Denis Wirtz

The physics of cancer: the role of physical interactions and mechanical forces in metastasis

Metastasis is a complex, multistep process responsible for >90% of cancer-related deaths. In addition to genetic and external environmental factors, the physical interactions of cancer cells with their microenvironment, as well as their modulation by mechanical forces, are key determinants of the metastatic process. We reconstruct the metastatic process and describe the importance of key physical and mechanical processes at each step of the cascade. The emerging insight into these physical interactions may help to solve some long-standing questions in disease progression and may lead to new approaches to developing cancer diagnostics and therapies.

Micro- and macrorheology of mucus.

Mucus is a complex biological material that lubricates and protects the human lungs, gastrointestinal (GI) tract, vagina, eyes, and other moist mucosal surfaces. Mucus serves as a physical barrier against foreign particles, including toxins, pathogens, and environmental ultrafine particles, while allowing rapid passage of selected gases, ions, nutrients, and many proteins. Its selective barrier properties are precisely regulated at the biochemical level across vastly different length scales. At the macroscale, mucus behaves as a non-Newtonian gel, distinguished from classical solids and liquids by its response to shear rate and shear stress, while, at the nanoscale, it behaves as a low viscosity fluid. Advances in the rheological characterization of mucus from the macroscopic to nanoscopic levels have contributed critical understanding to mucus physiology, disease pathology, and the development of drug delivery systems designed for use at mucosal surfaces. This article reviews the biochemistry that governs mucus rheology, the macro- and microrheology of human and laboratory animal mucus, rheological techniques applied to mucus, and the importance of an improved understanding of the physical properties of mucus to advancing the field of drug and gene delivery.

Mechanics of living cells measured by laser tracking microrheology

To establish laser-tracking microrheology (LTM) as a new technique for quantifying cytoskeletal mechanics, we measure viscoelastic moduli with wide bandwidth (5 decades) within living cells. With the first subcellular measurements of viscoelastic phase angles, LTM provides estimates of solid versus liquid behavior at different frequencies. In LTM, the viscoelastic shear moduli are inferred from the Brownian motion of particles embedded in the cytoskeletal network. Custom laser optoelectronics provide sub-nanometer and near-microsecond resolution of particle trajectories. The kidney epithelial cell line, COS7, has numerous spherical lipid-storage granules that are ideal probes for noninvasive LTM. Although most granules are percolating through perinuclear spaces, a subset of perinuclear granules is embedded in dense viscoelastic cytoplasm. Over all time scales embedded particles exhibit subdiffusive behavior and are not merely tethered by molecular motors. At low frequencies, lamellar regions (820 +/- 520 dyne/cm(2)) are more rigid than viscoelastic perinuclear regions (330 +/- 250 dyne/cm(2), p < 0.0001), but spectra converge at high frequencies. Although the actin-disrupting agent, latrunculin A, softens and liquefies lamellae, physiological levels of F-actin, alone (11 +/- 1.2 dyne/cm(2)) are approximately 70-fold softer than lamellae. Therefore, F-actin is necessary for lamellae mechanics, but not sufficient. Furthermore, in time-lapse of apparently quiescent cells, individual lamellar granules can show approximately 4-fold changes in moduli that last >10 s. Over a broad range of frequencies (0.1-30, 000 rad/s), LTM provides a unique ability to noninvasively quantify dynamic, local changes in cell viscoelasticity.

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.

Efficient active transport of gene nanocarriers to the cell nucleus

The intracellular transport of therapeutic gene carriers is poorly understood, limiting the rational design of efficient new vectors. We used live-cell real-time multiple particle tracking to quantify the intracellular transport of hundreds of individual nonviral DNA nanocarriers with 5-nm and 33-ms resolution. Unexpected parallels between several of natures most efficient DNA viruses and nonviral polyethylenimine/DNA nanocomplexes were revealed to include motor protein-driven transport through the cytoplasm toward the nucleus on microtubules. Active gene carrier transport led to efficient perinuclear accumulation within minutes. The results provide direct evidence to dispute the common belief that the efficiency of nonviral gene carriers is dramatically reduced because of the need for their relatively slow random diffusion through the cell cytoplasm to the nucleus and, instead, focuses the attention of rational carrier design on overcoming barriers downstream of perinuclear accumulation.

A perinuclear actin cap regulates nuclear shape

Defects in nuclear morphology often correlate with the onset of disease, including cancer, progeria, cardiomyopathy, and muscular dystrophy. However, the mechanism by which a cell controls its nuclear shape is unknown. Here, we use adhesive micropatterned surfaces to control the overall shape of fibroblasts and find that the shape of the nucleus is tightly regulated by the underlying cell adhesion geometry. We found that this regulation occurs through a dome-like actin cap that covers the top of the nucleus. This cap is composed of contractile actin filament bundles containing phosphorylated myosin, which form a highly organized, dynamic, and oriented structure in a wide variety of cells. The perinuclear actin cap is specifically disorganized or eliminated by inhibition of actomyosin contractility and rupture of the LINC complexes, which connect the nucleus to the actin cap. The organization of this actin cap and its nuclear shape-determining function are disrupted in cells from mouse models of accelerated aging (progeria) and muscular dystrophy with distorted nuclei caused by alterations of A-type lamins. These results highlight the interplay between cell shape, nuclear shape, and cell adhesion mediated by the perinuclear actin cap.

Particle-Tracking Microrheology of Living Cells: Principles and Applications

A multitude of cellular and subcellular processes depend critically on the mechanical deformability of the cytoplasm. We have recently introduced the method of particle-tracking microrheology, which measures the viscoelastic properties of the cytoplasm locally and with high spatiotemporal resolution. Here we establish the basic principles of particle-tracking microrheology, describing the advantages of this approach over more conventional approaches to cell mechanics. We present basic concepts of molecular mechanics and polymer physics relevant to the microrheological response of cells. Particle-tracking microrheology can probe the mechanical properties of live cells in experimentally difficult, yet more physiological, environments, including cells embedded inside a 3D matrix, adherent cells subjected to shear flows, and cells inside a developing embryo. Particle-tracking microrheology can readily reveal the lost ability of diseased cells to resist shear forces.

Micromechanical Mapping of Live Cells by Multiple-Particle-Tracking Microrheology

This paper introduces the method of live-cell multiple-particle-tracking microrheology (MPTM), which quantifies the local mechanical properties of living cells by monitoring the Brownian motion of individual microinjected fluorescent particles. Particle tracking of carboxylated microspheres imbedded in the cytoplasm produce spatial distributions of cytoplasmic compliances and frequency-dependent viscoelastic moduli. Swiss 3T3 fibroblasts are found to behave like a stiff elastic material when subjected to high rates of deformations and like a soft liquid at low rates of deformations. By analyzing the relative contributions of the subcellular compliances to the mean compliance, we find that the cytoplasm is much more mechanically heterogeneous than reconstituted actin filament networks. Carboxylated microspheres embedded in cytoplasm through endocytosis and amine-modified polystyrene microspheres, which are microinjected or endocytosed, often show directed motion and strong nonspecific interactions with cytoplasmic proteins, which prevents computation of local moduli from the microsphere displacements. Using MPTM, we investigate the mechanical function of alpha-actinin in non-muscle cells: alpha-actinin-microinjected cells are stiffer and yet mechanically more heterogeneous than control cells, in agreement with models of reconstituted cross-linked actin filament networks. MPTM is a new type of functional microscopy that can test the local, rate-dependent mechanical and ultrastructural properties of living cells.

Hypoxia and the extracellular matrix: drivers of tumour metastasis.

Of the deaths attributed to cancer, 90% are due to metastasis, and treatments that prevent or cure metastasis remain elusive. Emerging data indicate that hypoxia and the extracellular matrix (ECM) might have crucial roles in metastasis. During tumour evolution, changes in the composition and the overall content of the ECM reflect both its biophysical and biological properties and these strongly influence tumour and stromal cell properties, such as proliferation and motility. Originally thought of as independent contributors to metastatic spread, recent studies have established a direct link between hypoxia and the composition and the organization of the ECM, which suggests a new model in which multiple microenvironmental signals might converge to synergistically influence metastatic outcome.

Cell migration without a lamellipodium translation of actin dynamics into cell movement mediated by tropomyosin

The actin cytoskeleton is locally regulated for functional specializations for cell motility. Using quantitative fluorescent speckle microscopy (qFSM) of migrating epithelial cells, we previously defined two distinct F-actin networks based on their F-actin–binding proteins and distinct patterns of F-actin turnover and movement. The lamellipodium consists of a treadmilling F-actin array with rapid polymerization-dependent retrograde flow and contains high concentrations of Arp2/3 and ADF/cofilin, whereas the lamella exhibits spatially random punctae of F-actin assembly and disassembly with slow myosin-mediated retrograde flow and contains myosin II and tropomyosin (TM). In this paper, we microinjected skeletal muscle αTM into epithelial cells, and using qFSM, electron microscopy, and immunolocalization show that this inhibits functional lamellipodium formation. Cells with inhibited lamellipodia exhibit persistent leading edge protrusion and rapid cell migration. Inhibition of endogenous long TM isoforms alters protrusion persistence. Thus, cells can migrate with inhibited lamellipodia, and we suggest that TM is a major regulator of F-actin functional specialization in migrating cells.