Biomod/2011/Columbia/MotorProTeam:Results

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Alignment

Early experiments focused on using flow to align microtubules. The group performed motility assays, then flowed an antifade solution (a solution that removes unattached microtubules from the surface) containing AMP-PNP as a method of securing the microtubules in their positions for imaging. This method proved unreliable. While flow occasionally aligned a small proportion of the microtubules, in general this procedure did not work.

Image of flow test. Angle mean = 2.85°, sd = 55.145°‎

Currently, the team is investigating using a protein coated / blank surface boundary as a method to align microtubules. Microtubules that align themselves into the blank region of the flow cell (held at the positive end by a single kinesin motor protein) do not move out of alignment because the majority of their length is not acted upon by any motor proteins.

The team successfully created a barrier in a flow cell between an area with microtubules and an area without. This was performed by coating an isolated area (created using tape) with casein, and then removing the tape, attaching the top coverslip, and flowing kinesin through. When the motility solution and antifade were prepared with casein, the boundary was less defined when the solutions were prepared without.

Image of boundary with casein in solutions. Angle mean = -5.47°, sd = 50.65°

Image of boundary without casein in solutions. Angle mean on boundary = .14°, sd = 42.60°

Unfortunately, there was no alignment at the boundaries. The average angle of rotation was 8 degrees with a standard deviation of 48 degrees. The experiment was repeated with a higher concentration of kinesin and microtubules in order to make more microtubules at the boundary and to see if the concentration has an affect on the alignment of microtubules.

There was no difference in the alignment patterns between the highly concentrated microtubules and the lower concentrated ones.

Structure Formation

The first structure was built from a thin sheet of PDMS (100 μm). PDMS was spin-coated onto a patterned silicon wafer. Tape was wrapped around the circumference of the silicon wafer and the PDMS sheet was pulled off with the use of ethanol to help remove the thin layer from the surface.

(insert image of pattern here)

Polyurethane was poured inside the PDMS pattern and the top was scraped. The polymer was not viscous, allowing it to easily displace from the top of the PDMS mold. The polymer cured, and a flow cell was built using the pattern as the base. Microtubules stuck to the polymer.

Image of microtubules on polyurethane squares.

Microtubule Attachment

Microtubules successfully bound to a glass surface coated with casein and kinesin motors. Gluteraldehyde was used to cross link the microtubules, and BRB80 was flowed through after the application of gluteraldehyde in order to ensure that the microtubules did indeed adhere to the surface.

Based on this premise, we can assume that gluteraldehyde will bind microtubules to any casein coated surface, including the polyurethane squares that form the structure.