Journal: Nature Communications
Article Title: Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
doi: 10.1038/s41467-019-14087-0
Figure Lengend Snippet: a Experimental setup for b and c : non-phase-separated GFP-TPX2 (50 nM) and Alexa568-labeled tubulin mixed with Xenopus meiotic egg cytosol and imaged via oblique-TIRF microscopy. b GFP-TPX2 (green) localization to growing MT network imaged over time (minutes:seconds) in the cytosol (see Supplementary Movie ), representative of three experimental replicates. Microtubules are labeled red and growing plus-tips are blue. All scale bars are 3 µm, unless indicated. c GFP-TPX2 (green) - and Alexa568-labeled tubulin (red) colocalization in the cytosol treated with Nocodazole to prevent MT polymerization, representative of five experimental replicates. Images taken 10 min into reaction (see Supplementary Movie ). Scale bar is 3 µm. TPX2 was 50 nM and tubulin was 2 µM. d TIRF images (contrast-optimized), representative of four experimental replicates, and e quantification of GFP-TPX2 (green) and Cy5-labeled tubulin (magenta) (at indicated concentrations, both proteins equimolar) localized to pre-formed microtubules (Alexa568-labeled, GMPCPP stabilized—red) and spun down onto coverslips, fixed, and imaged; scale bar, 3 µm. f Oblique-TIRF images of only Cy5-labeled tubulin (magenta) condensed with GFP-TPX2 (not shown) either in solution (top panel) or on a pre-formed MT (lower panel—MT not shown) at concentrations shown. Images displayed at matched brightness and contrast. Enhanced contrast of 10 nM and 25 nM images shown is shown in the box. Scale bar, 2 µm. g Quantification of relative tubulin signal from TPX2-tubulin co-condensates in solution (blue curve) and on microtubules (purple curve) at concentrations shown in e . Mean (points) and SEM (error bars) of three replicate experiments (error bars) shown, n of microtubules per condition are: 145, 216, 114, 217, 243, 335, 309, and 309 for 1, 2.5, 5, 10, 25, 50, 100, and 250 nM [TPX2/TB], respectively; n of condensates per condition are: 280, 225, 266, and 282 for 10, 25, 50, 100, and 250 nM [TPX2/TB], respectively. Endogenous concentration range of TPX2 ( q = 25–100 nM) indicated. h Schematic of experimental setup—live observation of TPX2 (green) and soluble tubulin (magenta) localization to a stable microtubule (red) attached to a coverslip-bottomed well. i TIRF images of a time series of GFP-TPX2 (green) and soluble Cy5-labeled tubulin (magenta) localization to a GMPCPP-stabilized Alexa568-labeled microtubule seed (red) (see Supplementary Movie ). Images are contrast enhanced to show early non-association events and are representative of three experimental replicates. Arrowheads indicate fusion of two condensates. Time in minutes:seconds, 00:00 corresponds to addition of TPX2 into the well. Scale bar, 2 µm. Source data are provided as a Source Data file.
Article Snippet: The code for the custom MATLAB microtubule tracking software is published in and available through Thawani et al.
Techniques: Labeling, Microscopy, Concentration Assay
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![a Experimental setup for b and c : non-phase-separated GFP-TPX2 (50 nM) and Alexa568-labeled tubulin mixed with Xenopus meiotic egg cytosol and imaged via oblique-TIRF microscopy. b GFP-TPX2 (green) localization to growing MT network imaged over time (minutes:seconds) in the cytosol (see Supplementary Movie ), representative of three experimental replicates. Microtubules are labeled red and growing plus-tips are blue. All scale bars are 3 µm, unless indicated. c GFP-TPX2 (green) - and Alexa568-labeled tubulin (red) colocalization in the cytosol treated with Nocodazole to prevent MT polymerization, representative of five experimental replicates. Images taken 10 min into reaction (see Supplementary Movie ). Scale bar is 3 µm. TPX2 was 50 nM and tubulin was 2 µM. d TIRF images (contrast-optimized), representative of four experimental replicates, and e quantification of GFP-TPX2 (green) and Cy5-labeled tubulin (magenta) (at indicated concentrations, both proteins equimolar) localized to pre-formed microtubules (Alexa568-labeled, GMPCPP stabilized—red) and spun down onto coverslips, fixed, and imaged; scale bar, 3 µm. f Oblique-TIRF images of only Cy5-labeled tubulin (magenta) condensed with GFP-TPX2 (not shown) either in solution (top panel) or on a pre-formed MT (lower panel—MT not shown) at concentrations shown. Images displayed at matched brightness and contrast. Enhanced contrast of 10 nM and 25 nM images shown is shown in the box. Scale bar, 2 µm. g Quantification of relative tubulin signal from TPX2-tubulin co-condensates in solution (blue curve) and on microtubules (purple curve) at concentrations shown in e . Mean (points) and SEM (error bars) of three replicate experiments (error bars) shown, n of microtubules per condition are: 145, 216, 114, 217, 243, 335, 309, and 309 for 1, 2.5, 5, 10, 25, 50, 100, and 250 nM [TPX2/TB], respectively; n of condensates per condition are: 280, 225, 266, and 282 for 10, 25, 50, 100, and 250 nM [TPX2/TB], respectively. Endogenous concentration range of TPX2 ( q = 25–100 nM) indicated. h Schematic of experimental setup—live observation of TPX2 (green) and soluble tubulin (magenta) localization to a stable <t>microtubule</t> (red) attached to a coverslip-bottomed well. i TIRF images of a time series of GFP-TPX2 (green) and soluble Cy5-labeled tubulin (magenta) localization to a GMPCPP-stabilized Alexa568-labeled microtubule seed (red) (see Supplementary Movie ). Images are contrast enhanced to show early non-association events and are representative of three experimental replicates. Arrowheads indicate fusion of two condensates. Time in minutes:seconds, 00:00 corresponds to addition of TPX2 into the well. Scale bar, 2 µm. Source data are provided as a Source Data file.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9270/pmc06959270/pmc06959270__41467_2019_14087_Fig2_HTML.jpg)
