scholarly journals Two distinct functions of the carboxyl-terminal tail domain of NF-M upon neurofilament assembly: cross-bridge formation and longitudinal elongation of filaments.

1995 ◽  
Vol 129 (2) ◽  
pp. 411-429 ◽  
Author(s):  
T Nakagawa ◽  
J Chen ◽  
Z Zhang ◽  
Y Kanai ◽  
N Hirokawa
Keyword(s):  
Neuronal Cell ◽  
Molecular Architecture ◽  
Tail Domain ◽  
Bridge Formation ◽  
Cross Bridge ◽  
Filament Assembly ◽  
Bridge Structures ◽  
Carboxyl Terminal ◽  
Cross Bridges ◽  
10 Nm Filaments

Neurofilaments are the major cytoskeletal elements in the axon that take highly ordered structures composed of parallel arrays of 10-nm filaments linked to each other with frequent cross-bridges, and they are believed to maintain a highly polarized neuronal cell shape. Here we report the function of rat NF-M in this characteristic neurofilament assembly. Transfection experiments were done in an insect Sf9 cell line lacking endogenous intermediate filaments. NF-L and NF-M coassemble to form bundles of 10-nm filaments packed in a parallel manner with frequent cross-bridges resembling the neurofilament domains in the axon when expressed together in Sf9 cells. Considering the fact that the expression of either NF-L or NF-M alone in these cells results in neither formation of any ordered network of 10-nm filaments nor cross-bridge structures, NF-M plays a crucial role in this parallel filament assembly. In the case of NF-H the carboxyl-tail domain has been shown to constitute the cross-bridge structures. The similarity in molecular architecture between NF-M and NF-H suggests that the carboxyl-terminal tail domain of NF-M also constitutes cross-bridges. To examine this and to further investigate the function of the carboxyl-terminal tail domain of NF-M, we made various deletion mutants that lacked part of their tail domains, and we expressed these with NF-L. From this deletion mutant analysis, we conclude that the carboxyl-terminal tail domain of NF-M has two distinct functions. First, it is the structural component of cross-bridges, and these cross-bridges serve to control the spacing between core filaments. Second, the portion of the carboxyl-terminal tail domain of NF-M that is directly involved in cross-bridge formation affects the core filament assembly by helping them to elongate longitudinally so that they become straight.

Abstract P338: Myopeptide Improves Contractile Mechanics In A Mouse Model Of Heart Failure Expressing Phosphoablated Cardiac Myosin Binding Protein-C

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Rohit Singh ◽  
Sakthivel Sadayappan
Keyword(s):  
Heart Failure ◽  
Mouse Model ◽  
Protein C ◽  
Cardiac Myosin ◽  
Bridge Formation ◽  
Cross Bridge ◽  
Myosin Binding Protein ◽  
Myosin Binding Protein C ◽  
Myosin Binding ◽  
Cross Bridges

Rationale: Normal heart function depends on cardiac myosin binding protein-C (cMyBP-C) phosphorylation. Its decrease is associated with heart failure (HF) by inhibiting actomyosin interactions. In absence of cMyBP-C phosphorylation, the protein is bound to myosin S2, but released when phosphorylated, allowing myosin to form cross-bridges with actin. Challenging cMyBP-C/myosin S2 interaction by myopeptide (the first 126 amino acids of myosin S2) could promote actomyosin interaction in vitro , but its ability to improve contractility in HF remains untested. Objective: To test contractile function in skinned papillary fibers of a cMyBP-C dephosphorylated mouse model using myopeptide. Methods and Results: To mimic constitutive phosphoablation, a knock-in mouse model was established to express cMyBP-C in which serines 273, 282 and 302 were mutated to alanine (cMyBP-C AAA ). Western blotting revealed 50% and 100% of cMyBP-C AAA in het and homo mouse hearts, respectively. Echocardiography showed a decreased percentage of ejection fraction (28%, p<0.01) and fractional shortening (30%, p< 0.05) in both het and homo cMyBP-C AAA mice at 3 months of age, compared to knock-in negative controls. These mice also developed diastolic dysfunction with elevated ratio of E/A and E/e’ waves. Next, pCa-force measurements using skinned papillary fibers determined that maximal force (F max ) and rate of cross-bridge formation ( k tr ) were decreased in the cMyBP-C AAA groups, compared to the control. However, administration of dose-dependent myopeptide increased F max and k tr in wild-type and cMyBP-C AAA permeabilized skinned papillary fibers without affecting myofilament Ca 2+ sensitivity. Conclusions: Myopeptide can increase contractile force and rate of cross-bridge formation by releasing cMyBP-C/myosin S2 and promoting actomyosin formation of cross-bridges, thus validating its therapeutic potential.

Non-cross-bridge calcium-dependent stiffness in frog muscle fibers

2004 ◽  
Vol 286 (6) ◽  
pp. C1353-C1357 ◽  
Author(s):  
M. A. Bagni ◽  
B. Colombini ◽  
P. Geiger ◽  
R. Berlinguer Palmini ◽  
G. Cecchi
Keyword(s):  
Time Course ◽  
Frog Muscle ◽  
Static Stiffness ◽  
Bridge Formation ◽  
Calcium Dependent ◽  
Cross Bridge ◽  
Steady Level ◽  
Intracellular Ca ◽  
Butanedione Monoxime ◽  
Cross Bridges

At the end of the force transient elicited by a fast stretch applied to an activated frog muscle fiber, the force settles to a steady level exceeding the isometric level preceding the stretch. We showed previously that this excess of tension, referred to as “static tension,” is due to the elongation of some elastic sarcomere structure, outside the cross bridges. The stiffness of this structure, “static stiffness,” increased upon stimulation following a time course well distinct from tension and roughly similar to intracellular Ca2+ concentration. In the experiments reported here, we investigated the possible role of Ca2+ in static stiffness by comparing static stiffness measurements in the presence of Ca2+ release inhibitors (D600, Dantrolene, 2H2O) and cross-bridge formation inhibitors [2,3-butanedione monoxime (BDM), hypertonicity]. Both series of agents inhibited tension; however, only D600, Dantrolene, and 2H2O decreased at the same time static stiffness, whereas BDM and hypertonicity left static stiffness unaltered. These results indicate that Ca2+, in addition to promoting cross-bridge formation, increases the stiffness of an (unidentified) elastic structure of the sarcomere. This stiffness increase may help in maintaining the sarcomere length uniformity under conditions of instability.

scholarly journals The neurofilament middle molecular mass subunit carboxyl-terminal tail domains is essential for the radial growth and cytoskeletal architecture of axons but not for regulating neurofilament transport rate

2003 ◽  
Vol 163 (5) ◽  
pp. 1021-1031 ◽  
Author(s):  
Mala V. Rao ◽  
Jabbar Campbell ◽  
Aidong Yuan ◽  
Asok Kumar ◽  
Takahiro Gotow ◽  
...  
Keyword(s):  
Radial Growth ◽  
Average Rate ◽  
Embryonic Stem ◽  
Transport Rate ◽  
Tail Domain ◽  
Neurofilament Protein ◽  
Carboxyl Terminal ◽  
Cross Bridges ◽  
Neurofilament Transport

The phosphorylated carboxyl-terminal “tail” domains of the neurofilament (NF) subunits, NF heavy (NF-H) and NF medium (NF-M) subunits, have been proposed to regulate axon radial growth, neurofilament spacing, and neurofilament transport rate, but direct in vivo evidence is lacking. Because deletion of the tail domain of NF-H did not alter these axonal properties (Rao, M.V., M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.S. Calcutt, Y. Uchiyama, R.A. Nixon, and D.W. Cleveland. 2002. J. Cell Biol. 158:681–693), we investigated possible functions of the NF-M tail domain by constructing NF-M tail–deleted (NF-MtailΔ) mutant mice using an embryonic stem cell–mediated “gene knockin” approach that preserves normal ratios of the three neurofilament subunits. Mutant NF-MtailΔ mice exhibited severely inhibited radial growth of both motor and sensory axons. Caliber reduction was accompanied by reduced spacing between neurofilaments and loss of long cross-bridges with no change in neurofilament protein content. These observations define distinctive functions of the NF-M tail in regulating axon caliber by modulating the organization of the neurofilament network within axons. Surprisingly, the average rate of axonal transport of neurofilaments was unaltered despite these substantial effects on axon morphology. These results demonstrate that NF-M tail–mediated interactions of neurofilaments, independent of NF transport rate, are critical determinants of the size and cytoskeletal architecture of axons, and are mediated, in part, by the highly phosphorylated tail domain of NF-M.

scholarly journals The rod domain of NF-L determines neurofilament architecture, whereas the end domains specify filament assembly and network formation.

1993 ◽  
Vol 123 (6) ◽  
pp. 1517-1533 ◽  
Author(s):  
S Heins ◽  
P C Wong ◽  
S Müller ◽  
K Goldie ◽  
D W Cleveland ◽  
...  
Keyword(s):  
Network Formation ◽  
Molecular Model ◽  
Tail Domain ◽  
Lateral Association ◽  
Filament Assembly ◽  
Head Domain ◽  
Scanning Transmission ◽  
Bona Fide ◽  
10 Nm Filaments

Neurofilaments, assembled from NF-L, NF-M, and NF-H subunits, are the most abundant structural elements in myelinated axons. Although all three subunits contain a central, alpha-helical rod domain thought to mediate filament assembly, only NF-L self-assembles into 10-nm filaments in vitro. To explore the roles of the central rod, the NH2-terminal head and the COOH-terminal tail domain in filament assembly, full-length, headless, tailless, and rod only fragments of mouse NF-L were expressed in bacteria, purified, and their structure and assembly properties examined by conventional and scanning transmission electron microscopy (TEM and STEM). These experiments revealed that in vitro assembly of NF-L into bona fide 10-nm filaments requires both end domains: whereas the NH2-terminal head domain promotes lateral association of protofilaments into protofibrils and ultimately 10-nm filaments, the COOH-terminal tail domain controls lateral assembly of protofilaments so that it terminates at the 10-nm filament level. Hence, the two end domains of NF-L have antagonistic effects on the lateral association of protofilaments into higher-order structures, with the effect of the COOH-terminal tail domain being dominant over that of the NH2-terminal head domain. Consideration of the 21-nm axial beading commonly observed with 10-nm filaments, the approximate 21-nm axial periodicity measured on paracrystals, and recent cross-linking data combine to support a molecular model for intermediate filament architecture in which the 44-46-nm long dimer rods overlap by 1-3-nm head-to-tail, whereas laterally they align antiparallel both unstaggered and approximately half-staggered.

scholarly journals Cross-Bridges and Sarcomeric Non-cross-bridge Structures Contribute to Increased Work in Stretch-Shortening Cycles

2020 ◽  
Vol 11 ◽  
Author(s):  
André Tomalka ◽  
Sven Weidner ◽  
Daniel Hahn ◽  
Wolfgang Seiberl ◽  
Tobias Siebert
Keyword(s):  
Cross Bridge ◽  
Bridge Structures ◽  
Cross Bridges

Study of the Molecular Architecture of Keratin Filaments by Electron Microscopy

1982 ◽  
Vol 40 ◽  
pp. 118-119
Author(s):  
U. Aebi ◽  
P. Rew ◽  
T.-T. Sun
Keyword(s):  
Electron Microscopy ◽  
Structural Organization ◽  
Cell Types ◽  
Structural Features ◽  
Molecular Architecture ◽  
The Past ◽  
Keratin Filaments ◽  
Different Types ◽  
10 Nm Filaments ◽  
Different Cell Types

Various types of intermediate-sized (10-nm) filaments have been found and described in many different cell types during the past few years. Despite the differences in the chemical composition among the different types of filaments, they all yield common structural features: they are usually up to several microns long and have a diameter of 7 to 10 nm; there is evidence that they are made of several 2 to 3.5 nm wide protofilaments which are helically wound around each other; the secondary structure of the polypeptides constituting the filaments is rich in ∞-helix. However a detailed description of their structural organization is lacking to date.

Oxytocin-mediated recruitment of slowly cycling cross bridges and isometric force in rat myometrium

1996 ◽  
Vol 270 (2) ◽  
pp. E203-E208
Author(s):  
A. L. Ruzycky ◽  
B. T. Ameredes
Keyword(s):  
Isometric Force ◽  
Additional Stress ◽  
Shortening Velocity ◽  
Cycling Rate ◽  
Quick Release ◽  
Cross Bridge ◽  
Cross Bridges ◽  
Unit Stress ◽  
Release Method ◽  
The Relationship

The relationship between cross-bridge cycling rate and isometric stress was investigated in rat myometrium. Stress production by myometrial strips was measured under resting, K+ depolarization, and oxytocin-stimulated conditions. Cross-bridge cycling rates were determined from measurements of maximal unloaded shortening velocity, using the quick-release method. Force redevelopment after the quick release was used as an index of cross-bridge attachment. With maximal K+ stimulation, stress increased with increased cross-bridge cycling (+76%; P < 0.05) and attached cross bridges (+112%; P < 0.05). Addition of oxytocin during K+ stimulation further increased stress (+30%; P < 0.05). With this force component, the cross-bridge cycling rate decreased (-60%; P < 0.05) similar to that under resting conditions. Attached cross-bridges did not increase with this additional stress. The results suggest two distinct mechanisms mediating myometrial contractions. One requires elevated intracellular calcium and rapidly cycling cross bridges. The other mechanism may be independent of calcium and appears to be mediated by slowly cycling cross bridges, supporting greater unit stress.

Cross-bridge blocker BTS permits direct measurement of SR Ca2+ pump ATP utilization in toadfish swimbladder muscle fibers

2003 ◽  
Vol 285 (4) ◽  
pp. C781-C787 ◽  
Author(s):  
Iain S. Young ◽  
Claire L. Harwood ◽  
Lawrence C. Rome
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Direct Measurement ◽  
Motor Behavior ◽  
Force Generation ◽  
Skinned Fibers ◽  
Control Value ◽  
Cross Bridge ◽  
Direct Measurements ◽  
Hill Coefficients ◽  
Cross Bridges

Because the major processes involved in muscle contraction require rapid utilization of ATP, measurement of ATP utilization can provide important insights into the mechanisms of contraction. It is necessary, however, to differentiate between the contribution made by cross-bridges and that of the sarcoplasmic reticulum (SR) Ca2+ pumps. Specific and potent SR Ca2+ pump blockers have been used in skinned fibers to permit direct measurement of cross-bridge ATP utilization. Up to now, there was no analogous cross-bridge blocker. Recently, N-benzyl- p-toluene sulfonamide (BTS) was found to suppress force generation at micromolar concentrations. We tested whether BTS could be used to block cross-bridge ATP utilization, thereby permitting direct measurement of SR Ca2+ pump ATP utilization in saponin-skinned fibers. At 25 μM, BTS virtually eliminates force and cross-bridge ATP utilization (both <4% of control value). By taking advantage of the toadfish swimbladder muscle's unique right shift in its force-Ca2+ concentration ([Ca2+]) relationship, we measured SR Ca2+ pump ATP utilization in the presence and absence of BTS. At 25 μM, BTS had no effect on SR pump ATP utilization. Hence, we used BTS to make some of the first direct measurements of ATP utilization of intact SR over a physiological range of [Ca2+]at 15°C. Curve fits to SR Ca2+ pump ATP utilization vs. pCa indicate that they have much lower Hill coefficients (1.49) than that describing cross-bridge force generation vs. pCa (∼5). Furthermore, we found that BTS also effectively eliminates force generation in bundles of intact swimbladder muscle, suggesting that it will be an important tool for studying integrated SR function during normal motor behavior.

Heat production of rat anococcygeus muscle during isometric contraction

1988 ◽  
Vol 255 (4) ◽  
pp. C536-C542 ◽  
Author(s):  
J. S. Walker ◽  
I. R. Wendt ◽  
C. L. Gibbs
Keyword(s):  
Energy Expenditure ◽  
Heat Production ◽  
Progressive Increase ◽  
Isometric Contractions ◽  
Shortening Velocity ◽  
Anococcygeus Muscle ◽  
Cross Bridge ◽  
Time Integral ◽  
Rat Anococcygeus Muscle ◽  
Cross Bridges

Heat production, unloaded shortening velocity (Vus), and load-bearing capacity (LBC) were studied in the isolated rat anococcygeus muscle during isometric contractions at 27 degrees C. The relation between the total suprabasal heat produced and the stress-time integral for isometric contractions of various durations was curvilinear, demonstrating a decreasing slope as contractile duration increased. The rate of heat production at 600 s was approximately 68% of the peak value of 6.55 mW/g that occurred at 10 s. At the same time, force rose from a mean of 92 mN/mm2 at 10 s to a value of 140 mN/mm2 at 600 s. This produced a nearly threefold increase in the economy of force maintenance. The decline in the rate of heat production was accompanied by a decline in Vus from 0.56 Lo/s at 10 s to 0.28 Lo/s at 600 s, where Lo is the length for optimal force development. This suggests the fall in the rate of heat production was caused, at least in part, by a slowing of cross-bridge kinetics. The ratio of LBC to developed tension at 10 s was not significantly different from the ratio at 600 s, suggesting that the increase in tension was due to an increased number of attached cross bridges. The decline in heat production, therefore, appears contradictory, since an increased number of attached cross bridges would predict an increased rate of energy expenditure. The observations can be reconciled if either 1) the increase in force is caused by a progressive increase in the attachment time of a constant number of cross bridges that cycle at a lower frequency or 2) the decline in energy expenditure caused by the slowing of cross-bridge cycling is sufficient to mask the increase caused by the recruitment of additional cross bridges.

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