Research Article

Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics

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Science  28 Aug 2015:
Vol. 349, Issue 6251, aab3500
DOI: 10.1126/science.aab3500
  • Two approaches for improved live-cell imaging at sub-100-nm resolution.

    (Left) Association of cortical actin (purple) with clathrin-coated pits (green), the latter seen as rings (inset) at 84-nm resolution via a combination of total internal reflection fluorescence and structured illumination microscopy at ultrahigh numerical aperture (high-NA TIRF-SIM). (Right) Progression of resolution improvement across the actin cytoskeleton of a COS-7 cell, from conventional, diffraction-limited TIRF (220-nm resolution), to TIRF-SIM (97-nm resolution), and nonlinear SIM based on the patterned activation of a reversibly photoswitchable fluorescent protein (PA NL-SIM, 62 nm resolution). (Left and right represent single frames from time-lapse movies over 91 and 30 frames, respectively. Scale bars, 2 μm (left); 3 μm (right).

  • Fig. 1 High-speed live-cell imaging at 37°C of associations between proteins at sub-100-nm resolution.

    (A) Cytoskeletal proteins mApple-F-tractin (purple) and EGFP-myosin IIA (green) in a mouse embryonic fibroblast cell (Movie 1 and fig. S9). (B) Magnified view of the boxed region in (A), showing bipolar myosin IIA filaments with clearly resolved opposed head groups (for example, green arrowhead). (C) mApple-F-tractin (purple) and the focal adhesion protein mEmerald-paxillin (green) in a U2OS cell (movie S2). (D) Magnified view of the boxed region in (C), showing association of paxillin with smaller actin fibers fanning out from the ends of larger stress fiber cables. (E) Focal adhesion proteins mTagRFP-vinculin (purple) and mEmerald-paxillin (green) in a HFF-1 cell (Movie 2 and figs. S10 and S13). (F) Magnified view of the boxed region in (E), showing a gradient of increased paxillin concentration toward the cell periphery. Scale bars, 5 μm (A), (C), and (E); 1 μm (B), (D), and (F).

  • Fig. 2 Dynamics of clathrin-mediated endocytosis and the cortical actin cytoskeleton.

    (A) CCPs, resolved as rings (fig. S14 and movie S4) and color-coded according to their age since initial formation, at one time point from a movie of CCP dynamics in a BSC-1 cell at 37°C stably expressing EGFP-clathrin light chain a (Movie 3). (B) Histogram of maximum diameter of each CCP over its lifetime. (C) Plot of CCP overall lifetime versus CCP maximum diameter. (D) Sequential production of multiple CCPs at a CCP-generating “hot spot,” identified with green arrowheads (Movie 4 and fig. S16). (E) Formation, growth, and dissolution of a single CCP (right) and its relationship to cortical f-actin (left) in a COS-7 cell at 37°C transfected with mEmerald-clathrin light chain b and mCherry-Lifeact. Light blue arrowheads mark time points at which f-actin associates with the CCP. (F) Individual CCPs and clathrin plaques (green) and cortical f-actin (red) at one time point during their evolution in a COS-7 cell (Movie 5, figs. S17 and S18, and movie S5). (G) Formation of a nanoscale ring of f-actin (fig. S17B). Scale bars, 1 μm (A) and (F); and 200 nm (D), (E), and (G).

  • Fig. 3 Live-cell nonlinear structured illumination microscopy based on patterned photoactivation.

    (A) Single time point from a movie of the evolution of cortical f-actin in a COS-7 cell at 23°C transfected with Skylan-NS-Lifeact, seen at 62-nm resolution (Movie 6, fig. S31, and movie S8). (B) Magnified view from a different cell at 37°C, comparing diffraction-limited TIRF microscopy (top left), TIRF with deconvolution (top right), TIRF-SIM (bottom left), and nonlinear TIRF-SIM with patterned activation (PA NL-SIM, bottom right) (movies S9 and S10). (C) Caveolae in a COS-7 cell at 23°C transfected with Skylan-NS-caveolin, comparing TIRF with deconvolution (top left, 220-nm resolution), TIRF SIM (top right, 97-nm resolution), PA NL-SIM (bottom left, 62-nm resolution), and saturated PA NL-SIM (bottom right, 45-nm resolution). (Insets) A single caveolae pit eventually resolved as a ring by saturated PA NL-SIM (Movie 7, figs. S34 to S37, and movie S13. (D) Diversity of caveolae ring diameters as seen by means of PA NL-SIM. (E) Larger rings that may represent surface-docked vesicles. (F) Clusters of caveolae reminiscent of clathrin plaques. (D) to (F) are from a different cell at 37°C (fig. S33 and movie S12). Scale bars, 3 μm (A); 1 μm (B); 200 nm (C); and 100 nm (D), (E), (F), and (C), inset.

  • Fig. 4 Combined TIRF-SIM and PA NL-SIM of protein-pair dynamics in living cells.

    (A) Skylan-NS-Lifeact (orange, PA NL-SIM) and mCherry-Rab5a, a marker of early endosomes (green, TIRF-SIM) in a COS-7 cell at 23°C (Movie 8, figs. S38 and S39, and movie S14). (B) Comparison of EM images of early endosomes (37) with similarly shaped Rab5a patches seen in (A). (C) Magnified view at three successive time points, showing rapid transport of a Rab5a streak parallel to the cytoskeleton. (D) Skylan-NS-Lifeact (green, PA NL-SIM) and mCherry-α-actinin (purple, TIRF-SIM) in a COS-7 cell at 23°C (Movie 9, figs. S40 and S41, and movie S15). (E) Magnified view from (D), with Lifeact (top), α-actinin (middle), and overlay (bottom) showing paired association at focal adhesions and along the sides of large stress fibers. (F) Evolution of a membrane ruffle, showing α-actinin concentrated at the leading edge. Scale bars, 5 μm (A), (D); 200 nm (B); 1 μm (C) and (E); and 500 nm (F).

  • Fig. 5 Live-cell 3D PA NL-SIM via lattice light sheet microscopy.

    (A) (Top) Membrane marker Skylan-NS-TOM20 showing mitochondria in a COS-7 cell at 23°C, color-coded for distance from the substrate. (Bottom) Evolution of individual mitochondria, showing fission and fusion events, the former preceded by mitochondrial constriction (Movie 10 and fig. S44). (B) Time-lapse distribution of Golgi-resident enzyme Skylan-NS-Mann II in a U2OS cell at 23°C, showing centralized cisternae surrounded by vesicles. White arrowheads indicate a docking vesicle, and red arrowheads highlight rapid export of a long tubular vesicle (movies S17 and S18). Scale bars, 5 μm (A), top; 1 μm (A), bottom; and 3 μm (B).

  • Movie 1 Actomyosin dynamics in a MEF cell.

    Interaction of filamentous actin (mApple-F-tractin, purple) with myosin IIA paired head groups (EGFP, myosin IIA, green) at 20-s intervals for 100 time points, as seen with high-NA TIRF-SIM. (download hi-res file – 17.7 MB)

  • Movie 2 Focal adhesion dynamics at the periphery of an HFF-1 cell.

    Evolution and varying diatribution of mEmerald-paxillin (green) and mTagRFP-vinculin (purple) in peripheral focal adhesions at 20-s intervals for 70 time points, as seen with high-NA TIRF-SIM. (download hi-res file – 52.8 MB)

  • Movie 3 Clathrin-mediated endocytosis.

    Formation, maturation, and internalization of individual CCPs, resolved as rings, in a live BSC-1 cell expressing clathrin-EGFP as seen with high-NA TIRF-SIM for 200 time points at 3.5-s intervals. Color-coded lifetime analysis permits quantitative measurement and correlation of lifetimes to pit sizes and their spatial distribution. (download hi-res file – 27.6 MB)

  • Movie 4 Hot spot of clathrin pit formation.

    Sequential generation of multiple CCPs from a single subdiffractive patch of clathrin in a BSC-1 cell. Statistical analysis (fig. S16) reveals that sites of pit formation are not randomly distributed. (download hi-res file – 3 MB)

  • Movie 5 Dynamic interactions of cortical filamentous actin with CCPs.

    High-NA TIRF-SIM for 91 frames at 4-s intervals in a COS-7 cell expressing mEmerald-clthrin (green) and mCherry-Lifeact (orange). (download hi-res file – 34.3 MB)

  • Movie 6 Resolution comparisons and actin dynamics as seen with nonlinear SIM with patterned activation.

    Progressive resolution gain in a live COS-7 cell expressing Skylan-NS-Lifeact from diffraction-limited TIRF, to TIRF with deconvolution, linear SIM, and nonlinear SIM with patterned activation, followed by the dynamics of the actin cytoskeleton by means of PA NL-SIM for 30 time points at 20-s intervals. (download hi-res file – 43.6 MB)

  • Movie 7 Dynamics of caveolae as seen with different methods.

    Caveolae in a COS-7 cell expressing Skylan-NS-caveolin for 20 time points at 3-s intervals as seen with diffraction-limited TIRF, TIRF with deconvolution, linear SIM, and PA NL-SIM. (download hi-res file – 16.5 MB)

  • Movie 8 Dynamics of cortical actin and early endosomes by means of two-color combined linear and nonlinear SIM.

    A COS-7 cell expressing Skylan-NS-Lifeact (orange) as imaged by means of PA NL-SIM and mCherry-Rab5a (green), a marker of early endosomes, as imaged by means of linear SIM, both over 34 frames at 20-s intervals. (download hi-res file – 48.1 MB)

  • Movie 9 Dynamic interactions of f-actin and α-actinin by means of two-color combined linear and nonlinear SIM.

    A COS-7 cell expressing Skylan-NS-Lifeact (green) as imaged by means of PA NL-SIM and mCherry-α-actinin (purple) as imaged by means of linear SIM, both over 28 frames at 20-s intervals. (download hi-res file – 21.7 MB)

  • Movie 10 3D dynamics of mitochondria by means of lattice light sheet PA NL-SIM.

    2D xy scrolling orthoslice views at one time point, followed by the 3D evolution of mitochondria over 20 time points at 2-min intervals, in a COS-7 cell expressing Skylan-NS-TOMM20. (download hi-res file – 22.8 MB)

Supplementary Materials

  • Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics

    Dong Li, Lin Shao, Bi-Chang Chen, Xi Zhang, Mingshu Zhang, Brian Moses, Daniel E. Milkie, Jordan R. Beach, John A. Hammer III, Mithun Pasham, Tomas Kirchhausen, Michelle A. Baird, Michael W. Davidson, Pingyong Xu, Eric Betzig

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Supplementary Text
    • Figs. S1 to S55
    • Table S1 and S2

    Images, Video, and Other Other Media

    Higher quality videos available here
    Movie S1
    Phototoxicity of WF-RESOLFT vs. PA NL-SIM. Left: Behavior over time of a U2OS cell expressing Skylan-NS-Lifeact, at the excitation intensity and exposure time used by WF-RESOLFT (11). Right: Behavior of a similar cell under excitation and exposure conditions typical of PA NL-SIM.
    Movie S2
    Cytoskeletal attachment at focal adhesions in a live U2OS cell at 37°C. Dynamics of filamentous actin (mApple-F-tractin, magenta) and its interaction with mEmerald-paxillin (green) at focal adhesions, at 20 sec intervals for 81 time points, as seen by high NA TIRF-SIM.
    Movie S3
    Dynamics of clathrin-medited endocytosis of transferrin receptors at 37°C. High NA TIRF-SIM for 28 frames at 5 sec intervals of the interaction of clathrin coated pits (green) and transferrin cargo (magenta) in MEF cells labeled with mEmerald-CLTB and transferrin conjugated to Alex568.
    Movie S4
    Dynamics of clathrin coated pits at 37°C. Formation, maturation, and internalization of clathrin coated pits in a live BSC-1 cell expressing EGFP-CLTA as seen by high NA TIRF-SIM for 150 time points at 0.8 sec intervals.
    Movie S5
    Actin-clathrin interactions in a second cell. High NA TIRF-SIM for 181 frames at 5 sec intervals in another COS-7 cell at 37°C expressing mEmerald-CLTA (green) and mCherry-Lifeact (orange-red).
    Movie S6
    Molecular states during the phases of nonlinear SIM. Illustration of the sequential steps of activation, depletion, and read-out in three forms of nonlinear SIM: patterned activation, saturated patterned activation, and saturated depletion.
    Movie S7
    Actin dynamics as seen by SD NL-SIM. Dynamics of the actin cytoskeleton over 12 time points at 20 sec intervals by nonlinear SIM with saturated depletion, in a COS-7 cell at 23°C expressing Skylan-NS-Lifeact. The rapid decline in SNR compared to movie S11 is indicative of the relative photon inefficiency of SD NL-SIM compared to PA NL-SIM.
    Movie S8
    Actin rings and cytoskeletal dynamics as seen by PA NL-SIM. A COS-7 cell at 23°C expressing Skylan-NS-Lifeact as seen over 34 frames at 18 sec intervals by PA NL-SIM.
    Movie S9
    Actin dynamics as seen by high NA PA NL-SIM. Dynamics of the actin cytoskeleton over 26 time points at 15 sec intervals as seen by patterned activation nonlinear SIM, in a COS-7 cell at 37°C expressing Skylan-NS-Lifeact.
    Movie S10
    Actin dynamics as seen by high NA PA NL-SIM in a second cell. Dynamics of the actin cytoskeleton over 40 time points at 15 sec intervals as seen by patterned activation nonlinear SIM, in a COS-7 cell at 37°C expressing Skylan-NS-Lifeact.
    Movie S11
    Dynamics of keratin as seen by PA NL-SIM. Progressive resolution gain in a live COS-7 cell at 23°C expressing Skylan-NS-keratin from diffraction-limited TIRF, to TIRF with deconvolution, linear TIRF-SIM, and nonlinear SIM with patterned activation (PA NL-SIM), followed by the dynamics of keratin for 11 frames at 20 sec intervals by PA NL-SIM.
    Movie S12
    Dynamics of caveolae as seen by different methods. Caveolae in a COS-7 cell at 37°C expressing Skylan-NS-caveolin for 21 time points at 3 sec intervals as seen by diffraction-limited TIRF, TIRF with deconvolution, linear TIRF-SIM and PA NL-SIM.
    Movie S13
    Caveolae dynamics as seen by saturated PA NL-SIM. Caveolae in a COS-7 cell at 23°C expressing Skylan-NS-caveolin for 12 time points at 3 sec intervals as seen by TIRF with deconvolution, linear TIRF-SIM, PA NL-SIM, and saturated PA NL-SIM.
    Movie S14
    Cortical actin and early endosomes by TIRF-SIM and PA NL-SIM in a second cell. A COS-7 cell at 23°C expressing expressing Skylan-NS-Lifeact (orange-red) as imaged by PA NL-SIM and mCherry-Rab5a (green) as imaged by TIRF-SIM, both over 28 frames at 15 sec intervals.
    Movie S15
    Dynamic interactions of f-actin and ??-actinin by two-color combined TIRF-SIM and PA NL-SIM. A COS-7 cell at 23°C expressing expressing Skylan-NS-Lifeact (green) as imaged by PA NL-SIM and mCherry-??-actinin (magenta) as imaged by TIRF-SIM, both over 24 frames at 20 sec intervals.
    Movie S16
    3D dynamics of actin by lattice light sheet PA NL-SIM. 2D xy and xz scrolling orthoslice views at one time point, followed by the 3D evolution of the actin cytoskeleton over 18 time points at 2 min intervals, in a U2OS cell at 23°C expressing Skylan-NS-Lifeact.
    Movie S17
    3D dynamics of the Golgi apparatus by lattice light sheet PA NL-SIM. 3D evolution of a volume-rendered representation of the Golgi apparatus over 42 time points at 95 sec intervals, in a U2OS cell at 23°C expressing Skylan-NS-MannII, showing nuclear-facing hollowing structure and surrounding vesicles.
    Movie S18
    3D color-coded representation of Golgi dynamics by lattice light sheet PA NL-SIM. 3D evolution of a the Golgi apparatus, color coded by height above the cover slip, over 42 time points at 95 sec intervals, in a U2OS cell at 23°C expressing Skylan-NS-MannII.

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