Fast axonal transport in squid giant axon

Science ◽  
1982 ◽  
Vol 218 (4577) ◽  
pp. 1127-1129 ◽  
Author(s):  
R. Allen ◽  
J Metuzals ◽  
I Tasaki ◽  
S. Brady ◽  
S. Gilbert
Keyword(s):  
Axonal Transport ◽  
Giant Axon ◽  
Squid Giant Axon ◽  
Fast Axonal Transport

Fast axonal transport in extruded axoplasm from squid giant axon

Science ◽  
1982 ◽  
Vol 218 (4577) ◽  
pp. 1129-1131 ◽  
Author(s):  
S. Brady ◽  
R. Lasek ◽  
R. Allen
Keyword(s):  
Axonal Transport ◽  
Giant Axon ◽  
Squid Giant Axon ◽  
Fast Axonal Transport

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.

scholarly journals Monomer-polymer equilibria in the axon: direct measurement of tubulin and actin as polymer and monomer in axoplasm.

1984 ◽  
Vol 98 (6) ◽  
pp. 2064-2076 ◽  
Author(s):  
J R Morris ◽  
R J Lasek
Keyword(s):  
Axonal Transport ◽  
Direct Measurement ◽  
Giant Axon ◽  
Squid Giant Axon ◽  
The Other ◽  
Significant Fraction ◽  
Dissociation Reaction ◽  
Basic Principles

The monomer-polymer equilibria for tubulin and actin were analyzed for the cytoskeleton of the squid giant axon. Two methods were evaluated for measuring the concentrations of monomer, soluble (equilibrium) polymer, and stable polymer in extruded axoplasm. One method, the Kinetic Equilibration Paradigm ( KEP ), employs the basic principles of diffusion to distinguish freely diffusible monomer from proteins that are present in the form of polymer. The other method is pharmacological and employs either taxol or phalloidin to stabilize the microtubules and microfilaments, respectively. The results of the two methods agree and demonstrate that 22-36% of the tubulin and 41-47% of the actin are monomeric. The in vivo concentration of monomeric actin and tubulin were two to three times higher than the concentration required to polymerize these proteins in vitro, suggesting that assembly of these proteins is regulated by additional mechanisms in the axon. A significant fraction of the polymerized actin and tubulin in the axoplasm was stable microtubules and microfilaments, which suggests that the dissociation reaction is blocked at both ends of these polymers. These results are discussed in relationship to the axonal transport of the cytoskeleton and with regard to the ability of axons to change their shape in response to environmental stimuli.

scholarly journals Transport of ER vesicles on actin filaments in neurons by myosin V

1998 ◽  
Vol 111 (21) ◽  
pp. 3221-3234 ◽  
Author(s):  
J.S. Tabb ◽  
B.J. Molyneaux ◽  
D.L. Cohen ◽  
S.A. Kuznetsov ◽  
G.M. Langford
Keyword(s):  
Axonal Transport ◽  
Actin Filaments ◽  
Giant Axon ◽  
Vesicle Transport ◽  
Amino Acid Sequences ◽  
Dependent Manner ◽  
Tail Domain ◽  
Myosin V ◽  
Fast Axonal Transport

Axoplasmic organelles in the giant axon of the squid have been shown to move on both actin filaments and microtubules and to switch between actin filaments and microtubules during fast axonal transport. The objectives of this investigation were to identify the specific classes of axoplasmic organelles that move on actin filaments and the myosin motors involved. We developed a procedure to isolate endoplasmic reticulum (ER) from extruded axoplasm and to reconstitute its movement in vitro. The isolated ER vesicles moved on exogenous actin filaments adsorbed to coverslips in an ATP-dependent manner without the addition of soluble factors. Therefore myosin was tightly bound and not extracted during isolation. These vesicles were identified as smooth ER by use of an antibody to an ER-resident protein, ERcalcistorin/protein disulfide isomerase (EcaSt/PDI). Furthermore, an antibody to squid myosin V was used in immunogold EM studies to show that myosin V localized to these vesicles. The antibody was generated to a squid brain myosin (p196) that was classified as myosin V based on comparisons of amino acid sequences of tryptic peptides of this myosin with those of other known members of the myosin V family. Dual labeling with the squid myosin V antibody and a kinesin heavy chain antibody showed that the two motors colocalized on the same vesicles. Finally, antibody inhibition experiments were performed with two myosin V-specific antibodies to show that myosin V motor activity is required for transport of vesicles on actin filaments in axoplasm. One antibody was made to a peptide in the globular tail domain and the other to the globular head fragment of myosin V. Both antibodies inhibited vesicle transport on actin filaments by greater than 90% compared to controls. These studies provide the first direct evidence that ER vesicles are transported on actin filaments by myosin V. These data confirm the role of actin filaments in fast axonal transport and provide support for the dual filament model of vesicle transport.

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.

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