New insight into the dynamics of individual actin filaments

The assembly-disassembly dynamics of cytoskeletal polymers such as actin filaments and microtubules support motile and morphogenetic processes in eukaryotes. Understanding the detailed molecular mechanisms by which these dynamic processes are controlled is a challenging task that requires combined biochemical and biophysical approaches. New technological developments in monitoring single actin filament dynamics with unprecedented accuracy and temporal resolution allows quantitative theoretical analysis and mathematical modeling of the key regulatory mechanisms.

HFSP Program Grant holders Marie-France Carlier and Reinhard Lipowsky and HFSP Young Investigator Grant holder Guillaume Romet-Lemonne and colleagues
authored on Fri, 05 October 2012

In the past ten years, fluorescence microscopy observation of « live» individual actin filaments has opened perspectives in the analysis of regulatory mechanisms of actin dynamics, at the origin of cell motility. The recent development of microfluidics has greatly facilitated filament imaging, statistical analysis of the length changes while improving spatial and temporal resolution and avoiding artifacts inherent to some of the previous methods. 

Actin filaments are semi-flexible polymers, their fluctuations in shape complicate the analysis of length changes during assembly and disassembly monitored in conventional total internal reflection fluorescence microscopy (TIRFM).

The alignment of a large number of filaments in a microflow coupled with fluorescence microscopy (both epifluorescence and TIRF), and the very fast change in the solution composition offered by the microfluidic device greatly facilitate the observation of actin filament dynamics and enable accurate kinetic analyses of processes taking place on the filament following environmental changes with 1 to 2 s dead time. The method has been validated by solving two issues: first, to demonstrate that Pi release following cleavage of ATP associated with filament assembly occurs in a random fashion as the filament grows, which has important implications regarding the length fluctuations of the actin filament (PloSBiol 2011); second, in demonstrating that the depolymerization of individual filaments displays interruptions or pauses arising from the slow dissociation of covalent actin dimers formed upon photo-irradiation of fluorescently labeled subunits (PNAS 2012). The latter result, which was based on the statistical analysis of the interruption times, ruled out previous views of filament stabilization by ageing.

The combined experimental and theoretical method used in these two studies provides a unique probe for the interactions between actin protomers and, thus, for the stability and turnover of single filaments. This probe is rather sensitive and can detect changes of less than 0.6 kcal/mol in protomer interactions. Such changes should be induced by many  actin-binding molecules as well as regulatory proteins and can now be elucidated in a quantitative manner.

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Figure: Intermittent depolymerization of actin filaments: (a) Actin filaments are aligned by a continuous microfluidic flow, and depolymerization is induced by fast switching to a flow channel without actin; (b) Observed time evolution of the filament length during depolymerization; black data points correspond to a filament grown from MgATP-actin whereas red, green, and blue data points were obtained for three filaments grown from MgADP-actin. The interruption of the depolymerization process (white arrow) occurs when an actin dimer arrives at the filament end. The pause is terminated (black arrow) when the dimer finally dissociates from the filament; (c) The cumulative distribution for the interruption times has a sigmoidal shape which implies that the interruption process is both local and random. The theoretical analysis involves only a single fit parameter, the transition rate ω for the photo-induced dimerization process. This rate is proportional to the fraction of fluorescently labeled actin, see inset.


Intermittent depolymerization of actin filaments is caused by photo-induced dimerization of actin protomers. Niedermayer T, Jégou A, Chièze L, Guichard B, Helfer E, Romet-Lemonne G, Carlier MF, Lipowsky R.  Proc Natl Acad Sci U S A. 2012 Jul 3;109(27):10769-74. Epub 2012 Jun 13.

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