Conformational Heterogeneity of a Leucine Enkephalin Analog in Aqueous Solution and SDS Micelles: Comparison of Time- Resolved FRET and Molecular Dynamics Simulations

We have undertaken time-resolved Förster resonance energy transfer (FRET) and molecular dynamics simulations to analyze conformations and conformational heterogeneity of an analog of leucine enkephalin in solution and in the presence of SDS micelles. Enkephalins are opioid pentapeptides that interact with opioid receptors in the central nervous system. We used timecorrelated single-photon counting to detect energy transfer between the N-terminal tyrosine and a tryptophan residue substituted for phenylalanine at the 4 position. FRET from Tyr to Trp was measured over a temperature range from 5°C to 55°C in aqueous solution. By taking into account Tyr rotamer interconversion rates measured previously, we determined average distances between Tyr and Trp for the two populated rotameric conformations of Tyr. Molecular dynamics simulations (100 ns) support this analysis and indicate extensive conformational heterogeneity. The simulations also predict that the FRET orientational factor is correlated with the Tyr-Trp separation. Failure to account for the correlation between orientation and distance results in errors that appear to be largely offset in YGGWL by a weighting bias inherent in the R−6 dependence of the energy-transfer rate. The Tyr lifetimes decrease upon titration of the peptides with SDS, indicating formation of compact conformations of the peptide in the micelle environment. This result is consistent with the conjecture that the lipid environment may induce formation of bioactive conformations of the peptide

The stoichiometry of the outer kinetochore is modulated by microtubule-proximal regulatory factors

The kinetochore is a large molecular machine that attaches chromosomes to microtubules and facilitates chromosome segregation. The kinetochore includes submodules that associate with the centromeric DNA and submodules that attach to microtubules. Additional copies of several submodules of the kinetochore are added during anaphase, including the microtubule binding module Ndc80. While the factors governing plasticity are not known, they could include regulation based on microtubule–kinetochore interactions. We report that Fin1 localizes to the microtubule-proximal edge of the kinetochore cluster during anaphase based on single-particle averaging of super-resolution images. Fin1 is required for the assembly of normal levels of Dam1 and Ndc80 submodules. Levels of Ndc80 further depend on the Dam1 microtubule binding complex. Our results suggest the stoichiometry of outer kinetochore submodules is strongly influenced by factors at the kinetochore–microtubule interface such as Fin1 and Dam1, and phosphorylation by cyclin-dependent kinase. Outer kinetochore stoichiometry is remarkably plastic and responsive to microtubule-proximal regulation

The Membrane-Associated Proteins FCHo and SGIP Are Allosteric Activators of the AP2 Clathrin Adaptor Complex

The AP2 clathrin adaptor complex links protein cargo to the endocytic machinery but it is unclear how AP2 is activated on the plasma membrane. Here we demonstrate that the membrane-associated proteins FCHo and SGIP1 convert AP2 into an open, active conformation. We screened for C. elegans mutants that phenocopy the loss of AP2 subunits and found that AP2 remains inactive in fcho-1 mutants. A subsequent screen for bypass suppressors of fcho-1 nulls identified 71 compensatory mutations in all four AP2 subunits. Using a protease-sensitivity assay we show that these mutations restore the open conformation in vivo. The domain of FCHo that induces this rearrangement is not the F-BAR domain or the mu-homology domain, but rather is an uncharacterized 90 amino acid motif, found in both FCHo and SGIP proteins, that directly binds AP2. Thus, these proteins stabilize nascent endocytic pits by exposing membrane and cargo binding sites on AP2

Parallel control of mechanosensory hair cell orientation by the PCP and Wnt pathways

Cell polarity plays a crucial role during development of vertebrates and invertebrates. Planar Cell Polarity (PCP) is defined as the coordinated polarity of cells within a tissue axis and is essential for processes such as gastrulation, neural tube closure or hearing. Wnt ligands can be instructive or permissive during PCP-dependent processes, and Wnt pathway mutants are often classified as PCP mutants due to the complexity and the similarities between their phenotypes. Our studies of the zebrafish sensory lateral line reveal that disruptions of the PCP and Wnt pathways have differential effects on hair cell orientations. While mutations in PCP genes cause random orientations of hair cells, mutations in Wnt pathway members induce hair cells to adopt a concentric pattern. We show that PCP signaling is normal in hair cells of Wnt pathway mutants and that the concentric hair cell phenotype is due to altered organization of the surrounding support cells. Thus, the PCP and Wnt pathways work in parallel, as separate pathways to establish proper hair cell orientation. Our data suggest that coordinated support cell organization is established during the formation of lateral line primordia, much earlier than the appearance of hair cells. Together, these finding reveal that hair cell orientation defects are not solely explained by defects in PCP signaling and that some hair cell phenotypes warrant reevaluation

From planning to execution to the future: An overview of a concerted effort to enhance biological control in apple, pear, and walnut orchards in the western U.S.

We embarked on a large project designed to help enhance biological control in apple, pear and walnut orchards in the western U.S., where management programs were in the midst of a transition from older organo-phosphate insecticides to mating disruption and newer reduced-risk insecticides. A “pesticide replacement therapy” approach resulted in unstable management programs with unpredictable outbreaks of spider mites and aphids. Our project was designed to provide growers and pest managers with information on the effects of newer pesticide chemistries on a suite of representative natural enemies in both the laboratory and field, potential of new monitoring tools using herbivore-induced plant volatiles and floral volatiles, phenology of the key natural enemy species, economic consequences of using an enhanced biological control program, and value of an outreach program to get project outcomes into the hands of decision-makers. We present an overview of both the successes and failures of the project and of new projects that have spun off from this project to further enhance biological control in our systems in the near future

Structural plasticity of the living kinetochore

The kinetochore is a large, evolutionarily conserved protein structure that connects chromosomes with microtubules. During chromosome segregation, outer kinetochore components track depolymerizing ends of microtubules to facilitate the separation of chromosomes into two cells. In budding yeast, each chromosome has a point centromere upon which a single kinetochore is built, which attaches to a single microtubule. This defined architecture facilitates quantitative examination of kinetochores during the cell cycle. Using three independent measures-calibrated imaging, FRAP, and photoconversion-we find that the Dam1 submodule is unchanged during anaphase, whereas MIND and Ndc80 submodules add copies to form an "anaphase configuration" kinetochore. Microtubule depolymerization and kinesin-related motors contribute to copy addition. Mathematical simulations indicate that the addition of microtubule attachments could facilitate tracking during rapid microtubule depolymerization. We speculate that the minimal kinetochore configuration, which exists from G1 through metaphase, allows for correction of misattachments. Our study provides insight into dynamics and plasticity of the kinetochore structure during chromosome segregation in living cells

Pathologic polyglutamine aggregation begins with a self-poisoning polymer crystal

A long-standing goal of amyloid research has been to characterize the structural basis of the rate-determining nucleating event. However, the ephemeral nature of nucleation has made this goal unachievable with existing biochemistry, structural biology, and computational approaches. Here, we addressed that limitation for polyglutamine (polyQ), a polypeptide sequence that causes Huntington’s and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. To identify essential features of the polyQ amyloid nucleus, we used a direct intracellular reporter of self-association to quantify frequencies of amyloid appearance as a function of concentration, conformational templates, and rational polyQ sequence permutations. We found that nucleation of pathologically expanded polyQ involves segments of three glutamine (Q) residues at every other position. We demonstrate using molecular simulations that this pattern encodes a four-stranded steric zipper with interdigitated Q side chains. Once formed, the zipper poisoned its own growth by engaging naive polypeptides on orthogonal faces, in a fashion characteristic of polymer crystals with intramolecular nuclei. We further show that self-poisoning can be exploited to block amyloid formation, by genetically oligomerizing polyQ prior to nucleation. By uncovering the physical nature of the rate-limiting event for polyQ aggregation in cells, our findings elucidate the molecular etiology of polyQ diseases

Cohesin Proteins Promote Ribosomal RNA Production and Protein Translation in Yeast and Human Cells

Cohesin is a protein complex known for its essential role in chromosome segregation. However, cohesin and associated factors have additional functions in transcription, DNA damage repair, and chromosome condensation. The human cohesinopathy diseases are thought to stem not from defects in chromosome segregation but from gene expression. The role of cohesin in gene expression is not well understood. We used budding yeast strains bearing mutations analogous to the human cohesinopathy disease alleles under control of their native promoter to study gene expression. These mutations do not significantly affect chromosome segregation. Transcriptional profiling reveals that many targets of the transcriptional activator Gcn4 are induced in the eco1-W216G mutant background. The upregulation of Gcn4 was observed in many cohesin mutants, and this observation suggested protein translation was reduced. We demonstrate that the cohesinopathy mutations eco1-W216G and smc1-Q843Δ are associated with defects in ribosome biogenesis and a reduction in the actively translating fraction of ribosomes, eiF2α-phosphorylation, and 35S-methionine incorporation, all of which indicate a deficit in protein translation. Metabolic labeling shows that the eco1-W216G and smc1-Q843Δ mutants produce less ribosomal RNA, which is expected to constrain ribosome biogenesis. Further analysis shows that the production of rRNA from an individual repeat is reduced while copy number remains unchanged. Similar defects in rRNA production and protein translation are observed in a human Roberts syndrome cell line. In addition, cohesion is defective specifically at the rDNA locus in the eco1-W216G mutant, as has been previously reported for Roberts syndrome. Collectively, our data suggest that cohesin proteins normally facilitate production of ribosomal RNA and protein translation, and this is one way they can influence gene expression. Reduced translational capacity could contribute to the human cohesinopathies

The SUN Protein Mps3 Is Required for Spindle Pole Body Insertion into the Nuclear Membrane and Nuclear Envelope Homeostasis

The budding yeast spindle pole body (SPB) is anchored in the nuclear envelope so that it can simultaneously nucleate both nuclear and cytoplasmic microtubules. During SPB duplication, the newly formed SPB is inserted into the nuclear membrane. The mechanism of SPB insertion is poorly understood but likely involves the action of integral membrane proteins to mediate changes in the nuclear envelope itself, such as fusion of the inner and outer nuclear membranes. Analysis of the functional domains of the budding yeast SUN protein and SPB component Mps3 revealed that most regions are not essential for growth or SPB duplication under wild-type conditions. However, a novel dominant allele in the P-loop region, MPS3-G186K, displays defects in multiple steps in SPB duplication, including SPB insertion, indicating a previously unknown role for Mps3 in this step of SPB assembly. Characterization of the MPS3-G186K mutant by electron microscopy revealed severe over-proliferation of the inner nuclear membrane, which could be rescued by altering the characteristics of the nuclear envelope using both chemical and genetic methods. Lipid profiling revealed that cells lacking MPS3 contain abnormal amounts of certain types of polar and neutral lipids, and deletion or mutation of MPS3 can suppress growth defects associated with inhibition of sterol biosynthesis, suggesting that Mps3 directly affects lipid homeostasis. Therefore, we propose that Mps3 facilitates insertion of SPBs in the nuclear membrane by modulating nuclear envelope composition