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M Reedy

Publications and source records attributed to M Reedy.

9 recordsLinked to original sources
PubMed1995

Single-headed myosin II acts as a dominant negative mutation in Dictyostelium.

Conventional myosin II is an essential protein for cytokinesis, capping of cell surface receptors, and development of Dictyostelium cells. Myosin II also plays an important role in the polarization and movement of cells. All conventional myosins are double-headed molecules but the significance of this structure is not understood since single-headed myosin II can produce movement and force in vitro. We found that expression of the tail portion of myosin II in Dictyostelium led to the formation of single-headed myosin II in vivo. The resultant cells contain an approximately equal ratio of double- and single-headed myosin II molecules. Surprisingly, these cells were completely blocked in cytokinesis and capping of concanavalin A receptors although development into fruiting bodies was not impaired. We found that this phenotype is not due to defects in myosin light chain phosphorylation. These results show that single-headed myosin II cannot function properly in vivo and that it acts as a dominant negative mutation for myosin II function. These results suggest the possibility that cooperativity of myosin II heads is critical for force production in vivo.

Amino Acid Sequence↗
PubMed1995

Expression of light meromyosin in Dictyostelium blocks normal myosin II function.

The ability of myosin II to form filaments is essential for its function in vivo. This property of self association is localized in the light meromyosin (LMM) region of the myosin II molecules. To explore this property in more detail within the context of living cells, we expressed the LMM portion of the Dictyostelium myosin II heavy chain gene in wild-type Dictyostelium cells. We found that the LMM protein was expressed at high levels and that it folded properly into alpha-helical coiled-coiled molecules. The expressed LMM formed large cytoplasmic inclusions composed of entangled short filaments surrounded by networks of long tubular structures. Importantly, these abnormal structures sequestered the cell's native myosin II, completely removing it from its normal cytoplasmic distribution. As a result the cells expressing LMM displayed a myosin-null phenotype: they failed to undergo cytokinesis and became multinucleate, failed to form caps after treatment with Con A, and failed to complete their normal developmental cycle. Thus, expression of the LMM fragment in Dictyostelium completely abrogates myosin II function in vivo. The dominant-negative character of this phenotype holds promise as a general method to disrupt myosin II function in many cell types without the necessity of gene targeting.

Amino Acid Sequence↗
PubMed1993

Fast axonal transport is required for growth cone advance.

Growth cones are capable of advancing despite linkage to a stationary axonal cytoskeleton in chick and murine dorsal root ganglion neurites. Several lines of evidence point to the growth cone as the site of cytoskeletal elongation. Fast axonal transport is probably the means by which cytoskeletal elements or cofactors are rapidly moved through the axon. We report that direct, but reversible, inhibition of fast axonal transport with laser optical tweezers inhibits growth cone motility if cytoskeletal attachment to the cell body is maintained. Advancement ceases after a distance-dependent lag period which correlates with the rate of fast axonal transport. But severing the axonal cytoskeleton with the laser tweezers allows growth cones to advance considerably further. We suggest that axon elongation requires fast axonal transport but growth cone motility does not.

Animals↗
PubMed1991

Functional and ultrastructural effects of a missense mutation in the indirect flight muscle-specific actin gene of Drosophila melanogaster.

A single-site mutation of the flight-muscle-specific actin gene of Drosophila melanogaster causes a substitution of glutamic acid 93 by lysine in all the actin encoded in the indirect flight muscle (IFM). In these Act88FE93K mutants, myofibrillar bundles of thick and thin filaments are present but lack Z-discs and all sarcomeric repeats. Dense filament bundles, which are probably aberrant Z-discs, are seen in myofibrils of pupal flies, but early in adult life these move to the periphery of the fibrils and are not seen in skinned adult fibres. Consistent with this observation, alpha-actinin and other high molecular weight proteins, possibly associated with Z-discs, are not detected on SDS/polyacrylamide gels or Western blots of skinned adult IFM. The mutation lies at the beginning of a loop in the small domain of actin, near the myosin binding region. However, that the mutant actin binds myosin heads is shown by (1) rigor crossbridges in electron micrographs, (2) the appropriate rise in stiffness when ATP is withdrawn in mechanical experiments, and (3) equal protection against tryptic digestion provided by rigor binding between actin and myosin in both wild-type and mutant fibres. Reversal of rigor chevron angle along some thin filaments reflects reversal of thin-filament polarity due to lattice disorder. The absence of Z-discs, alpha-actinin and two high molecular weight proteins, and binding studies by others, suggest that the substitution at residue 93 affects the binding of the mutant actin to a protein, possibly alpha-actinin, which is necessary for Z-disc assembly or maintenance.

Actins↗
PubMed1990

Identification and localization of high molecular weight proteins in insect flight and leg muscle.

Thick and thin filaments in asynchronous flight muscle overlap nearly completely and thick filaments are attached to the Z-disc by connecting filaments. We have raised antibodies against a fraction of Lethocerus flight muscle myofibrils containing Z-discs and associated filaments and also against a low ionic strength extract of myofibrils. Monoclonal antibodies were obtained to proteins of 800 kd (p800), 700 kd (p700), 400 kd (p400) and alpha-actinin. The positions of the proteins in Lethocerus flight and leg myofibrils were determined by immunofluorescence and electron microscopy. p800 is in connecting filaments of flight myofibrils and in A-bands of leg myofibrils. p700 is in Z-discs of flight myofibrils and an immunologically related protein, p500, is in leg muscle Z-discs. p400 is in M-lines of both flight and leg myofibrils. Preliminary DNA sequencing shows that p800 is related to vertebrate titin and nematode twitchin. Molecules of p800 could extend from the Z-disc a short way along thick filaments, forming a mechanical link between the two structures. All three high molecular weight proteins probably stabilize the structure of the myofibril.

Amino Acid Sequence↗
PubMed1984

Structure of Limulus and other invertebrate thick filaments.

We have demonstrated remarkable similarity among the skeletal muscles of chelicerate arthropods with respect to the cross-bridge arrangement on the surface of their thick filaments. The latter, gently isolated from the muscles of three representative species (Limulus telson , tarantula leg and scorpion leg and tail) have been examined by electron microscopy and optical diffraction using both negatively stained and unidirectionally metal shadowed preparations. The filaments are highly periodic and produce clear and detailed diffraction patterns. The cross-bridge projections form integral surface helices, with an axial spacing of 14.5 nm between adjacent crowns and a major axial repeat every 43.5 nm. We have demonstrated previously that Limulus filaments are four-stranded and analysis of both electron micrographs and their transforms, as well as optical reconstructions of the arachnid filaments is consistent with their also having a four-start surface helix, which is right-handed in all cases. Of all those examined, thus far, only Limulus thick filaments have been demonstrated to change length under various conditions. Shortened Limulus filaments isolated from K+-stimulated fibers retain the 43.5 nm axial repeat periodicity and 14.5 nm axial spacing between crowns. In preliminary analysis of negatively stained and metal shadowed preparations, we see no systematic change with respect to screw or rotational symmetry in short as compared with long filaments. A few of the former have a very slightly increased diameter (3-4 nm) in the middle of each filament arm. This region often shows disorder on optical transforms. From our results we cannot rule out the possibility that disaggregation and reaggregation of thick filament proteins accompany the changes in length of Limulus thick filaments.

Animals↗
PubMedDate not supplied

Chromium determinations in a case of chromic acid ingestion.

A fatal case of acute chromic acid ingestion is reported. The case history and biological fluid concentrations of chromium are presented and discussed. Previous cases of acute chromium intoxication are reviewed and recommendations for the management of such patients are presented.

Adult↗