Correlative live-cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections - PubMed (original) (raw)

Correlative live-cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections

Štefan Bálint et al. Proc Natl Acad Sci U S A. 2013.

Abstract

Intracellular transport plays an essential role in maintaining the organization of polarized cells. Motor proteins tether and move cargos along microtubules during long-range transport to deliver them to their proper location of function. To reach their destination, cargo-bound motors must overcome barriers to their forward motion such as intersection points between microtubules. The ability to visualize how motors navigate these barriers can give important information about the mechanisms that lead to efficient transport. Here, we first develop an all-optical correlative imaging method based on single-particle tracking and superresolution microscopy to map the transport trajectories of cargos to individual microtubules with high spatiotemporal resolution. We then use this method to study the behavior of lysosomes at microtubule-microtubule intersections. Our results show that the intersection poses a significant hindrance that leads to long pauses in transport only when the separation distance of the intersecting microtubules is smaller than ∼100 nm. However, the obstructions are typically overcome by the motors with high fidelity by either switching to the intersecting microtubule or eventually passing through the intersection. Interestingly, there is a large tendency to maintain the polarity of motion (anterograde or retrograde) after the intersection, suggesting a high degree of regulation of motor activity to maintain transport in a given direction. These results give insights into the effect of the cytoskeletal geometry on cargo transport and have important implications for the mechanisms that cargo-bound motors use to maneuver through the obstructions set up by the complex cytoskeletal network.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Workflow of the all-optical correlative live-cell and superresolution imaging. A live-cell time-lapse movie is recorded at high temporal resolution. The sample is then fixed in situ and stained with antibodies conjugated with photoswitchable fluorophores for immunofluorescence and superresolution imaging (STORM). A STORM image of the microtubule network is then recorded. Single-particle tracking is used to obtain trajectories from the live-cell movie, and these trajectories are precisely aligned with the STORM image of the microtubules using fiduciary markers. IF, immunofluorescence.

Fig. 2.

Fig. 2.

Correlative live-cell and superresolution imaging allows interpreting cargo dynamics in the context of the cytoskeleton. (Upper) Multiple frames from a conventional dual-color movie of lysosomes (red dotted circle shows the position of a single lysosome as determined from the lysosome image) and microtubules (green). The trajectory (red line) of the lysosome is overlaid with the image of the microtubules at multiple times. (Lower) The same region as Upper but with the conventional microtubule image replaced by the end-point STORM image of microtubules. The lysosome trajectory can be mapped to the individual microtubules in the STORM image with high fidelity.

Fig. 3.

Fig. 3.

Lysosome behavior at microtubule intersections. (A) Examples showing the four different types of lysosome behavior at microtubule intersections. The lysosome trajectories have been color coded to show time according to the color scale bars. (Far Left) An example of pausing: The lysosome rapidly approaches the intersection (red arrow), indicated by the magenta-blue part of the trajectory, but spends extended time at the intersection, indicated by the blue-red part of the trajectory. (Middle Left) An example of passing: The lysosome moves with linear directed motion through the intersection (red arrow), indicated by the mostly uniform change of color of the trajectory. (Middle Right) Microtubule track switching at the intersection (red arrow). (Far Right) Direction reversing. (B) Histogram showing the percentage of lysosomes that pause (n = 108), pass (n = 70), switch track (n = 32), or reverse direction (n = 12) at microtubule intersections. (C) Histogram showing the lysosome behavior at microtubule intersections split into anterograde (red bars) and retrograde (blue bars) directions (anterograde: n = 44 pause, n = 21 pass, n = 16 switch, and n = 6 reverse; retrograde: n = 52 pause, n = 44 pass, n = 15 switch, and n = 4 reverse). (D) Histogram showing the secondary behavior of lysosomes after pausing. Passing (n = 36) and switching (n = 38) were equally likely; reversing (n = 16) was less common.

Fig. 4.

Fig. 4.

Correlation of lysosome behavior at microtubule intersections with the axial separation of microtubules. (A) Six frames from a time-lapse movie of lysosomes, in which the lysosome trajectory (red line) has been overlaid with 3D STORM image of microtubules. The 3D microtubule STORM image has been color coded to indicate _z_-position according to the color scale bar. The lysosome encounters three intersections (red arrows) in which the microtubules are axially separated by 250, 130, and less than 100 nm, respectively. The lysosome rapidly passes through the first intersection, arriving at the second intersection (first two frames). It pauses for 2.5 s at the second intersection before passing (frames 2–4). Finally it pauses for 1.5 s at the third intersection before passing (frames 4–6). (B) Another example showing a lysosome that encounters two intersections, both separated by less than 100 nm. Lysosome pauses and slowly passes through the first intersection (frames 1–4) and pauses for an extended period at the second intersection (frames 4–6). (C) Histogram showing the number of lysosomes that pause (red bar) or pass (green bars) versus the axial separation of microtubules. (D) Histogram showing the number of lysosomes that switch track versus the axial separation of microtubules.

Fig. 5.

Fig. 5.

Mechanistic model. When cargo arrives at a microtubule–microtubule intersection in which the axial separation of the intersecting microtubules is larger than 100 nm, there is minimal hindrance to the forward motion and transport continues on the same microtubule without disruption (A). When the axial separation of the intersecting microtubules is less than 100 nm, the intersection presents a major obstacle, stalling the motors and momentarily stopping forward motion until the obstacle is overcome (B). Cargos that are not positioned between the intersecting microtubules continue moving forward on the same microtubule without feeling the obstruction from the intersection (C).

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