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Mike Fainzilber

Publications and source records attributed to Mike Fainzilber.

At least 19 recordsLinked to original sources
PubMed2006

Vimentin binding to phosphorylated Erk sterically hinders enzymatic dephosphorylation of the kinase.

Cleavage fragments of de novo synthesized vimentin were recently reported to interact with phosphorylated Erk1 and Erk2 MAP kinases (pErk) in injured sciatic nerve, thus linking pErk to a signaling complex retrogradely transported on importins and dynein. Here we clarify the structural basis for this interaction, which explains how pErk is protected from dephosphorylation while bound to vimentin. Pull-down and ELISA experiments revealed robust calcium-dependent binding of pErk to the second coiled-coil domain of vimentin, with observed affinities of binding increasing from 180 nM at 0.1 microM calcium to 15 nM at 10 microM calcium. In contrast there was little or no binding of non-phosphorylated Erk to vimentin under these conditions. Geometric and electrostatic complementarity docking generated a number of solutions wherein vimentin binding to pErk occludes the lip containing the phosphorylated residues in the kinase. Binding competition experiments with Erk peptides confirmed a solution in which vimentin covers the phosphorylation lip in pErk, interacting with residues above and below the lip. The same peptides inhibited pErk binding to the dynein complex in sciatic nerve axoplasm, and interfered with protection from phosphatases by vimentin. Thus, a soluble intermediate filament fragment interacts with a signaling kinase and protects it from dephosphorylation by calcium-dependent steric hindrance.

Animals↗
PubMed2006

Building complex brains--missing pieces in an evolutionary puzzle.

The mechanisms underlying evolution of complex nervous systems are not well understood. In recent years there have been a number of attempts to correlate specific gene families or evolutionary processes with increased brain complexity in the vertebrate lineage. Candidates for evocation of complexity include genes involved in regulating brain size, such as neurotrophic factors or microcephaly-related genes; or wider evolutionary processes, such as accelerated evolution of brain-expressed genes or enhanced RNA splicing or editing events in primates. An inherent weakness of these studies is that they are correlative by nature, and almost exclusively focused on the mammalian and specifically the primate lineage. Another problem with genomic analyses is that it is difficult to identify functionally similar yet non-homologous molecules such as different families of cysteine-rich neurotrophic factors in different phyla. As long as comprehensive experimental studies of these questions are not feasible, additional perspectives for evolutionary and genomic studies will be very helpful. Cephalopod mollusks represent the most complex nervous systems outside the vertebrate lineage, thus we suggest that genome sequencing of different mollusk models will provide useful insights into the evolution of complex brains.

Animals↗
PubMed2006

Retrograde signaling in injured nerve--the axon reaction revisited.

Injury to axons elicits changes in macromolecule synthesis in the corresponding cell bodies that are critical for an effective regenerative response. For decades the most easily studied aspect of this phenomenon was the onset of chromatolysis, a suite of structural changes in the cell body characterized by swelling, shifting of the nucleus and dispersal of Nissl bodies. The question: 'what is the signal for chromatolysis?' received no less than 10 possible answers in a comprehensive review article published more than three decades ago. Here we come back to this 36 years old question, and review progress on understanding the mechanism of retrograde injury signaling in lesioned peripheral nerves. Recent work suggests that this is based on local axonal synthesis of critical carrier proteins, including importins and vimentin that link diverse signaling molecules to the dynein retrograde motor. A multiplicity of binding sites and of potential signaling molecules, including transcription factors and MAP kinases (Erk, Jnk), may allow diverse options for information-rich encoding of the injury status of the axon for transmission to the cell body.

Animals↗
PubMed2006

Tracking in the Wlds--the hunting of the SIRT and the luring of the Draper.

Wallerian degeneration of distal axons after nerve injury is significantly delayed in the Wlds mutant mouse. The Wlds protein is a fusion of nicotinamide mononucleotide adenyltransferase-1 (Nmnat1), an essential enzyme in the biosynthesis pathway of nicotinamide adenine dinucleotide (NAD), with the N-terminal 70 amino acids of the Ube4b ubiquitination assembly factor. The mechanism of Wlds action is still enigmatic, although recent efforts suggest that it is indirect and requires sequences flanking or linking the two fused open reading frames. Three papers in this issue of Neuron now show that Wlds action is conserved in Drosophila and that a critical role of Wlds may be the suppression of axonal self-destruct signals that induce Draper-mediated clearance of damaged axons by glial cells.

Animals↗
PubMed2005

Vimentin-dependent spatial translocation of an activated MAP kinase in injured nerve.

How are phosphorylated kinases transported over long intracellular distances, such as in the case of axon to cell body signaling after nerve injury? Here, we show that the MAP kinases Erk1 and Erk2 are phosphorylated in sciatic nerve axoplasm upon nerve injury, concomitantly with the production of soluble forms of the intermediate filament vimentin by local translation and calpain cleavage in axoplasm. Vimentin binds phosphorylated Erks (pErk), thus linking pErk to the dynein retrograde motor via direct binding of vimentin to importin beta. Injury-induced Elk1 activation and neuronal regeneration are inhibited or delayed in dorsal root ganglion neurons from vimentin null mice, and in rats treated with a MEK inhibitor or with a peptide that prevents pErk-vimentin binding. Thus, soluble vimentin enables spatial translocation of pErk by importins and dynein in lesioned nerve.

Amino Acid Sequence↗
PubMed2004

A genome wide screening approach for membrane-targeted proteins.

Membrane-associated proteins are critical for intra- and intercellular communication. Accordingly approaches are needed for rapid and comprehensive identification of all membrane-targeted gene products in a given cell or tissue. Here we describe a modification of the yeast Ras recruitment system to this end and designate the modified approach the Ras membrane trap (RMT). A pilot RMT screen was carried out on the central nervous system of the mollusk Lymnaea stagnalis, a model organism from a phylum that still lacks a representative with a sequenced genome. 112 gene products were identified in the screen of which 79 lack assignable homologs in available data bases. Currently available annotation tools predicted membrane association of only 45% of the 112 proteins, although experimental verification in mammalian cells confirmed membrane association for all clones tested. Thus, genome annotation using currently available tools is likely to underpredict representation of membrane-associated gene products. The 32 proteins with known homologies include many targeted to the endoplasmic reticulum or the nucleus, thus RMT provides a tool that can cover intracellular membrane proteomes. Two sequences were found to represent gene families not found to date in invertebrate genomes, emphasizing the need for whole genome sequences from mollusks and indeed from representatives of all major invertebrate phyla.

Animals↗
PubMed2004

Differential proteomics reveals multiple components in retrogradely transported axoplasm after nerve injury.

Information on axonal damage is conveyed to neuronal cell bodies by a number of signaling modalities, including the post-translational modification of axoplasmic proteins. Retrograde transport of a subset of such proteins is thought to induce or enhance a regenerative response in the cell body. Here we report the use of a differential 2D-PAGE approach to identify injury-correlated retrogradely transported proteins in nerves of the mollusk Lymnaea. A comprehensive series of gels at different pI ranges allowed resolution of approximately 4000 spots by silver staining, and 172 of these were found to differ between lesioned versus control nerves. Mass spectrometric sequencing of 134 differential spots allowed their assignment to over 40 different proteins, some belonging to a vesicular ensemble blocked by the lesion and others comprising an up-regulated ensemble highly enriched in calpain cleavage products of an intermediate filament termed RGP51 (retrograde protein of 51 kDa). Inhibition of RGP51 expression by RNA interference inhibits regenerative outgrowth of adult Lymnaea neurons in culture. These results implicate regulated proteolysis in the formation of retrograde injury signaling complexes after nerve lesion and suggest that this signaling modality utilizes a wide range of protein components.

Amino Acid Sequence↗
PubMed2004

From snails to sciatic nerve: Retrograde injury signaling from axon to soma in lesioned neurons.

The cell body of a lesioned neuron must receive accurate and timely information on the site and extent of axonal damage, in order to mount an appropriate response. Specific mechanisms must therefore exist to transmit such information along the length of the axon from the lesion site to the cell body. Three distinct types of signals have been postulated to underlie this process, starting with injury-induced discharge of axon potentials, and continuing with two distinct types of retrogradely transported macromolecular signals. The latter include, on the one hand, an interruption of the normal supply of retrogradely transported trophic factors from the target; and on the other hand activated proteins emanating from the injury site. These activated proteins are termed "positive injury signals", and are thought to be endogenous axoplasmic proteins that undergo post-translational modifications at the lesion site upon axotomy, which then target them to the retrograde transport system for trafficking to the cell body. Here, we summarize the work to date supporting the positive retrograde injury signal hypothesis, and provide some new and emerging proteomic data on the system. We propose that the retrograde positive injury signals form part of a complex that is assembled by a combination of different processes, including post-translational modifications such as phosphorylation, regulated and transient proteolysis, and local axonal protein synthesis.

Animals↗
PubMed2004

Neurotrophic activities of trk receptors conserved over 600 million years of evolution.

The trk family of receptor tyrosine kinases is crucial for neuronal survival in the vertebrate nervous system, however both C. elegans and Drosophila lack genes encoding trks or their ligands. The only invertebrate representative of this gene family identified to date is Ltrk from the mollusk Lymnaea. Did trophic functions of trk receptors originate early in evolution, or were they an innovation of the vertebrates? Here we show that the Ltrk gene conserves a similar exon/intron order as mammalian trk genes in the region encoding defined extracellular motifs, including one exon encoding a putative variant immunoglobulin-like domain. Chimeric receptors containing the intracellular and transmembrane domains of Ltrk undergo ligand-induced autophosphorylation followed by MAP kinase activation in transfected cells. The chimeras are internalized similarly to TrkA in PC12 cells, and their stimulation leads to differentiation and neurite extension. Knock-down of endogenous Ltrk expression compromises outgrowth and survival of Lymnaea neurons cultured in CNS-conditioned medium. Thus, Ltrk is required for neuronal survival, suggesting that trophic activities of the trk receptor family originated before the divergence of molluscan and vertebrate lineages approximately 600 million years ago.

Amino Acid Sequence↗
PubMed2004

Multi-tasking by the p75 neurotrophin receptor: sortilin things out?

Signalling by the p75 neurotrophin receptor has been implicated in diverse neuronal responses, including increased differentiation or survival, inhibition of regeneration, and initiation of apoptotic cell death. These numerous roles are matched by, but are not yet correlated with, a multiplicity of extracellular ligands and intracellular interactors. Membrane proteins such as sortilin, a member of the Vps10p family of sorting receptors, and the glycosylphosphatidylinositol-linked Nogo receptor (NgR) and the associated adaptor lingo 1 have recently been added to the list of p75-interacting modulators. Other studies have described intramembranal cleavage of p75 and the potential nuclear targeting of cleavage fragments or of the complete receptor after it has been internalized into a putative signalling endosome. These findings suggest that some of the diversity in p75 activities might be due to differential subcellular localization and transport of p75 receptor complexes. We therefore argue that cell-biology-driven approaches are now required to make sense of p75 signalling.

Adaptor Proteins, Vesicular Transport↗
PubMed2004

Integration of retrograde axonal and nuclear transport mechanisms in neurons: implications for therapeutics.

The elongated morphology of neuronal processes imposes a significant challenge for effective intracellular communication between the neurites and the cell body. This problem is especially acute upon injury, when the cell body must receive accurate and timely information on the site and extent of axonal damage to mount an appropriate response. Recent work has demonstrated that nuclear import factors from the importin (karyopherin) alpha and beta families provide a mechanism for retrograde injury signaling. Importins are found throughout axons and dendrites at significant distances from the cell body, and importin beta protein is increased after nerve lesion by local translation of axonal mRNA. This leads to formation of a high-affinity nuclear localization signal (NLS) binding complex that traffics retrogradely due to an interaction of importin alpha with the motor protein dynein. Disruption of the complex with excess NLS peptides delays regeneration of injured sensory neurons. The dual role of importins in retrograde transport in axons and nuclear import in cell bodies suggests new avenues for manipulating intrinsic regeneration mechanisms in the nervous system and may provide a novel route for drug delivery to the CNS.

Active Transport, Cell Nucleus↗
PubMed2003

Axoplasmic importins enable retrograde injury signaling in lesioned nerve.

Axoplasmic proteins containing nuclear localization signals (NLS) signal retrogradely by an unknown mechanism in injured nerve. Here we demonstrate that the importin/karyopherin alpha and beta families underlie this process. We show that importins are found in axons at significant distances from the cell body and that importin beta protein is increased after nerve lesion by local translation of axonal mRNA. This leads to formation of a high-affinity NLS binding complex that traffics retrogradely with the motor protein dynein. Trituration of synthetic NLS peptide at the injury site of axotomized dorsal root ganglion (DRG) neurons delays their regenerative outgrowth, and NLS introduction to sciatic nerve concomitantly with a crush injury suppresses the conditioning lesion induced transition from arborizing to elongating growth in L4/L5 DRG neurons. These data suggest a model whereby lesion-induced upregulation of axonal importin beta may enable retrograde transport of signals that modulate the regeneration of injured neurons.

Animals↗
PubMed2003

The prodomain of a secreted hydrophobic mini-protein facilitates its export from the endoplasmic reticulum by hitchhiking on sorting receptors.

Misfolded secretory proteins are retained in the endoplasmic reticulum (ER) by quality control mechanisms targeted to exposed hydrophobic surfaces. Paradoxically, certain conotoxins expose extensive hydrophobic surfaces upon folding to their bioactive structures. How then can such secreted mini-proteins traverse the secretory pathway? Here we show that secretion of the hydrophobic conotoxin-TxVI is strongly dependent on its propeptide domain, which enhances TxVI export from the ER. The propeptide domain interacts with sorting receptors from the sortilin Vps10p domain family. The sortilin-TxVI interaction occurs in the ER, and sortilin facilitates export of TxVI from the ER to the Golgi. Thus, the prodomain in a secreted hydrophobic protein acts as a tag that can facilitate its ER export by a hitchhiking mechanism.

Adaptor Proteins, Vesicular Transport↗
PubMed2003

Ligand-induced internalization of the p75 neurotrophin receptor: a slow route to the signaling endosome.

The nerve growth factor (NGF) family of neurotrophins binds two classes of cell-surface receptors, trk receptor tyrosine kinases and the shared p75 receptor. Rapid internalization and retrograde trafficking of neurotrophin-trk complexes have been demonstrated in a number of systems and are thought to transmit trophic signals from terminals to neuronal cell bodies. In contrast, the internalization and trafficking of neurotrophin-p75 complexes are not well understood. In this study, we used biotinylated NGF and a fluorescent-labeled anti-p75 antibody to follow the kinetics and route of ligand-induced internalization of the p75 receptor in cycling and differentiated PC12 cells. Binding of neurotrophins to p75 induced internalization at a rate approximately three times slower than that of transferrin and NGF-TrkA complexes in the same cells. The ligand-p75 complex was internalized via clathrin-coated pits into early endosomes and eventually accumulated in recycling endosomes in the cell body and vesicles colabeled by the cholera toxin B-subunit in the growth cones. Both internalized ligand and p75 were protected from proteolytic degradation and accumulated in vesicles that did not undergo acidification. Finally, NGF induced endosomal association of p75 and its MAGE interactors, necdin and NRAGE. These data suggest that signaling endosomes containing activated p75 are involved in neurotrophin signaling, and that such endosomes may be temporally and spatially distinct from those containing trk receptors.

Animals↗