Chiral growth of adherent filopodia.

Adherent filopodia are elongated finger-like membrane protrusions, extending from the edges of diverse cell types and participating in cell adhesion, spreading, migration, and environmental sensing. The formation and elongation of filopodia are driven by the polymerization of parallel actin filaments, comprising the filopodia cytoskeletal core. Here, we report that adherent filopodia, formed during the spreading of cultured cells on galectin-8-coated substrates, tend to change the direction of their extension in a chiral fashion, acquiring a left-bent shape. Cryoelectron tomography examination indicated that turning of the filopodia tip to the left is accompanied by the displacement of the actin core bundle to the right of the filopodia midline. Reduction of the adhesion to galectin-8 by treatment with thiodigalactoside abolished this filopodia chirality. By modulating the expression of a variety of actin-associated filopodia proteins, we identified myosin-X and formin DAAM1 as major filopodia chirality promoting factors. Formin mDia1, actin filament elongation factor VASP, and actin filament cross-linker fascin were also shown to be involved. Thus, the simple actin cytoskeleton of filopodia, together with a small number of associated proteins are sufficient to drive a complex navigation process, manifested by the development of left-right asymmetry in these cellular protrusions.

Differential cellular responses to adhesive interactions with galectin-8- and fibronectin-coated substrates.

The mechanisms underlying the cellular response to extracellular matrices (ECMs) that consist of multiple adhesive ligands are still poorly understood. Here, we address this topic by monitoring specific cellular responses to two different extracellular adhesion molecules - the main integrin ligand fibronectin and galectin-8, a lectin that binds ß-galactoside residues  - as well as to mixtures of the two proteins. Compared with cell spreading on fibronectin, cell spreading on galectin-8-coated substrates resulted in increased projected cell area, more-pronounced extension of filopodia and, yet, the inability to form focal adhesions and stress fibers. These differences can be partially reversed by experimental manipulations of small G-proteins of the Rho family and their downstream targets, such as formins, the Arp2/3 complex and Rho kinase. We also show that the physical adhesion of cells to galectin-8 was stronger than adhesion to fibronectin. Notably, galectin-8 and fibronectin differently regulate cell spreading and focal adhesion formation, yet act synergistically to upregulate the number and length of filopodia. The physiological significance of the coherent cellular response to a molecularly complex matrix is discussed. This article has an associated First Person interview with the first author of the paper.

The Actin Network Interfacing Diverse Integrin-Mediated Adhesions.

The interface between the cellular actin network and diverse forms of integrin-mediated cell adhesions displays a unique capacity to serve as accurate chemical and mechanical sensors of the cell's microenvironment. Focal adhesion-like structures of diverse cell types, podosomes in osteoclasts, and invadopodia of invading cancer cells display distinct morphologies and apparent functions. Yet, all three share a similar composition and mode of coupling between a protrusive structure (the lamellipodium, the core actin bundle of the podosome, and the invadopodia protrusion, respectively), and a nearby adhesion site. Cytoskeletal or external forces, applied to the adhesion sites, trigger a cascade of unfolding and activation of key adhesome components (e.g., talin, vinculin, integrin), which in turn, trigger the assembly of adhesion sites and generation of adhesion-mediated signals that affect cell behavior and fate. The structural and molecular mechanisms underlying the dynamic crosstalk between the actin cytoskeleton and the adhesome network are discussed.

A network of mixed actin polarity in the leading edge of spreading cells.

Physical interactions of cells with the underlying extracellular matrix (ECM) play key roles in multiple cellular processes. The actin cytoskeleton is a central driver and regulator of cellular dynamics, that produces membrane-protrusions such as lamellipodia and filopodia. Here, we examined actin organization in expanding lamellipodia during early stages of cell spreading. To gain insight into the 3D actin organization, we plated fibroblasts on galectin-8 coated EM grids, an ECM protein presents in disease states. We then combined cryo-electron tomography with advanced image processing tools for reconstructing the structure of F-actin in the lamellipodia. This approach enabled us to resolve the polarity and orientation of filaments, and the structure of the Arp2/3 complexes associated with F-actin branches. We show that F-actin in lamellipodial protrusions forms a dense network with three distinct sub-domains. One consists primarily of radial filaments, with their barbed ends pointing towards the membrane, the other is enriched with parallel filaments that run between the radial fibers, in addition to an intermediate sub-domain. Surprisingly, a minor, yet significant (~10%) population of actin filaments, are oriented with their barbed-ends towards the cell center. Our results provide structural insights into F-actin assembly and dynamic reorganization in the leading edge of spreading cells.

Unveiling the polarity of actin filaments by cryo-electron tomography.

The actin cytoskeleton plays a fundamental role in numerous cellular processes, such as cell motility, cytokinesis, and adhesion to the extracellular matrix. Revealing the polarity of individual actin filaments in intact cells would foster an unprecedented understanding of cytoskeletal processes and their associated mechanical forces. Cryo-electron tomography provides the means for high-resolution structural imaging of cells. However, the low signal-to-noise ratio of cryo-tomograms obscures the high frequencies, and therefore the polarity of actin filaments cannot be directly measured. Here, we developed a method that enables us to determine the polarity of actin filaments in cellular cryo-tomograms. We applied it to reveal the actin polarity distribution in focal adhesions, and show a linear relation between actin polarity and distance from the apical boundary of the adhesion site.