M phase phosphoprotein 1 is a human plus-end-directed kinesin-related protein required for cytokinesis.

International audienceThe human M phase phosphoprotein 1 (MPP1), previously identified through a screening of a subset of proteins specifically phosphorylated at the G2/M transition (Matsumoto-Taniura, N., Pirollet, F., Monroe, R., Gerace, L., and Westendorf, J. M. (1996) Mol. Biol. Cell 7, 1455-1469), is characterized as a plus-end-directed kinesin-related protein. Recombinant MPP1 exhibits in vitro microtubule-binding and microtubule-bundling properties as well as microtubule-stimulated ATPase activity. In gliding experiments using polarity-marked microtubules, MPP1 is a slow molecular motor that moves toward the microtubule plus-end at a 0.07 microm/s speed. In cycling cells, MPP1 localizes mainly to the nuclei in interphase. During mitosis, MPP1 is diffuse throughout the cytoplasm in metaphase and subsequently localizes to the midzone to further concentrate on the midbody. MPP1 suppression by RNA interference induces failure of cell division late in cytokinesis. We conclude that MPP1 is a new mitotic molecular motor required for completion of cytokinesis

Cap-Gly Proteins at Microtubule Plus Ends: Is EB1 Detyrosination Involved?

Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids −EEY. Detyrosination (cleavage of the terminal tyrosine) of α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of α-tubulin was only barely detyrosinated. Our results indicate that detyrosination regulates CLIPs interaction with α-tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin

Tubulin tyrosination regulates synaptic function and is disrupted in Alzheimer's disease

: Microtubules play fundamental roles in the maintenance of neuronal processes and in synaptic function and plasticity. While dynamic microtubules are mainly composed of tyrosinated tubulin, long-lived microtubules contain detyrosinated tubulin, suggesting that the tubulin tyrosination/detyrosination cycle is a key player in the maintenance of microtubule dynamics and neuronal homeostasis, conditions which go awry in neurodegenerative diseases. In the tyrosination/detyrosination cycle, the C-terminal tyrosine of α-tubulin is removed by tubulin carboxypeptidases and re-added by tubulin tyrosine ligase. Here we show that tubulin tyrosine ligase hemizygous mice exhibit decreased tyrosinated microtubules, reduced dendritic spine density, and both synaptic plasticity and memory deficits. We further report decreased tubulin tyrosine ligase expression in sporadic and familial Alzheimer's disease, and reduced microtubule dynamics in human neurons harboring the familial APP-V717I mutation. Finally, we show that synapses visited by dynamic microtubules are more resistant to oligomeric amyloid β peptide toxicity and that expression of tubulin tyrosine ligase, by restoring microtubule entry into spines, suppresses the loss of synapses induced by amyloid β peptide. Together, our results demonstrate that a balanced tyrosination/detyrosination tubulin cycle is necessary for the maintenance of synaptic plasticity, is protective against amyloid β peptide-induced synaptic damage, and that this balance is lost in Alzheimer's disease, providing evidence that defective tubulin retyrosination may contribute to circuit dysfunction during neurodegeneration in Alzheimer's disease

A Sensitive and Rapid Fluorescence-Based Assay for Determination of Tetanus Toxin Peptidase Activity

International audienceThe light chain of tetanus toxin (TeNT-L chain), endowed with a zinc metalloendopeptidase activity, cleaves specifically the vesicle-associated membrane protein (VAMP), also called synaptobrevin, at a single peptide bond (Gln76-Phe77), resulting in the blockade of neuroexocytosis. The 50-mer synaptobrevin peptide S 39-88, synthesized by solid-phase peptide synthesis, was determined to be the minimum substrate of TeNT still notably hydrolyzed by TeNT-L chain. In this peptide, Tyr88 was substituted by the highly fluorescent amino acid (L) pyrenylalanine (Pya) which was synthesized in good yields by an enantioselective method. The fluorescent substrate [Pya88] S 39-88 was cleaved four times more rapidly by TeNT-L chain than S 39-88 (kcat/Km = 9635 and 2455 M-1.min-1, respectively). One of the two metabolites formed by the action of TeNT L chain, [Pya88] S 77-88, was easily separated from the substrate in one step using Sep-Pak Vac C18 cartridges and its concentration quantified by fluorescence. This novel enzymatic assay, which could be easily extended to other clostridial neurotoxins, is a major improvement in term of sensitivity and time saving, compared to currently used methods (SDS-PAGE, HPLC). It lends itself readily to automation for large-scale screening of selective and potent inhibitors of these neurotoxins which remain to be developed

Analysis of developed anti-EB1 antibodies compared to the known anti-tubulin antibodies.

<p>(A) Western-blot analysis of the indicated proteins (15 ng) separated on 10% SDS-PAGE using a commercial anti-EB1 antibody (anti-total EB1, raised against amino-acids 107–268 of mouse EB1), the presently developed antibodies (anti-Tyr EB1 and anti-deTyr EB1), and tubulin antibodies. Detyrosinated EB1 was obtained from recombinant EB1 using carboxypeptidase A. Tyrosinated and detyrosinated tubulin were obtained from purified brain tubulin, using respectively TTL and carboxypeptidase A. Both anti-Tyr EB1 and anti-deTyr EB1 are highly specific. (B) Double immunostaining with anti-total EB1 antibody and anti-Tyr EB1 on fibroblasts after transfection of plasmids allowing expression of either tyrosinated or detyrosinated EB1 with EGFP at the N-terminus. The transfected cells were detected by EGFP signal. Anti-Tyr EB1 is highly specific of tyrosinated form of EB1. (C) Immunostaining of endogenous EB1 in astrocytes with anti-Tyr EB1 and anti-total EB1.</p

Analysis of C-termini of recombinant EB1 forms overexpressed in wild type and TTL-deficient fibroblasts.

<p>Western-blot analysis of the indicated control proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033490#pone-0033490-g001" target="_blank">figure 1</a>) and of fractions of immunoprecipitation experiments carried out on cells transfected with cDNA encoding different EB1 forms. EX: crude extract; SN: supernatant after immunoprecipitation; IP: immunoprecipitated fraction. (A) transfection of fibroblasts (NIH3T3) with plasmids encoding tyrosinated EB1 fused with GFP at the N-terminus (GFP-EB1), followed by immunoprecipitation using anti-GFP antibody and analysis using anti-Tyr EB1 (1∶15000) and anti-deTyr EB1 (1∶200). No detyrosinated GFP-EB1 could be detected. (B) Transfection of fibroblasts with cDNA encoding GFP-EB1 ending with the C-terminus of α-tubulin GEEEGEEY (GFP-EB1-CterTub), followed by immunoprecipitation with anti-GFP antibody and analysis using anti-Tyr Tub (1∶20,000) and anti-deTyr Tub (1∶20,000). NIH3T3 were used as TTL^+/+ cells and MEFs isolated from TTL null mice were used as TTL^−/−. A very low quantity of detyrosinated protein ending with α-tubulin residues was detected (upper band in IP fractions of lower panel).</p

Study of endogenous EB1 C-termini in fibroblasts and brain from wild type and TTL-deficient mouse.

<p>Western-blot analysis of the indicated control proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033490#pone-0033490-g001" target="_blank">figure 1</a>) or extracts. (A) Immunoprecipitation of endogenous EB1 from wild type (TTL^+/+) or TTL-deficient MEFs using anti-total EB1 antibody, and analysis with anti-Tyr EB1 (1∶15000), anti-detyr EB1 (1∶200), and anti-total EB1 (1∶2000). EX: crude extract; SN: supernatant after immunoprecipitation; IP: immunoprecipitated fraction. No detyrosinated EB1 could be detected in the IP fractions. Note that anti-total EB1 antibody being less sensitive than anti-Tyr EB1, EB1 failed to be detected in crude extract (upper panel). (B) Immunodepletion of tyrosinated EB1 with anti-Tyr EB1 (IP 1 to 4) in brain extracts from wild type and TTL-knockout mice, followed by immunoprecipitation of the remaining EB1 with anti-total EB1 (IP5), and analysis with anti-total EB1 (1∶2000). No remaining EB1 could be detected after tyrosinated-EB1 immunodepletion. (C) Tyrosinated and detyrosinated tubulin pools in brain extracts from wild type and TTL-deficient mice were analyzed using anti-α tubulin (1∶10,000), anti-tyrosinated tubulin (YL_1/2, 1∶20,000), and anti-detyrosinated tubulin (L₄, 1∶20,000).</p