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{"CAPTION FIG2-1.png": "'\\nFig. 2: **MINFLUX tracking of kinesin in live cells.** Panels (**A**) to (**D**) show tracking of full-length kinesin labeled N-terminally with a HaloTag bound to JF645 in live U20S cells. (**A**) Confocal images of GFP-a-tubulin in untreated live U20S cells and overlaid full-length human kinesin trajectories. (**B**) Knesin'", "CAPTION FIG2.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG1.png": "'vertical kinesin trajectories with color-coded walking directions. (**F**) Traads as indicated in (E) show side stepping. (**G**) Traads as done as 12 nm are clearly resolved and display lineshitching inter-family between neighboring microtubules or pretrellaments (movie S2). (**H**) Representative track and the corresponding time versus position pick at saturating ATP concentrations (>1 mM, here 6 mM) showing 8-nm making steps. (**I**) Histogram of step size at saturating ATP concentrations from seven experiments, (**A**) tracks, and 995 steps with a Gaussian fit (73 nm +- 2.7 SD +- 0.09 nm SEM red line). (**J**) Dual time histogram and H with a contribution of the experimental functions (average dwell time of 30.8 ms reci red line). (**K** and **L**) Representative track at low ATP concentrations (10 mM) and a corresponding time versus position, raw data (gray), and 20-ms running mean (black) clearly showing 8-nm walking steps (see movie S3). (**M**) Representative track showing a zigzag trajectory, indicating an asymmetric arrangement of the label within a kinesin molecule (see fig. S2, A and E, and movie S4; see fig. S3 for additional examples). Scale bars: (**L**) and (**M**), 10 nm (**F**) to (0), 100 nm; (**D**) and (**E**), 1 nm.\\n\\nFig. 1: **IMFLU tracking of kinesin in fixed cells. (A) Kiresin walls on microtubules in a hard-over-hard manner. The apparent step size is 8 nm when the label is attached to the C-terminal tail domain and 16 nm when it is attached to the N-terminal motor domain. (B) 20 MIFLUX tracking of a single molecule. A draw beam probes seven positions around a fluorophore to determine its location with nanometer precision. The scan pattern is iteratively centered on the fluorophore during tracking. (C) Micro-PMNT approach to track, kinesin in fixed cells. Cells are first permeabilized to extract cell contents and then gently feed to pressure microtubules. Purified fluorescently labeled kinesin (DmKHC (0-42L))-SNAP-tag-to-offel are added and tracked as they mask toward the plus ends of the microtubules (move S1). Parts (D) to (M) show MNFLUX motor-PMNT in fixed cells. (**D**) Confocal images of a neuron and overlaid kinesin trajectories in four neurites. Most of the neurites show kinesin trajectories in both directions, i.e., toward (magenta) and away (cyan) from the soma, as expected for dendrites. (**E**) Confocal microscopy images of green fluorescent protein (GFP)\u2013a-tubulin in U2OS cells showing what appears to be a centrosome and overlaid kinesin trajectories with color-coded walking directions. (**F**) Traads as indicated in (E) show side stepping. (**G**) Traads as done as 12 nm are clearly resolved and display lineshitching inter-family between neighboring microtubules or pretrellaments (movie S2). (**H**) Representative track and the corresponding time versus position pick at saturating ATP concentrations (>1 mM, here 6 mM) showing 8-nm making steps. (**I**) Histogram of step size at saturating ATP concentrations from seven experiments, (**A**) tracks, and 995 steps with a Gaussian fit (73 nm +- 2.7 SD +- 0.09 nm SEM red line). (**J**) Dual time histogram and H with a contribution of the experimental functions (average dwell time of 30.8 ms reci red line). (**K** and **L**) Representative track at low ATP concentrations (10 mM) and a corresponding time versus position, raw data (gray), and 20-ms running mean (black) clearly showing 8-nm walking steps (see movie S3). (**M**) Representative track showing a zigzag trajectory, indicating an asymmetric arrangement of the label within a kinesin molecule (see fig. S2, A and E, and movie S4; see fig. S3 for additional examples). Scale bars: (**L**) and (**M**), 10 nm (F**) to (0), 100 nm; (**D**) and (**E**), 1 nm.\\n\\n'", "CAPTION FIG2-2.png": "'track in which the localizations are rendered as a super-resolution image in the region indicated in (A). (**E**) Line plot connecting each localization. (**D**) Time versus position plot of the highlighted portion of the track in (C) showing steps of 16 nm. Panels (E) to (J) show tracking of truncated kinesin (HaloTag-K560) in Taxol-treated live U2OS cells. (**E**) Confocal images of GFP-a-tubulin and overlaid kinesin tracks. (**F** and **G**) The tracks indicated in (E) rendered as a super-resolution image (F) and line plots connecting each localization (G) (see movie S9) showing clear walking steps (localization precisier. 2 nm, temporal resolution 1 ms). (**H**) Time versus position picks of representative kinesin tracks as indicated in (E) showing clear 16 nm stepwise movements. (**J**) Step-sse histogram [6] experiments, 330 tracks, and 2887 steps) and a Gaussian fit (16.2 +- 3.8 SD +- 0.07 SEM rms). (**J**) Dual-time histogram, fit with a convolution of four exponential functions (average dwell time of 27.5 ms; red line). Panels (K) to (M) show tracking of kinesin (HaloTag-K560) in untreated live primary mouse cortical neurons. (**K**) Confocal images of GFP-a-tubulin and overlaid kinesin tracks. (**L** and **M**) Representative tracks corresponding to those indicated in (K) as line plots (L) and time versus position plots (M) showing 16-nm stepwise movements (see fig. S2, C and G, for step size and dwell time histograms). Scale bars: (B), (C), (F), (G), and (L), 100 nm; (A), (E), and (K), 1 nm.\\n\\n'", "CAPTION FIG3.png": "'switch microtubules (arrows). Panels (E) and (F) show 3D tracking in live cells. (**E**) Representative kinesin tracks in live U2DS cells in top and side views, showing stepwise movements both in the \\\\(x\\\\)-\\\\(y\\\\) plane and along the \\\\(z\\\\) axis (see fig. S2, D and H, for histograms and fig. S3 for confocal overview images; also see movie S10). (**F**) Position versus time plots of the tracks from (E) showing 16-nm steps. Scale bars: (E), 100 nm; (C) and (D), 200 nm.\\n\\n'"} |
