Cloning and characterization of AASPs: novel axon-associated SH3 binding-like proteins

Two cDNAs encoding closely related proteins were isolated from a crayfish nervous system lambdagtl0 cDNA library with a rat synapsin Ia cDNA probe. These proteins were expressed exclusively in neurons, were highly enriched in axons of the crayfish, and contained multiple, overlapping, putative Src homology 3 (SH3) binding sites. In concert with other proteins containing Src homology domains, SH3 binding proteins are thought to mediate protein-protein interactions in receptor signaling processes and with the cytoskeleton. We have named these proteins axon-associated SH3 binding-like proteins (AASPs). Except for these SH3 binding regions, which are also found in synapsins, AASPs were unlike any proteins in the database. AASPs were differentially expressed among motoneuron populations in crayfish and were found in growing axons and growth cones in culture. Affinity purified polyclonal antibodies to AASP-168 recognized immunoreactive proteins in rat and Xenopus, suggesting that AASPs may be conserved across species. Although the cellular function of AASPs is unclear at this time, they appear to be novel members of a neuron-specific SH3 binding protein family, which includes the synapsins.     

Exogenous synapsin I promotes functional maturation of developing neuromuscular synapses.

We have investigated the possible role of synapsin I, a nerve terminal-specific protein, in the maturation of neuromuscular synapses in Xenopus cell cultures. Purified synapsin I was loaded into embryonic spinal neurons by injection of the protein into one of the early blastomeres of a Xenopus embryo. At synapses made by synapsin I-loaded neurons, spontaneous synaptic currents occurred with higher frequency and amplitude, and the amplitude exhibited an earlier appearance of a bell-shaped distribution. These characteristics are indicative of more mature quantal secretion. Impulse-evoked synaptic currents also showed a significant increase in amplitude. Using cell manipulation techniques, enhanced transmitter release from synapsin I-loaded neurons was shown to occur at the onset of synaptogenesis, suggesting a presynaptic developmental action of synapsin I prior to synaptic contact. Taken together, these results suggest that endogenous synapsin I may participate in the functional maturation of synapses.     

Cloning and characterization of AASPs: Novel axon‐associated SH3 binding‐like proteins

Two cDNAs encoding closely related proteins were isolated from a crayfish nervous system λgt10 cDNA library with a rat synapsin Ia cDNA probe. These proteins were expressed exclusively in neurons, were highly enriched in axons of the crayfish, and contained multiple, overlapping, putative Src homology 3 (SH3) binding sites. In concert with other proteins containing Src homology domains, SH3 binding proteins are thought to mediate protein–protein interactions in receptor signaling processes and with the cytoskeleton. We have named these proteins axon‐associated SH3 binding‐like proteins (AASPs). Except for these SH3 binding regions, which are also found in synapsins, AASPs were unlike any proteins in the database. AASPs were differentially expressed among motoneuron populations in crayfish and were found in growing axons and growth cones in culture. Affinity purified polyclonal antibodies to AASP‐168 recognized immunoreactive proteins in rat and Xenopus, suggesting that AASPs may be conserved across species. Although the cellular function of AASPs is unclear at this time, they appear to be novel members of a neuron‐specific SH3 binding protein family, which includes the synapsins. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 581–594, 1999     

Phosphorylation-regulated inhibition of the Gz GTPase-activating protein activity of RGS proteins by synapsin I

Synapsins are neuronal proteins that bind and cluster synaptic vesicles in the presynaptic space, presumably by anchoring to actin filaments, but specific regulatory functions of the synapsins are unknown. We found that a sub-population of brain synapsin Ia, a splice variant of one of three synapsin isoforms, inhibits the GTPase-activating protein (GAP) activity of several RGS proteins. Inhibition is highly selective for Galphaz, a member of the Gi family that is found in neurons, platelets, adrenal chromaffin cells, and a few other neurosecretory cells. Gz has been indirectly implicated in the regulation of secretion. Synapsin Ia constitutes a major fraction of the total GAP-inhibitory activity in brain, and its inhibitory activity is absent from the brains of synapsin I(-/-)/II(-/-) mice. Inhibition depends on the cationic D/E domain of synapsin. Phosphorylation of synapsin Ia at serine 9 by either cyclic AMP-dependent protein kinase or p21-activated protein kinase (PAK1) attenuates its potency as a GAP inhibitor more than 7-fold. Synapsin can thus act as a phosphorylation-modulated mediator of feedback regulation of Gz signaling by the synaptic machinery.     

Crustacean frequenins: Molecular cloning and differential localization at neuromuscular junctions

Crustacean muscles are innervated by phasic and tonic motor neurons that display differential physiology and have morphologically distinct synaptic terminals. Phasic motor neurons release much more transmitter per impulse and have filiform terminals, whereas tonic motor neurons release less transmitter and have larger terminals with prominent varicosities. Using an antibody raised against Drosophila frequenin (frq), a calcium‐binding protein that enhances transmitter release in Drosophila synaptic terminals, we found that frq‐like immunoreactivity is prominent in many of the phasic, but not tonic nerve endings of crayfish motor neurons. In contrast, synapsin‐ and dynamin‐like immunoreactivities are strongly expressed in both types of terminal. The immunocytochemical findings strongly suggested the presence of an frq‐like molecule in crayfish, and its differential expression indicated a possible modulatory role in transmitter release. Therefore, we cloned the cDNA sequences for the crayfish and lobster homologues of Drosophila frq. Crustacean frequenins are very similar in sequence to their Drosophila counterpart, and calcium‐binding regions (EF hands) are conserved. The widespread occurrence of frq‐like molecules and their differential localization in crayfish motor neurons indicate a significant role in physiology or development of these neurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 165–175, 1999     

Localization of nuclear-encoded mRNAs to mitochondria outer surface

The diverse functions of mitochondria depend on hundreds of different proteins. The vast majority of these proteins is encoded in the nucleus, translated in the cytosol, and must be imported into the organelle. Import was shown to occur after complete synthesis of the protein, with the assistance of cytosolic chaperones that maintain it in an unfolded state and target it to the mitochondrial translocase of the outer membrane (TOM complex). Recent studies, however, identified many mRNAs encoding mitochondrial proteins near the outer membrane of mitochondria. Translation studies suggest that many of these mRNAs are translated locally, presumably allowing cotranslational import into mitochondria. Herein we review these data and discuss its relevance for local protein synthesis. We also suggest alternative roles for mRNA localization to mitochondria. Finally, we suggest future research directions, including revealing the significance of localization to mitochondria physiology and the molecular players that regulate it.     

Limited availability of ZBP1 restricts axonal mRNA localization and nerve regeneration capacity

Subcellular localization of mRNAs is regulated by RNA-protein interactions. Here, we show that introduction of a reporter mRNA with the 3'UTR of β-actin mRNA competes with endogenous mRNAs for binding to ZBP1 in adult sensory neurons. ZBP1 is needed for axonal localization of β-actin mRNA, and introducing GFP with the 3'UTR of β-actin mRNA depletes axons of endogenous β-actin and GAP-43 mRNAs and attenuates both in vitro and in vivo regrowth of severed axons. Consistent with limited levels of ZBP1 protein in adult neurons, mice heterozygous for the ZBP1 gene are haploinsufficient for axonal transport of β-actin and GAP-43 mRNAs and for regeneration of peripheral nerve. Exogenous ZBP1 can rescue the RNA transport deficits, but the axonal growth deficit is only rescued if the transported mRNAs are locally translated. These data support a direct role for ZBP1 in transport and translation of mRNA cargos in axonal regeneration in vitro and in vivo.     

Stable ribosome binding to the endoplasmic reticulum enables compartment-specific regulation of mRNA translation

In eukaryotic cells, protein synthesis is compartmentalized; mRNAs encoding secretory/membrane proteins are translated on endoplasmic reticulum (ER)-bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on free ribosomes. mRNA partitioning between the two compartments occurs via positive selection: free ribosomes engaged in the translation of signal sequence-encoding mRNAs are trafficked from the cytosol to the ER. After translation termination, ER-bound ribosomes are thought to dissociate, thereby completing a cycle of mRNA partitioning. At present, the physiological basis for termination-coupled ribosome release is unknown. To gain insight into this process, we examined ribosome and mRNA partitioning during the unfolded protein response, key elements of which include suppression of the initiation stage of protein synthesis and polyribosome breakdown. We report that unfolded protein response (UPR)-elicited polyribosome breakdown resulted in the continued association, rather than release, of ER-bound ribosomes. Under these conditions, mRNA translation in the cytosol was suppressed, whereas mRNA translation on the ER was sustained. Furthermore, mRNAs encoding key soluble stress proteins (XBP-1 and ATF-4) were translated primarily on ER-bound ribosomes. These studies demonstrate that ribosome release from the ER is termination independent and identify new and unexpected roles for the ER compartment in the translational response to induction of the unfolded protein response.     

Synapsin II. Mapping of a domain in the NH2-terminal region which binds to small synaptic vesicles

The synapsins are a family of neuron-specific phosphoproteins that selectively bind to small synaptic vesicles in the presynaptic nerve terminal. Using the cDNA encoding rat synapsin IIb, we employed an Escherichia coli expression system to synthesize a variety of fusion proteins containing a truncated protein A linked to different portions of the NH2-terminal region of synapsin II. The recombinant proteins were purified by IgG-Sepharose chromatography and tested in vitro for their ability to bind to purified synaptic vesicles. These experiments identified a region between amino acids 43 and 121 of the amino-terminal portion of synapsin II which binds to synaptic vesicles. Mild trypsinization of synaptic vesicles reduces binding of recombinant proteins to synaptic vesicles, suggesting that the interaction between synapsin II and the vesicles is in part mediated by a synaptic vesicle protein. The 42 NH2-terminal amino acids of synapsin II are not necessary for binding to synaptic vesicles, although this domain contains the phosphorylation site for cAMP-dependent protein kinase.     

Synaptic localization of growth‐associated protein 43 in cultured hippocampal neurons during synaptogenesis

Growth‐associated protein 43 (GAP‐43), a novel axonal phosphoprotein, is originally identified as a growth‐cone‐specific protein of developing neurons in vitro. The expression of GAP‐43 is also shown to be up‐regulated concomitant with increased synaptic plasticity in the brains in vivo, but how GAP‐43 is concerned with synaptic plasticity is not well understood. In the present study, therefore, we aimed to elucidate subcellular localization of GAP‐43 as culture development of rat hippocampal neurons. Western blotting showed that the expression of GAP‐43 in the cerebral and hippocampal tissues was prominently high at postnatal days 14 and 21 or the active period of synaptogenesis. Double‐labelling immunohistochemistry with an axonal marker Tau revealed that the immunoreactivity of GAP‐43 was seen throughout axons of cultured hippocampal neurons but stronger at axonal puncta of developing neurons than axonal processes. Double‐labelling immunohistochemistry with presynaptic terminal markers of synapsin and synaptotagmin revealed that the immunoreactivity of GAP‐43 was observed mostly at weak synapsin‐ and synaptotagmin‐positive puncta rather than strong ones. The quantitative analysis of immunofluorescent intensity showed a clear inverse correlation between GAP‐43 and either synapsin or synaptotagmin expression. These data indicate that GAP‐43 is highly expressed at immature growing axonal terminals and its expression is decreased along with the maturation of synaptogenesis. Copyright © 2012 John Wiley & Sons, Ltd.     

Crustacean frequenins: molecular cloning and differential localization at neuromuscular junctions.

Crustacean muscles are innervated by phasic and tonic motor neurons that display differential physiology and have morphologically distinct synaptic terminals. Phasic motor neurons release much more transmitter per impulse and have filiform terminals, whereas tonic motor neurons release less transmitter and have larger terminals with prominent varicosities. Using an antibody raised against Drosophila frequenin (frq), a calcium-binding protein that enhances transmitter release in Drosophila synaptic terminals, we found that frq-like immunoreactivity is prominent in many of the phasic, but not tonic nerve endings of crayfish motor neurons. In contrast, synapsin- and dynamin-like immunoreactivities are strongly expressed in both types of terminal. The immunocytochemical findings strongly suggested the presence of an frq-like molecule in crayfish, and its differential expression indicated a possible modulatory role in transmitter release. Therefore, we cloned the cDNA sequences for the crayfish and lobster homologues of Drosophila frq. Crustacean frequenins are very similar in sequence to their Drosophila counterpart, and calcium-binding regions (EF hands) are conserved. The widespread occurrence of frq-like molecules and their differential localization in crayfish motor neurons indicate a significant role in physiology or development of these neurons.     

Molecular Factors Affecting the Accumulation of Recombinant Proteins in the <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> Chloroplast

In an effort to develop microalgae as a robust system for the production of valuable proteins, we analyzed some of the factors affecting recombinant protein expression in the chloroplast of the green alga Chlamydomonas reinhardtii. We monitored mRNA accumulation, protein synthesis, and protein turnover for three codon-optimized transgenes including GFP, bacterial luciferase, and a large single chain antibody. GFP and luciferase proteins were quite stable, while the antibody was less so. Measurements of protein synthesis, in contrast, clearly showed that translation of the three chimeric mRNAs was greatly reduced when compared to endogenous mRNAs under control of the same atpA promoter/UTR. Only in a few conditions this could be explained by limited mRNA availability since, in most cases, recombinant mRNAs accumulated quite well when compared to the atpA mRNA. In vitro toeprint and in vivo polysome analyses suggest that reduced ribosome association might contribute to limited translational efficiency. However, when recombinant polysome levels and protein synthesis are analyzed as a whole, it becomes clear that other steps, such as inefficient protein elongation, are likely to have a considerable impact. Taken together, our results point to translation as the main step limiting the expression of heterologous proteins in the C. reinhardtii chloroplast.     

Messenger RNAs in dendrites: localization,stability, and implications for neuronal function

In the mammalian central nervous system (CNS), each neuron receives signals from other neurons through numerous synapses located on its cell body and dendrites. Molecules involved in the postsynaptic signaling pathways need to be targeted to the appropriate subcellular domains at the right time during both synaptogenesis and the maintenance of synaptic functions. The presence of messenger RNAs (mRNAs) in dendrites offers a mechanism for synthesizing the appropriate molecules at the right place in response to local extracellular stimuli. Several dendritic mRNAs have been identified, and the mechanisms controlling their localization are beginning to be understood. In many cell types, controls on mRNA stability play an important role in the regulation of gene expression, but it is unclear to what extent this type of control operates in dendrites. The regulation of protein synthesis and the control of mRNA stability in dendrites could have important implications for neuronal function. BioEssays 20:70–78, 1998. © 1998 John Wiley & Sons, Inc.     

Messenger RNA competition in living Xenopus oocytes.

When calf lens crystallin mRNA and rabbit globin mRNA are competing for factors limiting protein synthesis in living Xenopus oocytes, no mRNA species is preferentially selected for translation. Differences in the intrinsic translational efficiency of the mRNA species exist, but the relative efficiencies are the same at high and low mRNA concentrations. mRNAs already being translated, in particular endogenous oocyte mRNAs, are less sensitive to competitive inhibition by injected mRNAs. As injected mRNAs gradually become incorporated into the protein-synthesizing machinery of the oocyte, they acquire the same status as the oocyte's own active mRNAs. Exogenous mRNAs this become endogenous mRNAs. These results, together with previous estmates of the translational efficiency of injected heterologous mRNA species, are compatible with the assumption that a large proportion of the endogenous mRNAs is not competing for the translational apparatus of the oocyte and, therefore, probably is present in the temporarily inactivated form.