Analysis of the mechanism of fast axonal transport by intracellular injection of potentially inhibitory macromolecules: evidence for a possible role of actin filaments.

Goldberg, Daniel J.; Harris, David A.; Lubit, Beverly W.; Schwartz, James H.

doi:10.1073/pnas.77.12.7448

Cited by 47 publications

(28 citation statements)

References 39 publications

(29 reference statements)

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“…It had been shown that injection of DNase I into the cell body of the giant cerebral neuron (GCN) ofAplysia reduces the fast transport of serotonin (8).…”

Section: Resultsmentioning

confidence: 99%

“…Recently, Isenberg et al (7) and Goldberg et al (8) reported the use of "direct access" preparations to analyze the mechanism offast transport. Actin-specific agents were microinjected into the cell bodies of giant invertebrate neurons, and some were found to inhibit transport.…”

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confidence: 99%

“…Then, after the appropriate interval, the axon of R2 in the trypsinized segment of the right connective was impaled and 0.5 nl of the appropriate solution was injected into the axon. A precise volume could be injected because the injection electrode was calibrated before use (8). Preparations in which the resting potential recorded in the cell body was less negative than -38 mV or the resting potential recorded in the axon was less negative than -45 mV were discarded.…”

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confidence: 99%

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Microinjection into an identified axon to study the mechanism of fast axonal transport.

Goldberg¹

1982

Proc. Natl. Acad. Sci. U.S.A.

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Microinjection into an axon of an identified invertebrate neuron is shown to be a useful technique for analyzing the mechanism of fast axonal transport. It permits direct assessment ofthe effect ofagents that cannot permeate the plasma membrane on the translocation of material in the axon. The actin filament depolymerizer DNase I, when injected into the axon of the A3plysia neuron R2, caused a local block df fast transport of [H]glycoprotein. Two agents that should interfere with the functioning of actin filaments without causing extensive depolymerization, the N-ethylmaleimide-modified nuclease SI fragment of myosin (injected) and dihydrocytochalasin B (applied externally), had no effect. Together these results suggest that actin plays a structural role in the axonal cytoskeleton rather than a role in transport force generation, the effect of DNase I being mediated by structural disordering of the axoplasm. Experiments were also done with 'inhibitors of dynein, the microtubule-associated ATPase. erythro-9-[3-(2-Hydroxynonyl)]adenine blocked transport but vanadate was ineffective.The study of cell motility has been aided greatly by the use of preparations in which the experimenter has direct access to the cytoplasm.and, thus, the motile machinery. There are three general types of such preparations: (i) isolated cytoplasm, as used in the study ofameboid movement (1); (ii) cells with plasma membranes that have been permeabilized by treatment with detergent, as used to study mitosis (2); and (iii) large cells into which materials are introduced by injection through an impaling micropipette, such as amebae (3) and egg cells (4). One advantage of these preparations is that they permit analysis of the mechanism of motility by using agents that interact specifically with suspected constituents of the motile apparatus but do not readily permeate an intact plasma membrane. 'Evaluation ofthe role of actin in a motile process should especially benefit from accessibility to the cytoplasm, because the small repertory of actin-specific agents has lately been augmented by the introduction of several nonpermeant agents.Fast axonal transport is a motile process.in neurons in which organelles, such as transmitter storage vesicles, travel from their somal birthplace to areas ofutilization in the axon and synapses and lysosomes laden with cellular debris make the return trip (5). It may be a manifestation of the saltatory organelle movement that is seen in several other types of cells (6). The properties of fast transport are well described, but the molecular mechanism is not understood, although morphological and pharmacological data suggest a role for microtubules (5).Recently, Isenberg et al. (7) and Goldberg et al (8) reported the use of "direct access" preparations to analyze the mechanism offast transport. Actin-specific agents were microinjected into the cell bodies of giant invertebrate neurons, and some were found to inhibit transport. Injection into the cell body does not, however, allow one to distinguish an act...

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“…It had been shown that injection of DNase I into the cell body of the giant cerebral neuron (GCN) ofAplysia reduces the fast transport of serotonin (8).…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Microinjection into an identified axon to study the mechanism of fast axonal transport.

Goldberg¹

1982

Proc. Natl. Acad. Sci. U.S.A.

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“…The structural integrity of the MF that are associated with the microtubule bundles appears to be required for normal vesicle transport in the axon. For example, if axonal MF are depolymerized with DNase I or gelsolin, anterograde and retrograde vesicle transport is inhibited (Isenberg et al, 1980;Goldberg et al, 1980;Brady et al, 1984). Apparently, DNase I and gelsolin inhibit vesicle transport by disrupting the structural organization of the axoplasm, notably, the microtubule domains Nemhauser and Goldberg, 1985).…”

Section: Mf-microtubule Interactions In the Axonal Cytoskeletonmentioning

confidence: 99%

Two classes of actin microfilaments are associated with the inner cytoskeleton of axons.

Fath

Lasek

1988

The Journal of Cell Biology

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Abstract. The distribution and length of actin microfilaments (MF) was determined in axoplasm extruded from the giant axons of the squid (Loligo pealeii). Extruded axoplasm that was separated from the axonal cortex contains ,092% of the total axonal actin, and 60% of this actin is polymerized (Morris, J., and R. . J. Cell Biol. 98:2064-2076.Localization of MF with rhodamine-phalloidin indicated that the MF were organized in fine columns oriented longitudinally within the axoplasm. In the electron microscope, MF were surrounded by a dense matrix and they were associated with the microtubule domains of the axoplasm. The surrounding matrix tended to obscure the MF which may explain why MF have rarely been recognized before in the inner regions of the axon. The axoplasmic MF are relatively short (number average length of 0.55 gm). Length measurements of MF prepared either in the presence or absence of the actin-filament stabilizing drug phalloidin indicate that axoplasm contains two populations of MF: stable MF (number average length of 0.79 lxm) and metastable MF (number average length of 0.41 ltrn). Although individual axonal MF are much shorter than axonal microtubules, the combined length of the total MF is twice that of the total microtubules. Apparently, these numerous short MF have an important structural role in the architecture of the inner axonal cytoskeleton.TIN microfilaments (MF) 1 are essential components in acto-myosin-mediated motility and in the architecture of many kinds of cells. In axons, ultrastructural studies have suggested that MF are abundant in the cortical region subjacent to the axonal plasma membrane and that they are few in number in the central regions that constitute most of the axonal volume (Metuzals and Tasaki, 1978;Hirokawa, 1982;Hodge and Adelman, 1983;Letourneau, 1983;Tsukita et al., 1986). Furthermore, fluorescent probes for actin stain the axonal cortex much more intensely than the central axonal regions (Spooner and Holladay, 1981;Letourneau, 1983;Shaw et al., 1981;Hirokawa, 1982). These observations have led to the widely accepted view that axonal MF are primarily cortical and that few, if any, are located in the central regions of the axon (for review see Pachter et al., 1984).This idea that MF are negligible components of the central axonal cytoskeleton is not consistent with the results of physiological studies. For example, if MF in the central axon are disrupted, fast vesicle transport is impaired . Furthermore, autoradiographic analyses of radiolabeled slowly transported cytoskeletal elements indicate that MF and other components of slow component b are principally located in the central (noncortical) regions of axons 1. Abbreviations used in this paper: ME microfilaments; S-l, myosin subfragment. (Heriot et al., 1985). These studies indicate that actin MF have an important role in the dynamic architecture of the central axon.Quantitative biochemical analyses also indicate that MF are abundant in the central axonal cytoskeleton (Morris and Lasek, 1984). These studies s...

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“…Actin is a major cytoskeletal protein in neuronal cells (for review, see Puszkin and Schook, 1979;Bray and Gilbert, 1981) and in its filamentous form has been reported to be involved in different motile events such as growth cone motility (Bray, 1973;Wessels et al, 1978), axonal transport (Isenberg et al, 1980b;Goldberg et al, 1980), and neurotransmitter release (Pumplin and McClure, 1974). Though actin is present in nerve cell bodies as well as in axons and dendrites as shown by immunofluorescence microscopy (Isenberg et al, 1977;Isenberg and Small, 1978;Marchisio et al, 1978;Jockusch et al, 1979;Kuczmarski and Rosenbaum, 1979;Shaw et al, 1981;Jockusch and Jockusch, 1981), little is known about the organization of actin filaments in neurites and synaptic regions.…”

Section: Introductionmentioning

confidence: 99%

Actin filament capping protein from bovine brain.

Kilimann¹,

Isenberg²

1982

The EMBO Journal

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An actin filament capping protein has been purified from bovine brain. The protein has a native mol. wt. of 63 kilodaltons (kd) with subunits of 36 kd and 31 kd and is globular in shape. It nucleates actin polymerization, inhibits filament elongation and filament interactions, and decreases the steady state viscosity of F‐actin in substoichiometric amounts (molar ration 1:1000). In addition, the protein increases the critical concentration for actin polymerization. Neither Ca2+ nor calmodulin affects it action. All these effects can be explained by the binding of the protein to the ‘barbed’ end of actin filaments leading to a blockade of actin monomer addition at the preferred growing end. This is directly demonstrated by electron microscopy. Concerning the polypeptide composition, Ca2+‐independence, mode, and stoichiometry of actin interaction, the protein is similar to the capping protein, previously isolated from Acanthamoeba.

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Analysis of the mechanism of fast axonal transport by intracellular injection of potentially inhibitory macromolecules: evidence for a possible role of actin filaments.

Cited by 47 publications

References 39 publications

Microinjection into an identified axon to study the mechanism of fast axonal transport.

Microinjection into an identified axon to study the mechanism of fast axonal transport.

Two classes of actin microfilaments are associated with the inner cytoskeleton of axons.

Actin filament capping protein from bovine brain.

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