Justin E. Molloy

The gated gait of the processive molecular motor, myosin V.

Class V myosins are actin-based molecular motors involved in vesicular and organellar transport. Single myosin V molecules move processively along F-actin, taking several 36-nm steps for each diffusional encounter. Here we have measured the mechanical interactions between mouse brain myosin V and rabbit skeletal F-actin. The working stroke produced by a myosin V head is ∼25 nm, consisting of two separate mechanical phases (20 + 5 nm). We show that there are preferred myosin binding positions (target zones) every 36 nm along the actin filament, and propose that the 36-nm steps of the double-headed motor are a combination of the working stroke (25 nm) of the bound head and a biased, thermally driven diffusive movement (11 nm) of the free head onto the next target zone. The second phase of the working stroke (5 nm) acts as a gate — like an escapement in a clock, coordinating the ATPase cycles of the two myosin V heads. This mechanism increases processivity and enables a single myosin V molecule to travel distances of several hundred nanometres along the actin filament.

The motor protein myosin-I produces its working stroke in two steps

Many types of cellular motility, including muscle contraction, are driven by the cyclical interaction of the motor protein myosin with actin filaments, coupled to the breakdown of ATP. It is thought that myosin binds to actin and then produces force and movement as it ‘tilts’ or ‘rocks’ into one or more subsequent, stable conformations,. Here we use an optical-tweezers transducer to measure the mechanical transitions made by a single myosin head while it is attached to actin. We find that two members of the myosin-I family, rat liver myosin-I of relative molecular mass 130,000 (Mr 130K) and chick intestinal brush-border myosin-I, produce movement in two distinct steps. The initial movement (of roughly 6 nanometres) is produced within 10 milliseconds of actomyosin binding, and the second step (of roughly 5.5nanometres) occurs after a variable time delay. The duration of the period following the second step is also variable and depends on the concentration of ATP. At the highest time resolution possible (about 1 millisecond), we cannot detect this second step when studying the single-headed subfragment-1 of fast skeletal muscle myosin II. The slower kinetics of myosin-I have allowed us to observe the separate mechanical states that contribute to its working stroke.

Lights, action: optical tweezers

Optical tweezers were first realized 15 years ago by Arthur Ashkin and co-workers at the Bell Telephone Laboratories. Since that time there has been a steady stream of developments and applications, particularly in the biological field. In the last 5 years the flow of work using optical tweezers has increased significantly, and it seems as if they are set to become a mainstream tool within biological and nanotechnological fields. In this article we seek to explain the underpinning mechanism behind optical tweezers, to review the main applications of optical tweezers to date, to present some recent technological advances and to speculate on future applications within both biological and non-biological fields.

Physiological properties of the dorsal longitudinal flight muscle and the tergal depressor of the trochanter muscle of Drosophila melanogaster.

SummaryA prerequisite for using muscle mutants to study contraction inDrosophila melanogaster is a description of the mechanics of wild-type muscles. Here we describe the mechanics of two different wild-type muscles; the dorsal longitudinal flight muscle which is asynchronous (nerve impulses are not synchronised with each contraction), and a leg muscle, the tergal depressor of the trochanter, which is synchronous. We have compared their mechanics to those of the asynchronous flight and the synchronous leg muscle from the giant waterbugLethocerus indicus.We found that the mechanics of the asynchronous flight muscles from the two species were similar. At rest length both muscles had a high relaxed stiffness, were partially activated by Ca2+ (low steady-state active tension) and, once activated, had a large delayed increase in tension, which was well maintained, in response to a rapid stretch. The rate constant for the delayed increase in tension was about 10 times greater forD. melanogaster than forL. indicus under the same conditions. The mechanics of the synchronous leg muscles from both species were different from those of the flight muscles and resembled those of other synchronous muscles such as vertebrate striated muscle. At rest length, both muscles had a lower relaxed stiffness than the flight muscles, were fully activated by Ca2+ (high steady-state active tension) and, once activated, had a small delayed increase in tension, which was less well maintained, in response to a rapid stretch. The rate constant for the delayed increase in tension was similar for the leg muscles of both species.The different mechanical properties of the flight and leg muscles must arise from differences in their contractile proteins. The demonstration that satisfactory mechanical responses can be obtained from the small (less than 1 mm long) muscles ofD. melanogaster will enable future responses from mutant muscles to be tested.

Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy

Over the past 10 years, advances in laser and detector technologies have enabled single fluorophores to be visualized in aqueous solution. Here, we describe methods based on total internal reflection fluorescence microscopy (TIRFM) that we have developed to study the behavior of individual protein molecules within living mammalian cells. We have used cultured myoblasts that were transiently transfected with DNA plasmids encoding a target protein fused to green fluorescent protein (GFP). Expression levels were quantified from confocal images of control dilutions of GFP and cells with 1-100 nM GFP were then examined using TIRFM. An evanescent field was produced by a totally internally reflected, argon ion laser beam that illuminated a shallow region (50-100 nm deep) at the glass-water interface. Individual GFP-tagged proteins that entered the evanescent field appeared as individual, diffraction-limited spots of light, which were clearly resolved from background fluorescence. Molecules that bound to the basal cell membrane remained fixed in position for many seconds, whereas those diffusing freely in the cytoplasm disappeared within a few milliseconds. We developed automated detection and tracking methods to recognize and characterize the behavior of single molecules in recorded video sequences. This enabled us to measure the kinetics of photobleaching and lateral diffusion of membrane-bound molecules.

B Cells Use Mechanical Energy to Discriminate Antigen Affinities

B Cell Tug of War High-affinity, protective antibodies made by B cells are critical for providing long-term protection against reinfection. In order to produce antibodies, B cells must first bind to and extract antigens from the surface of antigen-presenting cells. Using an in vitro system that allows B cells to bind to antigenladen, flexible membranes, Natkanski et al. (p. 1587, published online 16 May) show that antigen extraction relies on myosin IIA–mediated contractile forces that pull upon the antigen-presenting membrane. These forces break the antigen-receptor bonds if affinity is low, thus ensuring that B cells only extract, internalize, and presumably respond to, high-affinity antigens. Mechanical forces allow immune B cells to extract high-affinity antigens from membrane surfaces. The generation of high-affinity antibodies depends on the ability of B cells to extract antigens from the surfaces of antigen-presenting cells. B cells that express high-affinity B cell receptors (BCRs) acquire more antigen and obtain better T cell help. However, the mechanisms by which B cells extract antigen remain unclear. Using fluid and flexible membrane substrates to mimic antigen-presenting cells, we showed that B cells acquire antigen by dynamic myosin IIa–mediated contractions that pull out and invaginate the presenting membranes. The forces generated by myosin IIa contractions ruptured most individual BCR-antigen bonds and promoted internalization of only high-affinity, multivalent BCR microclusters. Thus, B cell contractility contributes to affinity discrimination by mechanically testing the strength of antigen binding.

Myo1c is designed for the adaptation response in the inner ear

The molecular motor, Myo1c, a member of the myosin family, is widely expressed in vertebrate tissues. Its presence at strategic places in the stereocilia of the hair cells in the inner ear and studies using transgenic mice expressing a mutant Myo1c that can be selectively inhibited implicate it as the mediator of slow adaptation of mechanoelectrical transduction, which is required for balance. Here, we have studied the structural, mechanical and biochemical properties of Myo1c to gain an insight into how this molecular motor works. Our results support a model in which Myo1c possesses a strain‐sensing ADP‐release mechanism, which allows it to adapt to mechanical load.

Myosin-5, kinesin-1 and myosin-17 cooperate in secretion of fungal chitin synthase

Plant infection by pathogenic fungi requires polarized secretion of enzymes, but little is known about the delivery pathways. Here, we investigate the secretion of cell wall‐forming chitin synthases (CHSs) in the corn pathogen Ustilago maydis. We show that peripheral filamentous actin (F‐actin) and central microtubules (MTs) form independent tracks for CHSs delivery and both cooperate in cell morphogenesis. The enzyme Mcs1, a CHS that contains a myosin‐17 motor domain, is travelling along both MTs and F‐actin. This transport is independent of kinesin‐3, but mediated by kinesin‐1 and myosin‐5. Arriving vesicles pause beneath the plasma membrane, but only ∼15% of them get exocytosed and the majority is returned to the cell centre by the motor dynein. Successful exocytosis at the cell tip and, to a lesser extent at the lateral parts of the cell requires the motor domain of Mcs1, which captures and tethers the vesicles prior to secretion. Consistently, Mcs1‐bound vesicles transiently bind F‐actin but show no motility in vitro. Thus, kinesin‐1, myosin‐5 and dynein mediate bi‐directional motility, whereas myosin‐17 introduces a symmetry break that allows polarized secretion.

The SAH domain extends the functional length of the myosin lever

Stable, single alpha-helix (SAH) domains are widely distributed in the proteome, including in myosins, but their functions are unknown. To test whether SAH domains can act as levers, we replaced four of the six calmodulin-binding IQ motifs in the levers of mouse myosin 5a (Myo5) with the putative SAH domain of Dictyostelium myosin MyoM of similar length. The SAH domain was inserted between the IQ motifs and the coiled coil in a Myo5 HMM construct in which the levers were truncated from six to two IQ motifs (Myo5–2IQ). Electron microscopy of this chimera (Myo5–2IQ-SAH) showed the SAH domain was straight and 17 nm long as predicted, restoring the truncated lever to the length of wild-type (Myo5–6IQ). The powerstroke (of 21.5 nm) measured in the optical trap was slightly less than that for Myo5–6IQ but much greater than for Myo5–2IQ. Myo5–2IQ-SAH moved processively along actin at physiological ATP concentrations with similar stride and run lengths to Myo5–6IQ in in-vitro single molecule assays. In comparison, Myo5–2IQ is not processive under these conditions. Solution biochemical experiments indicated that the rear head did not mechanically gate the rate of ADP release from the lead head, unlike Myo5–6IQ. These data show that the SAH domain can form part of a functional lever in myosins, although its mechanical stiffness might be lower. More generally, we conclude that SAH domains can act as stiff structural extensions in aqueous solution and this structural role may be important in other proteins.

Actin residue glu(93) is identified as an amino acid affecting myosin binding.

Many mutants have been described that affect the function of the actin encoded by the Drosophila melanogaster indirect flight muscle-specific actin gene,Act88F. We describe the development of procedures for purification of this actin from the other isoforms expressed in the fly as well as in vitro motility, single molecule force/displacement measurements, and stop-flow solution kinetic studies of the wild-type actin and that of the E93K mutation of theAct88F gene. We show that this mutation affects in vitro motility of F-actin, in both the presence and absence of methylcellulose, and the ability of the ACT88F actin to bind the S1 fragment of rabbit skeletal myosin. However, optical tweezer measurements of the actomyosin working stroke and the force transmitted from the rabbit heavy meromyosin to and through F-actin are unchanged by the mutation. These results support the proposal (Holmes, K. C. (1995) Biophys J. 68, (suppl.) 2–7) that actin residue Glu93 is part of the secondary myosin binding site and suggest that myosin binding occurs first at the primary myosin binding site and then at the secondary site.