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TACC3 is a microtubule plus end-tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types.

Nwagbara BU, Faris AE, Bearce EA, Erdogan B, Ebbert PT, Evans MF, Rutherford EL, Enzenbacher TB, Lowery LA - Mol. Biol. Cell (2014)

Bottom Line: Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215.TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends.Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Boston College, Chestnut Hill, MA 02467.

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TACC3 localization on MT plus ends is more distal than EB1 and overlaps with XMAP215. (A–C) Representative time-lapse montage of GFP-TACC3 (A) and mKate2-EB1 (B) accumulation on growing MT plus end. MT is growing to the right. Merged image (C) highlights the spatial arrangement between GFP-TACC3 and mKate2-EB1 localizations. These images were compiled by translating the TACC3 channel on the x-axis, after calculating the frame-to-frame velocity of the growing MT plus end, in order to account for the 1-s time delay between channels, for each examined MT (using ImageJ Translate function). To further confirm correct translation, uncorrected time-lapse colocalizations were examined with both combinations of imaging—red channel first, green channel second; then green channel first, red channel second. (D) Fluorescence intensity profiles of GFP-TACC3 and mKate2-EB1. GFP-TACC3 and mKate2-EB1 signals from 12 individual MTs were quantified by intensity line scans to present the relative fluorescence intensity profiles, with the plus end of the MT toward the right. The highest-intensity peak of GFP-TACC3 is ∼0.5 μm distal to the peak of mKate2-EB1. (E–G) Representative time-lapse montage of mKate2-TACC3 (E) and XMAP215-GFP (F) accumulation on growing MT plus end. MT is growing to the right. Merged image (G) shows that mKate2-TACC3 and XMAP215-GFP localizations overlap. (H) Fluorescence intensity profiles of mKate2-TACC3 and XMAP215-GFP. mKate2-TACC3 and XMAP215-GFP signals from 11 individual MTs were quantified by intensity line scans to present the relative fluorescence intensity profiles. Note that peak intensities of mKate2-TACC3 and XMAP215-GFP closely align. Bar, 0.5 μm.
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Figure 6: TACC3 localization on MT plus ends is more distal than EB1 and overlaps with XMAP215. (A–C) Representative time-lapse montage of GFP-TACC3 (A) and mKate2-EB1 (B) accumulation on growing MT plus end. MT is growing to the right. Merged image (C) highlights the spatial arrangement between GFP-TACC3 and mKate2-EB1 localizations. These images were compiled by translating the TACC3 channel on the x-axis, after calculating the frame-to-frame velocity of the growing MT plus end, in order to account for the 1-s time delay between channels, for each examined MT (using ImageJ Translate function). To further confirm correct translation, uncorrected time-lapse colocalizations were examined with both combinations of imaging—red channel first, green channel second; then green channel first, red channel second. (D) Fluorescence intensity profiles of GFP-TACC3 and mKate2-EB1. GFP-TACC3 and mKate2-EB1 signals from 12 individual MTs were quantified by intensity line scans to present the relative fluorescence intensity profiles, with the plus end of the MT toward the right. The highest-intensity peak of GFP-TACC3 is ∼0.5 μm distal to the peak of mKate2-EB1. (E–G) Representative time-lapse montage of mKate2-TACC3 (E) and XMAP215-GFP (F) accumulation on growing MT plus end. MT is growing to the right. Merged image (G) shows that mKate2-TACC3 and XMAP215-GFP localizations overlap. (H) Fluorescence intensity profiles of mKate2-TACC3 and XMAP215-GFP. mKate2-TACC3 and XMAP215-GFP signals from 11 individual MTs were quantified by intensity line scans to present the relative fluorescence intensity profiles. Note that peak intensities of mKate2-TACC3 and XMAP215-GFP closely align. Bar, 0.5 μm.

Mentions: It is well established that the growing end of a MT is home to many different +TIPs, including EB1, CLASP, and XMAP215 (Akhmanova and Steinmetz, 2008). Different +TIPs have overlapping yet unique localizations on the ends of the MTs, depending on their particular binding affinities. XMAP215 is known as the distalmost +TIP, and EB1 is located directly behind it on MTs (Nakamura et al., 2012; Maurer et al., 2014). Most other +TIPs, including CLASP, partially overlap with EB1 and trail further behind it (Hur et al., 2011). Because the fly orthologue of TACC3 genetically interacts with CLASP (Long et al., 2013), yet TACC3 biochemically interacts with XMAP215 (Kinoshita et al., 2005; O'Brien et al., 2005; Peset et al., 2005), we examined where TACC3 specifically localized on the MT plus end. We performed sequential imaging of red and green channels in both time orders to compare colocalizations between the two proteins. Although this type of dual-image-comparison analysis allowed for the analysis of colocalization dynamics, we also calculated the frame-to-frame velocity of the growing MT plus end in order to account for the 1-s time delay between channels for each examined MT and used these measurements to translate one channel in the x-axis to obtain the final images in Figure 6. We also used these time-corrected images for measuring approximate distances between peak intensity values.


TACC3 is a microtubule plus end-tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types.

Nwagbara BU, Faris AE, Bearce EA, Erdogan B, Ebbert PT, Evans MF, Rutherford EL, Enzenbacher TB, Lowery LA - Mol. Biol. Cell (2014)

TACC3 localization on MT plus ends is more distal than EB1 and overlaps with XMAP215. (A–C) Representative time-lapse montage of GFP-TACC3 (A) and mKate2-EB1 (B) accumulation on growing MT plus end. MT is growing to the right. Merged image (C) highlights the spatial arrangement between GFP-TACC3 and mKate2-EB1 localizations. These images were compiled by translating the TACC3 channel on the x-axis, after calculating the frame-to-frame velocity of the growing MT plus end, in order to account for the 1-s time delay between channels, for each examined MT (using ImageJ Translate function). To further confirm correct translation, uncorrected time-lapse colocalizations were examined with both combinations of imaging—red channel first, green channel second; then green channel first, red channel second. (D) Fluorescence intensity profiles of GFP-TACC3 and mKate2-EB1. GFP-TACC3 and mKate2-EB1 signals from 12 individual MTs were quantified by intensity line scans to present the relative fluorescence intensity profiles, with the plus end of the MT toward the right. The highest-intensity peak of GFP-TACC3 is ∼0.5 μm distal to the peak of mKate2-EB1. (E–G) Representative time-lapse montage of mKate2-TACC3 (E) and XMAP215-GFP (F) accumulation on growing MT plus end. MT is growing to the right. Merged image (G) shows that mKate2-TACC3 and XMAP215-GFP localizations overlap. (H) Fluorescence intensity profiles of mKate2-TACC3 and XMAP215-GFP. mKate2-TACC3 and XMAP215-GFP signals from 11 individual MTs were quantified by intensity line scans to present the relative fluorescence intensity profiles. Note that peak intensities of mKate2-TACC3 and XMAP215-GFP closely align. Bar, 0.5 μm.
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Figure 6: TACC3 localization on MT plus ends is more distal than EB1 and overlaps with XMAP215. (A–C) Representative time-lapse montage of GFP-TACC3 (A) and mKate2-EB1 (B) accumulation on growing MT plus end. MT is growing to the right. Merged image (C) highlights the spatial arrangement between GFP-TACC3 and mKate2-EB1 localizations. These images were compiled by translating the TACC3 channel on the x-axis, after calculating the frame-to-frame velocity of the growing MT plus end, in order to account for the 1-s time delay between channels, for each examined MT (using ImageJ Translate function). To further confirm correct translation, uncorrected time-lapse colocalizations were examined with both combinations of imaging—red channel first, green channel second; then green channel first, red channel second. (D) Fluorescence intensity profiles of GFP-TACC3 and mKate2-EB1. GFP-TACC3 and mKate2-EB1 signals from 12 individual MTs were quantified by intensity line scans to present the relative fluorescence intensity profiles, with the plus end of the MT toward the right. The highest-intensity peak of GFP-TACC3 is ∼0.5 μm distal to the peak of mKate2-EB1. (E–G) Representative time-lapse montage of mKate2-TACC3 (E) and XMAP215-GFP (F) accumulation on growing MT plus end. MT is growing to the right. Merged image (G) shows that mKate2-TACC3 and XMAP215-GFP localizations overlap. (H) Fluorescence intensity profiles of mKate2-TACC3 and XMAP215-GFP. mKate2-TACC3 and XMAP215-GFP signals from 11 individual MTs were quantified by intensity line scans to present the relative fluorescence intensity profiles. Note that peak intensities of mKate2-TACC3 and XMAP215-GFP closely align. Bar, 0.5 μm.
Mentions: It is well established that the growing end of a MT is home to many different +TIPs, including EB1, CLASP, and XMAP215 (Akhmanova and Steinmetz, 2008). Different +TIPs have overlapping yet unique localizations on the ends of the MTs, depending on their particular binding affinities. XMAP215 is known as the distalmost +TIP, and EB1 is located directly behind it on MTs (Nakamura et al., 2012; Maurer et al., 2014). Most other +TIPs, including CLASP, partially overlap with EB1 and trail further behind it (Hur et al., 2011). Because the fly orthologue of TACC3 genetically interacts with CLASP (Long et al., 2013), yet TACC3 biochemically interacts with XMAP215 (Kinoshita et al., 2005; O'Brien et al., 2005; Peset et al., 2005), we examined where TACC3 specifically localized on the MT plus end. We performed sequential imaging of red and green channels in both time orders to compare colocalizations between the two proteins. Although this type of dual-image-comparison analysis allowed for the analysis of colocalization dynamics, we also calculated the frame-to-frame velocity of the growing MT plus end in order to account for the 1-s time delay between channels for each examined MT and used these measurements to translate one channel in the x-axis to obtain the final images in Figure 6. We also used these time-corrected images for measuring approximate distances between peak intensity values.

Bottom Line: Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215.TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends.Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Boston College, Chestnut Hill, MA 02467.

Show MeSH
Related in: MedlinePlus