Fast fluorescence laser tracking microrheometry, II: quantitative studies of cytoskeletal mechanotransduction.

Fluorescence laser tracking microrheometry (FLTM) is what we believe to be a novel method able to assess the local, frequency-dependent mechanical properties of living cells with nanometer spatial sensitivity at speeds up to 50 kHz. In an earlier article, we described the design, development, and optimization phases of the FLTM before reporting its performances in a variety of viscoelastic materials. In the work presented here, we demonstrate the suitability of FLTM to study local cellular rheology and obtain values for the storage and loss moduli G'(omega) and G''(omega) of fibroblasts consistent with past literature. We further establish that chemically induced cytoskeletal disruption is accompanied by reduced cellular stiffness and viscosity. Next, we provide a systematic study of some experimental variables that may critically influence microrheology measurements. First, we interrogate and justify the relevance of bead endocytosis as a method of cellular internalization of 1-microm probes in FLTM. Second, we show that as sample temperature increases, FLTM findings are elevated toward higher frequencies. Third, we confirm that relevant bead sizes (1 and 2 microm) have no effect on FLTM measurements. Fourth, we report the lack of influence of bead coatings (antiintegrin, antitransferrin, antidystroglycan, or uncoated tracers were surveyed) on their rheological readouts. Finally, we demonstrate the potential of FLTM in studying how substratum rigidity regulates cellular rheological properties. Interestingly, multiple, coupled strain relaxation mechanisms can be observed separated by two plateau moduli. Although these observations can be partly explained by rheological theories describing entangled actin filaments, there is a clear need to extend existing microrheology models to the cytoskeleton, including potentially important factors such as network geometry and remodeling.

Fast fluorescence laser tracking microrheometry. I: instrument development.

To gain insight into cellular mechanotransduction pathways, we have developed a fluorescence laser tracking microrheometer (FLTM) to measure material rheological features on micrometer length scales using fluorescent microspheres as tracer particles. The statistical analysis of the Brownian motion of a particle quantifies the viscoelastic properties of the probe's environment, parameterized by the frequency-dependent complex shear modulus G*(omega). This FLTM has nanometer spatial resolution over a frequency range extending from 1 Hz to 50 kHz. In this work, we first describe the consecutive stages of instrument design, development, and optimization. We subsequently demonstrate the accuracy of the FLTM by reproducing satisfactorily the known rheological characteristics of purely viscous glycerol solutions and cross-linked polyacrylamide polymer networks. An upcoming companion article will illustrate the use of FLTM in studying the solid-like versus liquid-like rheological properties of fibroblast cytoskeletons in living biological samples.

Detecting single quantum dot motion with nanometer resolution for applications in cell biology.

Quantum dots (QDs), semiconductor particles of nanometer dimension, have emerged as excellent fluorescent analogs in tracer experiments with single molecule sensitivity for bioassays. Cell imaging greatly benefits from the remarkable optical and physical properties of these inorganic nanocrystals: QDs are much brighter and exhibit a higher resistance to photobleaching than traditional fluorophores, and their narrow emission spectrum and flexible surface chemistry make them particularly suitable for multiplex imaging. Here, we have demonstrated the achievement of a nanometer spatial resolution on the position of a single QD in a simple optomechanical instrument using a high-sensitivity low-noise detector, an intensified CCD camera. Furthermore, nanometer variations in the amplitude of a QD's sinusoidal oscillations could be quantitatively distinguished after fast Fourier transform (FFT) based data processing. As confirmed by experiments where QDs were attached to the surface of bovine aortic endothelial cells, this method can be exploited in biology to assess molecular and subcellular contributions to responses such as motility, intracellular trafficking, and mechanotransduction, with high resolution and minimal disturbance to cells.

Mass spectrometry in high-throughput screening: a case study on acetyl-coenzyme a carboxylase using RapidFire--mass spectrometry (RF-MS).

In this review various technologies and approaches for the utilization of mass spectrometry in high-throughput analyses are discussed. The use of quadrupole-based mass spectrometry in the screening of chemical libraries against enzymatic targets for the identification of inhibitors and/or activators is highlighted. The RapidFire mass spectrometry system, an integrated on-line solid-phase extraction system interfaced to a triple-quadrupole mass spectrometer is described in detail, and the identification of a series of inhibitors of the acetyl-coenzyme A carboxylase (ACC) assay is described.

Cell mechanics at multiple scales.

The response of cells to mechanical stresses is a field of growing inquiry. It is well known that both the morphologic and molecular expression of cells depend, in part, on the local mechanical environment, especially for cells such as endothelial cells that experience shear stress, stretch, and pressures. To systematically study the large variety of responses of cells to physical forces (e.g., signaling, adhesion, or stiffness changes), a number of techniques have been developed and used. Here we present methods for three types of cell mechanical studies, from the multicellular to the subcellular scales, and describe the basic principle and main use of each technique along with some design and setup considerations.

Cell stiffness and receptors: evidence for cytoskeletal subnetworks.

Viscoelastic models of cells often treat cells as homogeneous objects. However, studies have demonstrated that cellular properties are local and can change dramatically on the basis of the location probed. Because membrane receptors are linked in various ways to the intracellular space, with some receptors linking to the cytoskeleton and others diffusing freely without apparent linkages, the cellular physical response to mechanical stresses is expected to depend on the receptor engaged. In this study, we tested the hypothesis that cellular mechanical stiffness as measured via cytoskeletally linked receptors is greater than stiffness measured via receptors that are not cytoskeletally linked. We used a magnetic micromanipulator to apply linear stresses to magnetic beads attached to living cells via selected receptors. One of the receptor classes probed, the dystroglycan receptors, is linked to the cytoskeleton, while the other, the transferrin receptors, is not. Fibronectin-coated beads were used to test cellular mechanical properties of the cytoskeleton without membrane dependence by allowing the beads to endocytose. For epithelial cells, transferrin-dependent stiffness and endocytosed bead-dependent stiffness were similar, while dystroglycan-dependent stiffness was significantly lower. For smooth muscle cells, dystroglycan-dependent stiffness was similar to the endocytosed bead-dependent stiffness, while the transferrin-dependent stiffness was lower. The conclusion of this study is that the measured cellular stiffness is critically influenced by specific receptor linkage and by cell type and raises the intriguing possibility of the existence of separate cytoskeletal networks with distinct mechanical properties that link different classes of receptors.