The use of gelatin substrates for traction force microscopy in rapidly moving cells.
The study of traction forces generated by rapidly moving cells requires the use of substrates that are highly elastic because these cells typically generate weaker traction forces than slower moving cells. Gelatin substrates are soft enough to allow deformation by rapidly moving cells such as fish epidermal keratocytes and Dictyostelium discoideum amoebas. In addition, gelatin substrates are thin (approximately 30-40 microm) and transparent, allowing them to be used in combination with high-resolution calcium imaging. Importantly, the responsiveness of gelatin substrates allows changes in traction force generation to be detected within seconds, corresponding to the timescale of calcium transients. Here we describe the manufacture and application of gelatin substrates to study the role of mechanochemical signaling in the regulation of keratocyte movement. We show how patterns of traction force generation can be analyzed from a time series of traction vector maps, and how to interpret them in relation to cell movement. In addition, we discuss how the gelatin traction force assay is being used to study the mechanics of Dictyostelium cell motility, and future applications such as the study of neuronal path finding.

title: The composition and dynamics of cell-substratum adhesions in locomoting fish keratocytes.
abstract: Close contacts are the predominant type of cell-substratum adhesion in rapidly moving cells yet little is known about their composition and dynamics. To address these issues we have attempted to identify the molecular components of close contacts formed in rapidly moving fish epithelial keratocytes. In addition we have utilized the simple shape of keratocytes to explore the relationship between close contact formation and rapid locomotion. Beta1 integrin and talin molecules were found to be localized within a narrow rim of very close contact along the leading edge. These molecules together with vinculin were also found within small foci distributed evenly throughout the lamella, corresponding to regions of variable close contact. Alpha-actinin was found in foci within older, more posteriorly located regions of the lamella and along stress fibers. In addition to close contacts, small focal adhesion-like structures which stained positively for all antibodies tested were found at the tips of stress fibers within retracting cell margins. Interference reflection and total internal reflection microscopy of moving keratocytes showed cell-substratum contacts to be organized into distinct patterns that appear to move forwards, in concert with the leading edge. A feature common to all cells is a rim of very close contact at the leading edge. This region is specialized for the formation of new cell-substratum adhesions and is the site where patterns of close contact are generated. We have found that cell locomotion is most rapid when a uniform contact pattern is present but cell speed is progressively reduced as the contact pattern becomes more irregular. Furthermore, the local rate of lamellar extension is most rapid when underlain by regions of intermediate closeness to the substratum, but is reduced or ceases if the underlying contact is either very close or more distant, respectively. Our results suggest that close contacts and focal adhesions are related structures formed from a common hierarchy of molecular interactions. In addition the relationship between close contact formation and lamellar extension indicates a direct coupling between these two processes at the leading edge. Furthermore, we can explain the dynamic behaviour of close contacts in terms of the relative rates of trapping and release of component molecules that is initiated at the leading edge. We suggest that regulation of the molecular dynamics involved in leading edge specialization determines both the pattern of cell-substratum contacts and the net rate of actin filament assembly.

title: Principles of locomotion for simple-shaped cells
abstract: MOVING cells display a variety of shapes and modes of locomotion1, but it is not clear how motility at the molecular level relates to the locomotion of a whole cell, a problem compounded in studies of cells with complex shapes2–5. A striking feature of fish epidermal keratocyte locomotion is its apparent simplicity6. Here we present a kinematic description of locomotion which is consistent with the semicircular shape and persistent 'gliding'; motion of fish epidermal keratocytes. We propose that extension of the front and retraction of the rear of these cells occurs perpendicularly to the cell edge, and that a graded distribution of extension and retraction rates along the cell margin maintains cell shape and size during locomotion. Evidence for this description is provided by the predicted circumferential motion of lamellar features and the curvature of 'photo-marked' lines within specific molecular components of moving keratocytes. Our description relates the dynamics of molecular assemblies to the movement of a whole cell.

title: Regulation of cell movement is mediated by stretch-activated calcium channels
abstract: Intracellular calcium regulates many of the molecular processes that are essential for cell movement. It is required for the production of actomyosin-based contractile forces, the regulation of the structure and dynamics of the actin cytoskeleton,, and the formation and disassembly of cell–substratum adhesions,. Calcium also serves as a second messenger in many biochemical signal-transduction pathways. However, despite the pivotal role of calcium in motile processes, it is not clear how calcium regulates overall cell movement. Here we show that transient increases in intracellular calcium, [Ca2+]i, during the locomotion of fish epithelial keratocytes, occur more frequently in cells that become temporarily ‘stuck’ to the substratum or when subjected to mechanical stretching. We find that calcium transients arise from the activation of stretch-activated calcium channels, which triggers an influx of extracellular calcium. In addition, the subsequent increase in [Ca2+]i is involved in detachment of the rear cell margin. Thus, we have defined a mechanism by which cells can detect and transduce mechanical forces into biochemical signals that can modulate locomotion.

title: Targeted cell immobilization by ultrasound microbeam.
abstract: Various techniques exerting mechanical stress on cells have been developed to investigate cellular responses to externally controlled stimuli. Fundamental mechanotransduction processes about how applied physical forces are converted into biochemical signals have often been examined by transmitting such forces through cells and probing its pathway at cellular levels. In fact, many cellular biomechanics studies have been performed by trapping (or immobilizing) individual cells, either attached to solid substrates or suspended in liquid media. In that context, we demonstrated two-dimensional acoustic trapping, where a lipid droplet of 125 µm in diameter was directed transversely toward the focus (or the trap center) similar to that of optical tweezers. Under the influence of restoring forces created by a 30 MHz focused ultrasound beam, the trapped droplet behaved as if tethered to the focus by a linear spring. In order to apply this method to cellular manipulation in the Mie regime (cell diameter > wavelength), the availability of sound beams with its beamwidth approaching cell size is crucial. This can only be achieved at a frequency higher than 100 MHz. We define ultrasound beams in the frequency range from 100 MHz to a few GHz as ultrasound microbeams because the lateral beamwidth at the focus would be in the micron range. Hence a zinc oxide (ZnO) transducer that was designed and fabricated to transmit a 200 MHz focused sound beam was employed to immobilize a 10 µm human leukemia cell (K-562) within the trap. The cell was laterally displaced with respect to the trap center by mechanically translating the transducer over the focal plane. Both lateral displacement and position trajectory of the trapped cell were probed in a two-dimensional space, indicating that the retracting motion of these cells was similar to that of the lipid droplets at 30 MHz. The potential of this tool for studying cellular adhesion between white blood cells and endothelial cells was discussed, suggesting its capability as a single cell manipulator.

title: Vessel Diameter Measurement from Intravital Microscopy
abstract: The blood vessel diameter is often measured in microcirculation studies to quantify the effects of various stimuli. Intravital video microscopy is used to measure the change in vessel diameter by first recording the video and analyzing it using electronic calipers or by using image shearing technique. Manual measurement using electronic calipers or image shearing is time-consuming and prone to measurement error, and automated measurement can serve as an alternative that is faster and more reliable. In this paper, a new feature-based tracking algorithm is presented for automatically measuring diameter of vessels in intravital video microscopy image sequences. Our method tracks the vessel diameter throughout the entire image sequence once the diameter is marked in the first image. The parameters were calibrated using the intravital videos with manual ground truth measurements. The expriment with 10 synthetic videos and 20 intravital microscopy videos, including 10 fluorescence confocal and 10 non-confocal transmission, shows that the measurement can be performed accurately.

title: Synthesis and evaluation of aza-peptidyl inhibitors of the lysosomal asparaginyl endopeptidase, legumain.
abstract: Legumain or asparaginly endopeptidase (AEP) is a lysosomal cysteine protease with a high level of specificity for cleavage of protein substrates after an asparagine residue. It is also capable of cleaving after aspartic acids sites when in the acidic environment of the lysosome. Legumain expression and activity is linked to a number of pathological conditions including cancer, atherosclerosis and inflammation, yet its biological role in these pathologies is not well-understood. Highly potent and selective inhibitors of legumain would not only be valuable for studying the functional roles of legumain in these conditions, but may have therapeutic potential as well. We describe here the design, synthesis and in vitro evaluation of selective legumain inhibitors based on the aza-asparaginyl scaffold. We synthesized a library of aza-peptidyl inhibitors with various non-natural amino acids and different electrophilic warheads, and characterized the kinetic properties of inactivation of legumain. We also synthesized fluorescently labeled inhibitors to investigate cell permeability and selectivity of the compounds. The inhibitors have second order rate constants of up to 5 × 10(4)M(-1)s(-1) and IC(50) values as low as 4 nM against recombinant mouse legumain. In addition, the inhibitors are highly selective toward legumain and have little or no cross-reactivity with cathepsins. Overall, we have identified several valuable new inhibitors of legumain that can be used to study legumain function in multiple disease models.

title: Microcantilever actuation via periodic internal heating.
abstract: This paper reports electrothermal actuation of silicon microcantilevers having integrated resistive heaters. Periodic electrical excitation induced periodic resistive heating in the cantilever, while the cantilever deflection was monitored with a photodetector. Excitation was either at the cantilever resonant frequency, f(0), f(0)/2, or f(0)/3. When the time averaged maximum cantilever temperature was 174 degrees C, the cantilever out-of-plane actuation amplitude was 484 nm near the cantilever resonance frequency of 24.9 kHz. This actuation was sufficiently large to operate the cantilever in intermittent contact mode and scan a calibration grating of height of 20 nm.

title: Free amino acid and cysteine sulfoxide composition of 11 garlic (Allium sativum L.) cultivars by gas chromatography with flame ionization and mass selective detection.
abstract: Two garlic subspecies (n = 11), Allium sativum L. var. opioscorodon (hardneck) and Allium sativum L. var. sativum (softneck), were evaluated for their free amino acid composition. The free amino acid content of garlic samples analyzed ranged from 1121.7 to 3106.1 mg/100 g of fresh weight (mean = 2130.7 +/- 681.5 mg/100 g). Hardneck garlic had greater methiin, alliin, and total free amino acids contents compared to softneck garlic. The major free amino acid present in all but one subspecies was glutamine (cv. Mother of Pearl had aspartic acid as the major free amino acid). Cv. Music Pink garlic (a rocambole hardneck variety) contained the most methiin, alliin, and total free amino acids. The solid-phase extraction, alkylchloroformate derivatization, GC-FID, and GC-MS methods used in this study were simple and rapid, allowing 18 free amino acids in garlic to be separated within 10 min.