Following injection into recombinase-activating gene-deficient (Rag1(-/-)) mice, pro-B cells lacking the Pax5 transcription factor (Pax5(-/-)) develop into most major hematopoietic lineages, with the notable exception of B cells. We assessed whether Pax5(-/-) pro-B cells that were also rendered deficient for the linker for activation of T cells (LAT), an adaptor essential for T cell receptor signaling, can be used for the rapid in vivo analysis of mutant forms of LAT. We showed that Pax5(-/-) Lat(-/-) pro-B cell lines can be infected with recombinant retroviruses expressing a LAT cDNA and sorted for the expression of LAT. When injected into Rag1(-/-) mice, they restore normal intrathymic T cell development and give rise to functional peripheral T cells. Considering that the handling of Pax5(-/-) pro-B cell lines is easier than that of bone marrow hematopoietic precursors, we used them for the rapid functional analysis of a novel Lat allelic series. When compared to knock-in and transgenic approaches, a major advantage of our Pax5(-/-) pro-B cell-based experimental approach consists in the production of mice bearing a given mutation within 2-3 months. Therefore, it constitutes a powerful first-line screen for mutations worth fastidious knock-in approaches.

s: 32 European Muscle Conference ‘A link between fundamental research and therapeutic trials’ The Annual Meeting of the European Society for Muscle Research MONTPELLIER, FRANCE, 6–10 SEPTEMBER 2003 Sunday, 7 September: Sarcomeric proteins and related pathologies SESSION I: STRUCTURAL AND REGULATORY MECHANISMS OF MUSCLE DYNAMICS I.1: Oral presentation 8:30–9:00 ADP and the strain sensing mechanism of myosin M. GEEVES Department of Biosciences, University of Kent, Canterbury Kent CT2 7NJ, UK I.2: Oral presentation 9:00–9:30 The pre-power stroke conformation of myosin V J. TRINICK School of Biomedical Sciences, University of Leeds, Leeds, LS2 9JT, UK I.3: Oral presentation 9:30–10:00 The myosin V motor visualized at 2.0Å without bound nucleotide reveals how myosin binds to F-actin PIERRE-DAMIEN COUREUX, AMBER L. WELLS, JULIE MÉNÉTREY, CHRIS M. YENGO, CARL A. MORRIS, H. LEE SWEENEY and ANNE HOUDUSSE Structural Motility, Institut Curie CNRS, UMR144, 26 rue d’Ulm, 75248 Paris Cedex 05, France; Department of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, PA 19104-6085 USA The lever arm hypothesis provides a possible mechanism for movement and force generation by the myosin superfamily of molecular motors. However, the structural details of the theory are largely based on high resolution structures of myosin that do not represent force generating states. Based on kinetic evidence that the processive unconventional myosin, myosin V, populates a unique state in the absence of nucleotide and actin, we hypothesized that its high-resolution structure could for the first time reveal features of a strong actin-binding, force generating state. We expressed and crystallized a myosin V containing only a motor domain and the first calmodulin-binding site. A 2.0 Å structure was obtained and revealed a novel conformation of myosin without bound nucleotide that has a number of key features not seen in previous myosin structures, many of which would be predicted to occur in the actin–myosin rigor complex. The nucleotide-binding site has adopted new conformations of the nucleotide-binding elements that reduces the affinity for nucleotide. The major cleft in the molecule has closed, and the lever arm has assumed a position consistent with that in an actomyosin rigor complex. These changes have been accomplished by relative movements of the subdomains of the molecule that were unexpected from all previous myosin structures. I.4: Oral presentation 10:00–10:15 (selected poster) Crossbridge and C-protein organisation in vertebrate muscle J.M. SQUIRE, H.A. AL-KHAYAT, R.W. KENSLER, C. KNUPP, P.K. LUTHER, E.P. MORRIS and M. ROESSLE Biological Structure & Function Section, Biomedical Sciences Division, Imperial College London, London SW7 2AZ, UK; Department of Anatomy, University of Puerto Rico Medical School, San Juan, 00936-5067, Puerto Rico; ESRF, 6 rue Jules Horowitz, 38043 Grenoble, France Electron microscopy and single particle analysis of isolated, negatively stained fish muscle myosin filaments have revealed the organisation of myosin heads in the relaxed state, including the myosin head perturbation, together with densities associated with non-myosin proteins such as C-protein (MyBP-C) and titin. Single particle analysis was carried out using the program IMAGIC. X-ray diffraction evidence from intact fish muscle (plaice) recorded at beamline ID02 at the ESRF, Grenoble has provided further evidence on C-protein organisation, including the possibility that some domains of C-protein that do not bind to myosin or titin on the filament backbone might bind to the neighbouring actin filaments. Sequence evidence identifies a possible actin-binding site in the Pro-Ala-rich region, N-terminal to domain C1, in the C-protein molecule. These results suggest that Cprotein might interconnect thick and thin filaments in relaxed muscle, Journal of Muscle Research and Cell Motility 24: 323–386, 2003. 323 2003 Kluwer Academic Publishers. Printed in the Netherlands.

Laminopathies are a group of disorders caused by mutations in the LMNA gene encoding A-type lamins, components of the nuclear lamina. Three of these disorders affect specifically the skeletal and/or cardiac muscles, and their pathogenic mechanisms are still unknown. We chose the LMNA H222P missense mutation identified in a family with autosomal dominant Emery-Dreifuss muscular dystrophy, one of the striated muscle-specific laminopathies, to create a faithful mouse model of this type of laminopathy. The mutant mice exhibit overtly normal embryonic development and sexual maturity. At adulthood, male homozygous mice display reduced locomotion activity with abnormal stiff walking posture and all of them die by 9 months of age. As for cardiac phenotype, they develop chamber dilation and hypokinesia with conduction defects. These abnormal skeletal and cardiac features were also observed in the female homozygous mice but with a later-onset than in males. Histopathological analysis of the mice revealed muscle degeneration with fibrosis associated with dislocation of heterochromatin and activation of Smad signalling in heart and skeletal muscles. These results demonstrate that LmnaH222P/H222P mice represent a good model for studying laminopathies affecting striated muscles as they develop a dystrophic condition of both skeletal and cardiac muscles similar to the human diseases.

The transmembrane adapter linker for activation of T cells (LAT) is thought to couple immunoreceptors to intracellular signaling pathways. In mice, its intracytoplasmic domain contains nine tyrosines which, when phosphorylated upon receptor aggregation, recruit Src-homology 2 domain-containing cytosolic enzymes and adapters. The four distal tyrosines are critical for both TCR and FcepsilonRI signaling. Unexpectedly, knock-in mice expressing LAT with a point mutation of the first or of the last three of these tyrosines exhibited an abnormal T cell development characterized by a massive expansion of TH2-like alphabeta or gammadelta T cells, respectively. This phenotype suggests that, besides positive signals, LAT might support negative signals that normally regulate terminal T cell differentiation and proliferation. We investigated here whether LAT might similarly regulate mast cell activation, by generating not only positive but also negative signals, following FcR engagement. To this end, we examined IgE- and/or IgG-induced secretory and intracellular responses of mast cells derived from knock-in mice expressing LAT with combinations of tyrosine mutations (Y136F, Y(175, 195, 235)F, or Y(136, 175, 195, 235)F). A systematic comparison of pairs of mutants enabled us to dissect the respective roles played by the five proximal and the four distal tyrosines. We found that LAT tyrosines differentially contribute to exocytosis and cytokine secretion and differentially regulate biological responses of mucosal- and serosal-type mast cells. We also found that, indeed, both positive and negative signals may emanate from distinct tyrosines in LAT, whose integration modulates mast cell secretory responses.

LAT (linker for activation of T cells) is essential for T cell receptor signaling. Mice homozygous for a mutation of the three C-terminal LAT tyrosine residues showed a block in αβ T cell development and a partially impaired γδ T cell development. Without intentional immunization, they accumulated γδ T cells in the spleen and lymph nodes that chronically produced T helper type 2 cytokines in large amounts, and caused the maturation of plasma cells secreting immunoglobulin E (IgE) and IgG1. These effects are very similar to that triggered in the αβ lineage by a mutation involving a distinct LAT tyrosine. Thus, LAT is an essential regulator of T cell homeostasis and terminal differentiation.

The transmembrane protein LAT (linker for activation of T cells) couples the T cell receptor (TCR) to downstream signaling effectors. Mice homozygous for a mutation of a single LAT tyrosine residue showed impeded T cell development. However, later they accumulated polyclonal helper T (T H ) cells that chronically produced type 2 cytokines in large amounts. This exaggerated T H 2 differentiation caused tissue eosinophilia and massive maturation of plasma cells secreting to immunoglobulins of the E and G1 isotypes. This paradoxical phenotype establishes an unanticipated inhibitory function for LAT that is critical for the differentiation and homeostasis of T H cells.

After stimulation of the T cell receptor (TCR), the tyrosine residues 292 and 315 in interdomain B of the protein tyrosine kinase ZAP-70 become phosphorylated and plausibly function as docking sites for Cbl and Vav1, respectively. The two latter proteins have been suggested to serve as substrates for ZAP-70 and to fine-tune its function. To address the role of these residues in T cell development and in the function of primary T cells, we have generated mice that express ZAP-70 molecules with Tyr to Phe substitution at position 292 (Y292F) or 315 (Y315F). When analyzed in a sensitized TCR transgenic background, the ZAP-70 Y315F mutation reduced the rate of positive selection and delayed the occurrence of negative selection. Furthermore, this mutation unexpectedly affected the constitutive levels of the CD3-ζ p21 phosphoisoform. Conversely, the ZAP-70 Y292F mutation upregulated proximal events in TCR signaling and allowed more T cells to produce interleukin 2 and interferon γ in response to a given dose of antigen. The observation that ZAP-70 Y292F T cells have a slower rate of ligand-induced TCR downmodulation suggests that Y292 is likely involved in regulating the duration activated TCR reside at the cell surface. Furthermore, we showed that Y292 and Y315 are dispensable for the TCR-induced tyrosine phosphorylation of Cbl and Vav1, respectively. Therefore, other molecules present in the TCR signaling cassette act as additional adaptors for Cbl and Vav1. The present in vivo analyses extend previous data based on transformed T cell lines and suggest that residue Y292 plays a role in attenuation of TCR signaling, whereas residue Y315 enhances ZAP-70 function.