Mobility
and
Conformational
Dynamics
of
large
DNA
diffusing
through
Cytoskeletal
Networks
Mobility and Conformational Dynamics of large DNA diffusing through Cytoskeletal Networks
Kathryn
Regan
Kathryn Regan
Navikka
Dasz,
Shea
Ricketts,
Rae
M.
Robertson-Anderson
Navikka Dasz, Shea Ricketts, Dr. Rae M. R. Anderson
University
of
San
Diego
University of San Diego
Abstract: The high concentrations of proteins crowding cells greatly influence intracellular DNA dynamics. These crowders, ranging from small mobile proteins to large cytoskeletal filaments such as semiflexible actin and rigid
The high concentrations of proteins crowding cells greatly influence intracellular DNA dynamics. These crowders, ranging from small mobile proteins to large cytoskeletal filaments such as semiflexible actin and rigid
microtubules, can hinder diffusion and induce conformational changes in DNA. Here, we use fluorescence microscopy and custom single-molecule conformational tracking algorithms to measure center-of-mass transport and timemicrotubules, can hinder diffusion and induce conformational changes in DNA. Here, we use fluorescence microscopy and custom single-molecule conformational tracking algorithms to measure center-of-mass transport
varying conformational sizes and shapes of single 115 kbp DNA molecules diffusing in networks of actin filaments and microtubules. We determine the role of protein concentration and rigidity (actin vs microtubules) on DNA
and time-varying conformational sizes and shapes of single 115 kbp DNA molecules diffusing in networks of actin filaments and microtubules. We determine the role of protein concentration and rigidity (actin vs
dynamics. Corresponding measurements with monomeric actin and tubulin identify the roles that network rigidity versus excluded volume play in transport. Initial results show that crowding by microtubules induces anomalous
microtubules) on DNA dynamics. Corresponding measurements with monomeric actin and tubulin identify the roles that network rigidity versus excluded volume play in transport. Initial results show that crowding by
transport and larger, slower conformational fluctuations of DNA.
microtubules induces anomalous, slower conformational fluctuations of DNA.
(α)
DNA Transport through the Cytoskeleton
The shape and structure of biological cells is maintained in part by a cytoskeleton composed
primarily of thick, rigid microtubules (LP = 1mm) made up of tubulin dimers, and thinner,
more flexible actin filaments (F-actin, LP = 17μm) comprised of actin monomers. These
proteins exist in both monomer and polymer form, depending on the requirements of the
cell, and contribute to cellular crowding. As such, the cytoskeleton greatly influences the
diffusion and conformational changes of nucleic acids and proteins in the cytoplasm.
MSD = t1
We track single DNA molecules diffusing in varying cytoskeleton environments
We label 115 kbp linear DNA with YOYO-I and image single We construct cytoskeleton networks with
DNA molecules using widefield fluorescence microscopy
varying degrees of rigidity
• Actin monomers (~5nm)
• Tubulin Monomers (~10nm)
• Actin Filaments (~10μm x 10nm)
• Microtubules (~10μm x 25nm)
We track single DNA molecules diffusing
in each network
Polymerized cytoskeleton proteins form
distinct rigid networks
• Actin (green): Thin, semiflexible
filaments, small mesh size
• Tubulin (red): thick, stiff filaments
(microtubules), large mesh size
We use high-speed fluorescence microscopy and novel particle-tracking techniques to
measure the transport and conformations of DNA crowded by cytoskeleton proteins
Create Samples:
• We embed 2.5ng/μL of labeled
DNA in 11.4μM solution of
tubulin or actin
• We polymerize actin or
tubulin by adding 2mM ATP
or GTP and incubating for 1 hr
at 37C
• We pipet solutions into sample
chamber and seal with epoxy
MSD = t0.72
Mean-squared displacements show
network-dependent DNA mobility
• Dilute DNA exhibits normal
diffusion  MSD ~ t
• Cytoskeleton networks exhibit
subdiffusive scaling: MSD ~ tα with
α<1
• Slope of MSDs determine diffusion
coefficients (MSD = 2Dt)
A.
Monomers
Polymers
Network Rigidity and Mesh Size determine
DNA mobility
• Polymers reduce mobility more than
monomers
• Smaller molecular spacing (actin) leads to
slower diffusion
• Actin filaments hinder DNA diffusion the
most  reduced to ~50% of diffusion in
microtubules
Reduced DNA diffusion is coupled to coil compaction
• Probability distributions of DNA coil diameters show
degree of coil compaction from dilute case
• Polymers compact DNA more than monomers due to
caging effects and depletion interactions
Actin compacts DNA more than microtubules
• Actin monomers and polymers have smaller
molecular spacing than microtubules
• Smaller spacing leads to more compaction and slower
diffusion
Compaction by
Tubulin Monomers
Data Acquisition:
• We image ~80 molecules in
each network using AIR
Nikon microscope with 60x
objective
• We track each molecule for
50 seconds at 10 fps
Center-of-Mass Tracking:
• We measure center-of-mass
position in each frame using
center-of-intensity algorithms
• We calculate COM meansquared displacements (MSD)
in x and y directions
• We calculate diffusion
coefficient (D) via
<x2 > = <y2> =2Dt
Coil Diameter (R) Tracking:
• We measure largest end-toend distance of DNA in
each frame
• Coil Diameter tracks DNA
coil size changes over time
• Changes in R can quantify
molecular compaction and
elongation
Crowding leads to subdiffusion
Crowding-induced subdiffusion enhanced by
polymers due to caging
• Actin has smaller cages than microtubules
 greater subdiffusive scaling
1.75 μm
2 μm
2 μm
Compaction by
Actin Monomers
Compaction by
Microtubules
1.75 μm
1.75
μm
1.75μm
Conclusion:
• Crowding by actin and tubulin induces compaction and decreased mobility of diffusing linear DNA.
• Crowding effects are increased with polymerization of cytoskeletal protein networks.
• Actin networks exhibit most dramatic crowding effects, due to their decreased molecular spacing.
Compaction by
Actin Filaments
1.65 μm
1.9 μm
1.9 μm
1.65
1.65μm
μm
References:
1. Chapman, Cole D., et al. “Crowding effects of molecular topology on
diffusion in entangled biopolymer blends.” Soft Matter 8.35 (2012): 91779182.
2. Gorzyca, S.M., et al. “Universal scaling of crowding-induced DNA
mobility is coupled with topology-dependent molecular compaction and
elongation.” Soft Matter 11 (2015): 7762-7768.
3. Hawkins, T., et al. “Mechanics of microtubules.” Journal of Biomechanics
1 (2010): 23-30.