Biochemical and immunocytochemical characterization and distribution of phosphorylated and nonphosphorylated subunits of neurofilaments in squid giant axon and stellate ganglion

Rs, Cohen; Pant, H. C.; House, Shirley B.; Gainer, Harold

doi:10.1523/jneurosci.07-07-02056.1987

Cited by 50 publications

(63 citation statements)

References 39 publications

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“…The gills were also dissected as a non-neural tissue control. Giant axons were carefully dissected from squid that had been killed by decapitation using previously described methods (Cohen et al 1987;Lasek et al 1977;Tytell and Lasek 1984). Each end of the axon was ligated with thread prior to excision and a dissecting microscope with dark-field illumination was used to clean the giant axon of adhering small axons and other extraneous tissues from its surface.…”

Section: Dissection Of Squid Tissues and Extraction Of Their Proteinsmentioning

confidence: 99%

“…Each end of the axon was ligated with thread prior to excision and a dissecting microscope with dark-field illumination was used to clean the giant axon of adhering small axons and other extraneous tissues from its surface. The giant fiber lobe (GFL), containing the cell bodies that give rise to the giant axon was dissected from the stellate ganglion by previously described methods (Cohen et al 1987;Lasek et al 1977;Tytell et al 1990). The optic lobes of the squid were dissected as described elsewhere (Cohen et al 1987;Szaro et al 1991).…”

Section: Dissection Of Squid Tissues and Extraction Of Their Proteinsmentioning

confidence: 99%

“…The incubations of the squid tissues in the above media was for 4 hours at the ambient, seawater temperature (18-21 °C), and was terminated by rapidly washing the axons and GFLs in several volumes of ASW to remove the unincorporated amino acids and the washed tissues were transferred to a glass-glass microhomogenizers (Micromedic, Cleveland) containing extraction buffers for SDS-PAGE and/or immunoprecipitation. The extraction buffer that was routinely used for of SDS-PAGE analysis contained 2% beta-mercaptoethanol, 8 M urea, 1% 9 sodium dodecyl sulfate (SDS) and 100 mM Tris-HCl, pH 7.2 (referred to as BUST in (Cohen et al 1987;Tytell and Lasek 1984). The extraction buffer used in immunoprecipitation experiments was a standard RIPA buffer with a composition of 150 mM NaCl,1% NP-40 ,0.5% sodium deoxycholate,0.1% SDS, and 50 mM Tris HCl, pH 8.0.…”

Section: Incorporation Of Radioactive Amino Acids Into Proteins In Sqmentioning

confidence: 99%

“…The homogenates were centrifuged at 10,000 g for 20 min at 20 °C to remove any insoluble debris and the supernatants were recovered & frozen until further use. The SDS-PAGE and Western blot procedures were done as previously described (Cohen et al 1987). …”

Section: Incorporation Of Radioactive Amino Acids Into Proteins In Sqmentioning

confidence: 99%

“…Our laboratory previously showed that neither NF mRNA (Way et al 1992) nor NF protein (Cohen et al 1987) was present in the squid giant axon but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia.…”

Section: Introductionmentioning

confidence: 95%

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Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins

et al. 2016

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When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein is present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon's neuronal cell bodies, but despite the presence of NF mRNA in the giant axon, no labeled NF protein is detected in the giant axon. This lends support to the Glia-Axon Protein Transfer Hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.

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Section: Dissection Of Squid Tissues and Extraction Of Their Proteinsmentioning

confidence: 99%

Section: Dissection Of Squid Tissues and Extraction Of Their Proteinsmentioning

confidence: 99%

Section: Incorporation Of Radioactive Amino Acids Into Proteins In Sqmentioning

confidence: 99%

Section: Incorporation Of Radioactive Amino Acids Into Proteins In Sqmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 95%

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Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins

et al. 2016

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Developmental increases in expression of neurofilament mRNA selectively in projection neurons of the lamprey CNS

1996

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Neurofilaments of the sea lamprey are unique in being homopolymers of a single subunit (NF-180). Digoxigenin-labeled RNA probes complementary to NF-180 were used to determine the distribution and timing of expression of neurofilament message in the brain and spinal cord of the lamprey. In the brainstem, detection of NF-180 mRNA was restricted to neurons with axons projecting to the spinal cord or the periphery. The majority of brainstem neurons, whose axons project locally, did not express NF-180 within the detection limits of this technique. NF-180-positive neurons included cells with a wide range of axon diameters, suggesting neurofilament mRNA expression was linked to axon length rather than caliber. To further evaluate this hypothesis, expression was studied in animals of different developmental stages between larvae and adults. In younger (shorter) larvae, the large Mauthner and rhombencephalic Müller cells did not express NF-180 mRNA, even though their axons are among the largest caliber in the animal and extend the entire length of the spinal cord. In contrast, many other reticulospinal neurons, whose axons are smaller in diameter than those of the Müller and Mauthner cells, expressed NF-180 message throughout larval development. Furthermore, neurons of the cranial motor nuclei did not express NF-180 until later developmental stages and the extraocular motor neurons did not label until metamorphosis. Therefore, while detectable neurofilament mRNA expression in the lamprey is restricted to neurons with long axons, its expression in this population of neurons appears to be developmentally regulated by factors still not determined. It is postulated that need for NF message is determined by a balance between the volume of axon to be filled and the rate of turnover of NF in that axon.

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Neurofilament spacing, phosphorylation, and axon diameter in regenerating and uninjured lamprey axons

et al. 1996

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It has been postulated that phosphorylation of the carboxy terminus sidearms of neurofilaments (NFs) increases axon diameter through repulsive electrostatic forces that increase sidearm extension and interfilament spacing. To evaluate this hypothesis, the relationships among NF phosphorylation, NF spacing, and axon diameter were examined in uninjured and spinal cord-transected larval sea lampreys (Petromyzon marinus). In untransected animals, axon diameters in the spinal cord varied from 0.5 to 50 microns. Antibodies specific for highly phosphorylated NFs labeled only large axons (> 10 microns), whereas antibodies for lightly phosphorylated NFs labeled medium-sized and small axons more darkly than large axons. For most axons in untransected animals, diameter was inversely related to NF packing density, but the interfilament distances of the largest axons were only 1.5 times those of the smallest axons. In addition, the lightly phosphorylated NFs of the small axons in the dorsal columns were widely spaced, suggesting that phosphorylation of NFs does not rigidly determine their spacing and that NF spacing does not rigidly determine axon diameter. Regenerating neurites of giant reticulospinal axons (GRAs) have diameters only 5-10% of those of their parent axons. If axon caliber is controlled by NF phosphorylation via mutual electrostatic repulsion, then NFs in the slender regenerating neurites should be lightly phosphorylated and densely packed (similar to NFs in uninjured small caliber axons), whereas NFs in the parent GRAs should be highly phosphorylated and loosely packed. However, although linear density of NFs (the number of NFs per micrometer) in these slender regenerating neurites was twice that in their parent axons, they were highly phosphorylated. Following sectioning of these same axons close to the cell body, axon-like neurites regenerated ectopically from dendritic tips. These ectopically regenerating neurites had NF linear densities 2.5 times those of uncut GRAs but were also highly phosphorylated. Thus, in the lamprey, NF phosphorylation may not control axon diameter directly through electrorepulsive charges that increase NF sidearm extension and NF spacing. It is possible that phosphorylation of NFs normally influences axon diameter through indirect mechanisms, such as the slowing of NF transport and the formation of a stationary cytoskeletal lattice, as has been proposed by others. Such a mechanism could be overridden during regeneration, when a more compact, phosphorylated NF backbone might add mechanical stiffness that promotes the advance of the neurite tip within a restricted central nervous system environment.

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Biochemical and immunocytochemical characterization and distribution of phosphorylated and nonphosphorylated subunits of neurofilaments in squid giant axon and stellate ganglion

Cited by 50 publications

References 39 publications

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins

Developmental increases in expression of neurofilament mRNA selectively in projection neurons of the lamprey CNS

Neurofilament spacing, phosphorylation, and axon diameter in regenerating and uninjured lamprey axons

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