10119469
Traction forces generated by locomoting keratocytes
Traction forces produced by moving fibroblasts have been observed as distortions in flexible substrata including wrinkling of thin, silicone rubber films. Traction forces generated by fibroblast lamellae were thought to represent the forces required to move the cell forwards. However, traction forces could not be detected with faster moving cell types such as leukocytes and growth cones (Harris, A. K., D. Stopak, and P. Wild. 1981. Nature (Lond.). 290:249-251). We have developed a new assay in which traction forces produced by rapidly locomoting fish keratocytes can be detected by the two-dimensional displacements of small beads embedded in the plane of an elastic substratum. Traction forces were not detected at the rapidly extending front edge of the cell. Instead the largest traction forces were exerted perpendicular to the left and right cell margins. The maximum traction forces exerted by keratocytes were estimated to be approximately 2 x 10(-8) N. The pattern of traction forces can be related to the locomotion of a single keratocyte in terms of lamellar contractility and area of close cell- substratum contact.
44856264
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.
20527996
The direction of membrane lipid flow in locomoting polymorphonuclear leukocytes.
The objective of this study was to determine the direction of membrane lipid flow in locomoting cells. The plasma membrane of human polymorphonuclear leukocytes was stained with a fluorescent lipid analog dihexadecanoyl indocarbocyanine. A line was photobleached on the cell surface perpendicular to the direction of cell motion. Low-light-level fluorescence microscopy and digital image-processing techniques were used to analyze a series of images taken at short intervals after photobleaching. The bleached line remained visible for about 5 seconds before being erased by diffusional recovery. Examination of fluorescence intensity profiles allowed a comparison to be made between the velocities of line and cell movement. Results indicate that the bleached line moves forward with the same velocity as the cell during locomotion, refuting the retrograde lipid flow model of locomotion. Instead, the plasma membrane lipid appears to move forward according to either the unit movement of membrane or the tank track model of locomotion.
4429405
Regulation of cell movement is mediated by stretch-activated calcium channels
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.
4366904
Principles of locomotion for simple-shaped cells
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.
20482730
The composition and dynamics of cell-substratum adhesions in locomoting fish keratocytes.
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.
32155732
Measurement issues across different cultures.
PURPOSE
The purposes of this methodologic paper are to (1) describe theoretical background in conducting research across different cultures; (2) address measurement issues related to instrument administration; and (3) provide strategies to deal with measurement issues.


METHODS
A thorough review of the literature was conducted. A theoretical background is provided, and examples of administering instrument in studies are described.


RESULTS
When applying an instrument to different cultures, both equivalence and bias need to be established. Three levels of equivalence, i.e., construct equivalence, measurement unit equivalence, and full score comparability, need to be explained to maintain the same concept being measured. In this paper, sources of bias in construct, method, and item are discussed. Issues related to instrument administration in a cross-cultural study are described.


CONCLUSION
Researchers need to acknowledge various group differences in concept and/or language that include a specific set of symbols and norms. There is a need to question the philosophical and conceptual appropriateness of an assessment measure that has been conceptualized and operationalized in a different culture. Additionally, testing different response formats such as narrowing response range can be considered to reduce bias.
9308687
Multidrug-resistant tuberculosis in a lung transplant recipient.
Tuberculosis infection has been a relatively rare complication after lung transplantation. However, as more countries in which Mycobacterium tuberculosis infection remains endemic embark on lung transplant programs, the occurrence of multidrug-resistant tuberculosis after transplantation is a genuine threat. We report the first case of multidrug-resistant tuberculosis in a double-lung transplant recipient who probably acquired the disease from the donor. We discuss the problems in clinical management of post-transplant tuberculosis infection and of drug-resistance.
1587472
Calibration of sound forces in acoustic traps
A two-dimensional or transverse acoustic trapping and its capability to noninvasively manipulate micrometersized particles with focused sound beams were experimentally demonstrated in our previous work. To apply this technique, as in optical tweezers, for studying mechanical properties of and interactions among biological particles such as cells, the trapping forces must be calibrated against known forces, i.e., viscous drag forces exerted by fluid flows. The trapping forces and the trap stiffness were measured under various conditions and the results were reported in this paper. In the current experimental arrangement, because the trapped particles were positioned against an acoustically transparent mylar membrane, the ultrasound beam intensity distribution near the membrane must be carefully considered. The total intensity field (the sum of incident and scattering intensity fields) around the droplet was thus computed by finite element analysis (FEA) with the membrane included, and it was then used in the ray acoustics model to calculate the trapping forces. The membrane effect on trapping forces was discussed by comparing effective beam widths with and without the membrane. The FEA results found that the broader beam width, caused by the scattered beams from the neighboring membrane and the droplet, resulted in the lower intensity, or smaller force, on the droplet. The experimental results showed that the measured forces were as high as 64 nN. The trap stiffness, approximated as a linear spring, was estimated by linear regressions and found to be 1.3 to 4.4 nN/μm, which was on a larger scale than that of optical trapping estimated for red blood cells, a few tenths of piconewtons/nanometer. The experimental and theoretical results were in good agreement.
23992310
Differential regulation of neuronal and inducible nitric oxide synthase (NOS) in the spinal cord of mutant SOD1 (G93A) ALS mice.
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by degeneration of motor neurons throughout the central nervous system. Mutations of the free radical scavenging enzyme superoxide dismutase-1 (SOD1) are a cause of familial ALS but it is not known how mutations lead to cell death. Free radicals such as nitric oxide (NO) are thought to play a key pathogenic role. NO is synthesized by NO synthases (NOSs) from arginine, which is a rate-limiting factor for NO production. We found that neuronal NOS (nNOS)-positive motor neurons were depleted while inducible NOS (iNOS)-positive activated glial cells were increased in transgenic mtSOD1 (G93A) ALS mice. iNOS expression was up regulated consistent with the increases of motor neuron loss and glial activation and citrulline and NO levels while nNOS expression was decreased in G93A ALS mice. Administration of l-arginine to G93A mice reduced the severity of motor neuron depletion and glial activation. In treated animals, nNOS expression was preserved while citrulline and NO were reduced, possibly due to reduced activation of glia expressing iNOS. Our findings show that high concentrations of NO correlate with iNOS expression rather than nNOS expression in G93A ALS mice. This suggests that therapy focused on iNOS inhibition might be a fruitful direction for future ALS therapeutic trials.
35904594
Targeted cell immobilization by ultrasound microbeam.
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.