Fig. 3
FP-tagged TgDCX generates curved microtubules in a heterologous system, Xenopus laevis S3 cells. a Deconvolved wide-field images of Xenopus S3 cells expressing EGFP-α-tubulin (green) and TgDCX-mCherryFP (red), showing that TgDCX-mCherryFP is localized to EGFP-α-tubulin containing fibers. TgDCX-mCherryFP expression drives the formation of curved fibers, many of which are short and share similar curvatures (also see Additional file 3: Movie S1). The areas within the dashed outlines are enlarged 1.5x in the small white boxes. b Deconvolved wide-field images of Xenopus S3 cells expressing EGFP-α-tubulin (green) and mCherryFP-TgDCX (red), showing that, similar to TgDCX-mCherryFP, mCherryFP-TgDCX expression drives the formation of curved fibers (also see Additional file 4: Movie S2). Arrows indicate microtubule bundles coated with mCherryFP-TgDCX. Note the fibers containing EGFP-tubulin only in the neighboring untransfected cell, which follow the meandering, gently curved, paths of typical cytoplasmic microtubules. Insets: 1.5x. c Histogram of radii of curvature of microtubules in Xenopus S3 cells, untransfected (green), or transfected with mCherryFP tagged TgDCX (red). Data include 844 microtubule segments (total length 7488 μm) for the untransfected cells and 599 segments (total length 2621 μm) for the transfected cells. All measurable microtubule segments in 8 representative untransfected cells and 7 transfected cells were included in the two histograms. Insets: fluorescent images of subregions of an untransfected (top) and a transfected cell (bottom). d Distribution of photon counts in images of Sindbis virus containing a capsid protein tagged with mCherryFP to be used as the fluorescence calibration standard for molecule counting. Y-axis: number of virions. X-axis: 104 photons/sec/virion. The images commonly include both single virus particles and a variable proportion of unresolved pairs, and occasionally a small number of larger aggregates. The single and double particles can be distinguished as two peaks in the histogram of measured intensities. The smooth blue and green curves overlying the histogram show the least squares fit to a Gaussian distribution with two components. The empirical means (sem) for the two components derived from this fitting are 38,318 (494) and 77,406 (683) photons/sec/virion. Inset: fluorescent image of a field of mCherryFP tagged Sindbis virus particles. e Photon count distribution in images of mCherryFP tagged TgDCX associated with “flexible” microtubules (green) or short curved arcs (red). Arrow indicates the expected photon count for FP tagged TgDCX molecules to decorate a single microtubule in a 1:1 TgDCX to tubulin dimer ratio. Y-axis: % of total. X-axis: 105 photons/sec/μm. Insets: fluorescent images containing “flexible” microtubules (bottom) or short curved arcs (top)