A nuclear localization signal targets proteins to the retrograde transport system,thereby evading uptake into organelles in aplysia axons

The turnover of soluble proteins in axons and terminals is effected by replacing used proteins with newly synthesized constituents from the cell body. To investigate this complex process, which is especially important during nerve regeneration, we microinjected proteins into varicosities on axons of Aplysia neurons in vitro. When human serum albumin (HSA) coupled to rhodamine (r) was injected, it initially filled the varicosity; within seconds, however, it began to accumulate in packets and by 15 min was punctate. A similar pattern was observed after injecting soluble proteins from extruded axoplasm. In contrast, when we injected rHSA covalently attached to the SV-40 nuclear localization sequence (sp), the distribution was never punctate and the rHSA-sp was retrogradely transported from the varicosity to the cell body and into the nucleus. Electron microscopy of varicosities injected with HSA-gold showed that >90% of the particles were inside vacuoles and multivesicular bodies. These organelles probably function as storage rather than degradatory sites since they did not contain acid phosphatase. In contrast, when HSA-sp-colloidal gold was injected, only 25% of the particles were in organelles. Thus, HSA and resident axonal proteins can be removed from axoplasm by uptake into organelles. The presence of a nuclear localization sequence (the sp) may avoid uptake by providing access to the retrograde transport/nuclear import pathway. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 151–160, 1997     

Factors affecting the expression of foreign proteins inEscherichia coli

Summary A variety of factors affect the expression of foreign proteins inEscherichia coli. These include: promoter strength, efficiency of ribosome binding, stability of the foreign protein inE. coli, location of the foreign protein inE. coli, the codons used to encode the foreign protein, the metabolic state of the cell, and the location, stability and copy number of the foreign gene. This paper contains a critical review of these factors with the idea that a detailed understanding of them is the key to the development of strategies for the efficient large-scale production of foreign proteins inE. coli.     

A dynamical system model of neurofilament transport in axons

We develop a dynamical system model for the transport of neurofilaments in axons, inspired by Brown's "stop-and-go" model for slow axonal transport. We use fast/slow time-scale arguments to lower the number of relevant parameters in our model. Then, we use experimental data of Wang and Brown to estimate all but one parameter. We show that we can choose this last remaining parameter such that the results of our model agree with pulse-labeling experiments from three different nerve cell types, and also agree with stochastic simulation results.     

NGF-stimulated retrograde transport of trkA in the mammalian nervous system

The present study was designed to clarify the in vivo function of trkA as an NGF receptor in mammalian neurons. Using the rat sciatic nerve as a model system, we examined whether trkA is retrogradely transported and whether transport is influenced by physiological manipulations. Following nerve ligation, trkA protein accumulates distal to the ligation site as shown by Western blot analysis. The distally accumulating trkA species were tyrosine phosphorylated. The trkA retrograde transport and phosphorylation were enhanced by injecting an excess of NGF in the footpad and were abolished by blocking endogenous NGF with specific antibodies. These results provide evidence that, upon NGF binding, trkA is internalized and retrogradely transported in a phosphorylated state, possibly together with the neurotrophin. Furthermore, our results suggest that trkA is a primary retrograde NGF signal in mammalian neurons in vivo.     

Products of endocytosis and autophagy are retrieved from axons by regulated retrograde organelle transport

Cellular homeostasis in neurons requires that the synthesis and anterograde axonal transport of protein and membrane be balanced by their degradation and retrograde transport. To address the nature and regulation of retrograde transport in cultured sympathetic neurons, I analyzed the behavior, composition, and ultrastructure of a class of large, phase-dense organelles whose movement has been shown to be influenced by axonal growth (Hollenbeck, P. J., and D. Bray. 1987. J. Cell Biol. 105:2827-2835). In actively elongating axons these organelles underwent both anterograde and retrograde movements, giving rise to inefficient net retrograde transport. This could be shifted to more efficient, higher volume retrograde transport by halting axonal outgrowth, or conversely shifted to less efficient retrograde transport with a larger anterograde component by increasing the intracellular cyclic AMP concentration. When neurons were loaded with Texas red- dextran by trituration, autophagy cleared the label from an even distribution throughout the neuronal cytosol to a punctate, presumably lysosomal, distribution in the cell body within 72 h. During this process, 100% of the phase-dense organelles were fluorescent, showing that they contained material sequestered from the cytosol and indicating that they conveyed this material to the cell body. When 29 examples of this class of organelle were identified by light microscopy and then relocated using correlative electron microscopy, they had a relatively constant ultrastructure consisting of a bilamellar or multilamellar boundary membrane and cytoplasmic contents, characteristic of autophagic vacuoles. When neurons took up Lucifer yellow, FITC-dextran, or Texas red-ovalbumin from the medium via endocytosis at the growth cone, 100% of the phase-dense organelles became fluorescent, demonstrating that they also contain products of endocytosis. Furthermore, pulse-chase experiments with fluorescent endocytic tracers confirmed that these organelles are formed in the most distal region of the axon and undergo net retrograde transport. Quantitative ratiometric imaging with endocytosed 8-hydroxypyrene-1,3,6- trisulfonic acid showed that the mean pH of their lumena was 7.05. These results indicate that the endocytic and autophagic pathways merge in the distal axon, resulting in a class of predegradative organelles that undergo regulated transport back to the cell body.     

Dynein mediates retrograde neurofilament transport within axons and anterograde delivery of NFs from perikarya into axons: regulation by multiple phosphorylation events

We examined the respective roles of dynein and kinesin in axonal transport of neurofilaments (NFs). Differentiated NB2a/d1 cells were transfected with green fluorescent protein-NF-M (GFP-M) and dynein function was inhibited by co-transfection with a construct expressing myc-tagged dynamitin, or by intracellular delivery of purified dynamitin and two antibodies against dynein's cargo domain. Monitoring of the bulk distribution of GFP signal within axonal neurites, recovery of GFP signal within photobleached regions, and real-time monitoring of individual NFs/punctate structures each revealed that pertubation of dynein function inhibited retrograde transport and accelerated anterograde, confirming that dynein mediated retrograde axonal transport, while intracellular delivery of two anti-kinesin antibodies selectively inhibited NF anterograde transport. In addition, dynamitin overexpression inhibited the initial translocation of newly-expressed NFs out of perikarya and into neurites, indicating that dynein participated in the initial anterograde delivery of NFs into neurites. Delivery of NFs to the axon hillock inner plasma membrane surface, and their subsequent translocation into neurites, was also prevented by vinblastine-mediated inhibition of microtubule assembly. These data collectively suggest that some NFs enter axons as cargo of microtubues that are themselves undergoing transport into axons via dynein-mediated interactions with the actin cortex and/or larger microtubules. C-terminal NF phosphorylation regulates motor association, since anti-dynein selectively coprecipitated extensively phosphorylated NFs, while anti-kinesin selectively coprecipitated less phosphorylated NFs. In addition, however, the MAP kinase inhibitor PD98059 also inhibited transport of a constitutively-phosphorylated NF construct, indicating that one or more additional, non-NF phosphorylation events also regulated NF association with dynein or kinesin.     

A rat liver cell-free system for the synthesis of proteins and their transport into mitochondria

A rat liver cytosol was used to study protein synthesis per se and also to study import of proteins into mitochondria since rat liver cytosol represents an environment closer to that of liver mitochondria than the generally used reticulocytes lysates. Two ATP-regenerating systems were compared. The creatine phosphate/creatine kinase yields higher protein synthesis than the phosphoenol pyruvate/pyruvate kinase system. Hemin, necessary to maintain synthesis by reticulocyte lysates, does not affect the rat liver cytosol. The level of protein synthesis obtained with this cell-free system is comparable to other eukaryotic systems described recently and to the expected value for "in vivo" conditions. Isolated mitochondria incorporated, under our standard conditions, newly synthesized proteins linearly up to 30 min, it ceases when a component(s) in the cytosol had been depleted; addition of freshly translated cytosol restores the import. The bulk of imported proteins are retained in mitoplasts or in mitochondria after treatment with trypsin. The cytosol system will be useful to study questions such as regulation of liver mRNA translation and mitochondrial protein turnover.     

Loss of material from the retrograde axonal transport system in frog sciatic nerve

Rapid axonal transport was studied in sciatic nerve preparations of the amphibian Xenopus laevis maintained in vitro at 23.0 +/- 0.2 degrees C. A pulse of [35S]methionine-labeled material was allowed to move in the anterograde direction until encountering a lesion, at which a portion of the pulse reversed directions and moved in the retrograde direction. By constricting the nerve during the course of the experiment, it was possible to prevent continuous return of label from the lesion, thus creating a retrogradely moving pulse that contained a defined quantity of radiolabel. Movement of both the anterograde and the retrograde pulse were monitored continuously for up to 24 h using a position-sensitive detector of ionizing radiation. The front and the back edge of the anterograde pulse were found to move at the rates of (mm/day) 179.9 +/- 3.9 (+/- SEM) and 149.9 +/- 5.9, respectively, and the front and the back edge of the retrograde pulse moved at the rates of 155.8 +/- 11.3 and 84.6 +/- 2.9, respectively. By comparison of the quantity of label lost to the stationary phase to the quantity of label calculated to have been present in the anterograde pulse, it was determined that 0.068 +/- 0.009 of the anterograde pulse is lost to each 3.18-mm region of nerve. Comparison of the quantity of label calculated to have been present in the retrograde pulse to that in the anterograde pulse revealed that 0.057 +/- 0.014 of the retrograde pulse is lost to each 3.18-mm region of nerve. It is concluded that protein originating in the cell body and which reverses its direction of transport at a lesion can be lost from the retrograde axonal transport system.     

The transport of organelles in axons

The fast anterograde and retrograde transport systems in axons convey organelles from the soma, where synthesis occurs, to the synaptic region and back. Studies of label incorporation into newly synthesized organelles show that they move along the axon with profiles resembling traveling waves. The underlying mechanism appears to be that cross-bridging “engines” attach to the organelles and this complex then attaches by the engines to the surface of microtubules, resulting in translocation of the organelles. A model incorporating this mechanism predicts traveling-wave-like profiles of labeled organelles and can serve to link mechanistic information with fast transport measurements in intact axons. Analysis of the simplest case provides insight into the factors determining the speed and shape of the wavelike profile.     

Fast axonal transport in central nervous system and peripheral nervous system axons following axotomy

After axotomy, changes in the composition of fast axonally transported proteins ( FTP ) within the peripheral nervous system (PNS) axons have been reported. The most significant and reproducible changes involved polypeptides found within the molecular weight range of 31.0 to 14.5 kilodaltons ( Bisby , 1980). We wished to determine whether similar changes following axotomy occur in axons of the central nervous system (CNS). Intracranial axotomy of the left optic tract was performed stereotaxically in rats. Six days post axotomy 50 muCi 35[S]-methionine was injected into the vitreous body of both eyes. FTP were isolated within the optic nerves 2 h after isotope injection. The nerve segments were processed for SDS-PAGE, fluorography, and compared to similarly prepared fluorographs of normal and eight day post-axotomy sciatic nerve segments. The labelling of 5 major polypeptide bands (S1, MW congruent to 28,000; S2a , MW congruent to 25,000; S2b , MW congruent to 23,000; T1, MW congruent to 20,200; and T2, MW congruent to 17,000) was studied by laser densitometry. Band S2b showed a highly significant (p less than 0.001) increase in concentration, while bands S1 and T1 demonstrated highly significant decreases in concentration following axotomy of the sciatic nerve. In contrast, after axotomy of the retinal ganglion cell axons the only significant change was a decrease (p less than 0.05) in T1. We suggest that failure of CNS axons to respond similarly to PNS axons following axotomy may be related to the failure of CNS axons to regenerate.     

Protein synthesis and transport in the regenerating goldfish visual system

The nature of the proteins synthesized in the goldfish retina and axonally transported to the tectum during optic nerve regeneration has been examined. Electrophoretic analysis of labeled soluble retinal proteins by fluorography verified our previous observation of a greatly enhanced synthesis of the microtubule subunits. In addition, labeling of a tubulin-like protein in the retinal particulate fraction was also increased during regeneration. Like soluble tubulin, the particulate material had an apparent MW of 53–55K and could be tyrosylated in the presence of cycloheximide and [³H]tyrosine. Comparison of post-crush and normal retinal proteins by two-dimensional gel electrophoresis also revealed a marked enhancement in the labeling of two acidic 68–70K proteins. Analysis of proteins slowly transported to the optic tectum revealed changes following nerve crush similar to those observed in the retina, with enhanced labeling of both soluble and particulate tubulin and of 68–70K polypeptides. The most striking change in the profile of rapidly transported protein was the appearance of a labeled 45K protein which was barely detectable in control fish.     

Labelling by axonal transport of myelin-associated proteins in the rabbit visual pathway.

After intraocular injections of [3H]leucine, six regions of the visual pathway of adult rabbit were used to study the spatio-temporal pattern of the slow anterograde axonal transport of radioactive proteins associated with the particulate fraction, the water-soluble fraction and the myelin fraction. Unlike other fractions, myelin-associated labelled proteins represented a time-constant (for a given region) percentage of total tissue radioactivity. This percentage increased from the first half to the second half of the optic nerve and remained high in the chiasma and tract. The peak specific radioactivity of myelin decreased in the same direction. Myelin proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the labelling patterns obtained in different regions and at different survival times were compared. At the peak of myelin radioactivity of a given region the label was typically associated with four protein bands, L1, L2, L3 and L4, of 40000, 44000, 62000, and 68000 mol.wts. respectively. The basic protein, the proteolipid protein and the W1 component (mol.wt. 51000-53000) of the Wolfgram proteins were not significantly labelled. The radioactivity associated with the W2 component (mol.wt 60000) of the Wolfgram proteins could be derived from the closely migrating L3 component. At shorter survival times no clear labelling pattern could be detected. At longer survival times radioactivity was almost totally localized around band L3. The results presented underline the importance of choosing appropriate experimental conditions to obtain a consistent labelling pattern of myelin-associated proteins and to investigate the possible mechanism responsible for this phenomenon.     

Cobalamin transport proteins and their cell-surface receptors

The primary function of cobalamin (Cbl; vitamin B12) is the formation of red blood cells and the maintenance of a healthy nervous system. Before cells can utilise dietary Cbl, the vitamin must undergo cellular transport using two distinct receptor-mediated events. First, dietary Cbl bound to gastric intrinsic factor (IF) is taken up from the apical pole of ileal epithelial cells via a 460 kDa receptor, cubilin, and is transported across the cell bound to another Cbl-binding protein, transcobalamin II (TC II). Second, plasma TC II-Cbl is taken up by cells that need Cbl via the TC II receptor (TC II-R), a 62 kDa protein that is expressed as a functional dimer in cellular plasma membranes. Human Cbl deficiency can develop as a result of acquired or inherited dysfunction in either of these two transmembrane transport events. This review focuses on the biochemical, cellular and molecular aspects of IF and TC II and their cell-surface receptors.     

Axonal transport of phospholipids in rat visual system.

Abstract— Seventeen day old rats were injected intraocularly with a phospholipid precursor, [³²P]phosphate, and a glycoprotein precursor, [³H]fucose. Animals were killed between 1 h and 21 days later, and structures of the visual pathway (retina, optic nerve, optic tract, lateral geniculate body, and superior colliculus) were dissected. Radioactivity in phospholipids ([³²P] in solvent-extracted material) and in glycoproteins ([³H] in solvent-extracted residue) was determined. Incorporation of [³H]fucose into retinal glycoproteins peaked at 6–8 h. Labelled glycoproteins were present in superior colliculus by 2h after injection, indicating a rapid rate of transport; maximal labelling was at 8–10 h after injection. Incorporation of [³²P]phosphate into retinal phospholipids peaked at 1 day after injection. Phospholipids were also rapidly transported since label was present in the superior colliculus by 3 h after injection: however, maximal labelling did not occur until 5–6 days. These results indicate that newly synthesized phospholipids enter a preexisting pool, part of which is later committed to transport at a rapid rate. Transported phospholipids were catabolized at the nerve endings with a maximum half-life of several days; there was minimal recycling of precursor label. Lipids were fractionated by thin-layer chromatography, and radioactivity in individual phospholipid classes determined. Choline and ethanolamine phosphoglycerides were the major transported phospholipids, together accounting for approx 85% of the total transported lipid radioactivity. At early time points, the ratio of radioactivity in choline phosphoglycerides to that in ethanolamine phosphoglycerides increased in structures progressively removed from the site of synthesis (retina) but by 2 days approached a constant value. In each structure, choline phosphoglyceride-ethanolamine phosphoglyceride radioactivity ratios decreased with time, rapidly at first, but plateaued by 2 days. These results indicate that choline phosphoglycerides are committed to transport sooner than ethanolamine phosphoglycerides. Some experiments were also conducted using [2-³H]glycerol as a phospholipid precursor. Results concerning incorporation of this precursor into individual phospholipid classes and their subsequent axonal transport were comparable to those obtained using [³²P]phosphate, with the following exceptions: (a) incorporation of [2-³H]glycerol into retinal phospholipids was relatively rapid (near-maximal levels at 1 h after injection) although transport to the superior colliculus showed an extended time course very similar to [³²P]-labelled lipids; (b) [2-³H]glycerol was somewhat less efficient than [³²P]phosphate in labelling lipids committed to transport relative to labelling those which remained in the retina; and (c) [2-³H]glycerol did not label plasmalogens.