Cytoplasmic dynein is the primary molecular motor responsible for transport of vesicles, organelles, proteins and RNA cargoes from the periphery of the cell towards the nucleus along the microtubule cytoskeleton of eukaryotic cells. Dynactin, a large multi-subunit activator of dynein, docks cargo to the motor and may enhance dynein processivity. Here, we show that individual fluorescently labelled dynein-dynactin complexes exhibit bidirectional and processive motility towards both the plus and minus ends of microtubules. The dependence of this activity on substrate ATP concentration, nucleotide analogues and inhibitors suggests that bidirectional motility is an active energy-transduction property of dynein-dynactin motor mechano-chemistry. The unique motility characteristics observed may reflect the flexibility of the dynein structure that leads to an enhanced ability to navigate around obstacles in the cell.

The ability to sense molecular tension is crucial for a wide array of cellular processes, including the detection of auditory stimuli, control of cell shape, and internalization and transport of membranes. We show that myosin-I, a motor protein which has been implicated in powering key steps in these processes, dramatically alters its motile properties in response to tension. We measured the displacement generated by single myosin-I molecules, and we determined the actin-attachment kinetics with varying tensions using an optical trap. The rate of myosin-I detachment from actin decreases > 75-fold within 2 pN of tension, resulting in myosin-I transitioning from a low (< 0.2) to a high duty-ratio motor (> 0.9). This impressive tension sensitivity supports a role for myosin-I as a molecular force sensor.Myosin-Is are the widely expressed, single-headed, and membrane-associated members of the myosin superfamily that participate in regulating membrane dynamics and structure in nearly all eukaryotic cells. Eight myosin-I isoforms are expressed in humans, making it the largest "unconventional" myosin family (1). One specific and well characterized molecular function of a myosin-I isoform (myo1c) is to dynamically provide tension to sensitize mechano-sensitive ion channels responsible for hearing (2-4). Myosin-Is also power the transport and deformation of membranes in the cell cortex and in apical cell projections (5-8). To perform these roles, myosin-Is have been proposed to act as tension-sensing proteins that alter their ATPase and mechanical properties in response to changes in loads imparted by their cellular cargos (3,9). Biochemical, structural, and single molecule experiments suggest that some myosin-I isoforms (myo1a, myo1b, myo1c) are adapted to sense tension. Specifically, it has been shown that myosin-I produces its working stroke displacement in two substeps (10). An initial displacement of the lever arm is followed by an additional ∼ 32° rotation that accompanies ADP release (11). Since ADP release kinetically limits the rate of the detachment of myosin from actin (12,13), the extra lever arm rotation has been proposed to be a force-sensing substep, with the presence of resisting loads preventing this lever arm rotation, and thus inhibiting ADP release and actin detachment. A similar model has been proposed for gating of myosin-V motor activity during processive motility (14).We characterized the motor activity of myosin-I by measuring single-molecule forcegenerating events using the three-bead configuration, in which a single actin filament, suspended between two beads held separate by optical traps, is brought close to the surface of a pedestal-bead that is sparsely coated with myosin (15). A recombinant myo1b splice isoform containing five IQ motifs and a C-terminal biotinylation tag (16) was attached to streptavidincoated pedestal beads (17). Single-molecule actomyosin interactions at low loads were acquired using low trap stiffness (∼ 0.022 pN/nm; Fig. 1A) in the presence of 1 -50 μM ATP. NI...

Approximately 60-70% of the total fiber calcium was localized in the terminal cisternae (TC) in resting frog muscle as determined by electron-probe analysis of ultrathin cryosections . During a 1 .2 s tetanus, 59% (69 mmol/kg dry TC) of the calcium content of the TC was released, enough to raise total cytoplasmic calcium concentration by -1 mM . This is equivalent to the concentration of binding sites on the calcium-binding proteins (troponin and parvalbumin) in frog muscle . Calcium release was associated with a significant uptake of magnesium and potassium into the TC, but the amount of calcium released exceeded the total measured cation accumulation by 62 mEq/kg dry weight . It is suggested that most of the charge deficit is apparent, and charge compensation is achieved by movement of protons into the sarcoplasmic reticulum (SR) and/or by the movement of organic co-or counterions not measured by energy dispersive electron-probe analysis . There was no significant change in the sodium or chlorine content of the TIC during tetanus. The unchanged distribution of a permeant anion, chloride, argues against the existence of a large and sustained transSR potential during tetanus, if the chloride permeability of the in situ SR is as high as suggested by measurements on fractionated SR .The calcium content of the longitudinal SR (LSR) during tetanus did not show the LSR to be a major site of calcium storage and delayed return to the TC . The potassium concentration in the LSR was not significantly different from the adjacent cytoplasmic concentration. Analysis of small areas of I-band and large areas, including several sarcomeres, suggested that chloride is anisotropically distributed, with some of it probably bound to myosin . In contrast, the distribution of potassium in the fiber cytoplasm followed the water distribution . The mitochondrial concentration of calcium was low and did not change significantly during a tetanus. The TIC of both tetanized and resting freeze-substituted muscles contained electron-lucent circular areas . The appearance of the TIC showed no evidence of major volume changes during tetanus, in agreement with the estimates of unchanged (-72%) water content of the TIC obtained with electron-probe analysis .The release of Ca from and its subsequent return to the triadic portion of the sarcoplasmic reticulum (SR) (28,80,82) are the major determinants of the contractile cycle of striated muscle (for review, see reference 24) . Since the demonstration of the SR as the ATP-dependent relaxing factor (57), a wealth of information has been accumulated about the kinetics and THE JOURNAL OF CELL BIOLOGY " VOLUME 90 SEPTEMBER 1981 577-594 © The Rockefeller University Press -0021-9525/81/09/0577/18 $1 .00 mechanisms of calcium uptake by the SR (e.g., 41,48,63,104,106, and for review, see references 62, 102). In contrast, comparatively little is known about the mechanism of release and associated ion movements, largely because isolated SR preparations do not lend themselves to reproduction of the ph...

A blood clot needs to have the right degree of stiffness and plasticity to stem the flow of blood and yet be digestable by lytic enzymes so as not to form a thrombus, causing heart attacks, strokes, or pulmonary emboli, but the origin of these mechanical properties is unknown. Clots are made up of a three-dimensional network of fibrin fibers stabilized through ligation with a transglutaminase, factor XIIIa. We developed methods to measure the elastic moduli of individual fibrin fibers in fibrin clots with or without ligation, using optical tweezers for trapping beads attached to the fibers that functioned as handles to flex or stretch a fiber. Here, we report direct measurements of the microscopic mechanical properties of such a polymer. Fibers were much stiffer for stretching than for flexion, as expected from their diameter and length. Elastic moduli for individual fibers in plasma clots were 1.7 ؎ 1.3 and 14.5 ؎ 3.5 MPa for unligated and ligated fibers, respectively. Similar values were obtained by other independent methods, including analysis of measurements of fluctuations in bead force as a result of Brownian motion. These results provide a basis for understanding the origin of clot elasticity.fibrinogen ͉ optical trap ͉ viscoelasticity ͉ microrheology ͉ cardiovascular B lood clots play an essential role by stopping bleeding, but they can also cause heart attacks and strokes. Clots are formed when the enzyme thrombin cleaves fibrinogen to generate fibrin monomers, which polymerize to produce a threedimensional network of fibers (1-8). Fibrin is stabilized by ligation, ¶ the formation of intermolecular covalent bonds at specific sites with a transglutaminase, factor XIIIa, rendering the whole clot stiffer and resistant to fibrinolytic dissolution (9, 10). The viscoelastic properties of clots and their major constituent fibrin are normally finely tuned to optimize how they stop bleeding while also minimizing their effect in cardiovascular disease, because bleeding occurs if clot stiffness is too low; a decreased rate of fibrinolysis and increased thrombosis and thromboembolism are generally associated with stiff and friable clots, although such relationships are complex (10 -14). Although much is known of fibrin assembly mechanisms (1)(2)(3)(4)(5)(6)(7)(8)(15)(16)(17)(18), the origin of clot viscoelasticity remains to be established.The elasticity of a fibrin clot, like that of rubber-like polymers, is characterized by very large deformability with essentially complete recovery (19). However, the elasticity of the fibrin clot cannot be rubber-like, because it is not a random-coil network made up of thin, highly flexible strands; instead, it is a network made up of thick branching fibers. As an example of how unrealistic such rubber-like models are, it can be calculated from clot stiffness that there would be an average of only one fibrin molecule per branch point for a rubber-like model (20), yet electron micrographs show that the clots used for these experiments commonly have Ϸ1 million fibrin molecules bet...

Myosins adjust their power outputs in response to mechanical loads in an isoform-dependent manner, resulting in their ability to dynamically adapt to a range of motile challenges. Here, we reveal the structural basis for force-sensing based on near-atomic resolution structures of one rigor and two ADP-bound states of myosin-IB (myo1b) bound to actin, determined by cryo-electron microscopy. The two ADP-bound states are separated by a 25° rotation of the lever. The lever of the first ADP state is rotated toward the pointed end of the actin filament and forms a previously unidentified interface with the N-terminal subdomain, which constitutes the upper half of the nucleotide-binding cleft. This pointed-end orientation of the lever blocks ADP release by preventing the N-terminal subdomain from the pivoting required to open the nucleotide binding site, thus revealing how myo1b is inhibited by mechanical loads that restrain lever rotation. The lever of the second ADP state adopts a rigor-like orientation, stabilized by class-specific elements of myo1b. We identify a role for this conformation as an intermediate in the ADP release pathway. Moreover, comparison of our structures with other myosins reveals structural diversity in the actomyosin binding site, and we reveal the high-resolution structure of actin-bound phalloidin, a potent stabilizer of filamentous actin. These results provide a framework to understand the spectrum of force-sensing capacities among the myosin superfamily.