Axoplasm Of Squid Giant Axon Research Articles

Dynamic instability and motile events of native microtubules from squid axoplasm.

Native microtubules from extruded axoplasm of squid giant axons were used as a paradigm to characterize the motion of organelles along free microtubules and to study the dynamics of microtubule length changes. The motion of large round organelles was visualized by AVEC-DIC microscopy and analyzed at a temporal resolution of 10 frames per second. The movements were smooth and showed no major changes in velocity or direction. During translocation, the organelles paused very rarely. Superimposed on the rather constant mean velocity was a velocity fluctuation, which indicated that the organelles are subject to considerable thermal motion during translocation. Evidence for a regular low-frequency oscillation was not found. The thermal motion was anisotropic such that axial motion was less restricted than lateral motion. We conclude that the crossbridge connecting the moving organelle to the microtubule has a flexible region that behaves like a hinge, which permits preferential movement in the direction parallel to the microtubule. The dynamic changes in length of native microtubules were studied at a temporal resolution of 1 Hz. About 98% of the native microtubules maintained their length ("stable" microtubules), while 2% showed phases of growing and/or shrinking typical for dynamic instability ("dynamic" microtubules). Gliding and organelle motion were not influenced by dynamic length changes. Transitions between growing and shrinking phases were low-frequency events (1-10 minutes per cycle). However, a new type of microtubule length fluctuation, which occurred at a high frequency (a few seconds per cycle), was detected. The length changes were in the 1-3 micron range. The latter events were very prominent at the (+) ends. It appears that the native axonal microtubules are much more stable than the purified microtubules and the microtubules of cultured cells that have been studied thus far. Potential mechanisms accounting for the three states of microtubule stability are discussed. These studies show that the native microtubules from squid giant axons are a very useful paradigm for studying microtubule-related motility events and microtubule dynamics.

Incorporation of 3H-amino acids into proteins in a partially purified fraction of axoplasm: evidence for transfer RNA-mediated, post- translational protein modification in squid giant axons

Transfer RNA (tRNA) has been demonstrated to be present in axons of both invertebrates and the higher vertebrates, but nothing is known of its role in the metabolism of the axon. The present experiments were performed to determine whether tRNA functions in axons as a participant in post-translational protein modification of endogenous proteins. RNA was extracted from the axoplasm of squid giant axons and incubated with a variety of 3H-amino acids, aminoacyl-tRNA synthetases (obtained from squid optic lobe), and an appropriate reaction mixture. All of the amino acids tested were bound to an RNA fraction, but this reaction did not occur when samples were incubated in the presence of ribonuclease or in the absence of axoplasmic RNA. When radioactive RNA was chromatographed by polyacrylamide gel electrophoresis, the radioactivity comigrated with known tRNA markers, suggesting the presence of 3H-aminoacylated tRNA. Aminoacylation of RNA could also be demonstrated by incubating fresh axoplasm with labeled amino acids and a reaction mixture, minus exogenous aminoacyl-tRNA synthetases. These findings indicate the presence in axoplasm of a variety of species of aminoacyl-tRNAs as well as their corresponding synthetase enzymes. In the latter experiment no radioactivity was found associated with the protein fraction. This was also the finding when 3H-aminoacylated tRNA was either injected directly into the axon or incubated with extruded axoplasm. Thus, under the conditions described above, there is no evidence of transfer of amino acids from tRNA to proteins. In other experiments, axoplasm was pooled to a volume of 50 to 100 microliters, homogenized gently, and centrifuged at 150,000 X g for 1 hr. Some of the high speed supernatant was incubated with labeled amino acids and an appropriate reaction mixture, and the remainder was passed through an S-200 Sephacryl column before incubation with the same reaction mixture. There was no incorporation of amino acids into protein in the high speed supernatant fraction. However, in the S-200 purified fraction 3H-labeled Arg, Lys, Tyr, Leu, and Asp were all incorporated into proteins in amounts of 44, 30, 7, 5 and 3.5 times heat-inactivated controls. The reaction is not inhibited by Ca2+ or Ca2+-activated proteases, but appears to be dependent on the presence of tRNA. The addition of amino acids to protein is not protein synthesis since the reactions occurred in a partially purified fraction of the 150,000 X g supernatant, a fraction devoid of ribosomes and free amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of diisopropylphosphorofluoridate‐hydrolyzing enzyme between sheath and axoplasm of squid giant axon

Distribution of diisopropylphosphorofluoridate‐hydrolyzing enzyme between sheath and axoplasm of squid giant axon

Identification of a calcium-binding, brain specific protein in the axoplasm of squid giant axons.

Abstract— An acidic protein has been isolated from the optic lobes of two cephalopods, Sepia officinalis and Loligo vulgaris. The protein has been obtained in pure form by fractionation with ammonium sulphate and chromatography on DEAE‐cellulose and Sephadex G 100. Its apparent molecular weight is 13,000–15,000. Glutamic and aspartic acids account for 35 per cent of the amino acid residues. The protein binds Ca2+ ions with an apparent dissociation constant of 2·5 × 10−5 M at physiological concentrations of KCI. Antibodies have been prepared against the protein purified from Sepia officinalis. By the micro‐complement fixation technique it has been shown that the protein is highly concentrated in the nervous system of cephalopods and that the amount in the axoplasm of squid giant axons is eight to nine‐fold higher than in the optic lobes of the same animal.

The influx of orthophosphate into squid giant axons.

1. The influx of (32)P, applied externally as orthophosphate, into the axoplasm of squid giant axons has been studied.2. An average orthophosphate influx of 20.9 f-mole/cm(2).sec is obtained if the (32)P found in the axoplasm is assumed to be indicative of orthophosphate which has crossed the axolemma.3. The influx does not show very much dependence on external orthophosphate concentration in the range 0.02-0.5 mM.4. The influx is reduced by cyanide, 2,4-dinitrophenol, ouabain and by the absence of external potassium.5. The (32)P appears to arrive in the axoplasm as orthophosphate.6. It is concluded that there is an inward movement of orthophosphate into the axons which is mediated by an active transport process and that this may have some connexion with the active transport of sodium and potassium.

Maintenance of axonal conduction and membrane permeability in presence of extensive phospholipid splitting

The importance of phospholipids for the maintenance of axonal conduction and membrane permeability has been investigated by treating squid giant axons with phospholipases, lysosphospholipids and fatty acids. It was found that phospholipase C, from Cloistridium welchii, induces an extensive splitting of axonal phospholipids, without however blocking conduction or increasing the penetration of a lipid-insoluble compound into the axoplasm of squid giant axons. These findings are in sharp contrast with the results obtained with phospholipase A, electrophoretically separated from Ringhals venom, where a close relationship between phospholipid splitting, block of axonal conduction, and increased penetration has been reported. Phospholipase B from bovine pancreas, and D from cabbage, did not markedly hydrolyze the axonal phospholipids nor did they affect conduction or penetration. Both purified lysolecithin and a mixture of lysophosphatides, prepared by the action of venom phospholipase A on the phospholipids of squid axons, blocked conduction and increased penetration into giant axons. In contrast to phospholipase A, which affects giant axons only if surrounded by adhering small nerve fibers, lysolecithin and the lysophosphatide mixture acted equally well on giant axons with or without adhering small nerve fibers. The other product of phospholipase A action, free fatty acids, was inert on the giant axon. On the basis of these and earlier findings, it is concluded that extensive splitting of axonal phospholipids by phospholipases is compatible with maintenance of normal function unless the hydrolytic products are themselves active. This conclusion would explain the marked differences in action found between phospholipases A and C.