Organogenesis of the somatic musculature in Drosophila is directed by the precise adhesion between migrating myotubes and their corresponding ectodermally derived tendon cells. Whereas the PS integrins mediate the adhesion between these two cell types, their extracellular matrix (ECM) ligands have been only partially characterized. We show that the ECM protein Thrombospondin (Tsp), produced by tendon cells, is essential for the formation of the integrin-mediated myotendinous junction. Tsp expression is induced by the tendon-specific transcription factor Stripe, and accumulates at the myotendinous junction following the association between the muscle and the tendon cell. In tsp mutant embryos, migrating somatic muscles fail to attach to tendon cells and often form hemiadherens junctions with their neighboring muscle cells, resulting in nonfunctional somatic musculature. Talin accumulation at the cytoplasmic faces of the muscles and tendons is greatly reduced, implicating Tsp as a potential integrin ligand. Consistently, purified Tsp C-terminal domain polypeptide mediates spreading of PS2 integrin-expressing S2 cells in a KGD-and PS2-integrin-dependent manner. We propose a model in which the myotendinous junction is formed by the specific association of Tsp with multiple muscle-specific PS2 integrin receptors and a subsequent consolidation of the junction by enhanced tendon-specific production of Tsp secreted into the junctional space.

Methyl-CpG binding domain (MBD) proteins in Arabidopsis thaliana bind in vitro methylated CpG sites. Here, we aimed to characterize the binding properties of AtMBDs to chromatin in Arabidopsis nuclei. By expressing in wild-type cells AtMBDs fused to green fluorescent protein (GFP), we showed that AtMBD7 was evenly distributed at all chromocenters, whereas AtMBD5 and 6 showed preference for two perinucleolar chromocenters adjacent to nucleolar organizing regions. AtMBD2, previously shown to be incapable of binding in vitro-methylated CpG, was dispersed within the nucleus, excluding chromocenters and the nucleolus. Recruitment of AtMBD5, 6, and 7 to chromocenters was disrupted in ddm1 and met1 mutant cells, where a significant reduction in cytosine methylation occurs. In these mutant cells, however, AtMBD2 accumulated at chromocenters. No effect on localization was observed in the chromomethylase3 mutant showing reduced CpNpG methylation or in kyp-2 displaying a reduction in Lys 9 histone H3 methylation. Transient expression of DDM1 fused to GFP showed that DDM1 shares common sites with AtMBD proteins. Glutathione S-transferase pull-down assays demonstrated that AtMBDs bind DDM1; the MBD motif was sufficient for this interaction. Our results suggest that the subnuclear localization of AtMBD is not solely dependent on CpG methylation; DDM1 may facilitate localization of AtMBDs at specific nuclear domains.

Correct muscle migration towards tendon cells, and the adhesion of these two cell types, form the basis for contractile tissue assembly in the Drosophila embryo. While molecules promoting the attraction of muscles towards tendon cells have been described, signals involved in the arrest of muscle migration following the arrival of myotubes at their corresponding tendon cells have yet to be elucidated. Here, we describe a novel tendon-specific transmembrane protein, which we named LRT due to the presence of a leucine-rich repeat domain (LRR) in its extracellular region. Our analysis suggests that LRT acts non-autonomously to better target the muscle and/or arrest its migration upon arrival at its corresponding tendon cell. Muscles in embryos lacking LRT exhibited continuous formation of membrane extensions despite arrival at their corresponding tendon cells, and a partial failure of muscles to target their correct tendon cells. In addition, overexpression of LRT in tendon cells often stalled muscles located close to the tendon cells. LRT formed a protein complex with Robo, and we detected a functional genetic interaction between Robo and LRT at the level of muscle migration behavior. Taken together, our data suggest a novel mechanism by which muscles are targeted towards tendon cells as a result of LRT-Robo interactions. This mechanism may apply to the Robo-dependent migration of a wide variety of cell types.

4-[4, 4-Dimethyl-3-(4-hydroxybutyl)-5-oxo-2-thioxo-1-imidazolidinyl]-2-+ ++trif luoromethylbenzonitrile (RU 59063) is a prototype of a new class of high-affinity nonsteroidal androgen receptor (AR) ligands. The search for a radioiodinated AR ligand prompted us to synthesize 4-[4, 4-dimethyl-3-(4-hydroxybutyl)-5-oxo-2-thioxo-1-imidazolidinyl]-2-i odo benzonitrile (DTIB) wherein the trifluoromethyl group of RU 59063 was substituted with the similarly hydrophobic iodine atom. DTIB displayed subnanomolar binding affinity (K(i) = 0.71 +/- 0.22 nM) for the rat AR in competitive binding assays. Additionally, DTIB demonstrated potent agonist activity, comparable to that of the natural androgen 5alpha-dihydrotestosterone (DHT), in a cell-based functional assay (cotransfection assay). DTIB represents a new lead for the development of high-affinity radioiodinated AR radioligands.

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