Cell migration requires the transmission of motion generated in the actin cytoskeleton to the extracellular environment through a complex assembly of proteins in focal adhesions. We developed correlational fluorescent speckle microscopy to measure the coupling of focal-adhesion proteins to actin filaments. Different classes of focal-adhesion structural and regulatory molecules exhibited varying degrees of correlated motions with actin filaments, indicating hierarchical transmission of actin motion through focal adhesions. Interactions between vinculin, talin, and actin filaments appear to constitute a slippage interface between the cytoskeleton and integrins, generating a molecular clutch that is regulated during the morphodynamic transitions of cell migration.

Crawling locomotion of eukaryotic cells is achieved by a process dependent on the actin cytoskeleton1: protrusion of the leading edge requires assembly of a network of actin filaments2, which must be disassembled at the cell rear for sustained motility. Although ADF/cofilin proteins have been shown to contribute to actin disassembly3, it is not clear how activity of these locally acting proteins could be coordinated over the whole-cell distance scale. Here we show that nonmuscle myosin II plays a direct role in actin network disassembly in crawling cells. In moving fish keratocytes, myosin II is concentrated in regions at the rear with high rates of network disassembly. Activation of myosin II by ATP in detergent-extracted cytoskeletons results in rear-localized disassembly of the actin network. Inhibition of myosin II activity and stabilization of actin filaments synergistically impede cell motility, suggesting the existence of two disassembly pathways, one of which requires myosin II activity. Our results establish the importance of myosin II as an enzyme for actin network disassembly; we propose that gradual formation and reorganization of an actomyosin network provides an intrinsic destruction timer, enabling long-range coordination of actin network treadmilling in motile cells.

Here we introduce plusTipTracker, a Matlab-based open source software package that combines automated tracking, data analysis, and visualization tools for movies of fluorescently-labeled microtubule (MT) plus end binding proteins (+TIPs). Although +TIPs mark only phases of MT growth, the plusTipTracker software allows inference of additional MT dynamics, including phases of pause and shrinkage, by linking collinear, sequential growth tracks. The algorithm underlying the reconstruction of full MT trajectories relies on the spatially and temporally global tracking framework described in (Jaqaman et al., 2008). Post-processing of track populations yields a wealth of quantitative phenotypic information about MT network architecture that can be explored using several visualization modalities and bioinformatics tools included in plusTipTracker. Graphical user interfaces enable novice Matlab users to track thousands of MTs in minutes. In this paper we describe the algorithms used by plusTipTracker and show how the package can be used to study regional differences in the relative proportion of MT subpopulations within a single cell. The strategy of grouping +TIP growth tracks for the analysis of MT dynamics has been introduced before (Matov et al., 2010). The numerical methods and analytical functionality incorporated in plusTipTracker substantially advance this previous work in terms of flexibility and robustness. To illustrate the enhanced performance of the new software we thus compare computer-assembled +TIP-marked trajectories to manually-traced MT trajectories from the same movie used in (Matov et al., 2010).

Regulation of microtubule dynamics is essential for many cell biological processes, and is likely to be variable between different subcellular regions. We describe a computational approach to analyze microtubule dynamics by detecting growing microtubule plus ends. Our algorithm tracks all EB1-EGFP comets visible in an image time-lapse sequence allowing the detection of spatial patterns of microtubule dynamics. We use spatiotemporal clustering of EB1-EGFP growth tracks to infer microtubule behaviors during phases of pause and shortening. The algorithm was validated by comparison to manually tracked, homogeneously labeled microtubules, and by analysis of the effects of well-characterized inhibitors of microtubule polymerization dynamics. We used our method to analyze spatial variations of intracellular microtubule dynamics in migrating epithelial cells.

The compliance and dimensionality of the ECM regulate distinct changes in microtubule growth speed and growth persistence.