Living cells are viscoelastic materials, with the elastic response dominating at long timescales (≳1 ms)1. At shorter timescales, the dynamics of individual cytoskeleton filaments are expected to emerge, but active microrheology measurements on cells accessing this regime are scarce2. Here, we develop high-frequency microrheology (HF-MR) to probe the viscoelastic response of living cells from 1Hz to 100 kHz. We report the viscoelasticity of different cell types and upon cytoskeletal drug treatments. At previously inaccessible short timescales, cells exhibit rich viscoelastic responses that depend on the state of the cytoskeleton. Benign and malignant cancer cells revealed remarkably different scaling laws at high frequency, providing a univocal mechanical fingerprint. Microrheology over a wide dynamic range up to the frequency of action of the molecular components provides a mechanistic understanding of cell mechanics.

We present a procedure that allows a reliable determination of the elastic (Young’s) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever’s spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions.

In eukaryotic cells, an actin-based cortex lines the inner leaflet of the plasma membrane, endowing the cells with crucial mechanical and functional properties. Unfortunately, it has not been possible to study the structural dynamics of the actin cortex at high lateral resolution in living cells. Here, we performed atomic force microscopy time-lapse imaging and mechanical mapping of actin in the cortex of living cells at high lateral and temporal resolution. Cortical actin filaments adopted discernible arrangements, ranging from large parallel bundles with low connectivity to a tight meshwork of short filaments. Mixing of these architectures resulted in attuned cortex networks with specific connectivity, mechanical responses, and marked differences in their dynamic behavior.

In multicellular organisms, cell shape and organization are dictated by cell-cell or cell-extracellular matrix adhesion interactions. Adhesion complexes crosstalk with the cytoskeleton enabling cells to sense their mechanical environment. Unfortunately, most of cell biology studies, and cell mechanics studies in particular, are conducted on cultured cells adhering to a hard, homogeneous, and unconstrained substrate with nonspecific adhesion sites, thus far from physiological and reproducible conditions. Here, we grew cells on three different fibronectin patterns with identical overall dimensions but different geometries (▽, T, and Y), and investigated their topography and mechanics by atomic force microscopy (AFM). The obtained mechanical maps were reproducible for cells grown on patterns of the same geometry, revealing pattern-specific subcellular differences. We found that local Young's moduli variations are related to the cell adhesion geometry. Additionally, we detected local changes of cell mechanical properties induced by cytoskeletal drugs. We thus provide a method to quantitatively and systematically investigate cell mechanics and their variations, and present further evidence for a tight relation between cell adhesion and mechanics.

Human Galectin-3 is found in the nucleus, the cytoplasm and at the cell surface. This lectin is constituted of two domains: an unfolded N-terminal domain and a C-terminal Carbohydrate Recognition Domain (CRD). There are still uncertainties about the relationship between the quaternary structure of Galectin-3 and its carbohydrate binding properties. Two types of self-association have been described for this lectin: a C-type self-association and a N-type self-association. Herein, we have analyzed Galectin-3 oligomerization by Dynamic Light Scattering using both the recombinant CRD and the full length lectin. Our results proved that LNnT induces N-type self-association of full length Galectin-3. Moreover, from Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance experiments, we observed no significant specificity or affinity variations for carbohydrates related to the presence of the N-terminal domain of Galectin-3. NMR mapping clearly established that the N-terminal domain interacts with the CRD. We propose that LNnT induces a release of the N-terminal domain resulting in the glycan-dependent self-association of Galectin-3 through N-terminal domain interactions.

The capacity of proteins to function relies on a balance between molecular stability to maintain their folded state and structural flexibility allowing conformational changes related to biological function. Among many others, four different examples can be chosen. The giant protein titin is stretched and can unfold during muscle contraction providing passive elasticity to muscle tissue; myoglobin adsorbs and releases oxygen molecules thank to conformational changes in its structure; the outer membrane protein G (OmpG) is a bacterial porin with a long and flexible loop that modulates gating; and the proton pump bacteriorhodopsin adapts its cytosolic half to allow proton pumping. All these conformational changes triggered either by chemical or by physical cues, require mechanical flexibility or elasticity of certain protein domains. While the methods to determine protein structure, X-ray crystallography above all, have been dramatically improved over the last decades, the number of tools that directly measure the mechanical flexibility of proteins and protein domains is still limited. In this tutorial, after a brief introduction to protein structure, we present some of the available techniques to estimate protein flexibility, then focusing on atomic force microscopy (AFM). We describe the principles of the technique and its various imaging and force spectroscopy modes of operation that allow probing the elasticity of proteins, protein domains and their surrounding environment.

In all organisms, replication impairment is a recognized source of genomic instability, raising an increasing interest in the fate of inactivated replication forks. We used Escherichia coli strains with a temperature-inactivated replicative helicase (DnaB) and in vivo single-molecule microscopy to quantify the detailed molecular processing of stalled replication forks. After helicase inactivation, RecA binds to blocked replication forks and is essential for the rapid release of hPol III. The entire holoenzyme is disrupted little by little, with some components lost in few minutes, while others are stable in 70% of cells for at least 1 hr. Although replisome dissociation is delayed in a recA mutant, it is not affected by RecF or RecO inactivation. RecFOR are required for full RecA filaments formation, and we propose that polymerase clearance can be catalyzed by short, RecFOR-independent RecA filaments. Our results identify a function for the universally conserved, central recombination protein RecA.

Cell motility is an essential cellular function required for tasks ranging from chemotaxis to the formation of multicellular structures. With motility being such a ubiquitous process, cells are outfitted with a vast yet well-conserved set of proteins that self-organize in order to form protrusions with a surprising range of shapes and sizes. One such hallmark shape is the lamellipodium: a long, flat, sheet-like protrusion used for migration along flat surfaces. At ten microns wide and two hundred nanometers tall, these high aspect ratio protrusions are formed by the coordinated force of thousands of actin polymers growing against the membrane at the leading edge. Even more surprising, these protrusions are stable on the order of minutes to hours. How are thousands of nanometer-sized actin filaments able to form a stable leading edge several orders of magnitude larger, in time and space, than the assembly of its individual components? Using high-speed, high-resolution phase imaging of a neutrophillike HL-60 cell line, I have discovered sub-second fluctuations along the leading edge of the lamellipodia. The time-evolution of these fluctuations is a direct readout of the self-correction mechanism used to create the smooth leading edge. High-resolution data and quantitative analysis now give us a handle to measure changes in leading-edge fluctuations in response to experimental perturbations. Using a cycle of theoretical modeling and experimental perturbation, I will tease apart the possible mechanisms for self-correction and the subsequent effects on the stability of the leading edge of motile cells. 667-Pos Board B447Separating Ciliary Beating Force into Dynein-Driven Force and Axoneme-Intrinsic Bending Force By 3-D Tracking Microscopy 5 JST-PRESTO, Saitama, Japan. Highly coordinated dyneins generate asymmetric ciliary beating composed of clearly distinguished effective and recovery strokes. We herein aim to understand the mechanism of asymmetric beating of a single cilium by combining multiple optical-microscope techniques: 3-D tracking; optical tweezers; DIC adjusted to the different focal planes. A fluorescent bead was attached to the tip of the surface-immobilized mouse-tracheal cilium that was reactivated with a 100 mM ATP-containing solution, and trapped at various points of the beating stroke to measure the 3-D force (F). Next, after the solution was replaced with 100 mM ATP and 50 mM Vanadate, we measured 2-D structural bending energy in a relaxed state at the same cilium. With the estimation of the 3-D force driven by dyneins (F D ) by subtraction of the effect of bending energy from F, the following were obtained: 1) F D periodically changed in the range from À75 to 35 pN, which is about 10 times larger than the force of viscous drag. 2) As the trapping point was displaced from the position of post-effective stroke, maximum values of F D (F DM ) of the effective stroke increased from 3 to 35 pN and F DM of the recovery stroke increased from À1 to À75 pN, suggesting that the change in the force depends on ...