Systems Biology (Methods in Molecular Biology)

Chapter 13

Spatiotemporal Fluctuation Analysis of Molecular Diffusion

Laws in Live-Cell Membranes

Francesco Cardarelli

Abstract

A present challenge of membrane biophysics is deciphering the dynamic behavior of molecules, such as
lipids and proteins, within the natural environment of a living-cell membrane. Here, a fluorescence
fluctuation-based approach will be described, which makes it possible to probe the “diffusion law” of
molecules directly from imaging, in the form of a mean square displacement vs time-delay plot (iMSD),
with no need for interpretative models. Of note, the presented approach does not require extraction of the
molecular trajectories nor the use of bright fluorophores. Conversely, it can be used at high fluorophore
density and with relatively dim fluorophores, such as GFP-tagged molecules transiently expressed within
cells. The ability of this approach to resolve average molecular dynamic properties well below the diffraction
limit will be discussed. Overall, this novel approach is proposed as a powerful tool for the determination of
kinetic and thermodynamic parameters over wide spatial and temporal scales.

Key wordsFluorescence correlation spectroscopy, Fluctuation analysis, Protein dynamics, Diffusion

law, Membrane heterogeneity, Transient confinement, Dynamic partitioning, GFP

1 Introduction

A major challenge of present (and future) membrane biophysics is

to quantitatively study how molecular ensembles dynamically inter-

act and exert their functional role in live-cell membranes. In this

context, high-speed single-particle tracking (SPT) techniques play a

crucial role: several individual molecular components (proteins,

lipids, etc.) can be typically produced (e.g., by cloning or synthesis),

purified, fluorescently labeled, and re-introduced within the living

cell for SPT analysis. Based on SPT, for instance, Kusumi and cow-

orkers quantitatively addressed the compartmentalization of the

fluid plasma memb into submicron domains by extracting the

diffusion law (in the form of the classical mean squared displace-

ment, or MSD) of many relevant membrane components (for a

reviewsee[1, 2]). Yet, the SPTapproach is inherently endowed with

challenging experimental requirements such as: (1) production,

Mariano Bizzarri (ed.),Systems Biology, Methods in Molecular Biology, vol. 1702,
https://doi.org/10.1007/978-1-4939-7456-6_13,©Springer Science+Business Media LLC 2018

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