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. 2009 Mar 31;7(3):e1000072. doi: 10.1371/journal.pbio.1000072

Figure 2. Truncation of the FHA+CC2 Region Relieves Autoinhibition of Microtubule Binding but Not Processive Motility.

Figure 2

(A) CC prediction for MmKIF1A using windows of 14 (gray line) or 21 (blue line) amino acids (COILS, Lupas Method).

(B) Schematic of mCit-tagged FL and truncated KIF1A constructs.

(C–E) Live-cell microtubule binding assay. COS cells expressing mCit-tagged KIF1A truncations were imaged during SLO permeabilization and AMPPNP treatment. (C) Still images. Scale bar indicates 20 μm. (D) Quantification of the percentage of KIF1A-mCit that changed from cytoplasmic to microtubule (MT)-associated localization (Relocation Index) over time. (1–726), n = 17 cells; (1–491), n = 11 cells; (1–393), n = 12 cells. (E) Percentage of cells whose KIF1A truncations localize to the indicated cellular locations before and after AMPPNP treatment. (Average = three to five experiments, 50–100 cells each).

(F) Processive motility in vivo. Representative images of differentiated CAD cells expressing mCit-tagged constructs. Scale bar indicates 20 μm.

(G and H) Processive motility in vitro. Single-molecule motility assays were carried out using COS cell lysates expressing 3xmCit-tagged constructs. Individual spots were tracked, and population velocities (G) and run lengths (H) were plotted as histograms.