Search

Rationale: We have previously shown that angiomotin (Amot) is essential for endothelial cell migration during mouse embryogenesis. However, approximately 5% of Amot knockout mice survived without any detectable vascular defects. Angiomotin-like protein 1 (AmotL1) potentially compensates for the absence of Amot as it is 62% homologous to Amot and exhibits similar expression pattern in endothelial cells., Objective: Here, we report the identification of a novel isoform of AmotL1 that controls endothelial cell polarization and directional migration., Methods and Results: Small interfering RNA-mediated silencing of AmotL1 in mouse aortic endothelial cells caused a significant reduction in migration. In confluent mouse pancreatic islet endothelial cells (MS-1), AmotL1 colocalized with Amot to tight junctions. Small interfering RNA knockdown of both Amot and AmotL1 in MS-1 cells exhibited an additive effect on increasing paracellular permeability compared to that of knocking down either Amot or AmotL1, indicating both proteins were required for proper tight junction activity. Moreover, as visualized using high-resolution 2-photon microscopy, the morpholino-mediated knockdown of amotl1 during zebrafish embryogenesis resulted in vascular migratory defect of intersegmental vessels with strikingly decreased junction stability between the stalk cells and the aorta. However, the phenotype was quite distinct from that of amot knockdown which affected polarization of the tip cells of intersegmental vessels. Double knockdown resulted in an additive phenotype of depolarized tip cells with no or decreased connection of the stalk cells to the dorsal aorta., Conclusions: These results cumulatively validate that Amot and AmotL1 have similar effects on endothelial migration and tight junction formation in vitro. However, in vivo Amot appears to control the polarity of vascular tip cells whereas AmotL1 mainly affects the stability of cell-cell junctions of the stalk cells.

To further characterize the molecular events supporting the tumor suppressor activity of Scrib in mammals, we aim to identify new binding partners. We isolated MCC, a recently identified binding partner for beta-catenin, as a new interacting protein for Scrib. MCC interacts with both Scrib and the NHERF1/NHERF2/Ezrin complex in a PDZ-dependent manner. In T47D cells, MCC and Scrib proteins colocalize at the cell membrane and reduced expression of MCC results in impaired cell migration. By contrast to Scrib, MCC inhibits cell directed migration independently of Rac1, Cdc42 and PAK activation. Altogether, these results identify MCC as a potential scaffold protein regulating cell movement and able to bind Scrib, beta-catenin and NHERF1/2.

Controlled regulation of Rho GTPase activity is an essential component mediating growth factor-stimulated migration. We have previously shown that angiomotin (Amot), a membrane-associated scaffold protein, plays a critical role during vascular patterning and endothelial migration during embryogenesis. However, the signaling pathways by which Amot controls directional migration are not known. Here we have used peptide pull-down and yeast 2-hybrid (Y2H) screening to identify proteins that interact with the C-terminal PDZ-binding motifs of Amot and its related proteins AmotL1 and 2. We report that Amot and its related proteins bind to the RhoA GTPase exchange factor (RhoGEF) protein Syx. We show that Amot forms a ternary complex together with Patj (or its paralogue Mupp1) and Syx. Using FRET analysis, we provide evidence that Amot controls targeting of RhoA activity to lamellipodia in vitro. We also report that, similar to Amot, morpholino knockdown of Syx in zebrafish results in inhibition of migration of intersegmental arteries. Taken together, our results indicate that the directional migration of capillaries in the embryo is governed by the Amot:Patj/Mupp1:Syx signaling that controls local GTPase activity.

Genetic studies have highlighted the key role of Scrib in the development of Metazoans. Deficiency in Scrib impairs many aspects of cell polarity and cell movement although the mechanisms involved remain unclear. In mammals, Scrib belongs to a protein complex containing betaPIX, an exchange factor for Rac/Cdc42, and GIT1, a GTPase activating protein for ARF6 implicated in receptor recycling and exocytosis. Here we show that the Scrib complex associates with PAK, a serine-threonine kinase family crucial for cell migration. PAK colocalizes with members of the Scrib complex at the leading edge of heregulin-treated T47D breast cancer cells. We demonstrate that the Scrib complex is required for epithelial cells and primary mouse embryonic fibroblasts to efficiently respond to chemoattractant cues. In Scrib-deficient cells, the pool of cortical PAK is decreased, thereby precluding its proper activation by Rac. Loss of Scrib also impairs the polarized distribution of active Rac at the leading edge and compromises the regulated activation of the GTPase in T47D cells and mouse embryonic fibroblasts. These data underscore the role of Scrib in cell migration and show the strong impact of Scrib in the function of PAK and Rac, two key molecules implicated in this process.

This prospective pilot study investigates the possibility of materno-fetal transmission of human coronaviruses (HCoV) responsible for cases of neonatal infection. This vertical transmission was studied with 159 samples from mother-child couples: maternal vaginal (MV) and respiratory (MR) samples during labor; and newborn gastric sample (NG) with detection of HCoV (229E, OC-43, NL-63, HKU1) via real time RT PCR. HCoV was detected in 12 samples (229E: 11; HKU1: 1) from seven mother-child couples. For three couples, only MR tested positive (cases 1-3). For two other couples all three samples (MV, MR and NG) tested positive (cases 4 and 5). For case 6, only MV and NG tested positive. In case 7, only MV was positive. Possible vertical transmission of HCoV was hypothesized in this pilot study and requires further investigation on a larger scale.

ICOS ligation in concert with TCR stimulation results in strong PI3K activation in T lymphocytes. The ICOS cytoplasmic tail contains an YMFM motif that binds the p85alpha subunit of class IA PI3K, similar to the YMNM motif of CD28, suggesting a redundant function of the two receptors in PI3K signaling. However, ICOS costimulation shows greater PI3K activity than CD28 in T cells. We show in this report that ICOS expression in activated T cells triggers the participation of p50alpha, one of the regulatory subunits of class IA PI3Ks. Using different T-APC cell conjugate systems, we report that p50alpha accumulates at the immunological synapse in activated but not in resting T cells. Our results demonstrate that ICOS membrane expression is involved in this process and that p50alpha plasma membrane accumulation requires a functional YMFM Src homology 2 domain-binding motif in ICOS. We also show that ICOS triggering with its ligand, ICOSL, induces the recruitment of p50alpha at the synapse of T cell/APC conjugates. In association with the p110 catalytic subunit, p50alpha is known to carry a stronger lipid kinase activity compared with p85alpha. Accordingly, we observed that ICOS engagement results in a stronger activation of PI3K. Together, these findings provide evidence that p50alpha is likely a determining factor in ICOS-mediated PI3K activity in T cells. These results also suggest that a differential recruitment and activity of class IA PI3K subunits represents a novel mechanism in the control of PI3K signaling by costimulatory molecules.

Molecular subtypes of breast cancer with relevant biological and clinical features have been defined recently, notably ERBB2-overexpressing, basal-like, and luminal-like subtypes. To investigate the ability of mass spectrometry-based proteomics technologies to analyze the molecular complexity of human breast cancer, we performed a SELDI-TOF MS-based protein profiling of human breast cell lines (BCLs). Triton-soluble proteins from 27 BCLs were incubated with ProteinChip arrays and subjected to SELDI analysis. Unsupervised global hierarchical clustering spontaneously discriminated two groups of BCLs corresponding to "luminal-like" cell lines and to "basal-like" cell lines, respectively. These groups of BCLs were also different in terms of estrogen receptor status as well as expression of epidermal growth factor receptor and other basal markers. Supervised analysis revealed various protein biomarkers with differential expression in basal-like versus luminal-like cell lines. We identified two of them as a carboxyl terminus-truncated form of ubiquitin and S100A9. In a small series of frozen human breast tumors, we confirmed that carboxyl terminus-truncated ubiquitin is observed in primary breast samples, and our results suggest its higher expression in luminal-like tumors. S100A9 up-regulation was found as part of the transcriptionally defined basal-like cluster in DNA microarrays analysis of human tumors. S100A9 association with basal subtypes as well as its poor prognosis value was demonstrated on a series of 547 tumor samples from early breast cancer deposited in a tissue microarray. Our study shows the potential of integrated genomics and proteomics profiling to improve molecular knowledge of complex tumor phenotypes and identify biomarkers with valuable diagnostic or prognostic values.

Unlabelled: Human coronaviruses (HCoV) have been implicated in neonatal nosocomial respiratory infection. Prior to our study, several cases of neonatal infection were observed in infants born at our hospital. This prospective pilot monocentric pilot study investigates the possibility of maternofetal transmission of HCoV responsible for cases of neonatal infection observed within the first 24 hours of life., Materials and Methods: Three samples from mother-child couples, maternal vaginal (VM) and respiratory (RM) samples during labor; newborn gastric sample (GNN), were assessed for viral analysis using real time RT-PCR for the detection of HCoV 229-E and OC43. Clinical follow-up of infants and mothers was up to Day 3 after birth., Results: One hundred (and) fifty-nine mother-child couples were included between July 2003 and August 2005. HCoV 229-E only was detected in 11 samples from 6 mother-child couples. For 2 couples, all 3 samples (VM, RM and GNN) were tested positive (cases 1 and 2). For case 3, both VM and GNN were positive. For 2 couples, only RM was positive (cases 4 and 5). In case 6, only VM was positive. Of the 3 positive GNN, no infant was symptomatic., Conclusion: Possible vertical transmission of HCoV was evidenced in this pilot study and requires further investigation on a larger scale. Equally indicated is the inclusion of tests to detect recently identified human coronaviruses HCoV NL63 and HKU1, as well as genomic profile analysis of HCoV 229-E detected in the 3 positive mother-child couples.

Background: Isolated tears of the subscapularis occur less commonly than those involving the superior and posterior components of the rotator cuff. The purpose of the present study was to evaluate the structural integrity and clinical outcomes after arthroscopic repair of isolated subscapularis tears., Methods: A prospective study of seventeen consecutive patients who were managed with an all-arthroscopic repair of the subscapularis tendon was performed. The study group included thirteen men and four women who had an average age of forty-seven years at the time of surgery. The average interval from the onset of symptoms to the time of surgery was twenty-four months. Thirteen tears were traumatic, and four were degenerative. Seven patients had a tear involving the superior third of the tendon, six had a tear involving the superior two-thirds of the tendon, and four had complete separation of the subscapularis from its insertion on the lesser tuberosity. Clinical findings were assessed for all patients preoperatively and postoperatively with use of the Constant and University of California at Los Angeles scoring systems, and all patients had postoperative computed tomographic arthrography studies to evaluate the structural integrity of the repair., Results: The average duration of follow-up was twenty-nine months. When the preoperative findings were compared with the most recent findings, the average relative Constant score had improved from 58% to 96% (p < 0.05), the average University of California at Los Angeles score had improved from 16 to 32 points (p < 0.05), the average pain score had improved from 5.9 to 13.5 points (p < 0.05), the average forward flexion had improved from 146 degrees to 175 degrees (p < 0.05), the average external rotation had improved from 50 degrees to 60.3 degrees (p < 0.05), the average internal rotation had improved from the level of the sacrum to L1-L2 (p < 0.05), and the average abduction strength had improved from 7.4 to 15.6 points (p < 0.05). The structural integrity of the repair was completely intact in fifteen patients and was partially reruptured in two patients on the basis of computed tomographic arthrography. Progression of fatty infiltration of the subscapularis was not observed in any patient. Subjectively, twelve patients were very satisfied with the result, four were satisfied, and one was not satisfied., Conclusions: Arthroscopic repair of an isolated subscapularis tear can yield marked improvements in shoulder function, can significantly reduce pain, and can result in a durable structural repair., Level of Evidence: Therapeutic Level IV.

We report clinical and molecular findings in 20 patients from 11 families with autosomal recessive congenital ichthyosis (ARCI) linked to chromosome 17p13, and attributed to mutations in the ALOX gene cluster, which includes three lipoxygenase genes, ALOXE3, ALOX12B, and ALOX15B. We identified six novel missense mutations and one novel deletion leading to a premature stop codon in ALOX12B in only six out of the 11 families which led us to investigate a possible implication of ALOX15B. Mutation analysis of this gene, as well as ALOXE3, which is known to be mutated in some cases of ARCI, failed to reveal causative mutations in the five remaining ARCI families, indicating that other genes on chromosome 17p13 may be involved in this disease. However, by adding new variants to the repertoire of ALOX12B mutations in non-bullous congenital ichthyosiform erythroderma, our data contribute to an enlargement of the spectrum of mutations for the development of efficient molecular genetic tests for analysis of at risk individuals whose carrier status is unknown.

In Drosophila, the tumor suppressor Scribble is localized at the septate junctions of epithelial cells. Its mammalian homologue, hScrib, is a basolateral protein likely associated to proteins of the cell-cell junctions. We report the direct interaction between hScrib and ZO-2, a junction-associated protein. This interaction relies on two PDZ domains of hScrib and on the C-terminal motif of ZO-2. Both proteins localise at cell-cell junctions of epithelial cells. A point mutation in the LRR of hScrib delocalises the protein from the plasma membrane and abrogates the interaction with ZO-2 but not with betaPIX. Tyrosine phosphorylation of hScrib does not impair the interaction with ZO-2. We show a direct link between two junctional proteins that are down-regulated during cancer progression.

Members of the LAP protein family, LET-413 in Caenorhabditis elegans, Scribble in Drosophila melanogaster, and Erbin, Lano, Densin-180 and hScrib in mammals, have conserved structural features. LET-413 and Scribble are junctional proteins involved in establishing and maintaining epithelial cell polarity. scribble also behaves as a neoplastic tumor suppressor gene. We show here that, in epithelial cells, hScrib is recruited at cell-cell junctions in an E-cadherin-dependent manner as shown by calcium switch assays in MDCK cells, re-expression of E-cadherin in MDA-231 cells treated by 5-Aza-2'-deoxycytidine (5Aza), and siRNA experiments. hScrib is restricted at the basolateral membrane of epithelial cells by its LRR domain, and is enriched in Triton X-100-insoluble fractions. In breast cancers, most lobular tumors did not express hScrib and E-cadherin while ductal tumors had a less frequent downregulation of hScrib. Our data provide additional insights on the modalities of recruitment of hScrib at the cell-cell junctions, and establish a potential link between the E-cadherin and hScrib tumor suppressors.

Purpose of the Study: Appropriate treatment of irreparable rotator cuff tears in patients without osteoarthritic shoulder joints remains a subject of debate. Medical treatment, a substitution muscle flap, and palliative arthroscopic treatment have been proposed. Arthroscopic tenotomy of the long head of the biceps brachii is warranted because this tendon is often the cause of part or all of the pain. If there is a full thickness tear of the rotator cuff, the exposed tendon of the long head of the biceps brachii can, because of its anterosuperior position, become impinged against the acromial vault during forward flexion. The purpose of this work was to evaluate the mid-term clinical and radiological results of arthroscopic tenotomy of the long head of the biceps brachii during treatment of full thickness tears of the rotator cuff., Material and Methods: The series included 40 shoulders operated on for tenotomy alone (n=32) or in combination with acromioplasty (n=8). The long head of the biceps brachii was in place in 23 shoulders (58%), displaced in seven and subluxed in five. The position was not determined in five. At last follow-up, the mean rough Constant score was 58 points, giving a gain of 20 points. The gain for pain was +7.1 points, +6.4 points for activity, and +6.6 points for motion. After the operation, muscle force for elbow flexion-supination was decreased 40% compared with an age-, sex- and dominance-matched control group. 86% of the patients were satisfied with the outcome and only two patients were disappointed by the asymmetry of arm muscle volume. Radiographically, at last follow-up there were no signs of superior excentration of the humeral head and the subacromial space, which measured 7.38 mm preoperatively was 7.19 mm postoperatively. Likewise only two shoulders progressed to excentered osteoarthritis at 41 and 72 months., Discussion: Mid-term results of arthroscopic tenotomy of the long head of the biceps brachii are satisfactory. The technique is simple and has limited functional consequences. The procedure has an undeniable impact on pain and has allowed a 34 degree gain in anterior flexion of the shoulder. Complementary acromioplasty was not found to provide a supplementary benefit in this series. Nevertheless, the degradation of the result in one female patient at six years suggests we should be prudent concerning the long-term benefit of this procedure which should be reserved for irreparable tears in patients with minimal functional demands.

Drosophila Scribble is implicated in the development of normal synapse structure and epithelial tissues, but it remains unclear how it plays a role and which process it controls. The mammalian homolog of Scribble, hScrib, has a primary structure and subcellular localization similar to that of its fly homolog, but its function remains unknown. Here we have used tandem mass spectrometry to identify major components of the hScrib network. We show that it includes betaPIX (also called Cool-1), a guanine nucleotide exchange factor (GEF), and its partner GIT1 (also called p95-APP1), a GTPase activating protein (GAP). betaPIX directly binds to the hScrib PDZ domains, and the hScrib/betaPIX complex is efficiently recovered in epithelial and neuronal cells and tissues. In cerebellar granule cell cultures, hScrib and betaPIX are both partially localized at neuronal presynaptic compartments. Furthermore, we show that hScrib is required to anchor betaPIX at the cell cortex and that dominant-negative betaPIX or hScrib proteins can each inhibit Ca2+-dependent exocytosis in neuroendocrine PC12 cells, demonstrating a functional relationship between these proteins. These data reveal the existence of a tight hScrib/betaPIX interaction and suggest that this complex potentially plays a role in neuronal transmission., (Copyright 2004 Elsevier Ltd.)

Nine cases of acquired metatarus elevatus or horizontalization of the first metatarsal with hallux flexus (dorsal bunion) were treated surgically associating: plantar wedge resection of the base of the first metatarsal or the first cuneiform; distal disinsertion of the long hallux flexor which was then positioned under the base of the first metatarsal and finally fixed on the distal dorsal segment of the metatarsophalangeal capsule; distal disinsertion of the anterior tibial tendon and tenodesis of the posterior tibial tendon. Weight bearing was allowed after pinning for one Month to position the axis of the first ray. Morphological results, recorded at 11 Years follow-up (mean) were satisfactory. There were no recurrent deformations and no residual instability of the first ray. The only observation was a minimal stiffness of the metatarsophalangeal joint with no tendency to degeneration.

Lamellar ichthyosis type 2 (LI2) is a rare autosomal recessive skin disorder for which a gene has been localized on chromosome 2q33-35. We report the identification of five missense mutations in the ABCA12 gene in nine families from Africa affected by LI2. The mutations were homozygous in eight consanguineous families and heterozygous in one non-consanguineous family. Four of these mutations are localized in the first ATP-binding domain (nucleotide-binding fold), which is highly conserved in all ABC proteins. The ABCA12 protein belongs to a superfamily of membrane proteins that translocate a variety of substrates across extra- and intracellular membranes. ABCA transporters have been implicated in several autosomal recessive disorders, notably of lipid metabolism. By analogy with ABCA3, a lamellar body membrane protein in lung alveolar type II cells, ABCA12 could function in cellular lipid trafficking in keratinocytes.

Mal de Meleda is a recessive, transgressive palmoplantar keratoderma for which we previously identified mutations in the gene encoding secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP-1). In this report we describe two new mutations: (i) a founder mutation, which changes a conserved cysteine residue to tyrosine (C99Y) in a large inbred Tunisian pedigree, and (ii) a signal sequence mutation (W15R), which was homozygous in a German family and heterozygous in a Scottish patient. Four ancestral haplotypes were observed in 69 patients from countries around the Mediterranean basin, and an additional haplotype was found in the German and Scottish patients.

Protein networks asymetrically distributed to basolateral and apical epithelial membranes maintain cell polarity and homeostasis of epithelial tissues. Genetic studies in non-vertebrates assigned two families of basolateral proteins, MAGUK (membrane-associated and guanylate kinase) and LAP (leucine-rich repeats and PDZ) proteins, to a common pathway crucial for the epithelial architecture and acting as a gatekeeper to malignancy. In mammals, three LAP proteins have been described, Densin-180, Erbin, and hScribble. Here, we identify a protein called Lano (LAP and no PDZ) only present in vertebrates and presenting strong identities with LAP proteins. Despite the lack of PDZ domain, Lano is located at the basolateral side of epithelial cells in a similar manner to Erbin and hScribble. Using in vitro and in vivo experiments, we demonstrate that Lano directly interacts with the PDZ domains of MAGUK proteins, including hDLG (human disc large), in epithelial cells. A second pool of Lano is complexed to Erbin. These LAP-MAGUK protein complexes coexist at the basolateral side of epithelial cells. We provide evidence for a direct interaction between LAP and MAGUK proteins, and we propose that various LAP-MAGUK networks targeted to the basolateral side of epithelial cells participate to homeostasis of epithelial tissues and tumor growth.

Flagellar motility is the result of specific interactions between axonemal microtubular proteins and the dynein motors. Tubulin, the main component of microtubule, is a very polymorphic protein resulting from the expression of several isogenes and from the existence of various post-translational modifications. In order to characterize tubulin isoforms and tubulin domains that are important for flagellar movement, we prepared monoclonal antibodies against axonemal proteins from whole sea-urchin sperm tails. The monoclonal antibodies obtained were screened for their potency to inhibit demembranated-reactivated sperm models and for their monospecific immunoreactivity on immunoblot. Among the different antibodies we obtained, D66 reacted specifically with a subset of beta-tubulin isoforms. Limited proteolysis, HPLC, peptide sequencing, mass spectroscopy and immunoblotting experiments indicated that D66 recognized an epitope localized in the primary sequence Gln423-Glu435 of the C-terminal domain of Lytechinus pictus beta2-tubulin, and that this sequence belongs to class IVb. The use of synthetic peptides and immunoblotting analysis further narrowed the amino acids important for antibody recognition to Asp427-Glu432. Because the primary effect of this antibody on sperm motility is to decrease the flagellar beat frequency, we suggest that this sequence is involved in the tubulin-dynein head interaction.

In this work, we report on a novel enzyme, tubulin polyglutamylase, which catalyzes the posttranslational formation of polyglutamyl side chains onto alpha- and beta-tubulin. The length of the polyglutamyl side chain regulates the interaction between tubulin and various microtubule-associated proteins. We first developed an in vitro glutamylation assay. Activity measured in brain, a tissue particularly enriched with glutamylated tubulin, decreases during postnatal development. Thus, brains from 3-day-old mice were chosen as the starting material, and the enzyme was purified approximately 1000-fold. Its Mr was estimated to be 360K and its sedimentation coefficient 10 s. The enzyme catalyzes the MgATP-dependent addition of l-glutamate onto tubulin subunits. Microtubules are much better substrates than unpolymerized tubulin, and the reaction is very specific for glutamate, other amino acids or glutamate analogues not being substrates. Moreover, glutamyl units are added sequentially onto tubulin, leading to progressive elongation of the polyglutamyl side chains. Side chains of one to six or seven glutamyl units were obtained with microtubules, whereas much longer side chains (up to 15-20 units) were formed with unpolymerized tubulin. Interestingly, such very long polyglutamyl side chains were recently detected in some situations in vivo.

In neuronal cells, microtubules are built from a very large number of alpha- and beta-tubulin variants. This diversity is due to the expression of a multigene family and to a combination of several original posttranslational modifications. Similarly, structural and motor microtubule-associated proteins, which regulate the assembly of microtubules, the modeling of their network and the mediation of their functions, are also very heterogeneous. As a consequence, mixing of these two protein polymorphisms leads to the formation of functionally-distinct microtubules. We have shown that polyglutamylation, the major posttranslational modification of neuronal tubulin, was used as a progressive regulator in the binding of structural and motor microtubule-associated proteins, in modulating gradually the conformation of the tubulin carboxy-terminal domain, playing thus a crucial role in microtubule dynamics.

A panel of monoclonal antibodies (mAbs) has been generated against sea urchin sperm axonemes and selected for their ability to inhibit the motility of sea urchin sperm models. The mAb C9 recognized a 50 kDa protein on blots of sea urchin sperm axonemes. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that C9 recognized isoforms of beta-tubulin. Low concentrations of C9 (0.1-1.0 microgram/ml) blocked the motility of sea urchin sperm models by decreasing the sliding velocity and frequency of flagellar beating to less than 1 Hz and by modifying the shear angle along the axoneme, especially the distal end. Other antitubulin antibodies had little effect on motility at concentrations 100-fold higher than those effective for C9. The effects on motility were not restricted to flagella of sea urchin spermatozoa. Flagellar beating of the dinoflagellate Oxyrrhis marina was completely blocked by C9 in a manner reminiscent of that of sea urchin sperm flagella. The mAb also inhibited the motility of human spermatozoa and Chlamydomonas reinhardtii. Immunofluorescence techniques revealed that C9 stains the whole axoneme of sea urchin spermatozoa and O. marina flagella together with the cortical network of O. marina cell body. C9 is the first antitubulin antibody reported to interfere with flagellar beat frequency. The observation that this antibody arrests the motility of flagella from sea urchin sperm along with that of dinoflagellate, algae, and human sperm flagella suggests that the epitope recognized by C9 is conserved over a long period of evolution and plays an important role in sperm motility.

Polyglutamylation is an important posttranslational modification of tubulin that is very active in nerve cells, where it accounts for the main factor responsible for tubulin heterogeneity. In the present work, we have analyzed quantitative and qualitative changes in glutamylated alpha- and beta-tubulin occurring during neuronal differentiation in culture. Glutamylated alpha- and beta-tubulin both markedly accumulate during this process with a time course remarkably similar to that observed in vivo during brain development. However, the characteristics of the glutamylation of the two subunits are not exactly the same. Glutamylated alpha-tubulin is already abundant in very young neurons and displays, at this stage, a wide range of its degree of glutamylation (1 to 6 glutamyl units present in the lateral polyglutamyl chain), which remains unchanged during the entire period of the culture. Glutamylated beta-tubulin is present at very low levels in young neurons and its accumulation during differentiation is accompanied by a progressive increase in its degree of glutamylation from 2 to 6 glutamyl units. Posttranslational incorporation of [3H]glutamate into alpha- and beta-tubulin decreases during differentiation, as well as the rate of the reverse deglutamylation reaction, suggesting that accumulation of glutamylated tubulin is accompanied by a decrease in the turnover of glutamyl units onto tubulin. Neuronal differentiation is also accompanied by an increase of other posttranslationally modified forms of tubulin, including acetylated and non-tyrosinatable alpha-tubulin, which can occur in combination with polyglutamylation and contributes to increase the complexity of tubulin in mature neurons.

The relationship between microtubule dynamics and polyglutamylation of tubulin was investigated in young differentiating mouse brain neurons. Selective posttranslational labeling with [3H]glutamate and immunoblotting with a specific monoclonal antibody (GT335) enabled us to analyze polyglutamylation of both alpha and beta subunits. Nocodazole markedly inhibited incorporation of [3H]glutamate into alpha- and beta-tubulin, whereas taxol had no effect for alpha-tubulin and a stimulating effect for beta-tubulin. These results strongly suggest that microtubule polymers are the preferred substrate for polyglutamylation. Chase experiments revealed the existence of a reversal reaction that, in the case of alpha-tubulin, was not affected by microtubule drugs, suggesting that deglutamylation of this subunit can occur on both polymers and soluble tubulin. Evidence was obtained that deglutamylation of alpha-tubulin operates following two distinct rates depending on the length of the polyglutamyl chain, the distal units (4th-6th) being removed rapidly whereas the proximal ones (1st-3rd) appearing much more resistant to deglutamylation. Partition of glutamylated alpha-tubulin isoforms was also correlated with the length of the polyglutamyl chain. Forms bearing four to six units were recovered specifically in the polymeric fraction, whereas those bearing one to three units were distributed evenly between polymeric and soluble fractions. It thus appears that the slow rate component of the deglutamylation reaction offers to neurons the possibility to maintain a basal level of glutamylated alpha-tubulin in the soluble pool independently of microtubule dynamics. Finally, some differences observed in the glutamylation of alpha- and beta-tubulin suggest that distinct enzymes are involved.

A monoclonal antibody (GT335) directed against polyglutamylated tubulin was obtained by immunization with a synthetic peptide which mimics the structure of the polyglutamylated site of alpha-tubulin. This peptide corresponds to the C-terminal sequence Glu441-Gly448 and was chemically modified by the addition of two glutamyl units at Glu445. The specificity of GT335 was assayed by direct and competitive enzyme-linked immunosorbent assay (ELISA) against tubulin and several synthetic peptides differing either by the structure of the added polyglutamyl chain or by their amino acid sequence. Further characterization was carried out by immunoblotting detection after one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The epitope appears to be formed by at least two constituents: a basic motif of monoglutamylation which is retained in the polyglutamylated forms independent of their degree of glutamylation, and some elements of the polypeptide chain close to the site of glutamylation. Given the specificity of GT335 and the delineation of its epitope, our results indicate that, in addition to alpha and beta' (class III)-tubulin, other beta-tubulin isotypes are also glutamylated. This antibody has been used to analyze the cell and tissue distributions of glutamylated tubulin. In mouse brain extracts, GT335 reacts strongly with alpha-tubulin and, to a lesser extent, with beta' (class III) and beta-tubulin. The same reactivity is also observed with cultured neurons whereas astroglial cells exhibit only low levels of glutamylated tubulin. In non-nervous mouse tissues such as spleen, lung or testis, glutamylation was shown to involve only beta-tubulin, but at far lower levels than in brain.