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.     

Direct observation of mobility state transitions in RNA trajectories by sensitive single molecule feedback tracking

Observation and tracking of fluorescently labeled molecules and particles in living cells reveals detailed information about intracellular processes on the molecular level. Whereas light microscopic particle observation is usually limited to two-dimensional projections of short trajectory segments, we report here image-based real-time three-dimensional single particle tracking in an active feedback loop with single molecule sensitivity. We tracked particles carrying only 1–3 fluorophores deep inside living tissue with high spatio-temporal resolution. Using this approach, we succeeded to acquire trajectories containing several hundred localizations. We present statistical methods to find significant deviations from random Brownian motion in such trajectories. The analysis allowed us to directly observe transitions in the mobility of ribosomal (r)RNA and Balbiani ring (BR) messenger (m)RNA particles in living Chironomus tentans salivary gland cell nuclei. We found that BR mRNA particles displayed phases of reduced mobility, while rRNA particles showed distinct binding events in and near nucleoli.     

Stochastically determined directed movement explains the dominant small-scale mitochondrial movements within non-neuronal tissue culture cells

The apparently stationary phase of mitochondrial motion was investigated in epithelial cells by spinning disk confocal light microscopy combined with image correlation based single particle tracking using custom software producing sub-pixel accuracy measurements (∼5 nm) at 10-12 Hz frame-rates. The analysis of these data suggests that the previously described stationary, or anchored phase, in mitochondrial movement actually comprise Brownian diffusion, interspersed with frequent and brief motor-driven events whose duration are stochastically determined. We have therefore discovered a new aspect of mitochondrial behavior, which we call stochastically determined, directed movement.     

Detection of temporary lateral confinement of membrane proteins using single-particle tracking analysis.

Techniques such as single-particle tracking allow the characterization of the movements of single or very few molecules. Features of the molecular trajectories, such as confined diffusion or directed transport, can reveal interesting biological interactions, but they can also arise from simple Brownian motion. Careful analysis of the data, therefore, is necessary to identify interesting effects from pure random movements. A method was developed to detect temporary confinement in the trajectories of membrane proteins that cannot be accounted for by Brownian motion. This analysis was applied to trajectories of two lipid-linked members of the immunoglobulin superfamily, Thy-1 and a neural cell adhesion molecule (NCAM 125), and the results were compared with those for simulated random walks. Approximately 28% of the trajectories for both proteins exhibited periods of transient confinement, which were < 0.07% likely to arise from random movements. In contrast to these results, only 1.5% of the simulated trajectories showed confined periods. Transient confinement for both proteins lasted on average 8 s in regions that were approximately 280 nm in diameter.     

Constrained diffusion or immobile fraction on cell surfaces: a new interpretation.

Protein lateral mobility in cell membranes is generally measured using fluorescence photobleaching recovery (FPR). Since the development of this technique, the data have been interpreted by assuming free Brownian diffusion of cell surface receptors in two dimensions, an interpretation that requires that a subset of the diffusing species remains immobile. The origin of this so-called immobile fraction remains a mystery. In FPR, the motions of thousands of particles are inherently averaged, inevitably masking the details of individual motions. Recently, tracking of individual cell surface receptors has identified several distinct types of motion (Gross and Webb, 1988; Ghosh and Webb, 1988, 1990, 1994; Kusumi et al. 1993; Qian et al. 1991; Slattery, 1995), thereby calling into question the classical interpretation of FPR data as free Brownian motion of a limited mobile fraction. We have measured the motion of fluorescently labeled immunoglobulin E complexed to high affinity receptors (Fc epsilon RI) on rat basophilic leukemia cells using both single particle tracking and FPR. As in previous studies, our tracking results show that individual receptors may diffuse freely, or may exhibit restricted, time-dependent (anomalous) diffusion. Accordingly, we have analyzed FPR data by a new model to take this varied motion into account, and we show that the immobile fraction may be due to particles moving with the anomalous subdiffusion associated with restricted lateral mobility. Anomalous subdiffusion denotes random molecular motion in which the mean square displacements grow as a power law in time with a fractional positive exponent less than one. These findings call for a new model of cell membrane structure.     

Quantitative Microscopy: Protein Dynamics and Membrane Organisation

The mobility of membrane proteins is a critical determinant of their interaction capabilities and protein functions. The heterogeneity of cell membranes imparts different types of motion onto proteins; immobility, random Brownian motion, anomalous sub-diffusion, 'hop' or confined diffusion, or directed flow. Quantifying the motion of proteins therefore enables insights into the lateral organisation of cell membranes, particularly membrane microdomains with high viscosity such as lipid rafts. In this review, we examine the hypotheses and findings of three main techniques for analysing protein dynamics: fluorescence recovery after photobleaching, single particle tracking and fluorescence correlation spectroscopy. These techniques, and the physical models employed in data analysis, have become increasingly sophisticated and provide unprecedented details of the biophysical properties of protein dynamics and membrane domains in cell membranes. Yet despite these advances, there remain significant unknowns in the relationships between cholesterol-dependent lipid microdomains, protein-protein interactions, and the effect of the underlying cytoskeleton. New multi-dimensional microscopy approaches may afford greater temporal and spatial resolution resulting in more accurate quantification of protein and membrane dynamics in live cells.     

Measuring Stepwise Binding of Thermally Fluctuating Particles to Cell Membranes without Fluorescence

Thermal motions enable a particle to probe the optimal interaction state when binding to a cell membrane. However, especially on the scale of microseconds and nanometers, position and orientation fluctuations are difficult to observe with common measurement technologies. Here, we show that it is possible to detect single binding events of immunoglobulin-G-coated polystyrene beads, which are held in an optical trap near the cell membrane of a macrophage. Changes in the spatial and temporal thermal fluctuations of the particle were measured interferometrically, and no fluorophore labeling was required. We demonstrate both by Brownian dynamic simulations and by experiments that sequential stepwise increases in the force constant of the bond between a bead and a cell of typically 20 pN/μm are clearly detectable. In addition, this technique provides estimates about binding rates and diffusion constants of membrane receptors. The simple approach of thermal noise tracking points out new strategies in understanding interactions between cells and particles, which are relevant for a large variety of processes, including phagocytosis, drug delivery, and the effects of small microplastics and particulates on cells.     

Prolonged activation of cAMP-dependent protein kinase during conditioning induces long-term memory in honeybees

To investigate the function cAMP-dependent protein kinase (PKA) exerts in the induction of long-term memory, changes in PKA activity induced by associative learning in vivo were measured in the antennal lobes (ALs) of honeybees. The temporal dynamics of PKA activation depend on both the sequence of conditioned and unconditioned stimuli and the number of conditioning trials. Only multiple-trial conditioning, which induces long-term memory (LTM), leads to a profound prolongation of PKA activation mediated by the NO/cGMP system. Imitation of this prolonged PKA activation in the ALs in combination with single-trial conditioning is sufficient to induce LTM. These findings not only demonstrate the close connection between conditioning procedure and temporal dynamics in PKA activation but also reveal that already during conditioning a distinct temporal pattern of PKA activation is critical for LTM induction in intact animals.     

3D Single Molecule Tracking with Multifocal Plane Microscopy Reveals Rapid Intercellular Transferrin Transport at Epithelial Cell Barriers

The study of intracellular transport pathways at epithelial cell barriers that line diverse tissue sites is fundamental to understanding tissue homeostasis. A major impediment to investigating such processes at the subcellular level has been the lack of imaging approaches that support fast three-dimensional (3D) tracking of cellular dynamics in thick cellular specimens. Here, we report significant advances in multifocal plane microscopy and demonstrate 3D single molecule tracking of rapid protein dynamics in a 10 micron thick live epithelial cell monolayer. We have investigated the transferrin receptor (TfR) pathway, which is not only essential for iron delivery but is also of importance for targeted drug delivery across cellular barriers at specific body sites, such as the brain that is impermeable to blood-borne substances. Using multifocal plane microscopy, we have discovered a cellular process of intercellular transfer involving rapid exchange of Tf molecules between two adjacent cells in the monolayer. Furthermore, 3D tracking of Tf molecules at the lateral plasma membrane has led to the identification of different modes of endocytosis and exocytosis, which exhibit distinct temporal and intracellular spatial trajectories. These results reveal the complexity of the 3D trafficking pathways in epithelial cell barriers. The methods and approaches reported here can enable the study of fast 3D cellular dynamics in other cell systems and models, and underscore the importance of developing advanced imaging technologies to study such processes.     

Morphogenetic movements during axolotl neural tube formation tracked by digital imaging

During neurulation in vertebrate embryos, epithelial cells of the neural plate undergo complex morphogenetic movements that culminate in rolling of the plate into a tube. Resolution of the determinants of this process requires an understanding of the precise movements of cells within the epithelial sheet. A computer algorithm that allows automated tracking of epithelial cells visible in digitized video images is presented. It is used to quantify the displacement field associated with morphogenetic movements in the axolotl (Ambystoma mexicanum) neural plate during normal neural tube formation. Movements from lateral to medial, axial elongations and area changes are calculated from the displacement field data and plotted as functions of time. Regional and temporal differences are identified. The approach presented is suitable for analyzing a wide variety of morphogenetic movements.     

Local small airway epithelial injury induces global smooth muscle contraction and airway constriction

Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (～250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ～70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca(2+) wave in the epithelium, and multiple Ca(2+) waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca(2+) or decreasing intracellular Ca(2+) both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca(2+)-dependent smooth muscle shortening.     

Generalized Scaling and the Master Variable for Brownian Magnetic Nanoparticle Dynamics

Understanding the dynamics of magnetic particles can help to advance several biomedical nanotechnologies. Previously, scaling relationships have been used in magnetic spectroscopy of nanoparticle Brownian motion (MSB) to measure biologically relevant properties (e.g., temperature, viscosity, bound state) surrounding nanoparticles in vivo. Those scaling relationships can be generalized with the introduction of a master variable found from non-dimensionalizing the dynamical Langevin equation. The variable encapsulates the dynamical variables of the surroundings and additionally includes the particles’ size distribution and moment and the applied field’s amplitude and frequency. From an applied perspective, the master variable allows tuning to an optimal MSB biosensing sensitivity range by manipulating both frequency and field amplitude. Calculation of magnetization harmonics in an oscillating applied field is also possible with an approximate closed-form solution in terms of the master variable and a single free parameter.     

An Intermittent Model for Intracellular Motions of Gold Nanostars by k-Space Scattering Image Correlation

Anisotropic metallic nanoparticles have been devised as powerful potential tools for in vivo imaging, photothermal therapy, and drug delivery thanks to plasmon-enhanced absorption and scattering cross sections, ease in synthesis and functionalization, and controlled cytotoxicity. The rational design of all these applications requires the characterization of the nanoparticles intracellular trafficking pathways. In this work, we exploit live-cell time-lapse confocal reflectance microscopy and image correlation in both direct and reciprocal space to investigate the intracellular transport of branched gold nanostars (GNSs). Different transport mechanisms, spanning from pure Brownian diffusion to (sub-)ballistic superdiffusion, are revealed by temporal and spatio-temporal image correlation spectroscopy on the tens-of-seconds timescale. According to these findings, combined with numerical simulations and with a Bayesian (hidden Markov model-based) analysis of single particle tracking data, we ascribe the superdiffusive, subballistic behavior characterizing the GNSs dynamics to a two-state switching between Brownian diffusion in the cytoplasm and molecular motor-mediated active transport. For the investigation of intermittent-type transport phenomena, we derive an analytical theoretical framework for Fourier-space image correlation spectroscopy (kICS). At first, we evaluate the influence of all the dynamic and kinetic parameters (the diffusion coefficient, the drift velocity, and the transition rates between the diffusive and the active transport regimes) on simulated kICS correlation functions. Then we outline a protocol for data analysis and employ it to derive whole-cell maps for each parameter underlying the GNSs intracellular dynamics. Capable of identifying even simpler transport phenomena, whether purely diffusive or ballistic, our intermittent kICS approach allows an exhaustive investigation of the dynamics of GNSs and biological macromolecules.     

Single particle tracking. Analysis of diffusion and flow in two-dimensional systems.

Analysis of the trajectories of small particles at high spatial and temporal resolution using video enhanced contrast microscopy provides a powerful approach to characterizing the mechanisms of particle motion in living cells and in other systems. We present here the theoretical basis for the analysis of these trajectories for particles undergoing random diffusion and/or systematic transport at uniform velocity in two-dimensional systems. The single particle tracking method, based on observations of the trajectories of individual particles, is compared with methods that characterize the motions of a large collection of particles such as fluorescence photobleaching recovery. Determination of diffusion coefficients or transport velocities either from correlation of positions or of velocities of the particles is discussed. A result of practical importance is an analysis of the dependence of the expected statistical uncertainty of these determinations on the number of position measurements. This provides a way of judging the accuracy of the diffusion coefficients and transport velocities obtained using this approach.     

Inheritable forms of medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) arises from parafollicular or C cells of the thyroid that produce calcitonin. It accounts for 5-10% of all thyroid cancers. Hereditary MTC represents 20-30% of all MTCs. It can be transmitted with an autosomal dominant pattern, either as a single entity, familial MTC, or it can arise as part of a multiple endocrine neoplasia (MEN) syndrome type 2A or 2B. The identification of hereditary MTC has been facilitated in recent years by the direct analysis of the ret proto-oncogene.     

Imaging of single fluorescent molecules using video-rate confocal microscopy

Single fluorescent molecules in aqueous solution were imaged for the first time at video-rate using Nipkow disk-type confocal microscopy. Performance of this method was evaluated by imaging single kinesin molecules labeled with fluorescent dyes of tetramethylrhodamine (TMR) or IC5. Photodecomposition lifetimes of the fluorophores were approximately 10 s for TMR and approximately 2 s for IC5 under the incident laser power of 0.5 W/mm(2). Both the fluorescence intensity and the photobleaching rate were proportional to the laser power from 0.65 to 3 W/mm(2). 2D sliding movement of single kinesin molecules along microtubules on glass surface and 3D Brownian motion of individual kinesin molecules in viscous solution could be observed using this microscopy. These results indicated that this method could be applicable to the study of single molecular events in living cells at real time.