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. 2014 Aug 26;26(8):3372–3386. doi: 10.1105/tpc.114.126789

Figure 5.

Figure 5.

ARK1-RFP Overexpression Leads to Increased Microtubule Catastrophe Frequencies.

The ARM domain is able to bind to microtubules. Data and bars represent the mean and sd, respectively. Asterisk represents a significant difference relative to the negative control (i.e., cells not expressing ARK1-RFP) (α = 0.05).

(A) Diagram of all the UBIQUITIN10 promoter translational fusion constructs used for transient expression in Arabidopsis cotyledons. RFP was tagged on the C terminus of all proteins.

(B) Confocal laser micrographs of the UBQ10pro:ARK1-RFP overexpression constructs in ark1-1; 35Spro:GFP-MBD plants. Negative control shows UBQ10pro:RFP expression. Bar = 10 μm.

(C) Cells transiently overexpressing ARK1-RFP have a significant increase in microtubule catastrophe frequency (0.034 ± 0.032 events/second) relative to cells not expressing ARK1-RFP (0.019 ± 0.011 events/second). For ARK1-RFP overexpression and the negative control, 26 and 42 microtubules were measured over a time period of 1700 and 2500 s, respectively.

(D) Distribution of time spent in each phase (growth, shrinkage, and pause) for cells overexpressing and not overexpressing ARK1-RFP in 35Spro:GFP-MBD plants. Microtubules in cells overexpressing ARK1-RFP spent a greater amount of time shrinking as a result of increased catastrophe frequency shown in (C). For ARK1-RFP overexpression and the negative control, 86 and 82 microtubules were visualized over a time period of 8000 and 5800 s, respectively.

(E) The mean microtubule growth and shrinkage velocities in cells overexpressing and not overexpressing ARK1-RFP. There is a significant reduction in growth velocities in ARK1-RFP overexpression cells (3.2 ± 0.1.6 μm/min) relative to the negative control (3.6 ± 1.8 μm/min). There is no significant difference in shrinkage velocities between the two treatments (ARK1-RFP, 7.4 ± 4.1 μm/min; negative control, 7.3 ± 4.2 μm/min). For each treatment, a minimum of 700 growth events and 150 shrinkage events were measured.

(F) Confocal laser micrographs of the various UBQ10pro overexpression constructs seen in (A) expressed in ark1-1; 35Spro:GFP-MBD plants. Microtubule labeling still occurs in constructs lacking the motor or ARM domain. Bars = 10 μm.