1

AU Zoghbi, ME, Woodhead, JL, Craig, R, Padron, R
TI Helical order in tarantula thick filaments requires the "closed" conformation of the myosin head
SO JOURNAL OF MOLECULAR BIOLOGY
DE striated muscle; myosin filaments; helical order; ATP analogs; temperature
ID X-RAY-DIFFRACTION; VERTEBRATE STRIATED-MUSCLE; INSECT FLIGHT-MUSCLE; MOTOR DOMAIN; SKELETAL-MUSCLE; CROSS-BRIDGES; FISH MUSCLE; ADENOSINE-TRIPHOSPHATE; ELECTRON-MICROSCOPY; STRUCTURAL-CHANGES
AB Myosin heads are helically ordered on the thick filament surface in relaxed muscle. In mammalian and avian filaments this helical arrangement is dependent on temperature and it has been suggested that helical order is related to ATP hydrolysis by the heads. To test this hypothesis, we have used electron microscopy and image analysis to study the ability and temperature dependence of analogs of ATP and ADP.Pi to induce helical order in tarantula thick filaments. ATP or
analogs were added to rigor myofibrils or purified thick filaments at 22 degreesC and 4 degreesC and the samples negatively stained. The ADP.Pi analogs ADP.AlF4 and ADP.Vi, and the ATP analogs ADP.BeFx, AMPPNP and ATPgammaNH(2), all induced helical order in tarantula thick filaments, independent of temperature. In the absence of nucleotide, or in the presence of ADP or the ATP analog, ATPgammaS, there was no helical ordering. According to crystallographic and tryptophan fluorescence studies, all of these analogs, except ATPgammaS and ADP, induce the "closed" conformation of the myosin head (in which the gamma phosphate pocket is closed). We suggest that helical order requires the closed conformation of the myosin head but is not dependent on the hydrolysis of ATP. (C) 2004 Elsevier Ltd. All rights reserved.
TC 0
PU ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD
PI LONDON
PA 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND
PD SEP 24
PY 2004
VL 342
IS 4
BP 1223
EP 1236
PG 14
UT ISI:000224005500013

[up]

2

AU Luther, PK, Padron, R, Ritter, S, Craig, R, Squire, JM
TI Heterogeneity of Z-band structure within a single muscle sarcomere: Implications for sarcomere assembly
SO JOURNAL OF MOLECULAR BIOLOGY
DE Z-line; Z-disc; fibre types; actin filament length; vertebrate muscle structure
ID ALPHA-ACTININ BINDING; VERTEBRATE Z-DISCS; 3-DIMENSIONAL STRUCTURE; SKELETAL-MUSCLE; STRIATED-MUSCLE; Z-LINE; FILAMENT LENGTH; FINE-STRUCTURE; Z-DISKS; TITIN
AB The vertebrate striated muscle Z-band connects actin filaments of opposite polarity from adjacent sarcomeres and allows tension to be transmitted along a myofibril during contraction. Z-bands in different muscles have a modular structure formed by layers of alpha-actinin molecules cross-linking actin filaments. Successive layers occur at 19 nm intervals and have 90 rotations between them. 3D reconstruction from electron micrographs show a two-layer "simple" Z-band in fish body fast muscle, a three-layer Z-band in fish fin fast muscle, and a six-layer Z-band in mammalian slow muscle. Related to the number of these layers, longitudinal sections of the Z-band show a number of zigzag connections between the oppositely oriented actin filaments. The number of layers also determines the axial width of the Z-band, which is a useful indicator of fibre type; fast fibres have narrow (similar to 30-50 nm) Z-bands; slow and cardiac fibres have wide (similar to100-140 nm) Z-bands. Here, we report the first observation of two different Z-band widths within a single sarcomere. By comparison with previous studies, the narrower Z-band comprises three layers. Since the increase in width of the wider Z-band is about 19 nm, we conclude that it comprises four layers. This finding is consistent with a Z-band assembly model involving molecular control mechanisms that can add additional layers of 19 nm periodicity. These multiple Z-band structures suggest that different isoforms of nebulin and titin with a variable number of Z-repeats could be present within a single sarcomere. (C) 2003 Elsevier Ltd. All rights reserved.
TC 1
PU ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD
PI LONDON
PA 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND
PD SEP 5
PY 2003
VL 332
IS 1
BP 161
EP 169
PG 9
UT ISI:000185034400014

[up]

3

[up]

4

AU Hidalgo, C, Craig, R, Ikebe, M, Padron, R
TI Mechanism of phosphorylation of the regulatory light chain of myosin from tarantula striated muscle
SO JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY
ID SMOOTH-MUSCLE; THICK FILAMENTS; SKELETAL-MUSCLE; PROTEIN-KINASE; CONTRACTION; FIBERS; CELLS; DEPHOSPHORYLATION; ARRANGEMENT; PHOSPHATASE
AB Contraction is modulated in many striated muscles by Ca2+-calmodulin dependent phosphorylation of the myosin regulatory light chain (RLC) by myosin light chain kinase. We have investigated the biochemical mechanism of RLC phosphorylation in tarantula muscle to better understand the basis of myosin-linked regulation. In an earlier study
it was concluded that the RLC occurred as two species, both of which could be phosphorylated, potentiating contraction. Here we present evidence that only a single species exists, and that this can be phosphorylated at one or two sites. In relaxed muscle we find evidence for a substantial level of basal phosphorylation at the first site. This is augmented on activation, followed by partial phosphorylation of the second site. We find in addition that Ca2+ has a dual effect on light chain phosphorylation, depending on its concentration. At low concentration (relaxing conditions) only basal phosphorylation is observed, while at higher concentrations (activating conditions) RLC phosphorylation is stimulated. At still higher Ca2+ concentrations we find partial inhibition of RLC phosphorylation, suggesting an additional mechanism by which the muscle cell can fine tune contractile activity by controlling the level of free Ca2+.
TC 2
PU KLUWER ACADEMIC PUBL
PI DORDRECHT
PA SPUIBOULEVARD 50, PO BOX 17, 3300 AA DORDRECHT, NETHERLANDS
PD JAN
PY 2001
VL 22
IS 1
BP 51
EP 59
PG 9
UT ISI:000169514000003

[up]

5

AU Offer, G, Knight, PJ, Burgess, SA, Alamo, L, Padron, R
TI A new model for the surface arrangement of myosin molecules in tarantula thick filaments
SO JOURNAL OF MOLECULAR BIOLOGY
DE myosin; thick filament; cross-bridges; three-dimensional reconstruction; muscle
ID CHANGES ACCOMPANYING PHOSPHORYLATION; OPTICAL DIFFRACTION ANALYSIS; SMOOTH-MUSCLE MYOSIN; X-RAY-SCATTERING; SCALLOP MYOSIN; 3-DIMENSIONAL RECONSTRUCTION; STRUCTURAL-CHANGES; REGULATORY DOMAIN; SCORPION MUSCLE; SKELETAL-MUSCLE
AB Three-dimensional reconstructions of the negatively stained thick filaments of tarantula muscle with a resolution of 50 Angstrom have previously suggested that the helical tracks of myosin heads are zigzagged, short diagonal ridges being connected by nearly axial links. However, surface views of lower contour levels reveal an additional J-shaped feature approximately the size and shape of a myosin head. We have modelled the surface array of myosin heads on the filaments
using as a building block a model of a two-headed regulated myosin molecule in which the regulatory light chains of the two heads together form a compact head-tail junction. Four parameters defining the radius, orientation and rotation of each myosin molecule were varied. In addition, the heads were allowed independently to bend in a plane perpendicular to the coiled-coil tail at three sites, and to tilt with respect to the tail and to twist at one of these sites. After low-pass filtering, models were aligned with the reconstruction, scored by cross-correlation and refined by simulated annealing. Comparison of the geometry of the reconstruction and the distance between domains in the myosin molecule narrowed the choice of models to two main classes. A good match to the reconstruction was obtained with a model in which each ridge is formed from the motor domain of a head pointing to the bare zone together with the head-tail junction of a neighbouring molecule. The heads pointing to the Z-disc intermittently occupy the J-position. Each motor domain interacts with the essential and regulatory light chains of the neighbouring heads. A near-radial spoke in the reconstruction connecting the backbone to one end of the
ridge can be identified as the start of the coiled-coil tail. (C) 2000 Academic Press.
TC 7
PU ACADEMIC PRESS LTD
PI LONDON
PA 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND
PD APR 28
PY 2000
VL 298
IS 2
BP 239
EP 260
PG 22
UT ISI:000086831200006

[up]

6

AU Padron, R, Alamo, L, Murgich, J, Craig, R
TI Towards an atomic model of the thick filaments of muscle
SO JOURNAL OF MOLECULAR BIOLOGY
DE muscle; thick filaments; myosin; structure; electron microscopy
ID DICTYOSTELIUM-DISCOIDEUM MYOSIN; X-RAY STRUCTURE; TARANTULA MUSCLE; SCALLOP MYOSIN; 3-DIMENSIONAL RECONSTRUCTION; CONFORMATIONAL-CHANGES; ANGSTROM RESOLUTION; STRIATED-MUSCLE; ACTIN FILAMENT; GENERAL MODEL
AB The thick filaments of muscle and non-muscle cells are polymers of myosin molecules whose energy-transducing heads lie on the filament surface, where they interact with actin to generate force. A key structural question is how the myosin heads are arranged in the relaxed state, and how this arrangement changes on activation of contraction. We have fitted the atomic structure of the myosin head to the three-dimensional structure of myosin filaments of tarantula muscle determined by electron microscopy to produce a near-atomic model of the head arrangement. A good fit is obtained only when the two heads from a myosin molecule run along the helical tracks antiparallel to each other. Oppositely oriented heads from axially adjacent molecules in a helix interact with each other, with their nucleotide-binding pockets opposed. This arrangement, supported also by crosslinking evidence, suggests a simple mechanism for the stabilization of myosin head helices in relaxed muscle via the formation of intermolecular "dimers" of heads from axially adjacent myosin molecules. (C) 1998 Academic
Press Limited.
TC 9
PU ACADEMIC PRESS LTD
PI LONDON
PA 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND
PD JAN 9
PY 1998
VL 275
IS 1
BP 35
EP 41
PG 7
UT ISI:000071392800005

[up]

7

AU Mateu, L, Moran, O, Padron, R, Borgo, M, Vonasek, E, Marquez, G, Luzzati, V
TI The action of local anesthetics on myelin structure and nerve conduction in toad sciatic nerve
SO BIOPHYSICAL JOURNAL
ID ORDER-DISORDER PHENOMENA; OPTIC NERVES; MEMBRANE; SHEATHS; RESOLUTION; MECHANISMS; MODEL
AB X-ray scattering and electrophysiological experiments were performed on toad sciatic nerves in the presence of local anesthetics. In vitro experiments were performed on dissected nerves superfused with Ringer's solutions containing procaine, lidocaine, tetracaine, or dibucaine. In vivo experiments were performed on nerves dissected from animals
anesthesized by targeted injections of tetracaine-containing solutions. In all cases the anesthetics were found to have the same effects on the x-ray scattering spectra: the intensity ratio of the even-order to the odd-order reflections increases and the lattice parameter increases. These changes are reversible upon removal of the anesthetic. The magnitude of the structural changes varies with the duration of the superfusion and with the nature and concentration of the anesthetic molecule. A striking quantitative correlation was observed between the structural effects and the potency of the anesthetic, Electron density profiles, which hardly showed any structural alteration of the unit membrane, clearly indicated that the anesthetics have the effect of moving the pairs of membranes apart by increasing the thickness of the cytoplasmic space. Electrophysiological measurements performed on the very samples used in the x-ray scattering experiments showed that the
amplitude of the compound action potential is affected earlier than the structure of myelin (as revealed by the x-ray scattering experiments), whereas conduction velocity closely follows the structural alterations.
TC 3
PU BIOPHYSICAL SOCIETY
PI BETHESDA
PA 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998
PD JUN
PY 1997
VL 72
IS 6
BP 2581
EP 2587
PG 7
UT ISI:A1997XA63600018

[up]

8

AU SanBlas, G, Padron, R, Alamo, L, SanBlas, F
TI Use of morphology index histograms to quantify populations of the fungal pathogen Paracoccidioides brasiliensis
SO MICROBIOLOGY-UK
DE Paracoccidioides brasiliensis; morphology index; dimorphism; morphogenesis
ID CELL; MUTANTS; GROWTH
AB To quantify the dimorphic process in wild and mutant strains of Paracoccidioides brasiliensis, we defined a morphology index (Mi) in terms of the maximum cell length (I), maximum cell diameter (d), and septal diameter (s), according to the equation Mi = 2 . 13 + 13 . log(10)(ls/d(2)), whose intercept and slope were such that Mi was around 1 for yeast (spherical) cells or 4 for hyphal (elongated) cells. This discriminatory power was used to quantify morphological population mixtures through Mi histograms. During the temperature-induced dimorphic transition (either way). mean Mi (<(Mi)over bar>) varied
linearly with time, suggesting a continuity in the process. Also, in wild strains and mutants thereof we found an inverse relationship between <(Mi)over bar> and content of both cell wall chitin and 1.3-alpha-glucan.
TC 2
PU SOC GENERAL MICROBIOLOGY
PI READING
PA MARLBOROUGH HOUSE, BASINGSTOKE RD, SPENCERS WOODS, READING, BERKS,
ENGLAND RG7 1AE
PD JAN
PY 1997
VL 143
PN Part 1
BP 197
EP 202
PG 6
UT ISI:A1997WE41100024

[up]

9

AU Padron, R, Alamo, L, Guerrero, JR, Granados, M, Uman, P, Craig, R
TI Three-dimensional reconstruction of thick filaments from rapidly frozen, freeze-substituted tarantula muscle
SO JOURNAL OF STRUCTURAL BIOLOGY
ID CHANGES ACCOMPANYING PHOSPHORYLATION; VERTEBRATE STRIATED-MUSCLE; INSECT FLIGHT-MUSCLE; X-RAY-DIFFRACTION; ELECTRON-MICROSCOPY; MYOSIN-FILAMENTS; SKELETAL-MUSCLE; ARRANGEMENT; HEADS; LIMULUS
AB We have applied three-dimensional helical reconstruction techniques to images of myosin filaments of tarantula leg muscle obtained from rapidly frozen, freeze-substituted specimens. Computed Fourier transforms of filaments selected from longitudinal sections show up to six layer lines indexing on the 43.5-nm helical repeat of myosin crossbridges. The three-dimensional reconstruction, performed after separation of overlapped Bessel functions, shows four continuous strands of density on the surface of the filament, modulated by density at 14.5-nm intervals, corresponding to the myosin heads aligned
approximately along the helical strands. In transverse view, the reconstruction shows four projections and is similar in profile to myosin filaments seen in thin transverse sections of rapidly frozen muscle. The reconstruction is similar to that of negatively stained, isolated tarantula filaments except that in the latter there is an additional modulation of the helix density, which better resolves the two heads of each myosin crossbridge. Thus, the general arrangement of the myosin heads in the freeze-substituted specimens is preserved, although finer details of structure such as individual myosin heads are lost. (C) 1995 Academic Press, Inc.
TC 6
PU ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS
PI SAN DIEGO
PA 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495
PD NOV-DEC
PY 1995
VL 115
IS 3
BP 250
EP 257
PG 8
UT ISI:A1995TM54800004

[up]

10

AU PADRON, R, PANTE, N, SOSA, H, KENDRICKJONES, J
TI X-RAY-DIFFRACTION STUDY OF THE STRUCTURAL-CHANGES ACCOMPANYING PHOSPHORYLATION OF TARANTULA MUSCLE
SO JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY
ID ACTIN-MYOSIN INTERACTION; LIMULUS THICK FILAMENTS; LIGHT-CHAIN; STRIATED-MUSCLE; GIZZARD MYOSIN; ACTIVATION; ARRANGEMENT; SCALLOP; PATTERN; KINASE
AB 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.
TC 11
PU CHAPMAN HALL LTD
PI LONDON
PA 2-6 BOUNDARY ROW, LONDON, ENGLAND SE1 8HN
PD JUN
PY 1991
VL 12
IS 3
BP 235
EP 241
PG 7
UT ISI:A1991FN32900003