Citations

The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin–SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortΔSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)ΔPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.

Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed cells such as leukocytes, macrophages, and osteoclasts. They are represented by columnar arrays of actin which are perpendicular to the substratum and contain tubular invaginations of the plasma membrane. Given the similarity of these tubules to those generated by dynamin around a variety of membrane templates, we investigated whether dynamin is present at podosomes. Immunoreactivities for dynamin 2 and for the dynamin 2–binding protein endophilin 2 (SH3P8) were detected at podosomes of transformed cells and osteoclasts. Furthermore, GFP wild-type dynamin 2aa was targeted to podosomes. As shown by fluorescence recovery after photobleaching, GFP-dynamin 2aa and GFP-actin had a very rapid and similar turnover at podosomes. Expression of the GFP-dynamin 2aaG273D abolished podosomes while GFP-dynaminK44A was targeted to podosomes but delayed actin turnover. These data demonstrate a functional link between a member of the dynamin family and actin at attachment sites between cells and the substratum.

Gle2p is implicated in nuclear export of poly(A)+ RNA and nuclear pore complex (NPC) structure and distribution in Saccharomyces cerevisiae. Gle2p is anchored at the nuclear envelope (NE) via a short Gle2p-binding motif within Nup116p called GLEBS. The molecular mechanism by which Gle2p and the Gle2p–Nup116p interaction function in mRNA export is unknown. Here we show that RAE1, the mammalian homologue of Gle2p, binds to a GLEBS-like NUP98 motif at the NPC through multiple domains that include WD-repeats and a COOH-terminal non–WD-repeat extension. This interaction is direct, as evidenced by in vitro binding studies and chemical cross-linking. Microinjection experiments performed in Xenopus laevis oocytes demonstrate that RAE1 shuttles between the nucleus and the cytoplasm and is exported from the nucleus in a temperature-dependent and RanGTP-independent manner. Docking of RAE1 to the NE is highly dependent on new mRNA synthesis. Overexpression of the GLEBS-like motif also inhibits NE binding of RAE1 and induces nuclear accumulation of poly(A)+ RNA. Both effects are abrogated either by the introduction of point mutations in the GLEBS-like motif or by overexpression of RAE1, indicating a direct role for RAE1 and the NUP98–RAE1 interaction in mRNA export. Together, our data suggest that RAE1 is a shuttling transport factor that directly contributes to nuclear export of mRNAs through its ability to anchor to a specific NUP98 motif at the NPC.

Local perturbations in material density induced in a material by a compressional wave give rise to local perturbations in refractive index. Accurate, high-resolution, three-dimensional, optical measurements of an instantaneous refractive index perturbation in a homogeneous, optically transparent medium may be obtained from measurements of scattered optical intensity alone. The method of generalized projections allows incorporation of optical intensity measurements into an iterative algorithm for computing the phase of the interrogating optical pulse as the solution of a fixed point equation. The complex optical field amplitude, computed in this manner, is unique up to a constant unit magnitude complex coefficient. The three-dimensional refractive index distribution may be computed via the Fourier slice reconstruction algorithm from the optical phase data under the assumption of weak optical scattering. The refractive index perturbation is related to local instantaneous pressure under a linear, small-displacement model for the mechanical wave. A numerical simulation of the measurement experiment, phase recovery, and reconstruction process for a plane piston ultrasound transducer with a semicircular aperture and center frequency of 1.5 MHz is described and corresponds very well with experiment. Experimental data obtained using an 810-nm laser source are used to reconstruct the three-dimensional pressure field from two elements of a 2.5-MHz linear array. Comparison with a measurement obtained via a 500-μm needle hydrophone shows excellent agreement.

It has been shown that acoustic ray paths in range-dependent ocean models exhibit chaotic behavior. Most of the investigations into the ray chaos phenomenon have been primarily numerical in nature. Analytical derivation of sufficient conditions for chaos in acoustic systems has been restricted to inherently discrete problems. This article reports a theoretical study of the existence of ray chaos in a class of continuous parabolic ray systems. This class of ray systems is indexed by a family of analytically prescribed double-channel sound-speed profiles perturbed by periodic range-dependent disturbances. The perturbed Hamiltonian ray systems are studied analytically via the Melnikov method. It is shown that, under certain conditions, ray trajectories of the systems are equivalent to trajectories of a classic chaotic system known as the horseshoe map when the perturbation is periodic and small. These conditions are sufficient for ray chaos and easily satisfied, thus explaining why double-channel propagation is very likely to exhibit chaotic behavior.

A novel algorithm based on the variational formulation is presented for the determination of the material parameters (λ+2μ)/ρ and μ/ρ throughout inhomogeneous media given volumetric displacement information. The performance of the inversion algorithm is subsequently investigated when applied to measured displacement data throughout an inhomogeneous test phantom. The vector displacement components throughout the test phantom subject to monochromatic shear excitation measured in time using MRI (Magnetic Resonance Imaging) were temporally Fourier transformed to extract the components of monochromatic excitation, and the data were delivered to the inversion algorithm. A sequence of studies is presented on the basis of subsequent wavenumber filtering, polarization selection, and variation in the size of the volume elements of resolution. The resulting performance is assessed and recommendations for future efforts are discussed. [Work supported by ONR and NIH.]

A technique for measuring instantaneous three-dimensional pressure in a wideband ultrasound pulse is presented. A collimated laser pulse impinges on the ultrasound field, and the optical intensity in a diffraction plane subsequent to this scattering event is imaged onto a CCD camera and stored. Timing is controlled precisely, allowing the acousto-optic interaction to occur at the same point in the temporal evolution of the sound field each time the experiment is performed. After interaction with the ultrasound pulse, the optical phase is approximately proportional to the ray sum of instantaneous pressure in the pulse (taken in the direction of optical propagation). Optical phase immediately subsequent to passage through the ultrasound pulse is computed from recorded optical intensity via a phase retrieval algorithm. The experiment is performed at several angles as the ultrasound transducer is rotated about an axis normal to the optical axis of the imaging system. Tomographic reconstruction algorithms are applied to the retrieved optical phase data, computing pressure at all points in the ultrasound pulse. The resulting pulse may be propagated forward or backward in time under a source-free model. Reconstructions of the field from a 2.5-MHz array transducer compare well with hydrophone measurements.

This investigation evaluated a possible source of reduced intelligibility in hypokinetic dysarthric speech, namely the mismatch between listeners’ perceptual strategies and the acoustic information available in the dysarthric speech signal. A paradigm of error analysis was adopted in which listener transcriptions of phrases were coded for the presence and type of word boundary errors. Seventy listeners heard 60 phrases produced by speakers with hypokinetic dysarthria. The six-syllable phrases alternated strong and weak syllables and ranged in length from three to five words. Lexical boundary violations were defined as erroneous insertions or deletions of lexical boundaries that occurred either before strong or before weak syllables. A total of 1596 lexical boundary errors in the listeners’ transcriptions was identified unanimously by three independent judges. The pattern of errors generally conformed with the predictions of the Metrical Segmentation Strategy hypothesis [Cutler and Norris, J. Exp. Psychol. 14, 113–121 (1988)] which posits that listeners attend to strong syllables to identify word onsets. However, the strength of adherence to this pattern varied across speakers. Comparison of acoustic evidence of syllabic strength to lexical boundary error patterns revealed a source of intelligibility deficit associated with this particular type of dysarthric speech pattern.

Many methods are available for transferring foreign genes into mammalian cells (transfection). The advantages of ultrasound-mediated transfection are that it can be controlled in both space, by focusing, and time, by exposure. It was tested whether ultrasound-mediated gene transfection in cultured immortalized human chondrocytes would be enhanced using artificial cavitation nuclei in the form of Albunex®. Here 1.0 MHz ultrasound transmitted through the bottoms of six-well culture plates containing media with green fluorescent protein reporter gene plasmids at a concentration of 40 μg/ml and Albunex® at 50×106 bubbles/ml produced a peak transfection efficiency of about 50% of the living cells when exposure was 4×105 P spatial average peak pressure (SAPP) for 20 mins. Using these parameters, transfection efficiency increased linearly with ultrasound exposure pressure with a transfection threshold observed at a SAPP of 105 P. Adding fresh Albunex® at 50×106 bubbles/ml prior to sequential one second 3.2 or 4.0×105 P exposures increased transfection efficiency by 15% for each of three consecutive exposures. Efficient in vitro and perhaps in vivo transfection appears possible with ultrasound providing both temporal and spatial control over the transfection process.

An optical spin micromotor operated by optical tweezers, described in the present study, converts radiant energy directly to mechanical energy. This conversion is of great practical and fundamental interest. The optical spin micromotor consists of a two-bead linkage. The large bead (2.0 μm in diameter) is trapped by the optical tweezers and rotates around the laser beam axis. The small bead (0.94 μm in diameter) is partially coated with gold/palladium for generating the spin torque through the change of momentum from gradient radiation pressure of the same laser applied on the coated small bead. With a laser power of 29 mW at the sample plane, the spin speed averaged 158.8±155.5 rpm (mean±standard deviation) and the torque was estimated as 101.9±99.8 pN nm. This optical spin micromotor will be useful in (1) providing a fundamental step toward developing optical-operated mechanical devices, (2) twisting macromolecules, and (3) generating vortex and shear force in medium at nanoscale.