Interferometric Scattering Microscopy for the Study of Molecular Motors.

Our understanding of molecular motor function has been greatly improved by the development of imaging modalities, which enable real-time observation of their motion at the single-molecule level. Here, we describe the use of a new method, interferometric scattering microscopy, for the investigation of motor protein dynamics by attaching and tracking the motion of metallic nanoparticle labels as small as 20nm diameter. Using myosin-5, kinesin-1, and dynein as examples, we describe the basic assays, labeling strategies, and principles of data analysis. Our approach is relevant not only for motor protein dynamics but also provides a general tool for single-particle tracking with high spatiotemporal precision, which overcomes the limitations of single-molecule fluorescence methods.

[Jawbone metastasis masquerading as dental pain].

Metastases to the oral cavity are rare. However, in 25% of cases, oral symptoms will be the first sign of metastatic disease. The incidence of jaws metastases is twice as high as the incidence of metastases to the soft tissues of the oral cavity. In some cases, jaws metastases can mimic dental or periodontal pain. We report a case of a 67 year old female who was referred to our clinic because of severe pain on her left posterior mandible which was not relieved by endodontic treatment of the first and second molar. She was diagnosed with breast cancer in 2005 and had been treated with surgery, chemotherapy and radiotherapy. Seven years later, lung metastases were found and she was treated with chemotherapy. Later on, brain metastases developed which had been treated with radiotherapy. On presentation, she complained of pain on the posterior left mandible which was accompanied by a burning sensation of the lower left lip and chin. CT scan revealed a soft tissue mass perforating the lingual and buccal plates of the posterior left mandible, which was compatible with a diagnosis of metastasis. Radiotherapy rapidly relieved the pain. Unfortunately, the patient passed away one month later. Dentists should be able to recognize the signs and symptoms associated with metastases to the jaws and should include it in the differential diagnosis, especially in patients with oncologic background.

Coupling between phosphate release and force generation in muscle actomyosin.

Energetic, kinetic and oxygen exchange experiments in the mid-1980s and early 1990s suggested that phosphate (Pi) release from actomyosin-adenosine diphosphate Pi (AM.ADP.Pi) in muscle fibres is linked to force generation and that Pi release is reversible. The transition leading to the force-generating state and subsequent Pi release were hypothesized to be separate, but closely linked steps. Pi shortens single force-generating actomyosin interactions in an isometric optical clamp only if the conditions enable them to last 20-40 ms, enough time for Pi to dissociate. Until 2003, the available crystal forms of myosin suggested a rigid coupling between movement of switch II and tilting of the lever arm to generate force, but they did not explain the reciprocal affinity myosin has for actin and nucleotides. Newer crystal forms and other structural data suggest that closing of the actin-binding cleft opens switch I (presumably decreasing nucleotide affinity). These data are all consistent with the order of events suggested before: myosin.ADP.Pi binds weakly, then strongly to actin, generating force. Then Pi dissociates, possibly further increasing force or sliding.

A small-molecule inhibitor of skeletal muscle myosin II.

We screened a small-molecule library for inhibitors of rabbit muscle myosin II subfragment 1 (S1) actin-stimulated ATPase activity. The best inhibitor, N-benzyl-p-toluene sulphonamide (BTS), an aryl sulphonamide, inhibited the Ca2+-stimulated S1 ATPase, and reversibly blocked gliding motility. Although BTS does not compete for the nucleotide-binding site of myosin, it weakens myosin's interaction with F-actin. BTS reversibly suppressed force production in skinned skeletal muscle fibres from rabbit and frog skin at micromolar concentrations. BTS suppressed twitch production of intact frog fibres with minimum alteration of Ca2+ metabolism. BTS is remarkably specific, as it was much less effective in suppressing contraction in rat myocardial or rabbit slow-twitch muscle, and did not inhibit platelet myosin II. The isolation of BTS and the recently discovered Eg5 kinesin inhibitor, monastrol, suggests that motor proteins may be potential targets for therapeutic applications.

A maximum entropy analysis of protein orientations using fluorescence polarization data from multiple probes.

Techniques have recently become available to label protein subunits with fluorescent probes at predetermined orientation relative to the protein coordinates. The known local orientation enables quantitative interpretation of fluorescence polarization experiments in terms of orientation and motions of the protein within a larger macromolecular assembly. Combining data obtained from probes placed at several distinct orientations relative to the protein structure reveals functionally relevant information about the axial and azimuthal orientation of the labeled protein segment relative to its surroundings. Here we present an analytical method to determine the protein orientational distribution from such data. The method produces the broadest distribution compatible with the data by maximizing its informational entropy. The key advantages of this approach are that no a priori assumptions are required about the shape of the distribution and that a unique, exact fit to the data is obtained. The relative orientations of the probes used for the experiments have great influence on information content of the maximum entropy distribution. Therefore, the choice of probe orientations is crucial. In particular, the probes must access independent aspects of the protein orientation, and two-fold rotational symmetries must be avoided. For a set of probes, a "figure of merit" is proposed, based on the independence among the probe orientations. With simulated fluorescence polarization data, we tested the capacity of maximum entropy analysis to recover specific protein orientational distributions and found that it is capable of recovering orientational distributions with one and two peaks. The similarity between the maximum entropy distribution and the test distribution improves gradually as the number of independent probe orientations increases. As a practical example, ME distributions were determined with experimental data from muscle fibers labeled with bifunctional rhodamine at known orientations with respect to the myosin regulatory light chain (RLC). These distributions show a complex relationship between the axial orientation of the RLC relative to the fiber axis and the azimuthal orientation of the RLC about its own axis. Maximum entropy analysis reveals limitations in available experimental data and supports the design of further probe angles to resolve details of the orientational distribution.

Effective elimination of lung metastases induced by tumor cells treated with hydrostatic pressure and N-acetyl-L-cysteine.

In previous studies, we have demonstrated that application of high hydrostatic pressure (P) to tumor cells in the presence of a slow-reacting membrane-impermeable cross-linker (CL), 2'-3'-adenosine dialdehyde, can rearrange cell surface proteins into immunogenic clusters. Here, we present evidence indicating that subsequent reduction of surface protein disulfides with N-acetyl-L-cysteine (NAC) further augments the immunogenic potential of PCL-modified tumor cells both in vitro and in vivo. Immunotherapy with PCL+NAC-modified 3LL-D122 Lewis lung carcinoma cells plus i.v. delivery of NAC in mice bearing established lung metastases provoked an antitumor response capable of eradicating the metastatic nodules as demonstrated by restoration of normal lung weight and histology. In addition, immunization with PCL+NAC-modified tumor cells gave rise to a strong delayed-type hypersensitivity recall response against parental D122 cells. We propose that this novel two-prong strategy, based on local immunization with autologous PCL+NAC-modified tumor cells and systemic boosting with NAC, could provide a practical, effective immunotherapeutic regimen for the treatment of human cancer.

Tomographic 3D reconstruction of quick-frozen, Ca2+-activated contracting insect flight muscle.

Motor actions of myosin were directly visualized by electron tomography of insect flight muscle quick-frozen during contraction. In 3D images, active cross-bridges are usually single myosin heads, bound preferentially to actin target zones sited midway between troponins. Active attached bridges (approximately 30% of all heads) depart markedly in axial and azimuthal angles from Rayment's rigor acto-S1 model, one-third requiring motor domain (MD) tilting on actin, and two-thirds keeping rigor contact with actin while the light chain domain (LCD) tilts axially from approximately 105 degrees to approximately 70 degrees. The results suggest the MD tilts and slews on actin from weak to strong binding, followed by swinging of the LCD through an approximately 35 degrees axial angle, giving an approximately 13 nm interaction distance and an approximately 4-6 nm working stroke.

Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction.

A new method is described for measuring motions of protein domains in their native environment on the physiological timescale. Pairs of cysteines are introduced into the domain at sites chosen from its static structure and are crosslinked by a bifunctional rhodamine. Domain orientation in a reconstituted macromolecular complex is determined by combining fluorescence polarization data from a small number of such labelled cysteine pairs. This approach bridges the gap between in vitro studies of protein structure and cellular studies of protein function and is used here to measure the tilt and twist of the myosin light-chain domain with respect to actin filaments in single muscle cells. The results reveal the structural basis for the lever-arm action of the light-chain domain of the myosin motor during force generation in muscle.

The ADP release step of the smooth muscle cross-bridge cycle is not directly associated with force generation.

When smooth muscle myosin subfragment 1 (S1) is bound to actin filaments in vitro, the light chain domain tilts upon release of MgADP, producing a approximately 3.5-nm axial motion of the head-rod junction (Whittaker et al., 1995. Nature. 378:748-751). If this motion contributes significantly to the power stroke, rigor tension of smooth muscle should decrease substantially in response to cross-bridge binding of MgADP. To test this prediction, we monitored mechanical properties of permeabilized strips of chicken gizzard muscle in rigor and in the presence of MgADP. For comparison, we also tested psoas and soleus muscle fibers. Any residual bound ADP was minimized by incubation in Mg2+-free rigor solution containing 15 mM EDTA. The addition of 2 mM MgADP, while keeping ionic strength and free Mg2+ concentration constant, resulted in a slight increase in rigor tension in both gizzard and soleus muscles, but a decrease in psoas muscle. In-phase stiffness monitored during small (<0.1%) 500-Hz sinusoidal length oscillations decreased in all three muscle types when MgADP was added. The changes in force and stiffness with the addition of MgADP were similar at ionic strengths from 50 to 200 mM and were reversible. The results with gizzard muscle were similar after thiophosphorylation of the regulatory light chain of myosin. These results suggest that the axial motion of smooth muscle S1 bound to actin, upon dissociation of MgADP, is not associated with force generation. The difference between the present mechanical data and previous structural studies of smooth S1 may be explained if geometrical constraints of the intact contractile filament array alter the motions of the myosin heads.

Trading force for speed: why superfast crossbridge kinetics leads to superlow forces.

Superfast muscles power high-frequency motions such as sound production and visual tracking. As a class, these muscles also generate low forces. Using the toadfish swimbladder muscle, the fastest known vertebrate muscle, we examined the crossbridge kinetic rates responsible for high contraction rates and how these might affect force generation. Swimbladder fibers have evolved a 10-fold faster crossbridge detachment rate than fast-twitch locomotory fibers, but surprisingly the crossbridge attachment rate has remained unchanged. These kinetics result in very few crossbridges being attached during contraction of superfast fibers (only approximately 1/6 of that in locomotory fibers) and thus low force. This imbalance between attachment and detachment rates is likely to be a general mechanism that imposes a tradeoff of force for speed in all superfast fibers.

Model-independent analysis of the orientation of fluorescent probes with restricted mobility in muscle fibers.

The orientation of proteins in ordered biological samples can be investigated using steady-state polarized fluorescence from probes conjugated to the protein. A general limitation of this approach is that the probes typically exhibit rapid orientational motion ("wobble") with respect to the protein backbone. Here we present a method for characterizing the extent of this wobble and for removing its effects from the available information about the static orientational distribution of the probes. The analysis depends on four assumptions: 1) the probe wobble is fast compared with the nanosecond time scale of its excited-state decay; 2) the orientational distributions of the absorption and emission transition dipole moments are cylindrically symmetrical about a common axis c fixed in the protein; 3) protein motions are negligible during the excited-state decay; 4) the distribution of c is cylindrically symmetrical about the director of the experimental sample. In a muscle fiber, the director is the fiber axis, F. All of the information on the orientational order of the probe that is available from measurements of linearly polarized fluorescence is contained in five independent polarized fluorescence intensities measured with excitation and emission polarizers parallel or perpendicular to F and with the propagation axis of the detected fluorescence parallel or perpendicular to that of the excitation. The analysis then yields the average second-rank and fourth-rank order parameters ( and ) of the angular distribution of c relative to F, and and , the average second-rank order parameters of the angular distribution for wobble of the absorption and emission transition dipole moments relative to c. The method can also be applied to other cylindrically ordered systems such as oriented lipid bilayer membranes and to processes slower than fluorescence that may be observed using longer-lived optically excited states.

Human tumor cells, modified by a novel pressure/crosslinking methodology, promote autologous lymphocyte proliferation and modulate cytokine secretion.

Hydrostatic pressure (P) combined with membrane protein crosslinking (CL) by adenosine dialdehyde (AdA) can render tumor cells immunogenic. We have recently shown that PCL treatment of murine tumor cells augmented the presentation of MHC-restricted tumor-associated antigens and enhanced cell-mediated immunity. In cancer patients inoculated with autologous PCL-modified tumor cells, a significant delayed-type hypersensitivity response was elicited. Since the balance between cell-mediated immunity and humoral immunity is reciprocally controlled by immunoregulatory cytokines, we have examined the proliferative response and cytokine secretion pattern in cultures of human peripheral blood mononuclear cells (PBMC) stimulated by autologous PCL-modified and unmodified tumor cells. These tumor cells were obtained from freshly resected tumor tissue of 16 patients with colon (8), lung (4) and renal (4) carcinomas. The results demonstrated that PCL-modified tumor cells promoted an increase in PBMC proliferation in 5 out of 8 (63%), 1 out of 4 (25%) and 4 out of 4 (100%) colon, lung and renal cell carcinomas. Fourteen of the above cultures were also analyzed for the secretion of interleukin-10 and interferon-gamma. Overall, a substantial decrease in IL-10 secretion was detected in 9 out of 14 (64%) cultures while a reciprocal increase in interferon-gamma secretion was noted in 8 out of 14 (57%) cultures. Our results confirmed that PCL-modified human tumor cells of different etiologies can modulate the pattern of cytokines released from stimulated autologous lymphocytes. Such a procedure could prove valuable in the production of autologous tumor vaccines.

Orientation changes of fluorescent probes at five sites on the myosin regulatory light chain during contraction of single skeletal muscle fibres.

Changes in the orientation of the myosin regulatory light chain (RLC) in single muscle fibres were measured using polarised fluorescence from acetamidotetramethylrhodamine (ATR). Mutants of chicken skeletal RLC containing single cysteine residues at positions 2, 73, 94, 126 and 155 were labelled with either the 5 or 6-isomer of iodo-ATR, giving ten different probes. The labelled RLCs were exchanged into demembranated fibres from rabbit psoas muscle without significant effect on active force generation. Fluorescence polarisation measurements showed that nine out of the ten probe dipoles were more perpendicular to the fibre axis in the absence of ATP (in rigor) than in either relaxation or active contraction. The orientational distribution of the RLC region of the myosin head in active contraction is closer to the relaxed than to the rigor orientation, and is not equivalent to a linear combination of the relaxed and rigor orientations. Rapid length steps were applied to the fibres to synchronise the motions of myosin heads attached to actin. In active contraction the fluorescence polarisation changed both during the step, indicating elastic distortion of the RLC region of the myosin head, and during the subsequent rapid force recovery that is thought to signal the working stroke. The peak change in fluorescence polarisation produced by an active release of 5 nm per half sarcomere indicates an axial tilt of less than 5 degrees for all ten probes, if all the myosin heads in the fibre respond to the length step. This tilting was towards the rigor orientation for all ten probes, and could be explained by 14% of the heads moving to the rigor orientation. An active stretch tilted the heads away from the rigor conformation by a similar extent.

Fluorescence polarization transients from rhodamine isomers on the myosin regulatory light chain in skeletal muscle fibers.

Fluorescence polarization was used to examine orientation changes of two rhodamine probes bound to myosin heads in skeletal muscle fibers. Chicken gizzard myosin regulatory light chain (RLC) was labeled at Cys108 with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLC (termed Cys108-5 or Cys108-6) was exchanged for the endogenous RLC in single, skinned fibers from rabbit psoas muscle. Three independent fluorescence polarization ratios were used to determine the static angular distribution of the probe dipoles with respect to the fiber axis and the extent of probe motions on the nanosecond time scale of the fluorescence lifetime. We used step changes in fiber length to partially synchronize the transitions between biochemical, structural, and mechanical states of the myosin cross-bridges. Releases during active contraction tilted the Cys108-6 dipoles away from the fiber axis. This response saturated for releases beyond 3 nm/half-sarcomere (h.s.). Stretches in active contraction caused the dipoles to tilt toward the fiber axis, with no evidence of saturation for stretches up to 7 nm/h.s. These nonlinearities of the response to length changes are consistent with a partition of approximately 90% of the probes that did not tilt when length changes were applied and 10% of the probes that tilted. The responding fraction tilted approximately 30 degrees for a 7.5 nm/h.s. release and traversed the plane perpendicular to the fiber axis for larger releases. Stretches in rigor tilted Cys108-6 dipoles away from the fiber axis, which was the opposite of the response in active contraction. The transition from the rigor-type to the active-type response to stretch preceded the main force development when fibers were activated from rigor by photolysis of caged ATP in the presence of Ca2+. Polarization ratios for Cys108-6 in low ionic strength (20 mM) relaxing solution were compatible with a combination of the relaxed (200 mM ionic strength) and rigor intensities, but the response to length changes was of the active type. The nanosecond motions of the Cys108-6 dipole were restricted to a cone of approximately 20 degrees half-angle, and those of Cys108-5 dipole to a cone of approximately 25 degrees half-angle. These values changed little between relaxation, active contraction, and rigor. Cys108-5 showed very small-amplitude tilting toward the fiber axis for both stretches and releases in active contraction, but much larger amplitude tilting in rigor. The marked differences in these responses to length steps between the two probe isomers and between active contraction and rigor suggest that the RLC undergoes a large angle change (approximately 60 degrees) between these two states. This motion is likely to be a combination of tilting of the RLC relative to the fiber axis and twisting of the RLC about its own axis.