Paul Matsudaira

Limited N-terminal sequence analysis.

Current methods in protein purification permit sequence analysis of picomole quantities of proteins and peptides. The key to obtaining sequence is to choose as the last step in the purification protocol the simplest (usually the fastest) method for isolating the protein or peptide.

Identification of an Actin Binding Region and a Protein Kinase C Phosphorylation Site on Human Fascin

Fascin is a 55-58-kDa actin-bundling protein, the actin binding of which is regulated by phosphorylation (Yamakita, Y., Ono, S., Matsumura, F., and Yamashiro, S. (1996) J. Biol. Chem. 271, 12632-12638). To understand the mechanism of fascin-actin interactions, we dissected the actin binding region and its regulatory site by phosphorylation of human fascin. First, we found that the C-terminal half constitutes an actin binding domain. Partial digestion of human recombinant fascin with trypsin yielded the C-terminal fragment with molecular masses of 32, 30, and 27 kDa. The 32- and 27-kDa fragments purified as a mixture formed a dimer and bound to F-actin at a saturation ratio of 1 dimer:11 actin molecules with an affinity of 1.4 × 106 M−1. Second, we identified the phosphorylation site of fascin as Ser-39 by sequencing a tryptic phosphopeptide purified by chelating column chromatography followed by C-18 reverse phase high performance liquid chromatography. Peptide map analyses revealed that the purified peptide represented the major phosphorylation site of in vivo as well as in vitro phosphorylated fascin. The mutation replacing Ser-39 with Ala eliminated the phosphorylation-dependent regulation of actin binding of fascin, indicating that phosphorylation at this site regulates the actin binding ability of fascin.

Macrophage podosomes assemble at the leading lamella by growth and fragmentation

Podosomes are actin- and fimbrin-containing adhesions at the leading edge of macrophages. In cells transfected with β-actin–ECFP and L-fimbrin–EYFP, quantitative four-dimensional microscopy of podosome assembly shows that new adhesions arise at the cell periphery by one of two mechanisms; de novo podosome assembly, or fission of a precursor podosome into daughter podosomes. The large podosome cluster precursor also appears to be an adhesion structure; it contains actin, fimbrin, integrin, and is in close apposition to the substratum. Microtubule inhibitors paclitaxel and demecolcine inhibit the turnover and polarized formation of podosomes, but not the turnover rate of actin in these structures. Because daughter podosomes and podosome cluster precursors are preferentially located at the leading edge, they may play a critical role in continually generating new sites of cell adhesion.

Passive and active microrheology with optical tweezers

Efforts at understanding the behaviour of complex materials at the micro scale have led to the development of many microrheological techniques capable of probing viscoelastic behaviour. Among these, optical tweezers have been extensively developed for biophysical applications: they offer several advantages over traditional techniques, and can be employed in both passive and active microrheology. In this report, we outline several methods that can be used with optical tweezers to measure the microrheological behaviour of materials such as glycerol, methylcellulose solutions, actin matrices, and cellular membranes. In addition, we quantify the effect that the index of refraction of the solution has on the stiffness of the optical trap. Our results indicate that optical tweezers force microscopy is a versatile tool for the exploration of viscoelastic behaviour in a range of substrates at the micro scale.

Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy

We developed a deep-ultraviolet (UV) microscope capable of imaging cell mitosis and motility at 280 nm for 45 min with minimal UV-induced toxicity, and for 6 h before the onset of visible cell death in cultured human and mouse cells. Combined with computational methods that convert the intensity of each pixel into an estimate of mass, deep-UV microscopy images generate maps of nucleic acid mass, protein mass and fluorescence yield in unlabeled cells.

Fimbrin in podosomes of monocyte‐derived osteoclasts

Fimbrin, an actin-bundling protein, is a component of the osteoclast adhesion complexes called podosomes. In this study, we (1) determined the localization of fimbrin in the mature rabbit osteoclast as well as in differentiating osteoclasts using the avian monocyte-derived osteoclast differentiation model, (2) characterized the distribution and accumulation of three fimbrin isotypes (T, L, and I) in avian monocytes as they fused to form multinucleate osteoclast-like cells, and (3) report for the first time, a close spatial relationship between podosomes and microtubules using fimbrin as a marker of the podosome. Immunofluorescence using anti-T-fimbrin, anti-L-fimbrin, and pan-isotype-anti-fimbrin antibodies, showed that fimbrin is an integral component of the podosome core in the mature rabbit osteoclast and in the monocyte-derived osteoclast throughout differentiation. Anti-I-fimbrin, however, did not show immunoreactivity in these cultures. These studies also show that in the avian model of monocyte-derived osteoclast differentiation, day 2 cells (D2) are predominantly mononucleate and have few podosomes. By days 4 and 6 in culture (D4 and D6), many cells have fused and punctate rows of podosomes are commonly observed at cell margins. Analysis by Western blot of protein accumulation showed that after an initial small rise from D2 to D4, L-fimbrin levels remained relatively constant from D4 to D6. However, T-fimbrin protein levels increase steadily from D2 to D6, suggesting that it may be related to the increase in podosome formation as monocytes fuse to form osteoclasts. Finally, we examined the distribution of podosomes relative to other cytoskeletal elements such as microtubules and intermediate filaments. Double immunofluorescence labeling using anti-fimbrin and anti-tubulin showed podosomes lying adjacent to microtubules at cell margins. When osteoclasts were treated with nocodazole (1 X 10(-6) M) to disrupt microtubules, the distribution of podosomes became more random and was no longer confined to the cell periphery. These results suggest that microtubule-podosome interactions may play a role in osteoclast adhesion.

Direct observation of stick-slip movements of water nanodroplets induced by an electron beam

Dynamics of the first few nanometers of water at the interface are encountered in a wide range of physical, chemical, and biological phenomena. A simple but critical question is whether interfacial forces at these nanoscale dimensions affect an externally induced movement of a water droplet on a surface. At the bulk-scale water droplets spread on a hydrophilic surface and slip on a nonwetting, hydrophobic surface. Here we report the experimental description of the electron beam-induced dynamics of nanoscale water droplets by direct imaging the translocation of 10- to 80-nm-diameter water nanodroplets by transmission electron microscopy. These nanodroplets move on a hydrophilic surface not by a smooth flow but by a series of stick-slip steps. We observe that each step is preceded by a unique characteristic deformation of the nanodroplet into a toroidal shape induced by the electron beam. We propose that this beam-induced change in shape increases the surface free energy of the nanodroplet that drives its transition from stick to slip state.

Understanding effects of matrix protease and matrix organization on directional persistence and translational speed in three-dimensional cell migration.

Recent studies have shown significant differences in migration mechanisms between two- and three-dimensional environments. While experiments have suggested a strong dependence of in vivo migration on both structure and proteolytic activity, the underlying biophysics of such dependence has not been studied adequately. In addition, the existing models of persistent random walk migration are primarily based on two-dimensional movement and do not account for the effect of proteolysis or matrix inhomogeneity. Using lattice Monte Carlo methods, we present a model to study the role of matrix metallo-proteases (MMPs) on directional persistence and speed. The simulations account for a given cell’s ability to deform as well as to digest the matrix as the cell moves in three dimensions. Our results show a bimodal dependence of speed and persistence on matrix pore size and suggest high sensitivity on MMP activity, which is in very good agreement with experimental studies carried out in 3D matrices.

Characterisation of the F-actin binding domains of villin: classification of F-actin binding proteins into two groups according to their binding sites on actin

The F‐actin binding properties of chicken villin, its headpiece and domains 2–3 (V2‐3) have been analysed to identify sites involved in bundle formation. Headpiece and V2‐3 bind actin with K d values of ~7 μM and ~0.3 μM, respectively, at low ionic strength. V2‐3 binding, like that of villin, is weakened with increasing salt concentration; headpiece binding is not. Competition experiments show that headpiece and V2‐3 bind to different sites on actin, forming the two cross‐linking sites of villin. Headpiece does not compete with the F‐actin binding domains of gelsolin or α‐actinin, but it dissociates actin depolymerizing factor. We suggest that the F‐actin binding domains of actin severing, crosslinking and capping proteins can be organized into two classes.

Detecting force-induced molecular transitions with fluorescence resonant energy transfer.

Single-molecule techniques have been responsible for substantial advances in the field of biophysics. Among these approaches, single-molecule fluorescence resonant energy transfer (FRET) spectroscopy provides an experimental view of the structural properties of individual molecules, whereas optical-tweezers force microscopy allows direct manipulation of the reaction coordinate of a single molecule. However, the simultaneous application of these techniques is complicated by optical-trap-induced photobleaching, which substantially reduces fluorophore longevity to unacceptably short time-scales. Herein, we describe a general solution to this problem and apply it to a novel force sensor based on a DNA hairpin, in the first successful combination of optical trapping and FRET. By alternately exposing the sample molecule to the optical-trapping and fluorescence-excitation lasers, we demonstrate the ability to reversibly manipulate a single molecule while simultaneously monitoring its structural configuration. This integrated measurement provides high-resolution mechanical control over molecular conformation with fluorescence-based structural reporting. The application of this technique for single-molecule exploration will lead to new experiments that employ combined optical trapping and single-molecule fluorescence for the simultaneous and active manipulation and monitoring of molecular structure in real time. Single-molecule force microscopy and fluorescence spectroscopy reveal individual molecular properties that are clouded by the inherent averaging of ensemble methods. However, the individual approaches of these techniques often fail to uncover the interplay between applied mechanical forces and structural changes. A single measurement of a force-sensing molecule connects these two perspectives by directly manipulating a molecular reaction coordinate while simultaneously detecting localized structural effects. Among the biophysical techniques capable of probing single-molecule properties, optical-tweezers force microscopy operates at piconewton force levels that are optimal for the detection of nanometer-scale conformational transitions. Likewise, single-molecule FRET spectroscopy provides complementary information about dynamic structural properties, including environment, orientation, and proximity, with comparable spatial resolution.[1] Previous efforts to combine these two techniques for a single, coincident measurement have been complicated by accelerated photobleaching rates induced by the high-intensity optical trap. Because of this effect, which is especially pronounced in common single-molecule FRET donor labels such as the dyes Cy3 and Alexa 555,[2] previous advances towards combining these techniques have spatially separated the fluorescent markers from the optical trap[3] or have employed uniquely robust chromophores.[4] We recently described a broadly applicable solution to this problem by alternately modulating the fluorescence-excitation and optical-trapping beams, which dramatically reduced this phenomenon without compromising trap integrity.[5] Herein, we show that such an optical modulation can be adapted to extend the emission times of FRET-paired labels without otherwise affecting their photophysical properties. To demonstrate this technique, we describe the first combination of optical-tweezers force microscopy with the single-molecule FRET detection of a novel force-sensing molecule into a single, integrated method capable of actively controlling molecular structure while simultaneously monitoring the conformational state of a single DNA hairpin molecule. The mechanics of DNA hairpins have been studied at the single-molecule level and, thus, offer a benchmark for examining optical tweezers and single-molecule FRET in a combined arrangement. These structures, which are commonly used to model secondary structure in nucleotides, are readily adapted for the mechanical exploration of conformational dynamics, as they undergo a sequence-dependent, reversible unzipping transition.[6,7] In addition, alternate constructs have been adapted for force-sensing applications.[8] The structure used in this work, which contains a 20-base-pair hairpin stem, is flanked by noncomplimentary sequences annealed to oligonucleotides functionalized with the fluorophores Cy3 and Alexa 647 (Figure 1). Complexes exhibiting single-molecule FRET emission were mechanically loaded with the optical trap, effectively reducing the energetic barrier to hairpin opening. This unzipping transition, which occurs at a force of approximately 18 pN, comparable to other similar measurements,[7] was reflected by the displacement of the bead toward the center of the trap. The conformational transition was accompanied by a simultaneous reduction in FRET efficiency caused by the increased physical separation of the Cy3 donor and the Alexa 647 acceptor, which indicated the precise location of the structural change caused by the translation of the mechanical load between the low-force (ca. 6 pN) and high-force (ca. 24 pN) states (Figure 2). The DNA complexes were moved through several transitions in a process corresponding to the reversible opening and closing of the hairpin segment, which demonstrated both the high degree of mechanical control and the simultaneous reporting by FRET emission. Furthermore, in the representative trace, single-step photobleaching of the donor after approximately 65 s verified the single-molecule measurement. Figure 1 Experimental assay design (see Experimental Section for details). DNA hairpin complexes, labeled with opposing Cy3 and Alexa 647 fluorophores, were mechanically loaded by translating the coverslip, as the position of the trapped bead and the emission ... Figure 2 Mechanically induced conformational changes monitored with FRET spectroscopy. A) A DNA hairpin was manipulated with optical tweezers between open or closed conformational states (black) that transition at loads of approximately 18 pN. The state of the ... This combination of optical-tweezers force microscopy and single-molecule FRET detection represents a significant advance for measuring the effects of structural changes on molecular function in a single molecule. By mechanically altering the conformational energy landscape, we actively induced a structural rearrangement pinpointed by strategically placed fluorescence labels. With minor modifications to existing assays, this approach can be extended beyond this model system to provide important new insight into the localized effects of mechanical force in biomolecular systems. For example, this combined technique can be adapted to monitor the intermolecular processes involved in the formation of a mechanically loaded protein complex,[9] the effects of mechanical deformation on single-enzyme catalysis,[10] or the intramolecular movements involved in biological-motor motility.[11,12] In addition, the presence of quantized single-molecule fluorescence signals can provide unambiguous verification of the size and location of a mechanical event, a critical tool for the design of often complex single-molecule assays. The new perspective that arises from this ability to physically deform single molecules while simultaneously measuring structural changes will allow the design of novel force-sensing molecules and will permit a new class of experiments for probing the interrelationship between molecular structure and biochemical function.