In vitro polymorphism and phase transitions of the neurofilamentous network isolated from the giant axon of the squid (Loligo pealei L.)

1988 ◽  
Vol 252 (2) ◽  
Author(s):  
J. Metuzals ◽  
H. Pant ◽  
H. Gainer ◽  
P.A.M. Eagles ◽  
N.S. White ◽  
...  
Keyword(s):  
Phase Transitions ◽  
Giant Axon ◽  
Neurofilamentous Network ◽  
Loligo Pealei

scholarly journals Stable complexes of axoplasmic vesicles and microtubules: protein composition and ATPase activity.

1986 ◽  
Vol 103 (3) ◽  
pp. 957-968 ◽  
Author(s):  
M M Pratt
Keyword(s):  
Atpase Activity ◽  
Protein Composition ◽  
Giant Axon ◽  
High Molecular Mass ◽  
Macromolecular Complexes ◽  
Distinct Subset ◽  
Associated Proteins ◽  
Fast Transport ◽  
Loligo Pealei

Fast transport of axonal vesicles and organelles is a microtubule-associated movement (Griffin, J. W., K. E. Fahnestock, L. Price, and P. N. Hoffman, 1983, J. Neuroscience, 3:557-566; Schnapp, B. J., R. D. Vale, M. P. Sheetz, and T. S. Reese, 1984, Cell, 40:455-462; Allen, R. D., D. G. Weiss, J. H. Hayden, D. T. Brown, H. Fujiwake, and M. Simpson, 1985, J. Cell Biol., 100:1736-1752). Proteins that mediate the interactions of axoplasmic vesicles and microtubules were studied using stable complexes of microtubules and vesicles (MtVC). These complexes formed spontaneously in vitro when taxol-stabilized microtubules were mixed with sonically disrupted axoplasm from the giant axon of the squid Loligo pealei. The isolated MtVCs contain a distinct subset of axoplasmic proteins, and are composed primarily of microtubules and attached membranous vesicles. The MtVC also contains nonmitochondrial ATPase activity. The binding of one high molecular mass polypeptide to the complex is significantly enhanced by ATP or adenyl imidodiphosphate. All of the axoplasmic proteins and ATPase activity that bind to microtubules are found in macromolecular complexes and appear to be vesicle-associated. These data allow the identification of several vesicle-associated proteins of the squid giant axon and suggest that one or more of these polypeptides mediates vesicle binding to microtubules.

Neurofilaments and Paired Helical Filaments

1985 ◽  
Vol 43 ◽  
pp. 740-743
Author(s):  
J. Metuzals
Keyword(s):  
Animal Model ◽  
Posttranslational Modifications ◽  
Posttranslational Modification ◽  
Giant Axon ◽  
Paired Helical Filaments ◽  
Squid Giant Axon ◽  
In Vitro Experiments ◽  
Thin Sections ◽  
Structural Relationships

It has been demonstrated that the neurofibrillary tangles in biopsies of Alzheimer patients, composed of typical paired helical filaments (PHF), consist also of typical neurofilaments (NF) and 15nm wide filaments. Close structural relationships, and even continuity between NF and PHF, have been observed. In this paper, such relationships are investigated from the standpoint that the PHF are formed through posttranslational modifications of NF. To investigate the validity of the posttranslational modification hypothesis of PHF formation, we have identified in thin sections from frontal lobe biopsies of Alzheimer patients all existing conformations of NF and PHF and ordered these conformations in a hypothetical sequence. However, only experiments with animal model preparations will prove or disprove the validity of the interpretations of static structural observations made on patients. For this purpose, the results of in vitro experiments with the squid giant axon preparations are compared with those obtained from human patients. This approach is essential in discovering etiological factors of Alzheimer's disease and its early diagnosis.

Electric current generated by squid giant axon sodium pump: external K and internal ADP effects

1978 ◽  
Vol 235 (1) ◽  
pp. C63-C68 ◽  
Author(s):  
R. F. Abercrombie ◽  
P. de Weer
Keyword(s):  
Sodium Pump ◽  
Giant Axon ◽  
Pump Current ◽  
Membrane Resistance ◽  
Extracellular Potassium ◽  
Sodium Efflux ◽  
Steroid Sensitive ◽  
External Potassium ◽  
Loligo Pealei ◽  
External K

The operation of the sodium pump of giant axons of the squid, Loligo pealei, has been studied simultaneously in two independent ways: 1) by measuring sodium efflux with 22Na, and 2) by calculating the transmembrane current generated by the pump from measurements of membrane resistance and digitalis-sensitive membrane potential. In normal, untreated axons, the effect of increasing the external potassium concentration on both sodium efflux and pump current is similar, which suggests that Na:K pump stoichiometry remains relatively constant in the range of 0-20 mM external K. The data are compatible with a 3:2 Na:K ratio. In axons whose intracellular ADP level has been elevated by injection of L-arginine, a large, electrically silent, cardiotonic steroid-sensitive sodium efflux takes place in the absence of external potassium; this suggests that pump-mediated Na:Na exchange is 1:1 or electroneutral. Finally, elevation of external potassium levels causes the appearance, in high-ADP axons, of electrogenic pumping, with little effect on sodium efflux; hence, in contrast to what is seen in normal (low-ADP) axons, the charge translocated, per sodium ion extruded, increases sharply with increasing extracellular potassium levels.

scholarly journals Subaxolemmal cytoskeleton in squid giant axon. II. Morphological identification of microtubule- and microfilament-associated domains of axolemma.

1986 ◽  
Vol 102 (5) ◽  
pp. 1710-1725 ◽  
Author(s):  
S Tsukita ◽  
S Tsukita ◽  
T Kobayashi ◽  
G Matsumoto
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Preceding Paper ◽  
Microscopic Analysis ◽  
Giant Axon ◽  
Squid Giant Axon ◽  
Molecular Organization ◽  
Fixation Method ◽  
Morphological Identification

In the preceding paper (Kobayashi, T., S. Tsukita, S. Tsukita, Y. Yamamoto, and G. Matsumoto, 1986, J. Cell Biol., 102:1710-1725), we demonstrated biochemically that the subaxolemmal cytoskeleton of the squid giant axon was highly specialized and mainly composed of tubulin, actin, axolinin, and a 255-kD protein. In this paper, we analyzed morphologically the molecular organization of the subaxolemmal cytoskeleton in situ. For thin section electron microscopy, the subaxolemmal cytoskeleton was chemically fixed by the intraaxonal perfusion of the fixative containing tannic acid. With this fixation method, the ultrastructural integrity was well preserved. For freeze-etch replica electron microscopy, the intraaxonally perfused axon was opened and rapidly frozen by touching its inner surface against a cooled copper block (4 degrees K), thus permitting the direct stereoscopic observation of the cytoplasmic surface of the axolemma. Using these techniques, it became clear that the major constituents of the subaxolemmal cytoskeleton were microfilaments and microtubules. The microfilaments were observed to be associated with the axolemma through a specialized meshwork of thin strands, forming spot-like clusters just beneath the axolemma. These filaments were decorated with heavy meromyosin showing a characteristic arrowhead appearance. The microtubules were seen to run parallel to the axolemma and embedded in the fine three-dimensional meshwork of thin strands. In vitro observations of the aggregates of axolinin and immunoelectron microscopic analysis showed that this fine meshwork around microtubules mainly consisted of axolinin. Some microtubules grazed along the axolemma and associated laterally with it through slender strands. Therefore, we were led to conclude that the axolemma of the squid giant axon was specialized into two domains (microtubule- and microfilament-associated domains) by its underlying cytoskeletons.

scholarly journals The Effects of External Potassium and Long Duration Voltage Conditioning on the Amplitude of Sodium Currents in the Giant Axon of the Squid, Loligo pealei

1969 ◽  
Vol 54 (5) ◽  
pp. 589-606 ◽  
Author(s):  
William J. Adelman ◽  
Yoram Palti
Keyword(s):  
Steady State ◽  
Time Constant ◽  
State Level ◽  
Giant Axon ◽  
Sodium Concentration ◽  
Quasi Steady State ◽  
Steady State Level ◽  
Long Duration ◽  
Time Courses ◽  
Loligo Pealei

Giant axons were voltage-clamped in solutions of constant sodium concentration (230 mM) and variable potassium concentrations (from 0 to 210 mM). The values of the peak initial transient current, Ip, were measured as a function of conditioning prepulse duration over the range from less than 1 msec to over 3 min. Prepulse amplitudes were varied from Em = -20 mv to Em = -160 mv. The attenuation of the Ip values in high [Ko] was found to vary as a function of time when long duration conditioning potentials were applied. In both high and low [Ko], Ip values which had reached a quasi-steady—state level within a few milliseconds following a few milliseconds of hyperpolarization were found to increase following longer hyperpolarization. A second plateau was reached with a time constant of about 100–500 msec and a third with a time constant in the range of 30 to 200 sec. The intermediate quasi-steady—state level was absent in K-free ASW solutions. Sodium inactivation curves, normalized to Ipmax values obtained at either the first or second plateaus, were significantly different in different [Ko]. The inactivation curves, however, tended to superpose after about 1 min of hyperpolarizing conditioning. The time courses and magnitudes of the intermediate and very slow sodium conductance restorations induced by long hyperpolarizing pulses are in agreement with those predicted from the calculated rates and magnitudes of [K+] depletion in the space between the axolemma and the Schwann layer.

scholarly journals The Influence of External Potassium on the Inactivation of Sodium Currents in the Giant Axon of the Squid, Loligo pealei

1969 ◽  
Vol 53 (6) ◽  
pp. 685-703 ◽  
Author(s):  
William J. Adelman ◽  
Yoram Palti
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Membrane Conductance ◽  
Absolute Magnitude ◽  
Giant Axon ◽  
Membrane Potentials ◽  
Initial Transient ◽  
External Potassium ◽  
Loligo Pealei ◽  
External K

Isolated giant axons were voltage-clamped in seawater solutions having constant sodium concentrations of 230 mM and variable potassium concentrations of from zero to 210 mM. The inactivation of the initial transient membrane current normally carried by Na+ was studied by measuring the Hodgkin-Huxley h parameter as a function of time. It was found that h reaches a steady-state value within 30 msec in all solutions. The values of h∞, τh, αh,and ßh as functions of membrane potential were determined for various [Ko]. The steady-state values of the h parameter were found to be inversely related, while the time constant, τh, was directly related to external K+ concentration. While the absolute magnitude as well as the slopes of the h∞ vs. membrane potential curves were altered by varying external K+, only the magnitude and not the shape of the corresponding τh curves was altered. Values of the two rate constants, αh and ßh, were calculated from h∞ and τh values. αh is inversely related to [Ko] while ßh is directly related to [Ko] for hyperpolarizing membrane potentials and is independent of [Ko] for depolarizing membrane potentials. Hodgkin-Huxley equations relating αh and ßh to Em were rewritten so as to account for the observed effects of [Ko]. It is concluded that external potassium ions have an inactivating effect on the initial transient membrane conductance which cannot be explained solely on the basis of potassium membrane depolarization.

Temperature and impulse velocity in giant axon of squid Loligo pealei

1975 ◽  
Vol 229 (5) ◽  
pp. 1249-1253 ◽  
Author(s):  
DM Easton ◽  
CE Swenberg
Keyword(s):  
Experimental Data ◽  
Fourth Order ◽  
Ionic Current ◽  
Membrane Conductance ◽  
Giant Axon ◽  
Velocity Versus ◽  
Stability Function ◽  
The Difference ◽  
Q10 Values ◽  
Loligo Pealei

Impulse propagation velocity as a function of temperature in the range 5--20degreesC was obtained by external recording from the giant axon of Loligo pealei. The stellar nerve was set into a chamber allowing continuous superfusion, temperature control, and double recording of the impulse. Velocity was calculated from the interval between the spike peaks. The Q10 of velocity was about 1.8. At all temperatures, the velocity increased with time so that only data obtained during the 1st h or 2 could be generally considered to be comparable. Impulse block occurred below --3.4degreesC, in contrast to the giant axon of L. vulgaris, which blocks at about 0degreeC, but at the higher range of temperatures, the velocity in the L. pealei axons was not as well sustained as in those of L. vulgaris. The expected impulse velocity was calculated from Huxley's stability function f(beta) by approximating that function to a fourth-order polynominal and by substituting into it suitable ratios of available Q10 values relating to membrane conductance, ionic current, capacitance, and axoplasmic resistance. The calculation provided an improved fit to published experimental data on L. vulgaris. The difference in slope of the log velocity versus temperature plots, between the presumably warm acclimatized L. vulgaris and the cold-acclimatized L. pealei, was present in both experimental and calculated curves.

scholarly journals Bidirectional transport of fluorescently labeled vesicles introduced into extruded axoplasm of squid Loligo pealei.

1984 ◽  
Vol 99 (2) ◽  
pp. 445-452 ◽  
Author(s):  
S P Gilbert ◽  
R D Sloboda
Keyword(s):  
Axonal Transport ◽  
Giant Axon ◽  
Epifluorescence Microscopy ◽  
Differential Interference Contrast Microscopy ◽  
Fast Axonal Transport ◽  
Bidirectional Transport ◽  
Interference Contrast Microscopy ◽  
The Mean ◽  
Loligo Pealei ◽  
Vesicle Movement

A reconstituted model was devised to study the mechanisms of fast axonal transport in the squid Loligo pealei. Axonal vesicles were isolated from axoplasm of the giant axon and labeled with rhodamine-conjugated octadecanol, a membrane-specific fluorescent probe. The labeled vesicles were then injected into a fresh preparation of extruded axoplasm in which endogenous vesicle transport was occurring normally. The movement of the fluorescent, exogenous vesicles was observed by epifluorescence microscopy for as long as 5 min without significant photobleaching, and the transport of endogenous, nonfluorescent vesicles was monitored by video-enhanced differential interference-contrast microscopy. The transport of fluorescent, exogenous vesicles was shown to be bidirectional and ATP-dependent and occurred at a mean rate of 6.98 +/- 4.11 micron/s (mean +/- standard deviation, n = 41). In comparison, the mean rate of transport of nonfluorescent, endogenous vesicles in control axoplasm treated with vesicle buffer alone was 4.76 +/- 1.60 micron/s (n = 64). These rates are slightly higher than the mean rate of endogenous vesicle movement in extruded axoplasm (3.56 +/- 1.05 micron/s, n = 40) not subject to vesicles or vesicle buffer. Not all vesicles and organelles, exogenous or endogenous, were observed to move. In experiments in which proteins of the surface of the fluorescent vesicles were digested with trypsin before injection, no movement of the fluorescent vesicles was observed, although the transport of endogenous vesicles and organelles appeared to proceed normally. The results summarized above indicate that isolated vesicles, incorporated into axoplasm, move with the characteristics of fast axonal transport. Because the vesicles are fluorescent, they can be readily distinguished from nonfluorescent, endogenous vesicles. Moreover, this system permits vesicle characteristics to be experimentally manipulated, and therefore may prove valuable for the elucidation of the mechanisms of fast axonal transport.

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