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Figure 1. TACC1 can act as a plusâend tracking protein in embryonic cells. Expression of mKate2âtubulin (A), GFPâTACC1 (B), and merge (C) in cultured mesenchymal cells derived from embryonic neural tube; see also Movies S1 in Supporting Information. (D) Magnified timeâlapse montages of the boxed region in panel C. The time interval between frames is 2.9 s. (E) Fluorescence intensity profile of GFPâTACC1. Signals from 20 individual MTs were quantified by intensity line scans to present the relative fluorescence intensity profiles, with the plusâend of the MT toward the right. Expression of mKate2âEB1 (F), GFPâTACC1 (G), and merge (H) in cultured embryonic mesenchymal cells; see also Movie S3 in Supporting Information. (I) Magnified timeâlapse montage of the boxed region in panel H. The time interval between frames is 2.7 s. For this panel, the green channel has been translated to the left by 0.27 µm to account for the different acquisition times of the red and green channels (the velocity of the plusâend was consistently moving at 0.27 µm/s in this time series, and the green channel was imaged 1 s after the red channel). This allows for a more accurate visualization of colocalization between the two +TIPs. This demonstrates that, even after correcting for the time displacement, GFPâTACC1 overlaps with and is distal to mKate2âEB1. (J) Fluorescence intensity profiles of GFPâTACC1 and mKate2âEB1. Signals from 18 MTs were quantified by intensity line scans to present the relative fluorescence intensity profiles, with the plusâend of the MT toward the right. For each measurement, the green channel has been translated to the left (approximately 0.2 µm) based on each individual MTs velocity, to account for the time displacement between channels. After the correction, the highest peak intensity of GFPâTACC1 is â¼0.4 μm distal to the peak of mKate2âEB1. Expression of mKate2âTACC3 (K), GFPâTACC1 (L), and merge (M) in cultured embryonic mesenchymal cells; see also Movie S4 in Supporting Information. (N) Magnified timeâlapse montage of the boxed region in panel M. The time interval between frames is 3.9 s. For this panel, the green channel has been translated to the left by 0.25 μm to account for the different acquisition times of the red and green channels (the velocity of the plusâend was consistently moving at 0.21 µm/s in this time series, and the green channel was imaged 1.2 s after the red channel). (O) Fluorescence intensity profiles of GFPâTACC1 and mKate2âTACC3. Signals from 12 MTs were quantified by intensity line scans to present the relative fluorescence intensity profiles, with the plusâend of the MT toward the right. For each measurement, the green channel has been translated to the left (approximately 0.1 µm) based on each individual MTs velocity, to account for the time displacement between channels. While mKate2âTACC3 and GFPâTACC1 mostly overlap, the peak of mKate2âTACC3 is just slightly distal to GFPâTACC1. Also, we found that GFPâTACC1 comets were consistently longer when mKate2âTACC3 was expressed. Note that mKate2âTACC3 is less effective at tracking MT plusâends than GFPâTACC3 [observed in Nwagbara et al., 2014]. One reason for this is that the presence of the mKate2 tag leads to more efficient cleavage of the Câterminal TACC domain (not shown), which is required for MT plusâend tracking. Moreover, note that in images KâM, yolk granules are strongly autofluorescing. Bar 5 µM.
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Figure 2. The Câterminal TACC domain is necessary and sufficient for MT plusâend tracking. (A) Schematic representation of GFPâtagged TACC1 protein and deletion constructs. The amino acid residue numbers refer to those listed in Fig S1A in Supporting Information. (B) Western blot of embryonic lysates following expression of constructs, blotted for GFP. This blot is intentionally overexposed in order to reveal the smaller band in the fullâlength construct lane. Expression of mKate2âEB1 (to identify MT plusâends; C, H), GFPâtagged TACC1 constructs (D, I), and merged images of both channels (E, J). Plusâend accumulation is apparent in I (with GFPâCtermTACC1) but not in D (with GFPâNtermTACC1); see also Movies S5 and S6 in Supporting Information. (F, K) Magnified timeâlapse montages of a representative EB1 comet from cell in (E, J) confirms that CtermTACC1 localizes to growing MT plusâends, while NtermTACC1 does not. Arrowhead in (K) points to MT lattice binding. The time interval between frames in panel F is 3.3 s and panel K is 2.1 s. (G, L) Fluorescence intensity profiles of GFPâTACC1 constructs and mKate2âEB1. For each plot, signals from 10 MTs were quantified by intensity line scans to present the relative fluorescence intensity profiles, with the plusâend of the MT toward the right. For K, the green channel has been translated to the left (approximately 0.1 µm) based on each individual MTs velocity, to account for the time displacement between channels. GFPâCterm TACC1 comets were consistently longer than fullâlength GFPâTACC1 (compare Figs. 2L to 1J). Bar 5 µM.
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Figure 3. TACC1 overexpression (OE) increases MT growth velocities. Quantification of MT growth track parameters after TACC1 and TACC3 overexpression in cultured embryonic mesenchymal cells derived from neural tube. TACC proteins are expressed approximately twice as much as in controls. mKate2âEB1 localizes to the ends of growing MTs and is thus a marker for MT polymerization. Automated tracking of mKate2âEB1 comets is used to calculate MT growth track velocity (A), MT growth track lifetime (B), and MT growth track length (C). Mean values for all the experiments combined (three were performed in total): MT growth velocity, control, 8.5 µm/min, TACC1 OE 10.1 µm/min, TACC3 OE 9.84 µm/min, double OE 10.1 µm/min; MT growth lifetime, control, 12.1 s, TACC1 OE 12.3 s, TACC3 OE 12.3 s, double OE 12.5 s; MT growth length, control, 1.8 µm, TACC1 OE 2.1 µm, TACC3 OE 2.1 µm, double OE 2.2 µm. Control data are the means of 26 cells, representing a total of 4292 analyzed MT growth tracks; TACC1 OE are from 35 cells and 7606 analyzed MT growth tracks; TACC3 OE are from 38 cells and 9138 MT growth analyzed tracks; double OE are from 30 cells and 7214 MT growth analyzed tracks. (DâF) Quantification of MT parameters after overexpression of TACC1 deletion constructs. Mean values: MT growth velocity, control, 7.7 µm/min, Câterm TACC1 8.1 µm/min, Nâterm TACC1 8.2 µm/min; MT growth lifetime, control, 12.7 s, Câterm TACC1 12.3 s, Nâterm TACC1 12.8 s; MT growth length, control, 1.7 µm, Câterm TACC1 1.7 µm, Nâterm TACC1 1.8 µm. Control data are the means of 40 cells (from 5 individual experiments), representing a total of 9425 analyzed MT growth tracks; Câterm TACC1 data are from 33 cells and 6960 analyzed MT growth tracks; Nâterm TACC1 data are from 46 cells and 9701 analyzed MT growth tracks. Boxâandâwhisker plots indicate the mean (diamond), median, extrema, and quartiles. An unpaired t test was performed to assess significance of overexpression conditions compared to control. **Pâ<â0.01, *Pâ<â0.05; n.s., not significant.
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Figure 4. TACC1 KD reduces MT growth velocities, while double KD of both TACC1 and TACC3 also reduces MT growth lifetime. Quantification of MT growth track parameters after TACC1 and TACC3 knockdown in cultured embryonic mesenchymal cells derived from neural tube. (A) RTâPCR demonstrates knockdown of normal TACC1. Automated tracking of mKate2âEB1 comets after TACC1 knockdown is used to calculate MT growth track velocity (B), MT growth track lifetime (C), and MT growth track length (D). Mean values: MT growth velocity, control, 10.9 µm/min, TACC1 KD 9.8 µm/min; MT growth lifetime, control, 12.1 s, TACC1 KD 11.8 s; MT growth length, control, 2.2 µm, TACC1 KD 2.0 µm. Control data are the means of 52 cells, representing a total of 12,947 analyzed MT growth tracks; TACC1 KD are from 24 cells and 9435 analyzed MT growth tracks. (EâG) Quantification of MT parameters after double TACC1/TACC3 knockdowns (TACC1 mRNA levels were reduced approximately 25% while TACC3 was reduced approximately 40%, not shown). Mean values: MT growth velocity, control, 10.5 µm/min, TACC1 partial KD 9.7 µm/min, TACC3 partial KD 10.6 µm/min, TACC1/TACC3 double partial KD 9.4 µm/min; MT growth lifetime, control, 12.0 s, TACC1 partial KD 12.1 s, TACC3 partial KD 11.8 s, TACC1/TACC3 double partial KD 11.4 s; MT growth length, control, 2.1 µm, TACC1 partial KD 2.0 µm, TACC3 partial KD 2.1 µm, TACC1/TACC3 double partial KD 1.8 µm. Control data are the means of 92 cells (from 5 individual experiments), representing a total of 25,266 analyzed MT growth tracks; TACC1 partial KD are from 80 cells and 22,637 analyzed MT growth tracks; TACC3 partial KD are from 31 cells and 8,143 analyzed MT growth tracks; TACC1/TACC3 double partial KD are from 44 cells and 21,531 analyzed MT growth tracks. Boxâandâwhisker plots indicate the mean (diamond), median, extrema, and quartiles. To determine statistical significance for panels EâG, an ANOVA test was performed first (which revealed that the means were not the same for any condition), followed by an unpaired tâtest to assess significance between conditions. **Pâ<â0.01, *Pâ<â0.05; n.s., not significant.
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Figure 5. Cartoon schematic of TACC localization at the MT plusâend. Our data suggest a model in which TACC3 (red) is at the distalâmost end, with TACC1 (green) overlapping with and slightly proximal to TACC3. EB1 (orange) lies further proximal to TACC1. Both TACC1 and TACC3 are required for maintaining normal rates of MT polymerization, while normal levels of either of TACC1 or TACC3 appears to be sufficient for preventing MT catastrophe. Note that it is still unclear whether TACC3 and TACC1 can bind to the plusâend independently of other factors, or whether other +TIPs are required (such as XMAP215, which interacts directly with TACC3). Not drawn to scale.
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