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Caroline Williams, Department of Integrative Biology, University of California, Berkeley

"Discovery and Characterization of Candidate Tardigrade Ice-Binding Proteins"

Abstract:

Tardigrades are known for their ability to survive a variety of stresses, including extreme temperatures, while in a desiccated tun state. Less well-known is how tardigrades respond to more ecologically relevant stressful temperatures in their active hydrated states, and very little has been done on how tardigrades survive cold. We are interested especially in the molecular mechanisms that allow resistance to freezing. One class of protective molecule found in several related taxa, including insects, is the ice-binding protein (IBP), which protects from freezing stress by binding to the faces of ice crystals, halting their growth and preventing them from damaging cells. Previous work has identified candidate IBPs in the tardigrade Hypsibius exemplaris. In this study we characterize these putative IBPs through molecular cloning and heterologous expression of the protein for in vitro functional assays. Our main ice-affinity assay is the ice shell, a setup to measure preferential binding of candidate proteins to ice by the relative concentration or fluorescence (via fusion proteins containing sfGFP) found in a growing ice fraction versus the leftover liquid fraction. We have estimated the IBP types of the candidates using structural similarity and putative functional motifs, and we test these hypotheses by comparing the candidate proteins’ ice-binding behaviors to several types of positive controls. We hope to find novel ice-binding properties in the H. exemplaris proteins, which may be of use in research, agricultural, or industrial IBP applications in preventing freeze damage.

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Eva Harris, Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley

"Comparing pathogenesis of non-structural protein 1 from the wild-type and attenuated vaccine-strain of yellow fever virus"

Abstract:

Yellow fever virus (YFV) 17D is an attenuated strain of YFV given as a live attenuated YFV vaccine; this YFV strain is also used in virology laboratories as a safe alternative to wild-type (WT) YFV. However, YFV 17D has major amino acid differences from WT YFV, which may lead to functional discrepancies in experimental models of YFV pathogenesis. Our laboratory uses YFV 17D non-structural protein 1 (NS1) in comparative flavivirus studies, as NS1 is a major determinant in flavivirus disease pathology, but we have not confirmed if YFV 17D NS1 reflects the pathologies of WT YFV. Furthermore, we have observed an unusually high cell binding pattern from YFV 17D NS1, but a lack of NS1-triggered pathology, which is normally mediated by NS1 binding. Thus, it is unclear whether attenuation has altered NS1 function or whether all YFV NS1 proteins display this discrepancy between cell binding and down-stream pathogenesis--atypical in other flavivirus NS1 proteins. In this study, we determined two amino acid residues of interest from a sequence comparison of YFV clinical isolate NS1s and YFV 17D NS1. These amino acid residues were mutated back to the WT variant in YFV 17D NS1, thus creating two single-reversion mutant proteins and one double-reversion mutant protein. We produced these mutant constructs using site-directed mutagenesis, then used PEI to transfect 293F cells with our three variants. The resulting mutant proteins were purified with Ni-NTA resin through affinity chromatography and then tested for purity. To test and compare function of these mutant proteins, we will use NS1 cell binding and endothelial dysfunction assays to determine whether YFV 17D NS1 is indeed attenuated in function. If our mutant proteins show divergence from the currently observed phenotype of YFV 17D NS1, this may indicate that changes to NS1 are integral in the attenuation process of YFV. This comparative study has the potential to either confirm our current understanding of YFV NS1-mediated endothelial dysfunction or reveal a mechanism by which YFV 17D has been attenuated through modification of YFV NS1 function.

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David Drubin, Department of Molecular and Cell Biology, University of California, Berkeley

"Mechanism of Myosin 1E and its Force Generation in Clathrin Mediated Endocytosis (CME)"

Abstract:

Clathrin-mediated endocytosis (CME) is a robust and relatively well-understood process at the molecular level. Through a series of time-sensitive steps, a cell is capable of packaging external cargo into a clathrin-coated vesicle (CCV). A eukaryotic process, CME is fundamental to cell survival as it is involved in regulating many activities around the plasma membrane. While the process is conserved in eukaryotic cells, there are slight but significant differences in yeast and mammalian CME. One of these differences is the varying degrees of importance for the actin assembly unit. In yeast, actin assembly is strictly necessary for CME. However, the role of actin in mammalian CME is somewhat unclear, with the most recent evidence leaning toward reliance on actin under conditions of increased plasma membrane tension. Another area of exploration is the relation of type I myosins with actin machinery. One such myosin is Myosin 1E (Myo1E), which is found in all mammalian cells and is the only long-tailed type I myosin found ubiquitously in mammalian cells. While Myo1E has not been as well-studied as its budding yeast homologue Myo3/5, recent studies are beginning to understand precise Myo1E mechanics in mammalian cells and its relationship to CME. To understand the relationship between Myo1E and membrane tension, I investigated lifetimes of clathrin-coated pit (CCP) lifetimes through knocking down fluorescently labeled Myo1E with hypotonic shock treatment. Furthermore, what also remains uninvestigated is the relationship between Myo1E and specific lipids on the plasma membrane, something that has been studied in its Myo3/5 counterpart. This will be done by purifying the TH1 domain of Myo1E and measuring association levels of the purified domain with different phosphoinositides.

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Adam Arkin, Lawrence Berkeley National Laboratory

"High-throughput Microfluidics to Isolate and Culture Microbes"

Abstract:

Many strains cannot be isolated without co-culturing or presence of specific media. It is also labor-intensive to prepare parallel cultures in order to study the growth of hundreds or thousands of strains in various growth conditions. Using microfluidics, microbes can be separated into droplets of medium suspended in oil and thus be isolated. The millions of droplets act as parallel cultures. Using microfluidics, the experimenter can not only observe growth of various microbes efficiently, but also correlate genotype with growth phenotype. Therefore, experiments were conducted to confirm the validity of this method.

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Eva Nogales, Department of Molecular and Cell Biology, University of California, Berkeley

"Architecture of the Chromatin Remodeler RSC and Insights into its Remodeling Mechanism"

Abstract:

Eukaryotic DNA is packaged into nucleosome arrays, which are repositioned by chromatin remodeling complexes to control DNA accessibility1,2. The Saccharomyces cerevisiae RSC (Remodeling the Structure of Chromatin) complex, a member of the SWI/SNF chromatin remodeler family, plays critical roles in genome maintenance, transcription, and DNA repair2–4. Here, we report cryo-electron microscopy (cryo-EM) and crosslinking mass spectrometry (CLMS) studies of yeast RSC complex and show that RSC is composed of a rigid tripartite core and two flexible lobes. The core structure is scaffolded by an asymmetric Rsc8 dimer and built with the evolutionarily conserved subunits Sfh1, Rsc6, Rsc9 and Sth1. The flexible ATPase lobe, composed of helicase subunit Sth1, Arp7, Arp9 and Rtt102, is anchored through the interactions between the N-terminus of Sth1 and the core. Our cryo-EM analysis also shows that in addition to the expected nucleosome-Sth1 interactions, RSC engages histones and nucleosomal DNA through one arm of the core structure, composed of Rsc8 SWRIM domains, Sfh1 and Npl6. Our findings provide structural insights into the conserved assembly process for all members of the SWI/SNF family of remodelers, and illustrate how RSC selects, engages, and remodels nucleosomes.

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Peter Sudmant, Department of Integrative Biology, University of California, Berkeley

"Using Copy Number Variation to Characterize Structural Variants in the Great Ape Lineage"

Abstract:

Copy number variation (CNV) has been frequently shown to cause rapid speciation and to give rise to adaptive phenotypes under different selective pressures. However, CNV and its effects have not been thoroughly studied in the great ape species. In collaboration with other lab members, I studied lineage-specific stratification and selection by exploring CNV in 58 chimpanzees, 13 bonobos, and 279 human genomes from the Great Ape Genome Diversity Project and the Simons Genome Diversity Project (SGDP). We developed a computational method that extracts read depth from short sequence reads and calculates copy number in sliding windows of fixed size and step across the genome. Using the generated copy numbers, we created copy number heat maps and plots of the population branch statistic (PBS) adapted for CNV. These visualizations, along with other computational methods, were used to determine regions of high variation among the great ape genomes. We constructed a list of genomic regions that have high CNV in chimpanzees, bonobos, and humans and identified population-stratified and fixed segmental duplications and deletions. Several of these regions have significant structural variation across species, indicating that these populations underwent similar selective pressures leading to convergent evolution. This adds insight into the evolutionary history of structural variants in great ape genomes and into the relationship between humans and their most closely related living species.

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Jamie Cate, Department of Molecular and Cell Biology, University of California, Berkeley

"Exploring Ribosomal Modifications to Accommodate Polymerization of New Monomers "

Abstract:

The ribosome is responsible for translating messenger RNA and assembling all proteins, which are subsequently made up of twenty naturally occurring amino acids. Currently, we are researching modifications that can be made to the ribosome structure that can allow for the accommodation of non-naturally occurring substrates, specifically non-amino acid monomers While early attempts at ribosome engineering mutated many highly conserved residues of the ribosome active site, we are using recent structures from our lab to target only a few ribosomal RNA bases. This should result in ribosomes with better overall activity and stability, avoiding assembly problems that have plagued previous efforts. We will mutate, purify, and test these ribosomes for translation with non-amino acid substrates. Our research represents the first steps toward a reprogrammed translation system capable of synthesizing non-peptide sequence defined polymers

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Jennifer Doudna, Department of Molecular & Cell Biology, University of California, Berkeley

"Examining CRISPR-Cas target search kinetics with yeast surface display"

Abstract:

DETECTR is a diagnostic technology that implements programmable RNA-guided Cas enzymes to detect specific nucleic acid sequences. DETECTR relies on the trans cleavage activity of Cas enzymes, where recognition of a specific DNA target by the Cas enzyme triggers indiscriminate ssDNA cleavage. Target DNA recognition by a trans cleaving Cas enzyme can be detected by the trans cleavage of a fluorescent ssDNA reporter molecule. The initial version of DETECTR was limited by the functionality of its Cas enzyme, Cas12a. The number of sequences detectable by Cas12a is restricted by the requirement for a protospacer adjacent motif (PAM) neighboring the target dsDNA sequence. While targeting ssDNA permits PAM-free DNA recognition, Cas12a is unable to effectively discriminate against off-target sequences in a ssDNA context. These deficiencies are remediated by Cas14a, a recently discovered trans cleaving Cas enzyme that can accurately recognize ssDNA without the requirement of a PAM. To integrate Cas14a with DETECTR, I developed techniques that amplify ssDNA from any nucleic acid template. I then demonstrated that Cas14a can accurately discriminate between single-nucleotide polymorphisms in ssDNA generated from human genomic dsDNA, and this detection was PAM-independent. These findings greatly increase the number of sequences detectable by DETECTR, widening the potential of this isothermal diagnostic technology.