Multiple Branch Points Research Articles

The splicing determinants of a regulated exon in the axonal MAP tau reside within the exon and in its upstream intron

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 6 of the gene is an alternatively spliced cassette whose expression profile is distinct from that of the other tau regulated exons, implying the utilization of distinct regulatory factors. Previous work had established the use of cryptic splice sites within exon 6 and the influence of flanking exons on the ratio of exon 6 variants. The present work shows that, in addition to the previously identified participants, the ratio of exon 6 isoforms is affected by: (1) suppression of the cryptic sites, (2) deletions of the upstream intron, and (3) the splicing regulators PTB and U2AF, both of which act on the branch point/polypyrimidine tract region. These results strongly suggest that factors binding immediately upstream of exon 6 are involved in regulation of this exon. They also lead to the conclusion that splicing of exon 6 is primarily governed by multiple branch points.

Native extracellular matrix induces a well-organized bipolar outgrowth pattern with neurite extension and retraction in cultured neurons

Cultured anterior pagoda (AP) neurons from the leech develop characteristic outgrowth patterns that depend on the molecular composition of the substrate. This article analyzes how native substrates from the central nervous system (CNS), such as the extracellular matrix (ECM) inside the capsules that enwrap the ganglia, determine the outgrowth patterns of AP neurons. When plated on the internal side of ganglion capsules, the remaining primary portion (stump) of AP neurons sprouted two main branches in opposite directions with bifurcations. This T-shaped pattern was distinctive for AP neurons and was different from the patterns of the same cell type plated on the external side of the capsule or on leech laminin extracts, in which they generated multiple neurites and branching points. AP neurons plated on tritonized CNS homogenates reproduced the outgrowth pattern displayed on ganglion capsules, in terms of the number of primary neurites, their length, their orientation, and the number of branch points. The development of the T-shaped outgrowth pattern of AP neurons on ganglion capsules and CNS homogenates started by the sprouting of one branch that later bifurcated, followed by a second branch in the opposite direction after a lag of several hours. Extension of the second branch and retraction of secondary neurites of the first were synchronous and contributed to refine the T-shaped pattern. These results suggest that during development or regeneration of the CNS, particular sets of ECM proteins have multiple effects regulating the number, direction, extension, and retraction of neurites.

The gene mod(mdg4) affects synapse specificity and structure in Drosophila.

The mechanisms by which synapse assembly and maturation are orchestrated during development are largely unknown. We used P-element mutagenesis and a larval anatomical screen to isolate mutants in which synapse structure was altered. Here, we describe a mutation isolated with this screen, branch point disrupted (bpd), in which both synapse specificity and synapse morphology were altered. Synaptic terminals in bpd mutants developed abnormally, forming multiple branch points, overgrowing to inappropriate neighboring muscles, and establishing aberrant folding of postsynaptic membranes. Ultrastructural characterization of synaptic boutons in bpd demonstrated abnormal layering of the postsynaptic specialization or subsynaptic reticulum (SSR). Genetic and molecular analyses revealed that bpd is an allele of mod(mdg4), a gene coding for a protein with many similarities to transcription factors, which has been implicated in the regulation of chromatin insulation. Our results suggest that mod(mdg4) may regulate a gene(s) essential to normal synapse formation.

Slow conduction in cardiac tissue, II: effects of branching tissue geometry.

In cardiac tissue, functional or structural current-to-load mismatches can induce local slow conduction or conduction block, which are important determinants of reentrant arrhythmias. This study tested whether spatially repetitive mismatches result in a steady-state slowing of conduction. Patterned growth of neonatal rat heart cells in culture was used to design unbranched cell strands or strands releasing branches from either a single point or multiple points at periodic intervals. Electrical activation was followed optically using voltage-sensitive dyes under control conditions and in elevated [K+]o (5.8 and 14.8 mmol/L, respectively; in the latter case, propagation was carried by the L-type Ca2+ current). Preparations with multiple branch points exhibited discontinuous and slow conduction that became slower with increasing branch length and/or decreasing inter-branch distance. Compared with unbranched strands, conduction was maximally slowed by 63% under control conditions (from 44.9+/-3.4 to 16.7+/-1.0 cm/s) and by 93% in elevated [K+]o (from 15.7+/-2.3 to 1.1+/-0.2 cm/s). Local activation delays induced at a single branch point were significantly larger than the delays per branch point in multiple branching structures. Also, selective inactivation of inward currents in the branches induced conduction blocks. These 2 observations pointed to a dual role of the branches in propagation: whereas they acted as current sinks for the approaching activation thus slowing conduction ("pull" effect), they supplied, once excited, depolarizing current supporting downstream activation ("push" effect). This "pull and push" action resulted in a slowing of conduction in which the safety was largely preserved by the "push" effect. Thus, branching microarchitectures might contribute to slow conduction in tissue with discontinuous geometry, such as infarct scars and the atrioventricular node.

Quenched approximation artifacts: A study in two-dimensional QED

The spectral properties of the Wilson-Dirac operator in 2-dimensional QED responsible for the appearance of exceptional configurations in quenched simulations are studied in detail. The mass singularity structure of the quenched functional integral is shown to be extremely compicated, with multiple branch points and cuts. The connection of lattice topological charge and exactly real eigenmodes is explored using cooling techniques. The lattice volume and spacing dependence of these modes is studied, as is the effect of clover improvement of the action. A recently proposed modified quenched approximation is applied to the study of meson correlators, and the results compared with both naive quenched and full dynamical calculations of the same quantity.

Molecular constitutive equations for a class of branched polymers: The pom-pom polymer

Polymer melts with long-chain side branches and more than one junction point, such as commercial low density polyethylene (LDPE), have extensional rheology characterized by extreme strain hardening, while the shear rheology is very shear thinning, much like that of unbranched polymers. Working with the tube model for entangled polymer melts, we propose a molecular constitutive equation for an idealized polymer architecture, which, like LDPE, has multiple branch points per molecule. The idealized molecule, called a “pom-pom,” has a single backbone with multiple branches emerging from each end. Because these branches are entangled with the surrounding molecules, the backbone can readily be stretched in an extensional flow, producing strain hardening. In start-up of shear, however, the backbone stretches only temporarily, and eventually collapses as the molecule is aligned, producing strain softening. Here we develop a differential/integral constitutive equation for this architecture, and show that it predicts rheology in both shear and extension that is qualitatively like that of LDPE, much more so than is possible with, for example, the K-BKZ integral constitutive equation.

A new ichnospecies of Spongeliomorpha from the Pleistocene of Sicily

A branched, scratch-ornamented burrow system is described from early Pleistocene sediments of Sicily, Italy. The trace fossil occurs in a bed of volcanic ash beneath a shallow marine silty unit and is remarkable in possessing enlarged, ovoid chambers above the multiple branching points of a maze. The structure, named Spongeliomorpha sicula, was probably produced by crustaceans. A likely interpretion is as an agrichnion, in which microbial gardening took place in the chambers.

Kinetic analysis of the formation of inositol 1:2-cyclic phosphate in carbachol-stimulated pancreatic minilobules: Half is formed by direct phosphodiesteratic cleavage of phosphatidylinositol

The issue as to whether there is direct phosphodiesteratic cleavage of phosphatidylinositol (PI), in addition to that of phosphatidylinositol 4,5-bisphosphate (PIP2), on agonist stimulation of cells has been controversial. In an attempt to resolve this issue, we have studied the kinetics of the formation and breakdown of the cyclic inositol phosphates. This approach is fairly straightforward, since the turnover of the cyclic inositol phosphates is very slow as compared to that of the noncyclic inositol phosphates and proceeds from inositol 1:2-cyclic 4,5-trisphosphate to inositol 1:2-cyclic phosphate (I(c1:2)P) directly by dephosphorylation without any branching pathways, in contrast to the multiple branchpoints of the noncyclic inositol phosphate pathway. Mouse pancreatic minilobules were prelabeled with [3H]inositol for 30 min, followed by washing to remove free inositol. They were then stimulated with carbachol for 30 min. The inositol cyclic polyphosphates reached steady state at 10-15 min, and I(c1:2)P reached steady state at 25 min. We blocked the action of carbachol by addition of an excess of atropine at 30 min, and the rate of disappearance of the three cyclic inositol phosphates was measured. From these data, the contribution of the inositol cyclic polyphosphate pathway to I(c1:2)P was calculated, which was 40-50% of total I(c1:2)P formation. Thus, 40-50% of the I(c1:2)P formed must have been derived from direct phosphodiesteratic cleavage of PI. This approach should prove useful in measuring the relative contributions of PI hydrolysis and PI phosphorylation (phosphatidylinositol 4,5-bisphosphate hydrolysis) in the overall PI cascade.

In vitro splicing of adenovirus E1A transcripts: characterization of novel reactions and of multiple branch points abnormally far from the 3' splice site.

During the analysis of the in vitro alternative splicing of the natural E1A transcript of adenovirus, other minor reactions were detected (Schmitt et al., 1987, Cell 50, 31-39). We report here their characterization. The first reaction concerns the excision of a 216 nucleotide intron delineated by the 9S 5' splice site and a 3' splice site 216 nucleotides downstream. It can occur on the premRNA transcript and the 13S and 12S mRNA species. Strikingly, the reaction uses one of 3 branch points located 51, 55 or 59 residues upstream of the 3' splice site, a distance which is unusually long since all the branch points mapped up to now are located between 18-37 nucleotides of the 3' splice site. The dramatic accumulation of the corresponding lariat intermediates, likely related to this long spacing indicates that the second splicing step is relatively unefficient. The second kind of reaction analysed is a cryptic splicing which uses a 3' splice site generated by the junction of the 13S mRNA exons, and leads to the formation of psi 12S and psi 9S mRNAs. In vitro, this reaction occurs only from a 13S mRNA transcript, and not from the 13S mRNA newly formed in the splicing assay, consistent with what has been observed in vivo. Thus, both the well known alternative and the minor reactions occurring in vivo from E1A premRNA and mRNAs are detected in vitro, implying that most of the alternative splicing machinery is reconstituted in the in vitro system.

The cytoskeleton of spreadingDictyostelium amoebae

The cytoarchitectural elements ofDictyostelium discoideum amoeba have been visualized by light and electron microscopy in cells prepared with mixtures of glutaraldehyde and Triton-X-100. After negative staining, the peripheral regions of spreading amoebae show a complex meshwork of actin filaments, the majority of which were less than 0.25 microns in length. Multiple branch points, end to side abutments and cross-overs were characteristic features of the actin meshworks. Filopodia extending from the cell periphery consisted of bundles of actin filaments that penetrated into and merged with the actin meshworks in the spreading lamellae. Microtubules emanating from the nucleus associated body penetrated to differing extents into the actin meshworks, sometimes extending close to the cell periphery.Dictyostelium cytoskeletons preparted as described here should prove useful for further studies on the locomotory mechanism.

Kinetic Aspects of Regulation of Metabolic Processes: THE HYSTERETIC ENZYME CONCEPT

Hysteretic enzymes are defined as those enzymes which respond slowly (in terms of some kinetic characteristic) to a rapid change in ligand, either substrate or modifier, concentration. Such slow changes, defined in terms of their rate relative to the over-all catalytic reaction, result in a lag in the response of the enzyme to changes in the ligand level. Several mechanisms, including ligand-induced isomerization of the enzyme, displacement of tightly bound ligands by other ligands, or polymerization and depolymerization, are discussed and it can be shown that the description of the time-dependent change in enzyme activity is similar for many different cases. Examination of the literature reveals that a large number of enzymes may fall into the category termed hysteretic, that such enzymes are frequently those which are important in metabolic regulation, that the time of conversion from one kinetic form to another may vary between seconds and minutes, and that there are experimental examples of all the mechanisms which are discussed theoretically. The possible relation between those enzymes which are hysteretic and regulation of complex metabolic processes is discussed in terms of the fact that the slow response of the hysteretic enzyme to changes in ligand level will lead to a time-dependent buffering of some metabolites and that this may be important with respect to pathways which utilize common intermediates or in which there are multiple branch points. It is suggested that the question of hysteresis in enzyme systems as defined here be systematically investigated in regulatory enzymes and that this concept may be of value in discussing the regulation of complex processes in vivo.