads and visualize the linkers, we polymerized microtubules which were labeled with both biotin linkers and rhodamine dye. This enabled us to both visualize the beads and the microtubules at the same time. However, with this system, we saw very few connections between the beads, due to the vast distances between beads. Even upon increasing the bead concentration many fold, the combination biotin-rhodamine microtubules still failed to form linkers. We conjecture that this system failed because (a) the combined biotin-rhodamine microtubules were not sticky enough (compared to only biotin-functionalized microtubules), and (b) the small beads offered only a very small surface area for the microtubules to bind to, with no possibility of wrapping.
We finally settled on a system involving 7 μm streptavidin coated nile red beads, linked via microtubules labeled with just biotin, which were visualized using an excess of GFP streptavidin to link to the unbound biotin sites. With the 7 μm beads, we were not only able to see attachment to the beads, but we also observed bridges and connections being created between the beads, forming the basis of networks. We hypothesize that the increased surface area of the larger beads, nearly 50 fold greater than that of the 1 μm beads allowed for greater attachment. The microtubules seemed to be overwhelmingly aligned in the horizontal direction due to the flow direction while loading the cell. In order to promote a more even alignment of tubes, the flow cell was placed in a shaking machine, and was shaken in a circular motion for 20 minutes, which resulted in an increased vertical attachment between the microtubules and beads. Furthermore, by increasing the 7 μm bead concentration, we attained more consistency in microtubule-bead connections.
Horizontal attachment observed before shaking.
Vertical and horizontal attachment observed after shaking, creating networks of beads.