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Vitro Motility (vitro + motility)

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Life Sciences	100%

Selected Abstracts

Actin-dependent motility of melanosomes from fish retinal pigment epithelial (RPE) cells investigated using in vitro motility assays

CYTOSKELETON, Issue 2 2004
E. L. McNeil
Melanosomes (pigment granules) within retinal pigment epithelial (RPE) cells of fish and amphibians undergo massive migrations in response to light conditions to control light flux to the retina. Previous research has shown that melanosome motility within apical projections of dissociated fish RPE cells requires an intact actin cytoskeleton, but the mechanisms and motors involved in melanosome transport in RPE have not been identified. Two in vitro motility assays, the Nitella assay and the sliding filament assay, were used to characterize actin-dependent motor activity of RPE melanosomes. Melanosomes applied to dissected filets of the Characean alga, Nitella, moved along actin cables at a mean rate of 2 ,m/min, similar to the rate of melanosome motility in dissociated, cultured RPE cells. Path lengths of motile melanosomes ranged from 9 to 37 ,m. Melanosome motility in the sliding filament assay was much more variable, ranging from 0.4,33 ,m/min; 70% of velocities ranged from 1,15 ,m/min. Latex beads coated with skeletal muscle myosin II and added to Nitella filets moved in the same direction as RPE melanosomes, indicating that the motility is barbed-end directed. Immunoblotting using antibodies against myosin VIIa and rab27a revealed that both proteins are enriched on melanosome membranes, suggesting that they could play a role in melanosome transport within apical projections of fish RPE. Cell Motil. Cytoskeleton 58:71,82, 2004. © 2004 Wiley-Liss, Inc. [source]

Regulation of monomeric dynein activity by ATP and ADP concentrations

CYTOSKELETON, Issue 4 2001
Katsuyuki Shiroguchi
Abstract Axonemal dyneins are force-generating ATPases that produce ciliary and flagellar movement. A dynein has large heavy chain(s) in which there are multiple (4,6) ATP-binding consensus sequences (P-loops) as well as intermediate and light chains, constituting a very large complex. We purified a monomeric form of dynein (dynein- a) that has at least three light chains from 14S dyneins of Tetrahymena thermophila and characterized it. In in vitro motility assays, dynein- a rotated microtubules around their longitudinal axis as well as translocated them with their plus-ends leading. ATPase activity at 1 mM ATP was doubled in the presence of a low level of ADP (, 20 ,M). Both ATPase activity and translocational velocities in the presence of ADP (, 20 ,M) fit the Michaelis-Menten equation well. However, in the absence of ADP (< 0.1 ,M), neither of the activities followed the Michaelis-Menten-type kinetics, probably due to the effect of two ATP-binding sites. Our results also indicate that dynein- a has an ATP-binding site that is very sensitive to ADP and affects ATP hydrolysis at the catalytic site. This study shows that a monomeric form of a dynein molecule regulates its activity by direct binding of ATP and ADP to itself, and thus the dynein molecule has an intramolecular regulating system. Cell Motil. Cytoskeleton 49:189,199, 2001. © 2001 Wiley-Liss, Inc. [source]

Different effects of cardiac versus skeletal muscle regulatory proteins on in vitro measures of actin filament speed and force

THE JOURNAL OF PHYSIOLOGY, Issue 3 2005
Emilie Warner Clemmens
Mammalian cardiac and skeletal muscle express unique isoforms of the thin filament regulatory proteins, troponin (Tn) and tropomyosin (Tm), and the significance of these different isoforms in thin filament regulation has not been clearly identified. Both in vitro and skinned cellular studies investigating the mechanism of thin filament regulation in striated muscle have often used heterogeneous mixtures of Tn, Tm and myosin isoforms, and variability in reported results might be explained by different combinations of these proteins. Here we used in vitro motility and force (microneedle) assays to investigate the influence of cardiac versus skeletal Tn and Tm isoforms on actin,heavy meromyosin (HMM) mechanics. When interacting with skeletal HMM, thin filaments reconstituted with cardiac Tn/Tm or skeletal Tn/Tm exhibited similar speed,calcium relationships and significantly increased maximum speed and force per filament length (F/l) at pCa 5 (versus unregulated actin filaments). However, augmentation of F/l was greater with skeletal regulatory proteins. Reconstitution of thin filaments with the heterogeneous combination of skeletal Tn and cardiac Tm decreased sliding speeds at all [Ca2+] relative to thin filaments with skeletal Tn/Tm. Finally, for filaments reconstituted with any heterogeneous mix of Tn and Tm isoforms, force was not potentiated over that of unregulated actin filaments. Combined the results suggest (1) that cardiac regulatory proteins limit the allosteric enhancement of force, and (2) that Tn and Tm isoform homogeneity is important when studying Ca2+ regulation of crossbridge binding and kinetics as well as mechanistic differences between cardiac and skeletal muscle. [source]