3 research outputs found
A novel 65 kDa RNA-binding protein in squid presynaptic terminals
Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Neuroscience 166 (2010): 73-83, doi:10.1016/j.neuroscience.2009.12.005.A polyclonal antibody (C4), raised against the head domain of chicken myosin Va,
reacted strongly towards a 65 kDa polypeptide (p65) on western blots of extracts from squid optic
lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by
other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to
identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase
chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from
SDS-PAGE gels. BLAST analysis and partial matching with ESTs from a Loligo pealei data bank
indicated that p65 contains consensus signatures for the hnRNP A/B family of RNA-binding
proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after
treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP
complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies
using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in
bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate
ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe
synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic
terminal within squid neurons and may have a role in synaptic localization of RNA and its
translation or processing.REL, JCR and JEM received
financial support from the Fundação de Amparo à Pesquisa do Estado de Sao Paulo (FAPESP), the
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação de
Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínicas da FMRP-USP (FAEPA). JAD
received financial support from the RI-INBRE Program Grant #P20RR016457 from the Nation
Center for Research Resources, NIH, Bethesda, MD. DTPL, LC, SBFT, EJRV and MMAB were
recipients of research fellowships from FAPESP and CNPq. REL and JEM received Productivityin-
Research fellowships from CNPq
Differential expression of the FMRF gene in adult and hatchling stellate ganglia of the squid Loligo pealei
The giant fiber system of the squid Loligo pealei mediates the escape response and is an important neurobiological model. Here, we identified an abundant transcript in the stellate ganglion (SG) that encodes a FMRFamide precursor, and characterized FMRFamide and FI/LRF-amide peptides. To determine whether FMRFamide plays a role in the adult and hatchling giant fiber system, we studied the expression of the Fmrf gene and FMRFamide peptides. In stage 29 embryos and stage 30 hatchlings, Ffmr transcripts and FMRFamide peptide were low to undetectable in the SG, in contrast to groups of neurons intensely expressing the Fmrf gene in several brain lobes, including those that innervate the SG. In the adult SG the Fmrf gene was highly expressed, but the FMRFamide peptide was in low abundance. Intense staining for FMRFamide in the adult SG was confined to microneurons and fibers in the neuropil and to small fibers surrounding giant axons in stellar nerves. This shows that the Fmrf gene in the SG is strongly regulated post-hatching, and suggests that the FMRFamide precursor is incompletely processed in the adult SG. The data suggest that the SG only employs the Fmrf gene post-hatching and restricts the biosynthesis of FMRFamide, demonstrating that this peptide is not a major transmitter of the giant fiber system. This contrasts with brain lobes that engage FMRFamide embryonically as a regulatory peptide in multiple neuronal systems, including the afferent fibers that innervate the SG. The biological significance of these mechanisms may be to generate diversity within Fmrf-expressing systems in cephalopods