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Biomedical subjects

W H Guilford

Publications and source records attributed to W H Guilford.

12 recordsLinked to original sources
PubMed2001

Quantitative comparison of algorithms for tracking single fluorescent particles.

Single particle tracking has seen numerous applications in biophysics, ranging from the diffusion of proteins in cell membranes to the movement of molecular motors. A plethora of computer algorithms have been developed to monitor the sub-pixel displacement of fluorescent objects between successive video frames, and some have been claimed to have "nanometer" resolution. To date, there has been no rigorous comparison of these algorithms under realistic conditions. In this paper, we quantitatively compare specific implementations of four commonly used tracking algorithms: cross-correlation, sum-absolute difference, centroid, and direct Gaussian fit. Images of fluorescent objects ranging in size from point sources to 5 microm were computer generated with known sub-pixel displacements. Realistic noise was added and the above four algorithms were compared for accuracy and precision. We found that cross-correlation is the most accurate algorithm for large particles. However, for point sources, direct Gaussian fit to the intensity distribution is the superior algorithm in terms of both accuracy and precision, and is the most robust at low signal-to-noise. Most significantly, all four algorithms fail as the signal-to-noise ratio approaches 4. We judge direct Gaussian fit to be the best algorithm when tracking single fluorophores, where the signal-to-noise is frequently near 4.

Algorithms↗
PubMed2001

Teaching peer review and the process of scientific writing.

Many undergraduate and graduate students understand neither the process of scientific writing nor the significance of peer review. In response, some instructors have created writing assignments that teach or mimic parts of the scientific publishing process. However, none fully reproduced peer review and revision of papers together with the writing and publishing process from research to final, accepted draft. In addition, most have been instituted at the graduate rather than undergraduate level. We present a detailed method for teaching undergraduate students the full scientific publishing process, including anonymous peer review, during the process of writing a "term paper." The result is a review article in the format for submission to a major scientific journal. This method has been implemented in the course Cell and Molecular Biology for Engineers at the University of Virginia. Use of this method resulted in improved grades, much higher quality in the final manuscript, greater objectivity in grading, and improved understanding of the importance of peer review.

Attitude↗
PubMed2000

The light chain binding domain of expressed smooth muscle heavy meromyosin acts as a mechanical lever.

Structural data led to the proposal that the molecular motor myosin moves actin by a swinging of the light chain binding domain, or "neck." To test the hypothesis that the neck functions as a mechanical lever, smooth muscle heavy meromyosin (HMM) mutants were expressed with shorter or longer necks by either deleting or adding light chain binding sites. The mutant HMMs were characterized kinetically and mechanically, with emphasis on measurements of unitary displacements and forces in the laser trap assay. Two shorter necked constructs had smaller unitary step sizes and moved actin more slowly than WT HMM in the motility assay. A longer necked construct that contained an additional essential light chain binding site exhibited a 1.4-fold increase in the unitary step size compared with its control. Kinetic changes were also observed with several of the constructs. The mutant lacking a neck produced force at a somewhat reduced level, while the force exerted by the giraffe construct was higher than control. The single molecule displacement and force data support the hypothesis that the neck functions as a rigid lever, with the fulcrum for movement and force located at a point within the motor domain.

Actins↗
PubMed1999

Two heads of myosin are better than one for generating force and motion.

Several classes of the myosin superfamily are distinguished by their "double-headed" structure, where each head is a molecular motor capable of hydrolyzing ATP and interacting with actin to generate force and motion. The functional significance of this dimeric structure, however, has eluded investigators since its discovery in the late 1960s. Using an optical-trap transducer, we have measured the unitary displacement and force produced by double-headed and single-headed smooth- and skeletal-muscle myosins. Single-headed myosin produces approximately half the displacement and force (approximately 6 nm; 0.7 pN) of double-headed myosin (approximately 10 nm; 1.4 pN) during a unitary interaction with actin. These data suggest that muscle myosins require both heads to generate maximal force and motion.

Actins↗
PubMed1998

The molecular mechanics of smooth muscle myosin.

Smooth muscle cells are capable of generating forces comparable to those of skeletal muscle cells but with far less myosin, the molecular motor that powers muscle contraction. This unique capability may be inherent to the myosin molecule. We have directly characterized the molecular mechanics of smooth muscle myosin using new technologies developed to measure the forces generated by these proteins. The data help explain the differences in force and velocity in whole smooth and skeletal muscles.

Animals↗
PubMed1997

Smooth muscle and skeletal muscle myosins produce similar unitary forces and displacements in the laser trap.

Purified smooth muscle myosin in the in vitro motility assay propels actin filaments at 1/10 the velocity, yet produces 3-4 times more force than skeletal muscle myosin. At the level of a single myosin molecule, these differences in force and actin filament velocity may be reflected in the size and duration of single motion and force-generating events, or in the kinetics of the cross-bridge cycle. Specifically, an increase in either unitary force or duty cycle may explain the enhanced force-generating capacity of smooth muscle myosin. Similarly, an increase in attached time or decrease in unitary displacement may explain the reduced actin filament velocity of smooth muscle myosin. To discriminate between these possibilities, we used a laser trap to measure unitary forces and displacements from single smooth and skeletal muscle myosin molecules. We analyzed our data using mean-variance analysis, which does not rely on scoring individual events by eye, and emphasizes periods in the data with constant properties. Both myosins demonstrated multiple but similar event populations with discrete peaks at approximately +11 and -11 nm in displacement, and 1.5 and 3.5 pN in force. Mean attached times for smooth muscle myosin were longer than for skeletal-muscle myosin. These results explain much of the difference in actin filament velocity between these myosins, and suggest that an increased duty cycle is responsible for the enhanced force-generating capacity of smooth over skeletal-muscle myosin.

Actins↗
PubMed1997

Actin filament mechanics in the laser trap.

Numerous biological processes, including muscular contraction, depend upon the mechanical properties of actin filaments. One such property is resistance to bending (flexural rigidity, EI). To estimate EI, we attached the ends of fluorescently labelled actin filaments to two microsphere 'handles' captured in independent laser traps. The positions of the traps were manipulated to apply a range of tensions (0-8 pN) to the filaments via the microsphere handles. With increasing filament tension, the displacement of the microspheres was inconsistent with a microsphere-filament system that is rigid. We maintain that this inconsistency is due to the microspheres rotating in the trap and the filaments bending near either attachments to accommodate this rotation. Fitting the experimental data to a simple model of this phenomena, we estimate actin's EI to be approximately 15 x 10(3) pNnm2, a value within the range of previously reported results, albeit using a novel method. These results both: support the idea that actin filaments are more compliant than historically assumed; and, indicate that without appropriately pretensioning the actin filament in similar laser traps, measurements of unitary molecular events (e.g. myosin displacement) may be significantly underestimated.

Actins↗
PubMed1995

The mechanics of arteriole-tissue interaction.

Arterioles are embedded in the extensive connective tissue matrix of the interstitium. Mechanical interactions with the interstitium may affect the length-tension characteristics of arterioles, and thus affect their reactivity. However, no studies have adequately characterized the coupling between arterioles and the interstitium or investigated how the interstitium might change the physiological expression of arterioles. Therefore, the goal of this project was to investigate the mechanical interactions between arterioles and the interstitium and then to predict the physiological consequences of these interactions. We measured in situ the mechanical coupling of arterioles to the interstitium, the mechanical properties of the interstitium, and the structure of the interstitium in the hamster cheek pouch. We demonstrated that there are mechanical interactions between arterioles and the interstitium that are mediated both through direct connections and through the movement of extracellular fluid through the connective tissue network. We also found that the elastic modulus of the interstitium increases in the vicinity of the arteriole. Finally, both the mechanical coupling of arterioles to the interstitium and the mechanical properties of the interstitium are explained by the structure of the connective tissue matrix. The arterioles appear to be connected to adjacent fibroblasts and fibrocytes by collagen fibrils. These cells are in turn connected to the fiber matrix of the interstitium. Furthermore, the presence of these cells may explain the mechanical heterogeneity of the interstitium. We propose that the physiological role of the interstitium surrounding arterioles is to protect arterioles from stretching and deformation of the tissue while allowing these vessels to constrict freely.

Animals↗
PubMed1995

Locomotive forces produced by single leukocytes in vivo and in vitro.

We report here the first time-resolved measurements of the forces produced during the migration of single leukocytes in vivo and in vitro. Pulmonary macrophages from hamsters and mice, in vitro, and Nembutal (pentobarbital sodium)-anesthetized hamster neutrophils, in vivo, generated maximum locomotive forces ranging from 1.9 to 10.7 nN or tenths of microdynes. Force production was periodic and correlated with the length of the leading lamellipod but not with generalized cell ruffling. Although the extension of the leading lamella is critical to locomotive force generation, these direct measurements suggest that lamellar extension may not arise from the same contractile processes driving forward motion of the cell mass. Indeed, cell ruffling, lamellar extension, and locomotive force generation may be differentially controlled and have different origins. This technique may be extended to test numerous hypotheses of how these and other nonmuscle cells crawl.

Actins↗
PubMed1995

Smooth muscle myosin: a high force-generating molecular motor.

Smooth muscle generates as much force per cross sectional area of muscle as skeletal muscle with only one-fifth the myosin content. Although this apparent difference could be explained at the tissue or cellular level, it is possible that at the molecular level smooth muscle cross-bridges generate greater average force than skeletal muscle cross-bridges. To test this hypothesis, we used an in vitro motility assay (VanBuren et al., 1994) in which either chicken thiophosphorylated gizzard smooth or pectoralis skeletal muscle monomeric myosin is adhered to a nitrocellulose surface. A fluorescently labeled actin filament, attached to an ultracompliant (50-200 nm/pN) glass microneedle, is brought in contact with the myosin surface. Isometric force, being generated by myosin cross-bridges pulling on the attached actin filament, is calculated from the extent to which the calibrated microneedle is deflected. By measuring the density of myosin adhered to the surface, we estimated the number of myosin cross-bridges that are able to interact with a length of actin filament in contact with the myosin surface. In a direct comparison between smooth and skeletal muscle myosin, the average force per cross-bridge was 0.8 and 0.2 pN, respectively. Surprisingly, smooth muscle myosin generates approximately 4 times greater average force per cross-bridge head than skeletal muscle myosin. Because average isometric force is the product of the cross-bridge unitary force and duty cycle, we are presently using a laser optical trap in an attempt to measure unitary events from single myosin molecules. This approach should allow us to determine whether an increase in unitary force, duty cycle, or both contribute to smooth muscle myosin's enhanced force-generating capacity compared with skeletal muscle myosin.

Actins↗
PubMed1992

A novel remote-sensing isometric force transducer for micromechanics studies.

We have developed an innovative transducer for measuring force with femtonewton-to-micronewton resolution in biological systems. A magnetic microsphere is attached to the specimen being studied and is positioned between two electromagnets. Video microscopy and edge detection are used to monitor small movements of the microsphere that occur when the specimen generates force. An automatic control system adjusts the current through the electromagnets to keep the microsphere stationary. Measured force is a linear function of this current. This transducer is unique in its combination of sensitivity and isometric properties and its ability to measure force without direct connections to the specimen. That is, the transducer is "remote sensing" and can measure force through intervening membrane or tissue. The transducer is isometric at steady state to the limit at which displacement of the microsphere can be resolved, which can be as low as 19 nm. The completed system is being used to study the mechanics of interstitial connective tissue but may also be used to study molecular generation of force.

Animals↗