Yunfeng Feng

A distinctive role for focal adhesion proteins in three- dimensional cell motility

Focal adhesions are large multi-protein assemblies that form at the basal surface of cells on planar dishes, and that mediate cell signalling, force transduction and adhesion to the substratum. Although much is known about focal adhesion components in two-dimensional (2D) systems, their role in migrating cells in a more physiological three-dimensional (3D) matrix is largely unknown. Live-cell microscopy shows that for cells fully embedded in a 3D matrix, focal adhesion proteins, including vinculin, paxillin, talin, α-actinin, zyxin, VASP, FAK and p130Cas, do not form aggregates but are diffusely distributed throughout the cytoplasm. Despite the absence of detectable focal adhesions, focal adhesion proteins still modulate cell motility, but in a manner distinct from cells on planar substrates. Rather, focal adhesion proteins in matrix-embedded cells regulate cell speed and persistence by affecting protrusion activity and matrix deformation, two processes that have no direct role in controlling 2D cell speed. This study shows that membrane protrusions constitute a critical motility/matrix-traction module that drives cell motility in a 3D matrix.

Ajuba LIM Proteins Are Negative Regulators of the Hippo Signaling Pathway

The mammalian Ajuba LIM proteins (Ajuba, LIMD1, and WTIP) are adaptor proteins that exhibit the potential to communicate cell adhesive events with nuclear responses to remodel epithelia. Determining their role in vivo, however, has been challenging due to overlapping tissue expression and functional redundancy. Thus, we turned to Drosophila, where a single gene, CG11063 or djub, exists. Drosophila lacking the djub gene or depleted of dJub by RNA interference identify djub as an essential gene for development and a novel regulator of epithelial organ size as a component of the conserved Hippo (Hpo) pathway, which has been implicated in both tissue size control and cancer development. djub-deficient tissues were small and had decreased cell numbers as a result of increased apoptosis and decreased proliferation, due to downregulation of DIAP1 and cyclin E. This phenocopies tissues deficient for Yorkie (Yki), the downstream target of the Hippo pathway. djub genetically interacts with the Hippo pathway, and epistasis suggests that djub lies downstream of hpo. In mammalian and Drosophila cells, Ajuba LIM proteins/dJub interact with LATS/Warts (Wts) and WW45/Sav to inhibit phosphorylation of YAP/Yki. This work describes a novel role for the Ajuba LIM proteins as negative regulators of the Hippo signaling pathway.

Actin cap associated focal adhesions and their distinct role in cellular mechanosensing

The ability for cells to sense and adapt to different physical microenvironments plays a critical role in development, immune responses, and cancer metastasis. Here we identify a small subset of focal adhesions that terminate fibers in the actin cap, a highly ordered filamentous actin structure that is anchored to the top of the nucleus by the LINC complexes; these differ from conventional focal adhesions in morphology, subcellular organization, movements, turnover dynamics, and response to biochemical stimuli. Actin cap associated focal adhesions (ACAFAs) dominate cell mechanosensing over a wide range of matrix stiffness, an ACAFA-specific function regulated by actomyosin contractility in the actin cap, while conventional focal adhesions are restrictively involved in mechanosensing for extremely soft substrates. These results establish the perinuclear actin cap and associated ACAFAs as major mediators of cellular mechanosensing and a critical element of the physical pathway that transduce mechanical cues all the way to the nucleus.

The LIM protein Ajuba influences p130Cas localization and Rac1 activity during cell migration

Cell migration requires extension of lamellipodia that are stabilized by formation of adhesive complexes at the leading edge. Both processes are regulated by signaling proteins recruited to nascent adhesive sites that lead to activation of Rho GTPases. The Ajuba/Zyxin family of LIM proteins are components of cellular adhesive complexes. We show that cells from Ajuba null mice are inhibited in their migration, without associated abnormality in adhesion to extracellular matrix proteins, cell spreading, or integrin activation. Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes. In response to migratory cues Rac activation is blunted in Ajuba null cells, as detected biochemically and by FRET analysis. Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.

Ajuba LIM proteins are snail/slug corepressors required for neural crest development in Xenopus.

Snail family transcriptional repressors regulate epithelial mesenchymal transitions during physiological and pathological processes. A conserved SNAG repression domain present in all vertebrate Snail proteins is necessary for repressor complex assembly. Here, we identify the Ajuba family of LIM proteins as functional corepressors of the Snail family via an interaction with the SNAG domain. Ajuba LIM proteins interact with Snail in the nucleus on endogenous E-cadherin promoters and contribute to Snail-dependent repression of E-cadherin. Using Xenopus neural crest as a model of in vivo Snail- or Slug-induced EMT, we demonstrate that Ajuba LIM proteins contribute to neural crest development as Snail/Slug corepressors and are required for in vivo Snail/Slug function. Because Ajuba LIM proteins are also components of adherens junctions and contribute to their assembly or stability, their functional interaction with Snail proteins in the nucleus suggests that Ajuba LIM proteins are important regulators of epithelia dynamics communicating surface events with nuclear responses.

α-Catenin mediates initial E-cadherin-dependent cell–cell recognition and subsequent bond strengthening

α-Catenin is essential in cadherin-mediated epithelium development and maintenance of tissues and in cancer progression and metastasis. However, recent studies question the conventional wisdom that α-catenin directly bridges the cadherin adhesion complex to the actin cytoskeleton. Therefore, whether α-catenin plays a direct role in cadherin-dependent cell adhesion is unknown. Here, single-molecule force spectroscopy measurements in cells depleted of α-catenin or expressing the hereditary diffuse gastric cancer associated V832M E-cadherin germ-line missense mutation show that α-catenin plays a critical role in cadherin-mediated intercellular recognition and subsequent multibond formation within the first 300 ms of cell contact. At short contact times, α-catenin mediates a 30% stronger interaction between apposing E-cadherin molecules than when it cannot bind the E-cadherin–β-catenin complex. As contact time between cells increases, α-catenin is essential for the strengthening of the first intercellular cadherin bond and for the ensuing formation of additional bonds between the cells, all without the intervention of actin. These results suggest that a critical decision to form an adhesion complex between 2 cells occurs within an extremely short time span and at a single-molecule level and identify a previously unappreciated role for α-catenin in these processes.

Actin stress fiber pre-extension in human aortic endothelial cells

Actin stress fibers (SFs) enable cells to sense and respond to mechanical stimuli and affect adhesion, motility and apoptosis. We and others have demonstrated that cultured human aortic endothelial cells (HAECs) are internally stressed so that SFs are pre-extended beyond their unloaded lengths. The present study explores factors affecting SF pre-extension. In HAECs cultured overnight the baseline pre-extension was 1.10 and independent of the amount of cell shortening. Decreasing contractility with 30 mM BDM or 10 microM blebbistatin decreased pre-extension to 1.05 whereas increasing contractility with 2 nM calyculin A increased pre-extension to 1.26. Knockdown of alpha-actinin-1 with an interfering RNA increased pre-extension to 1.28. None of these affected the wavelength of the buckled SFs. Pre-extension was the same in unperturbed cells as in those in which the actin cytoskeleton was disrupted by both chemical and mechanical means and then allowed to reassemble. Finally, disrupting MTs or IFs did not affect pre-extension but increased the wavelength. Taken together, these results suggest that pre-extension of SFs is determined primarily by intrinsic factors, i.e. the level of actin-myosin interaction. This intrinsic control of pre-extension is sufficiently robust that pre-extension is the same even after the actin cytoskeleton has been disrupted and reorganized. Unlike pre-extension, the morphology of the compressed SFs is partially determined by MTs and IFs which appear to support the SFs along their lengths.

Dimensional and temporal controls of three-dimensional cell migration by zyxin and binding partners

Spontaneous molecular oscillations are ubiquitous in biology. But to our knowledge, periodic cell migratory patterns have not been observed. Here we report the highly regular, periodic migration of cells along rectilinear tracks generated inside three-dimensional matrices, with each excursion encompassing several cell lengths, a phenotype that does not occur on conventional substrates. Short hairpin RNA depletion shows that these one-dimensional oscillations are uniquely controlled by zyxin and binding partners α-actinin and p130Cas, but not vasodilator-stimulated phosphoprotein and cysteine-rich protein 1. Oscillations are recapitulated for cells migrating along one-dimensional micropatterns, but not on two-dimensional compliant substrates. These results indicate that although two-dimensional motility can be well described by speed and persistence, three-dimensional motility requires two additional parameters, the dimensionality of the cell paths in the matrix and the temporal control of cell movements along these paths. These results also suggest that the zyxin/α-actinin/p130Cas module may ensure that motile cells in a three-dimensional matrix explore the largest space possible in minimum time.

Loss of α-Catenin Decreases the Strength of Single E-cadherin Bonds between Human Cancer Cells

The progression of several human cancers correlates with the loss of cytoplasmic protein α-catenin from E-cadherin-rich intercellular junctions and loss of adhesion. However, the potential role of α-catenin in directly modulating the adhesive function of individual E-cadherin molecules in human cancer is unknown. Here we use single-molecule force spectroscopy to probe the tensile strength, unstressed bond lifetime, and interaction energy between E-cadherins expressed on the surface of live human parental breast cancer cells lacking α-catenin and these cells where α-catenin is re-expressed. We find that the tensile strength and the lifetime of single E-cadherin/E-cadherin bonds between parental cells are significantly lower over a wide range of loading rates. Statistical analysis of the force displacement spectra reveals that single cadherin bonds between cancer cells feature an exceedingly low energy barrier against tensile forces and low molecular stiffness. Disassembly of filamentous actin using latrunculin B has no significant effect on the strength of single intercellular E-cadherin bonds. The absence of α-catenin causes a dominant negative effect on both global cell-cell adhesion and single E-cadherin bond strength. These results suggest that the loss of α-catenin alone drastically reduces the adhesive force between individual cadherin pairs on adjoining cells, explain the global loss of cell adhesion in human breast cancer cells, and show that the forced expression of α-catenin in cancer cells can restore both higher intercellular avidity and intercellular E-cadherin bond strength.

Effect of Focal Adhesion Proteins on Endothelial Cell Adhesion, Motility and Orientation Response to Cyclic Strain

Focal adhesion proteins link cell surface integrins and intracellular actin stress fibers and therefore play an important role in mechanotransduction and cell motility. When endothelial cells are subjected to cyclic mechanical strain, time-lapse imaging revealed that cells underwent significant morphological changes with their resultant long axes aligned away from the strain direction. To explore how this response is regulated by focal adhesion-associated proteins the expression levels of paxillin, focal adhesion kinase (FAK), and zyxin were knocked down using gene silencing techniques. In addition, rescue of endogenous and two mutant zyxins were used to investigate the specific role of zyxin interactions. Cells with decreased zyxin expression levels and rescue with the mutant lacking zyxin/α-actinin binding exhibited lower orientation angles after comparable times of stretching as compared to normal and control cells. However, knockdown of the expression levels of paxillin and FAK and rescue with the mutant lacking zyxin/VASP (vasodilator-stimulated phosphoprotein) binding did not significantly affect the degree of cell orientation. In addition, wound closure speed and cell–substratum adhesive strength were observed to be significantly reduced only for cells with zyxin depletion and the mutation lacking zyxin/α-actinin binding. These results suggest that zyxin and its interaction with α-actinin are important in the regulation of endothelial cell adhesive strength, motility and orientation response to mechanical stretching.