Figure 4.
Figure 4. Dimerized NOD tip tracks on dynamic MTs and directly interacts with EB1 through a new motif. (A) A mitotic cell coexpressing NOD485CC-EGFP (green) and EB1-mCherry (red). Kymographs of dynamic astral MTs within the white box reveal subpopulations of NOD puncta tip-tracking coincident with EB1, remaining associated with depolymerizing plus ends, and walking toward the MT plus ends. (B) Kymograph of motile and tip-tracking NOD485CC (green) dimers on dynamic MTs (red, plus and minus ends labeled in MT kymograph) incubated in cell lysate and subjected to TIRF imaging. (C) Coomassie-stained SDS-PAGE gels showing purified MBP-GFP and MBP-NOD485CC-EGFP (left) and Drosophila GST-EB1-TagRFP-T (right) used in the pulldown assays. (D) Western blot for TagRFP of pulldown assay showing Drosophila GST-EB1-TagRFP-T specifically interacts with MBP-NOD485CC-EGFP, but not with MBP-EGFP (left blot). Asterisks denote αTagRFP cross-reactivity with MBP-EGFP and MBP-NOD485CC-EGFP, which were detected by Western blot with α-GFP (right blot). (E) Coomassie-stained SDS-PAGE gel showing the purified Drosophila EB1-TagRFP-T (GST cleaved off) used to probe the SPOT peptide arrays. (F) SPOT peptide arrays of NOD 400–485, and including a “perfect” SxIP peptide as a positive control, probed with anti-EB1 serum (control) or purified EB1-TagRFP-T followed by incubation with the anti-EB1 serum. Two peptides with positive EB1 binding are highlighted in red (PT motif-1) and blue (PT motif-2). (G) Alanine scans of the two peptides highlighted in F. (H) Coomassie-stained SDS-PAGE gel showing the purified Drosophila GST-EB1 used in MST. (I) MST was done by titrating GST-EB1 while maintaining a constant concentrations of FITC-labeled “perfect” SxIP aptamer (left) or NOD “PT” motif-1 (right) resulting in measurable changes in the fluorescence signal within a temperature gradient that can be used to calculate dissociation constants (SxIP Kd = 807 ± 86 nM and NOD PT motif-1 Kd = 725 ± 77 nM). Error bars represent standard deviation of n = 3 (PT motif-1) and 2 (SxIP motif) MST runs. The Kd values are reported as mean ± SEM. Horizontal bars: 5 µm (A); 1 µm in all kymographs (A and B). Vertical bars: 20 s.

Dimerized NOD tip tracks on dynamic MTs and directly interacts with EB1 through a new motif. (A) A mitotic cell coexpressing NOD485CC-EGFP (green) and EB1-mCherry (red). Kymographs of dynamic astral MTs within the white box reveal subpopulations of NOD puncta tip-tracking coincident with EB1, remaining associated with depolymerizing plus ends, and walking toward the MT plus ends. (B) Kymograph of motile and tip-tracking NOD485CC (green) dimers on dynamic MTs (red, plus and minus ends labeled in MT kymograph) incubated in cell lysate and subjected to TIRF imaging. (C) Coomassie-stained SDS-PAGE gels showing purified MBP-GFP and MBP-NOD485CC-EGFP (left) and Drosophila GST-EB1-TagRFP-T (right) used in the pulldown assays. (D) Western blot for TagRFP of pulldown assay showing Drosophila GST-EB1-TagRFP-T specifically interacts with MBP-NOD485CC-EGFP, but not with MBP-EGFP (left blot). Asterisks denote αTagRFP cross-reactivity with MBP-EGFP and MBP-NOD485CC-EGFP, which were detected by Western blot with α-GFP (right blot). (E) Coomassie-stained SDS-PAGE gel showing the purified Drosophila EB1-TagRFP-T (GST cleaved off) used to probe the SPOT peptide arrays. (F) SPOT peptide arrays of NOD 400–485, and including a “perfect” SxIP peptide as a positive control, probed with anti-EB1 serum (control) or purified EB1-TagRFP-T followed by incubation with the anti-EB1 serum. Two peptides with positive EB1 binding are highlighted in red (PT motif-1) and blue (PT motif-2). (G) Alanine scans of the two peptides highlighted in F. (H) Coomassie-stained SDS-PAGE gel showing the purified Drosophila GST-EB1 used in MST. (I) MST was done by titrating GST-EB1 while maintaining a constant concentrations of FITC-labeled “perfect” SxIP aptamer (left) or NOD “PT” motif-1 (right) resulting in measurable changes in the fluorescence signal within a temperature gradient that can be used to calculate dissociation constants (SxIP Kd = 807 ± 86 nM and NOD PT motif-1 Kd = 725 ± 77 nM). Error bars represent standard deviation of n = 3 (PT motif-1) and 2 (SxIP motif) MST runs. The Kd values are reported as mean ± SEM. Horizontal bars: 5 µm (A); 1 µm in all kymographs (A and B). Vertical bars: 20 s.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal