CRISPR/Cas9-mediated knockout of Populus BRANCHED1 and BRANCHED2 orthologs reveals a major function in bud outgrowth control.

The TCP-type transcription factors BRANCHED1 and BRANCHED2 shape plant architecture by suppressing bud outgrowth, with BRANCHED2 only playing a minor role in Arabidopsis. Here, we investigated the function of orthologs of these genes in the model tree Populus. We used CRISPR/Cas9 to generate loss-of-function mutants of previously identified Populus BRANCHED1-1 and BRANCHED2-1 candidate genes. BRANCHED1-1 mutants exhibited strongly enhanced bud outgrowth. BRANCHED2-1 mutants had an extreme bud outgrowth phenotype and possessed two ectopic leaves at each node. While BRANCHED1 function is conserved in poplar, BRANCHED2, in contrast to its Arabidopsis counterpart, plays an even more critical role in bud outgrowth regulation. In addition, we identified a new, not yet reported association of this gene to leaf development.

A Two-Component Adhesive: Tau Fibrils Arise from a Combination of a Well-Defined Motif and Conformationally Flexible Interactions.

Fibrillar aggregates of Aß and Tau in the brain are the major hallmarks of Alzheimer's disease. Most Tau fibers have a twisted appearance, but the twist can be variable and even absent. This ambiguity, which has also been associated with different phenotypes of tauopathies, has led to controversial assumptions about fibril constitution, and it is unclear to-date what the molecular causes of this polymorphism are. To tackle this question, we used solid-state NMR strategies providing assignments of non-seeded three-repeat-domain Tau3RD with an inherent heterogeneity. This is in contrast to the general approach to characterize the most homogeneous preparations by construct truncation or intricate seeding protocols. Here, carbon and nitrogen chemical-shift conservation between fibrils revealed invariable secondary-structure properties, however, with inter-monomer interactions variable among samples. Residues with variable amide shifts are localized mostly to N- and C-terminal regions within the rigid beta structure in the repeat region of Tau3RD. By contrast, the hexapeptide motif in repeat R3, a crucial motif for fibril formation, shows strikingly low variability of all NMR parameters: Starting as a nucleation site for monomer-monomer contacts, this six-residue sequence element also turns into a well-defined structural element upon fibril formation. Given the absence of external causes in vitro, the interplay of structurally differently conserved elements in this protein likely reflects an intrinsic property of Tau fibrils.

Knockdown of strigolactone biosynthesis genes in Populus affects BRANCHED1 expression and shoot architecture.

Plant architecture is modified by a regulatory system that controls axillary bud outgrowth. Key components in this system are strigolactones (SLs) and BRANCHED1, which inhibit bud outgrowth. Their role has been described in herbaceous model systems, including Arabidopsis, rice and pea. However, a role in woody perennial species, including the model tree poplar, has not been unequivocally proven. In this study, we tested a role for SLs in Populus × canescens by treatment with the synthetic SL GR24. We generated MORE AXILLARY BRANCHING4 (MAX4) knockdown lines to study the architectural phenotype of poplar SL biosynthesis mutants and the expression of SL-regulated genes. We show that GR24 is perceived by the model tree poplar. MAX4 knockdown lines exhibit typical SL deficiency symptoms. The observed changes in branching pattern, internode length and plant height can be rescued by grafting. We identified putative poplar BRANCHED1 and BRANCHED2 genes and provide evidence for a regulation of BRANCHED1 by SLs. Our results suggest a conservation of major regulatory mechanisms in bud outgrowth control in the model tree poplar. This may facilitate further research, pinpointing the role of SLs and BRANCHED1 in the complex regulation of bud outgrowth in trees.

Allosteric switch regulates protein-protein binding through collective motion.

Many biological processes depend on allosteric communication between different parts of a protein, but the role of internal protein motion in propagating signals through the structure remains largely unknown. Through an experimental and computational analysis of the ground state dynamics in ubiquitin, we identify a collective global motion that is specifically linked to a conformational switch distant from the binding interface. This allosteric coupling is also present in crystal structures and is found to facilitate multispecificity, particularly binding to the ubiquitin-specific protease (USP) family of deubiquitinases. The collective motion that enables this allosteric communication does not affect binding through localized changes but, instead, depends on expansion and contraction of the entire protein domain. The characterization of these collective motions represents a promising avenue for finding and manipulating allosteric networks.

Small molecule-mediated stabilization of vesicle-associated helical α-synuclein inhibits pathogenic misfolding and aggregation.

α-synuclein is an abundant presynaptic protein that is important for regulation of synaptic vesicle trafficking, and whose misfolding plays a key role in Parkinson's disease. While α-synuclein is disordered in solution, it folds into a helical conformation when bound to synaptic vesicles. Stabilization of helical, folded α-synuclein might therefore interfere with α-synuclein-induced neurotoxicity. Here we show that several small molecules, which delay aggregation of α-synuclein in solution, including the Parkinson's disease drug selegiline, fail to interfere with misfolding of vesicle-bound α-synuclein. In contrast, the porphyrin phtalocyanine tetrasulfonate directly binds to vesicle-bound α-synuclein, stabilizes its helical conformation and thereby delays pathogenic misfolding and aggregation. Our study suggests that small-molecule-mediated stabilization of helical vesicle-bound α-synuclein opens new possibilities to target Parkinson's disease and related synucleinopathies.