Posttranslational modification of neurofilament polypeptides in rabbit retina

Three polypeptides that compose neurofilaments, designated H, M, and L, are synthesized in the cell bodies of neurons and subsequently conveyed down their axons by the process of slow axonal transport. The axonal form of H, which is a component of the cross bridges between the neurofilaments, is antigenically different from the form in the cell bodies and dendrites. To understand how this special form of H is directed to the axon, and more generally how intracellular differentiation is established and maintained by the selective delivery of different molecular species to different compartments of a cell, we have studied the events that occur immediately after the synthesis of the three neurofilament polypeptides in the retinas of rabbits. We observed that H and M are synthesized in the retina as precursor polypeptides, EH and EM, that migrate markedly faster on SDS polyacrylamide gels than their mature axonal forms. The maturation of these precursors requires more than one day and appears to involve their phosphorylation. Only the electrophoretically mature forms appear in the axons of the retinal ganglion cells in the optic nerve. We consider the following interpretation of these observations. Shortly after they are translated in the cell body, the neurofilament polypeptides become phosphorylated at multiple sites. However, only after they have moved a distance of several hundred micrometers down the axon, H and M are phosphorylated at additional sites, causing their conformation or binding properties to change. This change, which is reflected in the reduction of their electrophoretic mobility and the appearance of new antigenic determinants, may function to alter the H-mediated crossbridges and produces the morphological and structural properties of the neurofilament lattice that is characteristic of axons.     

Rhythmic chloroplast migration in the green alga Ulva: dissection of movement mechanism by differential inhibitor effects

Rhodopseudomonas viridis thylakoid membrane polypeptides were characterised by SDS gels, 2 D gels and surface-specific iodination. Four polypeptides with apparent molecular weights of 38 000, 33 000, 27 000, and 24 000 (reaction centre) and three low molecular weight polypeptides 11 000, 8000 and 6000 (probably light harvesting polypeptides) were identified. Antibodies were produced against the polypeptides eluted from SDS gels and tested for specificity by an immunoblotting assay. The antibodies were bound to the membranes and viewed by electron microscopy using a modification of the ferritin labelling technique. It is suggested that antigenic determinants for the 38 000, 33 000, and 27 000 reaction centre polypeptides and the 11 000 and 8000 low molecular weight polypeptides are present on the cytoplasmic membrane surface. The 33 000, 27 000, 11 000 and 6000 polypeptides appear to have surface-located residues which can be iodinated. The photosynthetic membrane of Rps. viridis appears to be a highly asymmetrical membrane.     

Compositional analysis of growing axons from rat sympathetic neurons

We describe culture systems for neurons of an adrenergic autonomic ganglion which: (a) permit cultivation of neurons without supporting cells, (b) permit separate harvest of somal and axonal material, and (c) permit direct access to the neuronal surface. The antimetabolites used to suppress supporting cell growth did not have demonstrable effects on neuronal polypeptide synthesis. Rapid neurite outgrowth, which characterized these cultures, was prevented by colchicine or cycloheximide and resumed promptly after their withdrawal. Axons separated from cell bodies showed no incorporation of label from leucine or fucose, but did exhibit incorporation of glucosamine. The major polypeptides present in this neuron, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, are described. No major differences in polypeptide content were observed when soma and axons were compared. Likewise, there were no differences detected in polypeptides synthesized by neurons in suspension or neurons actively extending processes. Analysis of the polypeptides within the neurites after labeling with amino acids indicated transport at a number of different rates; certain of these polypeptides corresponded in size and transport characteristics to polypeptides observed in the rabbit optic nerve after labeling of retinal ganglion cells. Tubulin and actin have been definitively identified in this cell type (18); we found proteins similar in size and proportionate amounts to be among the rapidly transported soluble polypeptides. The prominent polypeptides observed after several methods of surface labeling are described.     

Nerve-specific enolase and creatine phosphokinase in axonal transport: soluble proteins and the axoplasmic matrix

The axonal transport of two soluble enzymes of intermediary metabolism was evaluated: the nerve-specific form of the glycolytic enzyme enolase (NSE) and the brain isozyme of creatine phosphokinase (CPK). Previously, little was known about the intracellular movements of the soluble proteins of the cell. Although the soluble enzymes of glycolysis and other pathways of intermediary metabolism have been thought to be freely diffusing in the cytosol, many are required in the axonal extremities of the neuron and must be transported to the sites of utilization. Comigration of purified enzymes with radioactive polypeptides associated with specific rate components of axonal transport in two-dimensional gel electrophoresis indicates that both NSE and CPK move in the axon solely as part of the group of proteins known as slow component b (SCb) at a rate of 2 mm/day. Peptide mapping following limited proteolysis confirmed identification of NSE and CPK in SCb. Materials associated with SCb have been shown to move coherently along the axon and to behave as a discrete cellular structure, the axoplasmic matrix. Association of two soluble enzymes, NSE and CPK, with the SCb complex of proteins requires a reevaluation of the assumption that these and other soluble proteins of the axon are freely diffusible.     

Actin Depolymerizing Factor Is a Component of Slow Axonal Transport

We examined the low molecular weight proteins transported with actin in the chicken sciatic nerve after injection of [35S]methionine into the lumbar spinal cord. A prominent component of slow axonal transport with apparent molecular mass 19 kDa comigrated on two-dimensional gels with chicken actin depolymerizing factor (ADF), previously shown to be a major actin-binding protein in brain. There was comparatively little radioactivity associated with the actin monomer sequestering proteins, profilin or cofilin, and examination of the rapid component of axonal transport failed to reveal appreciable quantities of actin, ADF, profilin, or cofilin. These results show that both actin and ADF are carried by slow axonal transport and raise the possibility that actin travels within the axon in an unpolymerized form in a complex with ADF.     

The purification and polypeptide composition of avian infectious bronchitis virus.

Purification of egg-grown infectious bronchitis virus (IBV) by sucrose density gradient centrifugation alone, or sucrose density gradient centrifugation plus pH 8.0 treatment, concanavalin A precipitation or metrizamide density gradient centrifugation, failed to produce any differences in the virus polypeptide pattern following polyacrylamide gel electrophoresis in the presence of SDS(SDS-PAGE). SDS-PAGE of purified IBV on 7.5% acrylamide gels separated 16 polypeptides which were detectable by staining with Coomassie blue or measurement of radioactivity following electrophoresis of (3H)-leucine labelled IBV. The molecular weights of the polypeptides were within the range 15,000-135,000. The polypeptides of egg and chick kidney (CK) cell-grown IBV were identical in both size and number but quantitative differences were detected. In particular the relative proportion of the major 52,000 molecular weight polypeptide was greatly reduced in IBV grown in CK cells. SDS-PAGE of purified IBV and staining with Schiff's reagent to detect carbohydrate revealed four.bands with molecular weights of 128,000, 86,000, 67,500 and 37,000. The 128,000 band did not correspond to any of the detected polypeptides. Use of 5% acrylamide gels for SDS-PAGE of IBV failed to resolve all the minor polypeptides and only seven bands were detected.     

Slow components of axonal transport: two cytoskeletal networks

We have identified two slowly moving groups of axonally transported proteins in guinea pig retinal ganglion cell axons (4). The slowest group of proteins, designated slow component a (SCa), has a transport rate of 0.25 mm/d and consists of tubulin and neurofilament protein. The other slowly transported group of proteins, designated slow components b (SCb), has a transport rate of 2-3 mm/d and consists of many polypeptides, one of which is actin (4). Our analyses of the transport kinetics of the individual polypeptides of SCa and SCb indicate that (a) the polypeptides of SCa are transported coherently in the optic axons, (b) the polypeptides of SCb are also transported coherently but completely separately from the SCa polypeptides, and (c) the polypeptides of SCa differ completely from those comprising SCb. We relate these results to our general hypothesis that slow axonal transport represents the movements of structural complexes of proteins. Furthermore, it is proposed that SCa corresponds to the microtubule-neurofilament network, and that SCb represents the transport of the microfilament network together with the proteins complexed with microfilaments.     

Actin-Based Gelation of Cytoplasmic Extracts from Brain Tissue

Incubation of rat brain cytoplasmic extracts under the conditions described in this paper results in the formation of three-dimensional gels. Ultrastructurally, these gels correspond to complex supramolecular structures formed by single microfilaments and by microfilament bundles. Analysis of protein composition indicates that cytoplasmic gels are composed of actin and several associated proteins. Among the latter class of components, we have identified polypeptides with molecular weights of 55,000 (55K), 140,000 (140K), and a set of two or three polypeptides with molecular weights in the range of 235,000-245,000 (235-245K). The 55K and 140K components do not seem to correspond to any previously identified actin-associated proteins, while the 235-245K polypeptides may correspond to the protein known as fodrin.     

A Comparison of In Vitro- and In Vivo Phosphorylated Neurofilament Polypeptides

Abstract: Neurofilament polypeptides phosphorylated in vitro by incubation of neurofilament-enriched preparations from rat CNS with [γ-³²P]ATP were compared with the corresponding polypeptides labeled in vivo by injection of ³²P_i into the lateral ventricles of rats. Autoradiography of sodium dodecyl sulfate (SDS)-polyacrylamide gels revealed that the major phosphorylated species in both preparations were the three neurofilament subunits, which have molecular weights of 200K, 145K, and 68K. However, the relative levels of ³²P detected in the three in vitro -labeled subunits differed from the relative in vivo levels. The two larger neurofilament polypeptides displayed similar ³²P isoprotein distribution patterns on two-dimensional gels, whereas additional isoproteins were seen in the in vitro -labeled 68K species. Limited proteolysis in SDS-polyacrylamide gels revealed the presence of common phosphopeptides in the corresponding pairs of in vitro- and in vivo-labeled subunits, but the in vivo -labeled 145K and in vitro -labeled 200K polypeptides contained additional digestion products. Two-dimensional peptide mapping of the 68K polypeptide digested with a mixture of trypsin and chymotrypsin indicated that this component was phosphorylated at a single, identical site, both in vivo and in vitro. These results indicate that the protein kinase that copurifies with neurofilament preparations may be involved in their in vivo phosphorylation.     

Abnormal neurofilament transport caused by targeted disruption of neuronal kinesin heavy chain KIF5A

To test the hypothesis that fast anterograde molecular motor proteins power the slow axonal transport of neurofilaments (NFs), we used homologous recombination to generate mice lacking the neuronal-specific conventional kinesin heavy chain, KIF5A. Because null KIF5A mutants die immediately after birth, a synapsin-promoted Cre-recombinase transgene was used to direct inactivation of KIF5A in neurons postnatally. Three fourths of such mutant mice exhibited seizures and death at around 3 wk of age; the remaining animals survived to 3 mo or longer. In young mutant animals, fast axonal transport appeared to be intact, but NF-H, as well as NF-M and NF-L, accumulated in the cell bodies of peripheral sensory neurons accompanied by a reduction in sensory axon caliber. Older animals also developed age-dependent sensory neuron degeneration, an accumulation of NF subunits in cell bodies and a reduction in axons, loss of large caliber axons, and hind limb paralysis. These data support the hypothesis that a conventional kinesin plays a role in the microtubule-dependent slow axonal transport of at least one cargo, the NF proteins.     

Identification of cytoskeletal components involved in attachment of L929 cells and macrophages to polystyrene

We have previously shown that lactoperoxidase (LPO) covalently coupled to polystyrene tissue culture flasks can be used to radioiodinate monolayer cell proteins that come into intimate contact with the LPO- polystyrene surface. These studies have now been extended to include a detailed examination of the class of iodinated polypeptides migrating with apparent molecular weights of 50,000 and 55,000 in SDS polyacrylamide gels. Whereas in cultured L929 cells the 55,000 band is predominantly iodinated, in thioglycollate-activated murine peritoneal macrophages the 55,000 and 50,000 bands are of equal intensity. It is possible that the marked degree of exposure of the 50,000 mol wt polypeptide to immobilized LPO is related to the unique strength of macrophages attachment. After labeling of both L929 cells and macrophages with immobilized LPO, all polypeptides in this molecular weight region were subjected to peptide mapping by simultaneous limited proteolysis and electrophoresis in a second SDS polyacrylamide slab gel. The results clearly show that the two major polypeptides in this region are identical within the limits of resolution of this technique. The 55,000 mol wt polypeptide can also be identified in Triton X-100 cytoskeletons from L929 cells after labeling with soluble LPO either before or after detergent lysis. We conclude that this cell surface polypeptide is in continuity with the cytoskeleton and is preferentially exposed to the substratum during attachment to polystyrene.     

Outer membrane proteins of Escherichia coli. II. Heterogeneity of major outer membrane polypeptides

When E. coli outer membrane protein is dissolved in sodium dodecyl sulfate (SDS) solution and boiled briefly, a single major peak (peak B) with a molecular weight of 42,000 daltons is observed on SDS-containing polyacrylamide gels. If the protein is dissolved in SDS solution at 37 °C and applied to gels without further treatment, peak B disappears and two other major peaks appear: Peak A, which is composed of aggregates and migrates more slowly than peak B, and peak C which is composed of monomeric protein not fully reacted with SDS and which migrates faster than peak B. When cyanogen bromide peptides of protein from peak A and peak C were compared, it was evident that peak A and peak C contained entirely different polypeptides. This was further confirmed by differential labeling studies with methionine and leucine. The cyanogen bromide peptide profiles of protein from peak A suggested that this peak was composed of two polypeptides, and this was confirmed by electrophoresis in an alkaline gel system which resolves peak B into three subcomponents. Two of these were derived from peak A and the third was derived from peak C. These results indicate that the outer membrane of E. coli contains at least three nonidentical major polypeptides, each of which has a nearly identical molecular weight of about 42,000 daltons. These polypeptides are present in identical proportions in the soluble and insoluble fractions obtained when the outer membrane is treated with Triton X-100 plus EDTA.     

Application of sodium dodecyl sulfate-gel electrophoresis to low molecular weight polypeptides

Experiments with nine polypeptides with molecular weights between 2000 and 10,760 confirm the value of sodium dodecy sulfate (SDS)-gel electrophoresis for separating polypeptides in this molecular weight range. In one case, electrophoretic blotting and microsequencing were successfully carried out. However, molecular weight determination in the low molecular weight range (less than 10,000) is much less reliable than that in the conventional molecular weight range (greater than 10,000) for SDS gels. Information provided by suppliers of horse heart myoglobin fragment kits is potentially misleading.     

Molecular motors in axonal transport

Neurons require a large amount of intracellular transport. Cytoplasmic polypeptides and membrane-bounded organelles move from the perikaryon, down the length of the axon, and to the synaptic terminals. This movement occurs at distinct rates and is termed axonal transport. Axonal transport is divided into the slow transport of cytoplasmic proteins including glycolytic enzymes and cytoskeletal structures and the fast transport of membrane-bounded organelles along linear arrays of microtubules. The polypeptide compositions of the rate classes of axonal transport have been well characterized, but the underlying molecular mechanisms of this movement are less clear. Progress has been particularly slow toward understanding force-generation in slow transport, but recent developments have provided insight into the molecular motors involved in fast axonal transport. Recent advances in the cellular and molecular biology of one fast axonal transport motor, kinesin, have provided a clearer understanding of organelle movement along microtubules. The availability of cellular and molecular probes for kinesin and other putative axonal transport motors have led to a reevaluation of our understanding of intracellular motility.     

Genetic polymorphism of erythrocyte histone H1 in Japanese quail

Histone H1 from erythrocytes of Japanese quail was resolved in a sodium dodecyl sulfate (SDS)-polyacrylamide gel into five fractions differing in apparent molecular weights. A polymorphism of histone H1.1, H1.2, and H1.3 bands was detected among quail individuals. While some birds possessed either a high (phenotype .3+) or a low (phenotype .3+/.3-) level of H1.3, at least half of the quail population lacked this H1 band (phenotype .3-). Appropriate genetic crosses demonstrated that H1.3 behaved as though it was coded by a gene with two codominant alleles at an autosomal locus. Using two-dimensional polyacrylamide gel electrophoresis (acid-urea followed by SDS gels), it was found that birds .3+ contained polypeptides H1.b1 and H1.b'1; birds .3-, polypeptides H1.b2 and H1.b'2 with lower apparent molecular weights; and birds .3+/.3-, both types of polypeptides in equal proportions. The H1.b2 + H1.b'2 complement was not discernible in SDS gels, for it migrated together with H1.c' within band H1.4. It was found that a small number of birds lacking the H1.2 band in SDS gels failed to express histone H1.a. Since birds with phenotype .2- with a defective allele of the gene H1.a were simultaneously lacking the H1.3 band, it seems that the imperfect allele of the H1.a gene might be closely linked to the alleles producing H1.b2 + H1.b'2.     

Components of fast and slow axonal transport in the goldfish optic nerve

The composition of the fast and slow components of axonal transport in the goldfish optic nerve was investigated, using specific radioactive precursors injected into the eye. Tritiated glucosamine and fucose label macromolecules, presumably glycoproteins, which are rapidly transported from the eye to the optic tectum. Material labeled with these precursors is not evident in the slowly transported component. Glucosamine and fucose incorporation are blocked when a protein synthesis inhibitor, acetoxycycloheximide, is injected into the eye concurrently with the precursors. As well as labeling macromolecules, ³H-glucosamine and ³H-N-acetylmannosamine ( a precursor of sialic acids) also label rapidly-transported chloroform-methanol-extractable material which may contain transported glycolipids. Two procedures were used to show that the slow component of axonal transport contains tubulin, a protein characteristic of the microtubules:

• (a) Tracer doses of tritiated colchicine injected into the eye label a wave of radioactivity which moves 0.5 mm/day, the rate of slow axonal transport in the goldfish optic nerve. We believe this wave represents the movement of colchicine which is bound to colchicine-binding protein moving in the slow component of axonal transport.
• (b) Tritiated proline labels a slowly transported protein which is precipitated by vinblastine and has a mobility on polyacrylamide gels comparable to authentic tubulin. These results indicate that the fast and slow components of axonal transport each provide specific chemical substances to the nerve endings.

     

Multiple phosphorylated variants of the high molecular mass subunit of neurofilaments in axons of retinal cell neurons: characterization and evidence for their differential association with stationary and moving neurofilaments

The 200-kD subunit of neurofilaments (NF-H) functions as a cross-bridge between neurofilaments and the neuronal cytoskeleton. In this study, four phosphorylated NF-H variants were identified as major constituents of axons from a single neuron type, the retinal ganglion cell, and were shown to have characteristics with different functional implications. We resolved four major Coomassie Blue-stained proteins with apparent molecular masses of 197, 200, 205, and 210 kD on high resolution one- dimensional SDS-polyacrylamide gels of mouse optic axons (optic nerve and optic tract). Proteins with the same electrophoretic mobilities were radiolabeled within retinal ganglion cells in vivo after injecting mice intravitreally with [35S]methionine or [3H]proline. Extraction of the radiolabeled protein fraction with 1% Triton X-100 distinguished four insoluble polypeptides (P197, P200, P205, P210) with expected characteristics of NF-H from two soluble neuronal polypeptides (S197, S200) with few properties of neurofilament proteins. The four Triton- insoluble polypeptides displayed greater than 90% structural homology by two-dimensional alpha-chymotryptic iodopeptide map analysis and cross-reacted with four different monoclonal and polyclonal antibodies to NF-H by immunoblot analysis. Each of these four polypeptides advanced along axons primarily in the Group V (SCa) phase of axoplasmic transport. By contrast, the two Triton-soluble polypeptides displayed only a minor degree of alpha-chymotryptic peptide homology with the Triton-insoluble NF-H forms, did not cross-react with NF-H antibodies, and moved primarily in the Group IV (SCb) wave of axoplasmic transport. The four NF-H variants were generated by phosphorylation of a single polypeptide. Each of these polypeptides incorporated 32P when retinal ganglion cells were radiolabeled in vivo with [32P]orthophosphate and each cross-reacted with monoclonal antibodies specifically directed against phosphorylated epitopes on NF-H. When dephosphorylated in vitro with alkaline phosphatase, the four variants disappeared, giving rise to a single polypeptide with the same apparent molecular mass (160 kD) as newly synthesized, unmodified NF-H. The NF-H variants distributed differently along optic axons. P197 predominated at proximal axonal levels; P200 displayed a relatively uniform distribution; and P205 and P210 became increasingly prominent at more distal axonal levels, paralleling the distribution of the stationary neurofilament network.(ABSTRACT TRUNCATED AT 400 WORDS)     

Radiolabelling of DNA/polypeptide complexes in isolated bulk DNA and in residual nuclear matrix DNA by nick-translation

Conditions are described that allow 32P-radiolabelling and detection of tight complexes between DNA and polypeptides by nick-translation. Prolonged nick-translation of purified bulk DNA results in radiolabelled complexes migrating on SDS-polyacrylamide gels with apparent molecular weights of 68 kd and 54 kd respectively. Residual nuclear matrix DNA which is not accessible to DNase I on the nuclear level becomes accessible to radiolabelling by nick-translation on the nuclear matrix level. In this case the in situ radiolabelled complexes migrate on SDS-polyacrylamide gels with apparent molecular weights of 68 kd and 100 kd. The DNA/polypeptide complexes are stable during treatments with SDS, beta-mercapto ethanol and alkali which points to covalent bonds between the polypeptides and DNA strands.     

Effect of various detergents on protein migration in the second dimension of two-dimensional gels.

Two-dimensional gel electrophoresis (2D)1 is a powerful technique used to separate complex protein mixtures. The technique involves the separation of proteins by charge in the first dimension and by molecular weight in the second dimension. The effect of substituting various detergents for sodium dodecyl sulfate (SDS) in the second dimension (PAGE) was investigated. Individual C-10 through C-14 alkyl sulfates, C-11 through C-14 alkyl sulfonates, sodium N-lauroyl-N-methyl-taurine, N-lauroylsarcosine, sodium laurate, or benzyldimethyl-n-hexadecylammonium chloride were substituted for SDS in equilibration buffer, gel buffer, and upper running buffer. The cationic benzyldimethyl-n-hexadecylammonium chloride system was run with reversed polarity. Dramatic effects on protein migration from human mesothelial cell extracts were observed when different detergents were utilized. The C-12 (SDS) through C-14 alkyl sulfates and sulfonates resulted in anomalous migration of the simple epithelial keratins. Unlike SDS, the C-10 and C-11 alkyl sulfates and C-11 sulfonate resulted in gels in which the keratins were separated accurately with respect to their gene sequence-determined molecular weights. However, with these shorter chain alkyl sulfates and sulfonate, resolution was compromised, especially with respect to the high-molecular-weight polypeptides. The C-12 alkyl sulfate (SDS) and alkyl sulfonate provided the best resolution of polypeptides. Mixtures of C-11 sulfate and SDS resulted in gels with better sequence molecular weight estimates and high resolution. In addition, trace amounts of sodium tetradecyl sulfate/sodium heptadecyl sulfate in commercial SDS preparations had an effect on polypeptide resolution.     

DNA-dependent RNA polymerases from Acanthamoeba castellanii. Comparative subunit structures of the homogeneous enzymes.

The constituent polypeptides of the three classes of DNA-dependent RNA polymerase from Acanthamoeba castellanii were compared by several electrophoretic methods. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) reveals that a number of polypeptide components of the isozymes have identical molecular weights. Two-dimensional electrophoresis (isoelectric focusing in 8 M urea:SDS-polyacrylamide gel electrophoresis) demonstrates that the polypeptides of identical molecular weights also have identical isoelectric pH values. These polypeptides were also coincident after electrophoresis in 8 M urea at acidic or basic pH values followed by a second electrophoretic separation in the presence of SDS. By these criteria, subunits of molecular weight 13,300, 15,500, 17,500, 22,500, 37,000, and 39,000 are indistinguishable in polymerase I and III. The 13,300, 15,500, and 22,500 subunits are also shared by the class II polymerase. In addition, electrophoresis in 8 M urea under basic conditions reveals microheterogeneity in the 17,500 molecular weight subunit. The strikingly similar pattern of common subunits between yeast and Acanthamoeba suggests that a universal arrangement of functional units may be an essential feature of the eukaryotic polymerases.