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High-speed single-particle tracking of GM1 in model membranes reveals anomalous diffusion due to interleaflet coupling and molecular pinning.

ABSTRACT: The biological functions of the cell membrane are influenced by the mobility of its constituents, which are thought to be strongly affected by nanoscale structure and organization. Interactions with the actin cytoskeleton have been proposed as a potential mechanism with the control of mobility imparted through transmembrane "pickets" or GPI-anchored lipid nanodomains. This hypothesis is based on observations of molecular mobility using various methods, although many of these lack the spatiotemporal resolution required to fully capture all the details of the interaction dynamics. In addition, the validity of certain experimental approaches, particularly single-particle tracking, has been questioned due to a number of potential experimental artifacts. Here, we use interferometric scattering microscopy to track molecules labeled with 20-40 nm scattering gold beads with simultaneous <2 nm spatial and 20 μs temporal precision to investigate the existence and mechanistic origin of anomalous diffusion in bilayer membranes. We use supported lipid bilayers as a model system and demonstrate that the label does not influence time-dependent diffusion in the small particle limit (≤40 nm). By tracking the motion of the ganglioside lipid GM1 bound to the cholera toxin B subunit for different substrates and lipid tail properties, we show that molecular pinning and interleaflet coupling between lipid tail domains on a nanoscopic scale suffice to induce transient immobilization and thereby anomalous subdiffusion on the millisecond time scale.

SUBMITTER: Spillane KM

PROVIDER: S-EPMC4160260 | biostudies-other | 2014 Sep

REPOSITORIES: biostudies-other

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Project description:Single-particle tracking is an important technique in the life sciences to understand the kinetics of biomolecules. The analysis of apparent diffusion coefficients in vivo, for example, enables researchers to determine whether biomolecules are moving alone, as part of a larger complex, or are bound to large cellular components such as the membrane or chromosomal DNA. A remaining challenge has been to retrieve quantitative kinetic models, especially for molecules that rapidly switch between different diffusional states. Here, we present analytical diffusion distribution analysis (anaDDA), a framework that allows for extracting transition rates from distributions of apparent diffusion coefficients calculated from short trajectories that feature less than 10 localizations per track. Under the assumption that the system is Markovian and diffusion is purely Brownian, we show that theoretically predicted distributions accurately match simulated distributions and that anaDDA outperforms existing methods to retrieve kinetics, especially in the fast regime of 0.1-10 transitions per imaging frame. AnaDDA does account for the effects of confinement and tracking window boundaries. Furthermore, we added the option to perform global fitting of data acquired at different frame times to allow complex models with multiple states to be fitted confidently. Previously, we have started to develop anaDDA to investigate the target search of CRISPR-Cas complexes. In this work, we have optimized the algorithms and reanalyzed experimental data of DNA polymerase I diffusing in live Escherichia coli. We found that long-lived DNA interaction by DNA polymerase are more abundant upon DNA damage, suggesting roles in DNA repair. We further revealed and quantified fast DNA probing interactions that last shorter than 10 ms. AnaDDA pushes the boundaries of the timescale of interactions that can be probed with single-particle tracking and is a mathematically rigorous framework that can be further expanded to extract detailed information about the behavior of biomolecules in living cells.

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High-speed single-particle tracking of GM1 in model membranes reveals anomalous diffusion due to interleaflet coupling and molecular pinning.

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