Electrical activity and postsynapse formation in adult muscle: gamma-AChRs are not required.

Skeletal muscle fibers will not accept hyperinnervation by foreign motor axons unless they are paralyzed, suggesting that paralysis makes them receptive to innervation, e.g., by upregulating extrasynaptic expression of gamma-AChRs and/or of the agrin receptor MuSK. To examine the involvement of these parameters in paralysis-mediated synapse induction, ectopic expression of agrin, a factor from motor neurons controlling neuromuscular synapse formation, was made dependent on the administration of doxycycline in innervated adult muscle fibers. In response to doxycycline-induced agrin secretion, adult fibers did form ectopic postsynaptic specializations, even when they were electrically active, lacked fetal AChRs, and expressed normal low levels of MuSK. These data demonstrate that paralysis and changes associated with it are not required for agrin-induced postsynapse formation. They suggest that paralyzed muscle induces synapse formation via the release of factors that make motor neurites contact muscle fibers and secrete agrin.

Constitutively active MuSK is clustered in the absence of agrin and induces ectopic postsynaptic-like membranes in skeletal muscle fibers.

In skeletal muscle fibers, neural agrin can direct the accumulation of acetylcholine receptors (AChR) and transcription of AChR subunit genes from the subsynaptic nuclei. Although the receptor tyrosine kinase MuSK is required for AChR clustering, it is less clear whether MuSK regulates gene transcription. To elucidate the role of MuSK in these processes, we constructed a constitutively active MuSK receptor, MuSKneuTMuSK, taking advantage of the spontaneous homodimerization of the transmembrane domain of neuT, an oncogenic variant of the neu/erbB2 receptor. In the extrasynaptic region of innervated muscle fibers, MuSKneuTMuSK formed highly concentrated aggregates that colocalized with AChR clusters. Associated with MuSK-induced AChR clusters was a normal complement of synaptic proteins. Moreover, transcription of the AChR-epsilon subunit gene was increased, albeit via an indirect mechanism by MuSK-induced aggregation of erbB receptors and neuregulin. Although neural agrin was not required, the activity of MuSKneuTMuSK was nevertheless potentiated by ectopic expression of a muscle agrin isoform inactive in AChR clustering. To define the role of the kinase domain in the formation of a postsynaptic-like membrane, a second fusion receptor, neuneuTMuSK, which included the MuSK kinase but not the MuSK extracellular domain, was expressed. Significantly, neuneuTMuSK induced AChR clusters that colocalized with aggregates of endogenous MuSK. Taken together, it was concluded that the MuSK kinase domain is sufficient to initiate the recruitment of additional MuSK receptors, which then develop into highly concentrated aggregates by means of a positive feedback loop to induce a postsynaptic membrane in the absence of neural agrin.

Rapamycin inhibition of the G1 to S transition is mediated by effects on cyclin D1 mRNA and protein stability.

The immunosuppressant rapamycin has been shown previously to inhibit the G1/S transition in several cell types by prolonging the G1 phase of the cell cycle. This process appears to be controlled, in part, by the rapamycin-sensitive FK506-binding protein-rapamycin-associated protein-p70 S6 kinase (p70(S6k)) pathway and the cyclin-dependent kinases (Cdk). We now show that in serum-stimulated NIH 3T3 cells, rapamycin treatment delays the accumulation of cyclin D1 mRNA during progression through G1. Rapamycin also appears to affect stability of the transcript. The combined transcriptional and post-transcriptional effects of the drug ultimately result in decreased levels of cyclin D1 protein. Moreover, degradation of newly synthesized cyclin D1 protein is accelerated by rapamycin, a process prevented by inclusion of the proteasome inhibitor, N-acetyl-Leu-Leu-norleucinal. The overall effect of rapamycin on cyclin D1 leads, in turn, to impaired formation of active complexes with Cdk4, a process which triggers retargeting of the p27(Kip1) inhibitor to cyclin E/Cdk2. In view of this novel experimental evidence, we discuss a possible mechanism for the rapamycin-induced cell cycle arrest at the G1/S transition.

Characterization of the helper proteins for the assembly of tail fibers of coliphages T4 and lambda.

Assembly of tail fibers of coliphage T4 requires the action of helper proteins. In the absence of one of these, protein 38 (p38), p37, constituting the distal part of the long tail fiber, fails to oligomerize. In the absence of the other, p57, p34 (another component of the long tail fiber), p37, and p12 (the subunit of the short tail fiber) remain unassembled. p38 can be replaced by the Tfa (tail fiber assembly) protein (pTfa) of phage lambda, which has the advantage of remaining soluble even when produced in massive amounts. The mechanisms of action of the helpers are unknown. As a first step towards elucidation of these mechanisms, p57 and pTfa have been purified to homogeneity and have been crystallized. The identity of gene 57 (g57), not known with certainty previously, has been established. The 79-residue protein p57 represents a very exotic polypeptide. It is oligomeric and acidic (an excess of nine negative charges). It does not contain Phe, Trp, Tyr, His, Pro, and Cys. Only 25 N-terminal residues were still able to complement a g57 amber mutant, although with a reduced efficiency. In cells overproducing the protein, it assumed a quasi-crystalline structure in the form of highly ordered fibers. They traversed the cells longitudinally (and thus blocked cell division) with a diameter approaching that of the cell and with a hexagonal appearance. The 194-residue pTfa is also acidic (an excess of 13 negative charges) and is likely to be dimeric.

Determinants of receptor specificity of coliphages of the T4 family. A chaperone alters the host range.

E. coli phages of the T4 family (T4, TuIa, TuIb) recognize their cellular receptors with a C-terminal region of protein 37. This protein, common to all three phages, is present as a dimer located at the distal part of the long tail fibers and possesses a C-terminal domain consisting of 40 to 70 highly conserved C-terminal residues, followed by a variable region of 50 to 80 residues which is again followed by a highly conserved area. Protein 38, not being a component of the mature virion, is required for dimerization of protein 37; this represents a non-covalent association of a structural protein. Seven host range mutants of TuIa or TuIb were analyzed which were able to use proteinaceous receptors other than those recognized by their parents. All had suffered amino acid substitutions within the variable region. It is concluded that in all probability it is this region which interacts directly with the cellular receptors. Conditional mutants of T4 are known which, when propagated at the non-permissive temperature (42 degrees C), yield phage of normal morphology but these are more or less unable to adsorb to cells. The causative amino acid substitutions were found both downstream and upstream from the variable area. Distortion of it in the mutants could suggest a "snap-back" conformation of the tail fiber; the conserved C-terminal region may fold back and expose the variable region as a loop at the tip of the fiber. One of the phage mutants (L93), when grown at the permissive temperature, had lost the ability to use the OmpC porin (a receptor for T4) as a receptor. A secondary mutant, able to do so, was isolated. An additional mutation, leading to one amino acid substitution, had occurred in gene 38. This mutant gene acted in trans and caused a much enhanced temperature-sensitivity of infectivity without conferring temperature-sensitivity per se, i.e. the mutant protein 38 apparently altered the conformation of the receptor-recognizing area of the dimer of protein 37. A gene from phage lambda, about 40% identical to gene 38 of T4, complements gene 38 amber mutants. The corresponding protein also restored the ability of L93 to recognize OmpC but did not cause any such temperature-sensitivity. Hence, protein 38, classifying as a chaperone, appears to act instructively in conveying steric information to the target polypeptide.

Alterations of receptor specificities of coliphages of the T2 family.

The T-even type coliphage M1 uses the outer membrane protein OmpA as a receptor. Host range mutants were isolated in two sequential steps, the first resulting in strains able to use the porin OmpC, the second in mutants using the outer membrane protease OmpT as receptor. The mutational alterations in the receptor-recognizing protein of the phage (a component of the long tail fibers) have been determined. Their character and that of such previously described alterations suggest an antibody-type of ligand binding.

Receptor-recognizing proteins of T-even type bacteriophages. The receptor-recognizing area of proteins 37 of phages T4 TuIa and TuIb.

Escherichia coli phages of the T4 family (T4, TuIa, TuIb) recognize their cellular receptors by means of a C-terminal region of protein 37; a dimer of this polypeptide (1026 residues in T4) is located at the distal part of the long tail fibers. Virions of the T2 family use protein 38 (which is attached to the free end of protein 37) for this purpose. The corresponding areas of genes 37 belonging to TuIa and TuIb were cloned and sequenced. Comparison of the deduced protein primary structures, including those of T4 and lambda Stf (Stf most likely representing a subunit of the side tail fibers of phage lambda) showed that an area of 70 to 100 residues is characterized by very variable sequences, while the sequences of the adjacent 43 to 44 C-terminal residues as well as those upstream from the variable region are highly homologous. The variable regions are flanked and interrupted seven or eight times by the motif His-x-His-y, with x and y most often being Ser or Thr; furthermore, the locations of these repeated tetrapeptides are conserved. Using hybrid phages obtained by recombination of one phage with cloned fragments of gene 37 of another, it could be shown that the area of this gene encoding receptor specificity includes the variable area. The situation is analogous to the known receptor-recognizing region of proteins 38 belonging to the T2-type family, except that the repeating sequence is of a different nature. In T4, receptor specificity is coded for by 382 base-pairs of the 3'-end of the gene, starting exactly at the variable area. It was found that T4 can use the outer membrane protein OmpC or lipopolysaccharide as receptors with the same efficiency, and it is proposed that the 70 residues of the variable part of the protein serve to bind to both ligands.