Biomedical subjects

H Sosa

Publications and source records attributed to H Sosa.

17 recordsLinked to original sources

PubMed2001

Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules.

Kinesin is a molecular motor that interacts with microtubules and uses the energy of ATP hydrolysis to produce force and movement in cells. To investigate the conformational changes associated with this mechanochemical energy conversion, we developed a fluorescence polarization microscope that allows us to obtain information on the orientation of single as well as many fluorophores. We attached either monofunctional or bifunctional fluorescent probes to the kinesin motor domain. Both types of labeled kinesins show anisotropic fluorescence signals when bound to axonemal microtubules, but the bifunctional probe is less mobile resulting in higher anisotropy. From the polarization experiments with the bifunctional probe, we determined the orientation of kinesin bound to microtubules in the presence of AMP-PNP and found close agreement with previous models derived from cryo-electron microscopy. We also compared the polarization anisotropy of monomeric and dimeric kinesin constructs bound to microtubules in the presence of AMP-PNP. Our results support models of mechanochemistry that require a state in which both motor domains of a kinesin dimer bind simultaneously with similar orientation with respect to the microtubule.

Actin Cytoskeleton↗

PubMed2001

ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy.

Kinesin is an ATP-driven molecular motor protein that moves processively along microtubules. Despite considerable research, the detailed mechanism of kinesin motion remains elusive. We applied an enhanced suite of single- and multiple-molecule fluorescence polarization microscopy assays to report the orientation and mobility of kinesin molecules bound to microtubules as a function of nucleotide state. In the presence of analogs of ATP, ADP-Pi or in the absence of nucleotide, the kinesin head maintains a rigid orientation. In the presence of ADP, the motor domain of kinesin, still bound to the microtubule, adopts a previously undescribed, highly mobile state. This state may be general to the chemomechanical cycle of motor proteins; in the case of kinesin, the transition from a highly mobile to a rigid state after ADP release may contribute to the generation of the 8 nm step.

Adenosine Diphosphate↗

PubMed1998

Relationship between moiré patterns, tubulin shape, and microtubule polarity.

The polarity of microtubules is reflected in cryo-electron microscope images and in three-dimensional reconstructions [Chrétien et al., 1996: Structure 4:1031-1040; Sosa and Milligan, 1996: J. Mol. Biol. 260:743-755]. This paper shows how the directionality of the moiré patterns and the shape of the tubulin subunits are related. Microtubules observed by cryo-electron microscopy show an arrowhead moiré pattern that points toward the plus end of microtubules with a right-handed protofilament skew and toward the minus end of microtubules with a left-handed protofilament skew. On the other hand, three-dimensional reconstructions of microtubules observed from the plus end reveal a radial counterclockwise slew of the tubulin subunits. We show how these polar features are related and present unambiguous rules for determining the polarity on longitudinal and axial views of microtubules.

Computer Simulation↗

PubMed1997

A model for the microtubule-Ncd motor protein complex obtained by cryo-electron microscopy and image analysis.

Kinesin motors convert chemical energy from ATP hydrolysis into unidirectional movement. To understand how kinesin motors bind to and move along microtubules, we fit the atomic structure of the motor domain of Ncd (a kinesin motor involved in meiosis and mitosis) into three-dimensional density maps of Ncd-microtubule complexes calculated by cryo-electron microscopy and image analysis. The model reveals that Ncd shares an extensive interaction surface with the microtubule, and that a portion of the binding site involves loops that contain conserved residues. In the Ncd dimer, the microtubule-bound motor domain makes intimate contact with its partner head, which is dissociated from the microtubule. This head-head interaction may be important in positioning the dissociated head to take a step to the next binding site on the microtubule protofilament.

Adenosine Triphosphatases↗

PubMed1997

Three different approaches for calculating the three-dimensional structure of microtubules decorated with kinesin motor domains.

We have used three different electron microscopy approaches to calculate three-dimensional maps of tubulin assemblies decorated with the motor domain of kinesin. The approaches used were: (1) Tilt series reconstruction of negatively stained tubulin sheets. (2) Back-projection reconstruction of microtubules in ice. (3) Helical reconstruction of microtubules in ice. The calculated maps show the overall configuration of the protofilaments and the interactions between the motor and the protofilaments at a resolution of 2-4 nm. The three methods revealed a similar binding configuration of the kinesin motor domain to the protofilament. We also found that seams can be present in potentially helical microtubules, limiting the use of helical reconstruction algorithms. Advantages and disadvantages of each of the three approaches are discussed.

Animals↗

PubMed1996

Three-dimensional structure of ncd-decorated microtubules obtained by a back-projection method.

We have used cryo-electron microscopy and image analysis to obtain the three-dimensional (3D) structure of 11, 12, 14 and 15 protofilament microtubules decorated with the motor domain of ncd. To obtain the 3D maps, we developed a back-projection method that does not require a helical arrangement of the tubulin heterodimers. This method allows the calculation of 3D maps even when lattice discontinuities (seams) are present. The maps show that the microtubules we studied conform to a B-type lattice with one or more seams. In the presence of 5'-adenylim-idodiphosphate (AMP-PNP), the motor domain of ncd binds to the microtubule protofilament crest interacting with only one protofilament. Viewing the structures along the microtubule axis shows that the ncd motor domain and the tubulin are titled in opposite directions. We determined that a clockwise tilt of the tubulin subunits corresponds to a view from the minus end towards the plus end of the microtubule.

Drosophila Proteins↗

PubMed1994

Ultrastructure of skeletal muscle fibers studied by a plunge quick freezing method: myofilament lengths.

We have set up a system to rapidly freeze muscle fibers during contraction to investigate by electron microscopy the ultrastructure of active muscles. Glycerinated fiber bundles of rabbit psoas muscles were frozen in conditions of rigor, relaxation, isometric contraction, and active shortening. Freezing was carried out by plunging the bundles into liquid ethane. The frozen bundles were then freeze-substituted, plastic-embedded, and sectioned for electron microscopic observation. X-ray diffraction patterns of the embedded bundles and optical diffraction patterns of the micrographs resemble the x-ray diffraction patterns of unfixed muscles, showing the ability of the method to preserve the muscle ultrastructure. In the optical diffraction patterns layer lines up to 1/5.9 nm-1 were observed. Using this method we have investigated the myofilament lengths and concluded that there are no major changes in length in either the actin or the myosin filaments under any of the conditions explored.

Actin Cytoskeleton↗

PubMed1994

X-ray diffraction measurements of the extensibility of actin and myosin filaments in contracting muscle.

We have used a small angle scattering system assembled on the high flux multipole wiggler beam line at CHESS (Cornell) to make very accurate spacing measurements of certain meridional and layer-line reflections from contracting muscles. During isometric contraction, the actin 27.3 A reflection increases in spacing from its resting value by approximately 0.3%, and other actin reflections, including the 59 and 51 A off-meridional reflections, show corresponding changes in spacing. When tension is augmented or diminished by applying moderate speed length changes to a contracting muscle, changes in spacing in the range of 0.19-0.24% (when scaled to full isometric tension) can be seen. The larger difference between the resting and isometric spacings suggests either nonlinearity at low tension levels or the presence of a component related to activation itself. Myosin filaments also show similar increases in axial period during slow stretch, in addition to the well known larger change associated with activation. An actin spacing change of 0.25-0.3% can also be measured during a 2 ms time frame immediately after a quick release, showing that the elastic behavior is rapid. These observations of filament extensions totaling 2-3 nm per half-sarcomere may necessitate some significant revision of the interpretation of a number of mechanical experiments in muscle, in which it has usually been assumed that virtually all of the elasticity resides in the cross-bridges.

Actins↗

PubMed1994

[The atrioventricular canal and vascular ring. The surgical treatment of a rare anatomical association].

We report the clinical and surgical data of 11 patients with atrioventricular canal and vascular ring. Down syndrome was present in 8 cases (72.7%); 8 patients were female (72.7%). Seven patients underwent, at the same time, correction of vascular ring and banding of the pulmonary artery with 4 death. Two children underwent correction of vascular ring and subsequent correction of atrioventricular canal without mortality. Two patients underwent correction of atrioventricular canal and subsequent correction of the vascular ring with one death. The mortality of the 6 patients operated after the age of 6 months was in measure of 16.6%. This association of malformations may be due to anomalies of neural crest and, although rare, should be particularly kept in mind referring to female patients with Down syndrome.

Aorta, Thoracic↗

PubMed1991

X-ray diffraction study of the structural changes accompanying phosphorylation of tarantula muscle.

Electron microscopy of negatively stained isolated thick filaments of tarantula muscle has revealed that phosphorylation of myosin regulatory light chains is accompanied by a loss of the helical order of myosin heads. From equatorial X-ray diffraction patterns of tarantula muscles in the phosphorylated state we have detected a mass movement in the myosin filaments that supports this finding.

Actin Cytoskeleton↗

PubMed1988