Nodes of Ranvier and Paranodes in Chronic Acquired Neuropathies

Chronic acquired neuropathies of unknown origin are classified as chronic inflammatory demyelinating polyneuropathies (CIDP) and chronic idiopathic axonal polyneuropathies (CIAP). The diagnosis can be very difficult, although it has important therapeutic implications since CIDP can be improved by immunomodulating treatment. The aim of this study was to examine the possible abnormalities of nodal and paranodal regions in these two types of neuropathies. Longitudinal sections of superficial peroneal nerves were obtained from biopsy material from 12 patients with CIDP and 10 patients with CIAP and studied by immunofluorescence and in some cases electron microscopy. Electron microscopy revealed multiple alterations in the nodal and paranodal regions which predominated in Schwann cells in CIDP and in axons in CIAP. In CIDP paranodin/Caspr immunofluorescence was more widespread than in control nerves, extending along the axon in internodes where it appeared intense. Nodal channels Nav and KCNQ2 were less altered but were also detected in the internodes. In CIAP paranodes, paranodin labeling was irregular and/or decreased. To test the consequences of acquired primary Schwann cells alteration on axonal proteins, we used a mouse model based on induced deletion of the transcription factor Krox-20 gene. In the demyelinated sciatic nerves of these mice we observed alterations similar to those found in CIDP by immunofluorescence, and immunoblotting demonstrated increased levels of paranodin. Finally we examined whether the alterations in paranodin immunoreactivity could have a diagnosis value. In a sample of 16 biopsies, the study of paranodin immunofluorescence by blind evaluators led to correct diagnosis in 70±4% of the cases. This study characterizes for the first time the abnormalities of nodes of Ranvier in CIAP and CIDP, and the altered expression and distribution of nodal and paranodal proteins. Marked differences were observed between CIDP and CIAP and the alterations in paranodin immunofluorescence may be an interesting tool for their differential diagnosis

Drosophila stathmins bind tubulin heterodimers with high and variable stoichiometries

International audienceIn vertebrates, stathmins form a family of proteins possessing two tubulin binding repeats (TBRs), which each binds one soluble tubulin heterodimer. The stathmins thus sequester two tubulins in a phosphorylation-dependent manner, providing a link between signal transduction and microtubule dynamics. In Drosophila, we show here that a single stathmin gene (stai) encodes a family of D-stathmin proteins. Two of the D-stathmins are maternally deposited and then restricted to germ cells, and the other two are detected in the nervous system during embryo development. Like in vertebrates, the nervous system-enriched stathmins contain an N-terminal domain involved in subcellular targeting. All the D-stathmins possess a domain containing three or four predicted TBRs, and we demonstrate here, using complementary biochemical and biophysical methods, that all four predicted TBR domains actually bind tubulin. D-stathmins can indeed bind up to four tubulins, the resulting complex being directly visualized by electron microscopy. Phylogenetic analysis shows that the presence of regulated multiple tubulin sites is a conserved characteristic of stathmins in invertebrates and allows us to predict key residues in stathmin for the binding of tubulin. Altogether, our results reveal that the single Drosophila stathmin gene codes for a stathmin family similar to the multigene vertebrate one, but with particular tubulin binding properties

Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain

International audienceMicrotubules are cytoskeletal polymers of tubulin involved in many cellular functions. Their dynamic instability is controlled by numerous compounds and proteins, including colchicine and stathmin family proteins. The way in which microtubule instability is regulated at the molecular level has remained elusive, mainly because of the lack of appropriate structural data. Here, we present the structure, at 3.5 A resolution, of tubulin in complex with colchicine and with the stathmin-like domain (SLD) of RB3. It shows the interaction of RB3-SLD with two tubulin heterodimers in a curved complex capped by the SLD amino-terminal domain, which prevents the incorporation of the complexed tubulin into microtubules. A comparison with the structure of tubulin in protofilaments shows changes in the subunits of tubulin as it switches from its straight conformation to a curved one. These changes correlate with the loss of lateral contacts and provide a rationale for the rapid microtubule depolymerization characteristic of dynamic instability. Moreover, the tubulin-colchicine complex sheds light on the mechanism of colchicine's activity: we show that colchicine binds at a location where it prevents curved tubulin from adopting a straight structure, which inhibits assembly

A synergistic relationship between three regions of stathmin family proteins is required for the formation of a stable complex with tubulin.

Stathmin is a ubiquitous 17 kDa cytosolic phosphoprotein proposed to play a general role in the integration and relay of intracellular signalling pathways. It is believed to regulate microtubule dynamics by sequestering tubulin in a complex made of two tubulin heterodimers per stathmin molecule (T2S complex). The other proteins of the stathmin family can also bind two tubulin heterodimers through their SLD (stathmin-like domain), but the different tubulin:SLD complexes display varying stabilities. In this study, we analysed the relative influence of three regions of SLDs on the interaction with tubulin and the mechanistic processes that lead to its sequestration. Tubulin-binding properties of fragments and chimaeras of stathmin and RB3(SLD) were studied in vitro by tubulin polymerization, size-exclusion chromatography and surface plasmon resonance assays. Our results show that the N-terminal region of SLDs favours the binding of the first tubulin heterodimer and that the second C-terminal tubulinbinding site confers the specific stability of a given tubulin:SLD complex. Our results highlight the molecular processes by which tubulin co-operatively interacts with the SLDs. This knowledge may contribute to drug development aimed at disturbing microtubules that could be used for the treatment of cancer

Alpha-fetoprotein binding and uptake by primary cultures of human skeletal muscle

α-Fetoprotein (AFP), a serum a-globulin mainly synthesized by the fetal liver and the yolk sac, is the major carrier of polyunsaturated fatty acids during embryo-fetal development. One property characteristic of fetal cells undergoing growth and differentiation is their ability to bind and internalize AFP. In the present work, we have studied the binding and endocytosis of AFP by human muscular cells developing in vitro. Primary cultures of human skeletal muscle, obtained from biopsies and examined at two stages of differentiation (myoblasts and myotubes), were incubated for different times, at 0 and 37°C, with a colloidal-gold-conjugated human AFP probe and studied by light and electron microscopy, as well as by laser scanning confocal microscopy in the reflection mode. The results obtained show that (a) human myoblasts in primary culture bind and internalize the protein, probably through specific AFP receptors, (b) this property is strongly reduced or lost in well-differentiated myotubes, and (c) AFP is also bound, throughout culture development, to the extracellular matrix of fusing myoblasts and differentiated myotubes. The physiological significance of AFP uptake by human myoblasts undergoing growth and differentiation may be based on the ability of AFP to carry and deliver fatty acids to fetal cells.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Identification of Small Molecules Affecting the Secretion of Therapeutic Antibodies with the Retention Using Selective Hook (RUSH) System

Unlocking cell secretion capacity is of paramount interest for the pharmaceutical industry focused on biologics. Here, we leveraged retention using a selective hook (RUSH) system for the identification of human osteosarcoma U2OS cell secretion modulators, through automated, high-throughput screening of small compound libraries. We created a U2OS cell line which co-expresses a variant of streptavidin addressed to the lumen-facing membrane of the endoplasmic reticulum (ER) and a recombinant anti-PD-L1 antibody. The heavy chain of the antibody was modified at its C-terminus, to which a furin cleavage site, a green fluorescent protein (GFP), and a streptavidin binding peptide (SBP) were added. We show that the U2OS cell line stably expresses the streptavidin hook and the recombinant antibody bait, which is retained in the ER through the streptavidin–SBP interaction. We further document that the addition of biotin to the culture medium triggers the antibody release from the ER, its trafficking through the Golgi where the GFP-SBP moiety is clipped off, and eventually its release in the extra cellular space, with specific antigen-binding properties. The use of this clone in screening campaigns led to the identification of lycorine as a secretion enhancer, and nigericin and tyrphostin AG-879 as secretion inhibitors. Altogether, our data support the utility of this approach for the identification of agents that could be used to improve recombinant production yields and also for a better understanding of the regulatory mechanism at work in the conventional secretion pathway

N-Terminal Stathmin-like Peptides Bind Tubulin and Impede Microtubule Assembly

International audienceMicrotubules are major cytoskeletal components involved in numerous cellular functions such as mitosis, cell motility, or intracellular traffic. These cylindrical polymers of alphabeta-tubulin assemble in a closely regulated dynamic manner. We have shown that the stathmin family proteins sequester tubulin in a nonpolymerizable ternary complex, through their stathmin-like domains (SLD) and thus contribute to the regulation of microtubule dynamics. We demonstrate here that short peptides derived from the N-terminal part of SLDs impede tubulin polymerization with various efficiencies and that phosphorylation of the most potent of these peptides reduces its efficiency as in full-length stathmin. To understand the mechanism of action of these peptides, we undertook a NMR-based structural analysis of the peptide-tubulin interaction with the most efficient peptide (I19L). Our results show that, while disordered when free in solution, I19L folds into a beta-hairpin upon binding to tubulin. We further identified, by means of saturation transfer difference NMR, hydrophobic residues located on the beta2-strand of I19L that are involved in its tubulin binding. These structural data were used together with tubulin atomic coordinates from the tubulin/RB3-SLD crystal structure to model the I19L/tubulin interaction. The model agrees with I19L acting through an autonomous tubulin capping capability to impede tubulin polymerization and provides information to help understand the variation of efficiency against tubulin polymerization among the peptides tested. Altogether these results enlighten the mechanism of tubulin sequestration by SLDs, while they pave the way for the development of protein-based compounds aimed at interfering with tubulin polymerization