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. 2017 Mar 21;8:14772.
doi: 10.1038/ncomms14772.

Probing Cytoskeletal Modulation of Passive and Active Intracellular Dynamics Using Nanobody-Functionalized Quantum Dots

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Free PMC article

Probing Cytoskeletal Modulation of Passive and Active Intracellular Dynamics Using Nanobody-Functionalized Quantum Dots

Eugene A Katrukha et al. Nat Commun. .
Free PMC article

Abstract

The cytoplasm is a highly complex and heterogeneous medium that is structured by the cytoskeleton. How local transport depends on the heterogeneous organization and dynamics of F-actin and microtubules is poorly understood. Here we use a novel delivery and functionalization strategy to utilize quantum dots (QDs) as probes for active and passive intracellular transport. Rapid imaging of non-functionalized QDs reveals two populations with a 100-fold difference in diffusion constant, with the faster fraction increasing upon actin depolymerization. When nanobody-functionalized QDs are targeted to different kinesin motor proteins, their trajectories do not display strong actin-induced transverse displacements, as suggested previously. Only kinesin-1 displays subtle directional fluctuations, because the subset of microtubules used by this motor undergoes prominent undulations. Using actin-targeting agents reveals that F-actin suppresses most microtubule shape remodelling, rather than promoting it. These results demonstrate how the spatial heterogeneity of the cytoskeleton imposes large variations in non-equilibrium intracellular dynamics.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Non-isotropic diffusion of intracellular QDs.
(a) COS-7 cell fixed 30 min after electroporation with QDs and stained with phalloidin. Maximum projection of a z-stack acquired with the spinning disk microscope. Scale bar, 10 μm. (b) Lateral YZ (left) and XZ maximum projection views of cross sections along the boxes depicted in a. Scale bar, 5 μm. (c) Stills from a stream recording of GFP-actin expressing COS-7 cell electroporated with QDs. The interval between frames is 2.4 ms. Blue and red arrows indicate slow and fast cytosolic diffusion of QDs, respectively. The complete trajectories (66 frames, 156 ms) are depicted on the right panel. Scale bar, 2 μm. (d) Distribution of the mean square displacement at one frame (2.4 ms) delay for the trajectories of QDs in control (black, N=485, 10 cells) and latrunculin A treated cells (red, N=474, 13 cells). Dashed line marks the threshold used to separate trajectories of fast and slow diffusing particles in f. (e) Example trajectories of intracellular QDs, color-coded according to the corresponding value of mean square displacement at one frame (2.4 ms) delay. (f) Average MSD of slow (blue solid line, N=318) and fast (red solid line, N=167) fractions of QDs trajectories. Error bars represent s.e.m. Dashed line shows fit MSD(τ)=4+dx2 where offset dx2=(35 × 35) nm2 reflects squared average localization precision. (g) Density of fast and slow diffusing subpopulation of QDs in cells, imaged either near the coverslip or 1 μm above (N=6 cells per each condition). Error bars represent s.e.m. (h) Proposed origin of slow and fast diffusing subpopulations of QDs.
Figure 2
Figure 2. Probing specific motor proteins using nanobody-conjugated QDs reveals limited transverse fluctuations during microtubule-based runs.
(a) Linkage of QDs to GFP-fused motor proteins through GFP nanobody (top) and expected movement of individual QDs-kinesin complexes along microtubules (bottom). (b) Distribution of electroporated QDs–VHHGFP inside COS-7 cell expressing kinesin-1-GFP (cyan) and electroporated with QDs–VHHGFP (yellow). White curve indicates cell outline. Scale bar, 5 μm. (c) QDs–VHHGFP colocalize with microtubules decorated by kinesin-1 (KIF5B-GFP-FRB). Single frames from TIRFM stream recordings of COS-7 cell expressing kinesin-1-GFP (cyan/right) and electroporated with QDs–VHHGFP (yellow). Scale bar, 2 μm. (dg) Example trajectories (top row) and kymographs (bottom row) for QDs coupled to kinesins from different families. Scale bars, 2 μm and 2 s. (h) Average MSD of longitudinal (top) and transverse (bottom) components of directed motion segments of different kinesins (n=104, 146, 151, 144 for Kinesin-1,2,3,4) decomposed using B-spline trajectory fitting with 1 μm control points distance. (ik) Average speed i, run length j and run duration k of the individual motor runs for the different kinesins. Error bars represent s.e.m. See Supplementary Table 3 for numeric values.
Figure 3
Figure 3. Kinesin-1 preferably walks on acetylated microtubules that are highly curved and undergo bending deformations.
(a) Example of two trajectories with the same radius of curvature R, but different average angle between the directions of consecutive displacements. (b) Distribution of local curvatures of directed motion segments in trajectories of QDs coupled to different kinesins. (n=4,247, 8,922, 7,965, 6,041 for Kinesin-1,2,3,4). (c) Characteristic decay values from exponential fits to distributions in b. Error bars represent s.e. of fitting. (d) Average directional persistence (cosine between consecutive displacements) as a function of displacement, assuming constant average speed of kinesins (n is the same as in Fig. 2h, see also Supplementary Fig. 3a). Error bars represent s.e.m. (e) COS-7 cell expressing KIF5B-GFP (green) stained for tyrosinated (blue) and acetylated (red) tubulin. Scale bar, 10 μm. (f,g) Stills from a time lapse recording of a COS-7 cell expressing KIF5B-GFP (f) or KIF21B-GFP (g) (green) and Tubulin-TagRFP (red). Shapes of microtubules highlighted by white arrows are traced over time (s) and color-coded as indicated. Scale bars, 2 μm.
Figure 4
Figure 4. Analysis of microtubules bending deformations under the treatment of F-actin modifying drugs.
(a) Still from a spinning disk movie of COS-7 cells expressing mCherry-tubulin. Dashed yellow lines mark areas used to build kymographs. Scale bar, 5 μm. (b) Representative kymographs built from spinning disk movies of COS-7 cells transfected with mCherry-tubulin under the treatment of indicated drugs. Scale bars, 60 s (vertical) and 5 μm (horizontal). (c) Plot of average iMSD versus time derived from kymograph analysis (N=11, 10, 14 and 13 for control, 10 μM latrunculin A, 10 μM jasplakinolide and 50 μM blebbistatin treatment). Error bars represent s.e.m. (d) Average diffusion coefficient derived from individual fitting of iMSD curves for indicated conditions. **P<0.01 (two-tailed Mann–Whitney test), n>=9, each group. Error bars represent s.e.m. (e) Average confinement size derived from individual fitting of iMSD curves for indicated conditions ***P<0.001 (two-tailed Mann–Whitney test), n≥9, each group). Error bars represent s.e.m. (f) Hypothetical phase diagram reflecting the behaviour of intracellular particles and cellular components of different sizes on different timescales.

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