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Document Type

Campus-Only Access for One (1) Year

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Physics

Year Degree Awarded

2019

Month Degree Awarded

February

First Advisor

Lori Goldner

Second Advisor

Tobias Baskin

Third Advisor

Anthony Dinsmore

Fourth Advisor

Jonathan Machta

Subject Categories

Biological and Chemical Physics | Biophysics | Statistical, Nonlinear, and Soft Matter Physics

Abstract

The polysaccharide cellulose is the main component of plant cell walls, so it is the most abundant polymer on Earth. While it is widely used in industry due to its remarkable properties, such as renewability and biodegradability, its biosynthesis is still not well understood. The large transmembrane protein Cellulose Synthase Complex (CSC) is responsible for synthesizing cellulose by polymerizing UDP glucose into the constituent glucan chains of cellulose.

In this project, I used variable angle epi-fluorescence microscopy (VAEM) in combination with single-particle tracking to characterize the motion of GFP labeled CSCs in the hypocotyl of Arabidopsis thaliana (A. thaliana) seedlings that are 3 to 4 days old. The CSCs’ motion is known to be guided by cortical microtubules, but no molecular motors are involved. Rather, the motion is thought to be driven by the polymerization and crystallization of the cellulose. A mean-squared displacement analysis shows that CSCs move sub-diffusively on short time scales and undergo a transition to super-diffusive motion on a time scale of about 10 s. The sub-diffusive motion might be explained by trapping. It has been proposed that a Brownian ratchet model can explain super-diffusive motion at longer time scales. This may be true for the mean-squared displacement of the particles; however, I show that the step-size distribution from CSC trajectories is not Gaussian and therefore not consistent with a simple Brownian ratchet model.

I also characterized CSCs’ motility in the model grass, Brachypodium distachyon (B. distachyon). The Baskin lab generated lines in which the CSCs in the B. distachyon are tagged with GFP. The GFP labeled CSCs were imaged in the mesocotyl and root using VAEM then a particle tracking algorithm was used to calculate the average speed of the CSCs. The average speed of the CSCs in the mesocotyl was 164 ± 78 nm min-1 (n = 1451 particles). The average speed in the root was similar. For comparison, average speed of GFP labeled CSCs in the A. thaliana hypocotyl was 184 ± 86 nm min-1 (n = 2755). I also quantified the speed of CSCs in response to inhibitors of cellulose (dichlorobenylnitrile; DCB), microtubules (oryzalin), and actin (latrunculin B). Neither oryzalin nor latrunculin affected the speed of CSCs; whereas, DCB reduced the average speed by about 50% in B. distachyon and by about 35% in A. thaliana.

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