By Simone Siegel and Avery Baker (Dr. Shubeita) Molecular Motors By Simone Siegel and Avery Baker (Dr. Shubeita)

Molecular Motors Move lipid droplets Have two “heads” Walk along microtubules Take steps, like humans Study kinesin and dynein

Kinesin vs. Dynein Well known Harder to study because of speed Transports lipid droplets to positive end of nuclei Has a step size of 8nm Harder to study because of speed Transports lipid droplets to negative end of nuclei Has an unknown step size http://bernstein.harvard.edu/research/motor_protein.htm

Overall Goal: slow down dynein and measure its step size It is easier to see individual steps when dynein is slowed down Study at colder temps. In vivo (inside the cell) Reducing the temp. breaks ATP bonds less often, less energy released, should move slower

Procedure: fly embryos Learned fruit fly embryo preparation techniques (staging, peeling, mounting) -Peel when about 2.5hrs old -nuclei are arranged on outside of embryo -microtubules run to and away from the nuclei -place on a flow cell, not flattened so embryo doesn’t know it is dead http://www.nature.com/nature/journal/v414/n6864/fig_tab/414593a_F1.html

Obtaining Data Record videos on cold DIC (Differential Interface Contrast) microscope -you can see the lipid droplets moving -their direction indicates whether kinesin or dynein is moving them

http://www.biologicalprocedures.com/bpo/arts/1/138/m138.htm

Analyzing With video, position of lipid droplets can be tracked Excel spreadsheet used to graph the data from the lipid droplet tracked Data shows the speed of the lipid droplet

Graph of distance (nm) vs. time (sec) -Plateaus indicate one or two dynein molecules -Difficulty finding the velocity -Could show dynein taking steps

Graph of slopes of multiple lipid droplet tracks -The y-axis is the velocity of the lipid droplet -Several moved fairly slowly -Higher speeds indicate that more dynein molecules were moving the same lipid droplet -The cooler temperature could have slowed down the speed of movement

http://www.bio.brandeis.edu/flyweb/