Research ArticleBIOPHYSICS

Topology-dependent anomalous dynamics of ring and linear DNA are sensitive to cytoskeleton crosslinking

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Science Advances  13 Dec 2019:
Vol. 5, no. 12, eaay5912
DOI: 10.1126/sciadv.aay5912
  • Fig. 1 Experimental approach to elucidating the effect of DNA topology on the transport and conformational properties of DNA diffusing through model cytoskeleton composites.

    (A) Cartoon of fluorescent-labeled 115-kbp ring and linear DNA molecules embedded in composite networks of actin and microtubules that are either entangled or crosslinked by biotin-NeutrAvidin crosslinkers. L is the DNA contour length, R0 is the topology-dependent mean end-to-end length of the DNA coils, lp is persistence length, and ξ is the composite mesh size. Not drawn to scale. (B) Single-molecule analysis (1) tracks the center-of-mass (COM) position and the lengths of the major and minor axes (Rmax and Rmin) of each DNA molecule for every frame of the time series to quantify the transport and conformational dynamics of individual DNA molecules. From (1), the COM MSD (2) and probability distributions of Rmax, Rmin, and Rcoil = [½(Rmax2 + Rmin2)]1/2 (3) are computed. (C) From the differences in images separated by a given lag time (1), DDM analysis computes the matrix D(q,t), where q is the magnitude of the wave vector (2). The intermediate scattering functions (ISFs) f(q,t) versus lag time for each spatial frequency q describes the ensemble dynamics (3).

  • Fig. 2 Ring DNA in cytoskeleton composites exhibits unique two-phase subdiffusion distinct from linear DNA and amplified by cytoskeleton crosslinking.

    (A) MSDs versus time for ring (open circles) and linear (closed squares) DNA in entangled (cyan; E) and crosslinked (magenta; XL) actin-microtubule networks. Horizontal dashed line denotes where MSD = ξ2. Black lines represent power-law scaling with exponents listed. Fits of the MSDs to the power-law relation MSD = Ktα yield transport coefficients K (B) and scaling exponents α (C) for linear (closed squares) and ring (open and crossed circles) DNA. MSDs for linear DNA obey a single power law over the entire measurement time [squares in (C)], while rings exhibit a second slower phase with lower α values [crossed circles in (C)] starting at ~0.4 μm2. (C) Linear DNA exponents are determined from fits over t = 0.1 to 4 s (closed squares), while ring DNA exhibits two different exponents with values determined from fits over t = 0.1 to 2 s (open circles) and t = 2 to 4 s (crossed circles). As shown, linear DNA exhibits faster transport and less subdiffusion in crosslinked compared to entangled networks, while crosslinking has the opposite effect on ring DNA.

  • Fig. 3 Both DNA topology and cytoskeleton crosslinking affect the conformational dynamics of ring and linear DNA in cytoskeleton composites.

    (A) Probability distributions of the coil sizes Rcoil for every frame of every molecule. Rcoil is rescaled by the expected dilute-limit end-to-end distance R0, which we denote as rcoil. Distributions show compaction of linear DNA (squares; distribution centered at <1) from normal R0 values, while ring DNA (open circles) swells and accesses a broader range of coil sizes (distribution centered at >1, broader than linear DNA distributions). (B) Mean rescaled coil sizes <rcoil> quantify the swelling of rings (circles) and compaction of linear DNA (squares) in entangled (E; cyan) and crosslinked (XL; magenta) networks. (C) FWHM of rcoil distributions shown in (A), displaying the topology-dependent range of conformational states accessed by DNA. (D) Fractional fluctuation length Lf(t) = <|Rmax(0) − Rmax(t)|>/<Rmax> for linear and ring DNA with black lines denoting power-law scaling with exponent listed. Linear DNA fluctuates more quickly and over a larger range than ring DNA, approaching steady-state values in contrast to the slow power-law rise of ring DNA. (E) The final fractional fluctuation length Lf,f plotted alongside the time τ at which molecules reach 90% of Lf,f. As shown, ring DNA fluctuates more slowly and over a smaller range than linear DNA in both entangled and crosslinked networks.

  • Fig. 4 DDM reveals heterogeneous slow transport of ring DNA with unique sensitivity to crosslinking.

    (A) Average ISF f(q,t) with q = 2.53 rad μm−1 for ring (circles) and linear (squares) DNA in entangled (cyan) and crosslinked (magenta) cytoskeleton composites. Displayed curves are averages over 20 regions of interest (ROIs). ISFs for linear DNA decay much faster than for ring DNA and exhibit expected exponential decay not seen for rings. While crosslinking slows the decay for rings, it has a negligible effect on the linear DNA ISF. (B and C) All individual ISFs (gray) comprising the average ISF (color coded) for linear and ring DNA in entangled (B) and crosslinked (C) composites. The substantial spread in ISF curves for ring DNA and the slow decay to zero—both features absent for linear DNA—indicate heterogeneous or multimode transport and anomalous slow diffusion, respectively. ISFs for crosslinked networks show a slightly smaller spread for ring topologies, while the spread for linear topologies is slightly larger. (D) That spread in ISFs is quantified by taking the difference between the maximum and minimum values of f(q,t), Δf(q,t), among the multiple ROIs at q = 2.53 rad μm−1. (E) The average Δf(q,t) over the range of time lags is greater with ring than with linear DNA for both networks. For rings, moving from an entangled to a crosslinked network decreases the spread. Conversely, for linear DNA, the spread increases slightly upon crosslinking.

  • Fig. 5 Ring DNA molecules in cytoskeleton networks adopt multiple modes of transport that are not accessible to linear DNA and are affected by cytoskeleton crosslinking.

    Cartoon of ring (green) and linear (red) DNA diffusing through entangled and crosslinked networks of actin (purple) and microtubules (blue). Time scale is arbitrary, and cartoons are not drawn to scale. Each panel is a depiction of a slice in the xy plane with DNA aligned in the plane and actin and microtubule constraints oriented along z. In right-hand panels (Δt = 2), lighter shaded circles denote the two previous positions (Δt = 0, 1) of the corresponding constraints. Within entangled networks, linear DNA can reptate through the network, while ring DNA adopts branched, folded, or threaded conformations. Reptation of the entangled cytoskeleton filaments allows threaded rings to become unthreaded via constraint release of the threading filaments. Crosslinking suppresses the mobility of cytoskeleton filaments that can cause rings to become permanently threaded, slowing their transport, while, at the same time, increasing the mobility of linear DNA as described in text (Fig. 2).

Supplementary Materials

  • Supplementary Materials

    This PDF file includes:

    • Table S1. Quantities derived from single-molecule conformational dynamics analysis.
    • Fig. S1. Distribution of individual MSDs from SMCT analysis.
    • Fig. S2. Fits to D(q,t) from DDM analysis.

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