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James H. Hurley , Department of Molecular & Cell Biology
In Vitro Characterization of VPS37A's Interaction with ATG8 Proteins
Abstract:
Macroautophagy is an essential intracellular process by which macromolecules and damaged organelles are recycled via degradation. The formation of autophagosome, a double-membrane structure surrounding the cargo, requires the endosomal sorting complex required for transport (ESCRT) for the nal step of closing the phagophore. A genetic screen revealed a requirement for the ESCRT-I subunit VPS37A in the closure of the phagophore. The putative ubiquitin E2 variant (PUEV) domain of VPS37A is hypothesized to be required for the phagophore localization of the VPS37A-containing ESCRT-I complex. The putative ubiquitin E2 variant (PUEV) domain of VPS37A is hypothesized to be required for the phagophore localization of the VPS37A-containing ESCRT-I complex. Furthermore, two putative LC3 interacting regions (pLIR) are pinpointed through PSSM socring. In addition, curvature sensing is a possible mechanism of recruitment that is observed in other proteins in autophagosome biogenesis. This study aims to explore how VPS37A is recruited to late-stage autophagosomes.
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Megan Martik , Department of Molecular & Cell Biology, University of California Berkeley
Understanding Axial Regionalization and Gene Regulation of Neural Crest Cells in Agnathans
Abstract:
The neural crest is a multipotent stem cell population present in early development that gives rise to various derivatives along the anteroposterior axis. The multipotency of the neural crest in jawed vertebrates is restricted to distinct populations: cranial, vagal, trunk, and sacral, each having its own distinct derivatives and molecular features. The acquisition and elaboration of these axial regions plays a crucial role in the evolution of morphological novelties such as the jaw, which facilitated the transition from filter-feeding to active predation. Insight into the diversification of the vertebrate lineage can be best understood by examining the sea lamprey– a basal jawless vertebrate that possesses only cranial and trunk neural crest subpopulations with less defined boundaries than later branching jawed vertebrates. Analyzing the molecular state of these populations provides significant insights into the state of the ancestral neural crest. By collecting and analyzing single-cell gene expression data from the lamprey cranial and trunk populations of the neural crest at various stages of development, my analysis identifies novel candidate genes implicated in the evolutionary complexification of neural crest axial identities. Sfrp2, associated with the downregulation of Wnt signaling, was found to be cranial-specific in the data, consistent with the established practice of using WNT/𝛽-catenin signaling to trigger more posterior neural crest fates in cell culture. Additionally, two transcription factors, Sall2 and Sall3 are restricted to cranial and trunk regions respectively, whereas other transcription factors, such as Sp8 and Sp9, are homogeneously distributed throughout the neural crest clusters. Additionally, I have generated single-cell ATAC data and SoxE1 knockout lamprey crispants to test chromatin accessibility and regulatory linkages along the axial levels. The combined gene expression and ATAC profiles provide high-resolution data to expand on the evolution of the molecular circuits that regulate the axial specification of neural crest cells.
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Dr. Deborah Dean , Children's Hospital Oakland Research Institute
Understanding the Diversity of Chlamydia-Like Organisms Among Individuals with Trachoma Residing in the Endemic Region of Amhara, Ethiopia
Abstract:
Trachoma is a chronic eye disease that is caused by repeated conjunctival infection with Chlamydia trachomatis and possibly other chlamydial species infections. The overall objective of this project was to determine the role of Chlamydia-like organisms in trachoma in Ethiopia. We identified the diversity of Chlamydia-like organisms that may facilitate infection, reinfection and transmission among infected individuals from villages in the trachoma hyperendemic Amhara region of Ethiopia and may enhance the eye infection and disease among the population.
There were 1008 participants with samples available for analysis. DNA was extracted with a lysozyme cocktail. Real-Time (RT)-quantitative (q)PCR was employed to detect the presence of the family Chlamydiaceae using the pORF2 gene, which has seven to 10 copies in the chlamydial plasmid. If negative, RT-PCR was run to detect the presence of the phylum Chlamydiae using a 200bp region of Chlamydiae-specific 16S rRNA. For Chlamydiae-positive samples, an 800bp region of Chlamydiae-specific 16S rRNA was amplified by PCR, sequenced using Sanger sequencing, and analyzed with the NCBI BLAST-n and Taxonomy Browser. The trachoma grades of these samples were analyzed with Chi-square test and Fisher Exact test.
106 of 1008 samples were Chlamydiaceae-positive. Of the Chlamydiaceae-negative samples, 240 (26.6%) of 902 samples were positive for Chlamydiae, of which 93 belong to the family Parachlamydiaceae, 18 to Rhabdochlamydiaceae and two to Simkaniaceae. The presence of Parachlamydiales was significantly associated with active trachoma. However, the presence of Chlamydiales, Parachlamydiales, Candidatus Protochlamydia sp. CRIB40, or Neochlamydia hartmannellae was negatively significantly associated with severe trachoma. This demonstrates that CLOs are likely to play a role in trachoma and its severity.
We hope to develop novel therapeutic approaches and prevention strategies to decrease/prevent infection, transmission and the devastating sequelae of blindness in Ethiopia and other parts of Africa where trachoma is endemic.
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Yangnan Gu , Department of Plant & Microbial Biology
Elucidating the Mechanism of PNET1 during Cell Cycle Regulation at the Nuclear Pore Complex
Abstract:
Cell division and expansion underlie plant growth and development. Among the myriad of cell cycle regulators, nucleoporins (Nups) residing in the nuclear pore complex (NPC) represent a group of emerging players that facilitate cell cycle progression across eukaryotes. However, the functional significance of Nups in cell cycle-related processes is often less well-established in plants than in their animal counterparts. Here, we investigate the potential role of a newfound Nup, Plant Nuclear Envelope Transmembrane 1 (PNET1) in mediating cell cycle progression in Arabidopsis thaliana. We found that pnet1 knockout exhibits defective root apical meristem size and that PNET1 transcript level significantly affects expression of multiple cell cycle-associated genes. We also showed that PNET1 physically and genetically interacts with another plant-specific Nup, Constitutive expresser of PR genes 5 (CPR5), which is implicated in both cell cycle regulation and plant immunity. Additionally, exploration of the cellular and genetic interplay between PNET1 and SIAMESE (SIM), one of the cyclin-dependent kinase inhibitors (CKI) from the CPR5-regulatory pathway and is reported to interact with CPR5 at the NPC, did not reveal salient differences in phenotypes and expression patterns between wild-type and pnet1 in the overexpression background of SIM. Interestingly, the cross of cpr5 with the cell cycle indicator line PlaCCI produces a similar phenotype as the pnet1 cpr5 double mutant, indicating that the overexpression of cell cycle-related genes including CDT1a, H3.1, and CYCB1;1 in PlaCCI might be interfering with the cell cycle pathway in a similar way as the loss of PNET1 in cpr5. Therefore, these genes were individually transformed into cpr5 to understand which specific gene is involved in the rescue of cpr5, which can also elucidate PNET1’s function during cell cycle.
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Hillel Adesnik , Department of Molecular & Cell Biology
Investigating the Role of Downstream Neural Targets of Primary Visual Cortex (V1) in Conscious Vision and Blindsight
Abstract:
The phenomenon of blindsight describes individuals who have lost the conscious experience of seeing, but are still able to localize visual stimuli. This condition occurs after the primary visual cortex (V1) is damaged. To identify the neurons within V1 that are crucial for conscious vision, we developed a mouse model of blindsight. Mice are trained to run to visual targets in a virtual reality behavioral assay. Following neural manipulation, changes in this visual behavior indicate whether the mouse is completely blind or blind with blindsight. Previous work in our lab showed that removal of V1 caused blindsight-like behavior in mice. However, it remained unknown which brain areas downstream of V1 are important for conscious vision, in humans and mice. Several brain areas receive input from V1, such as Anterior Cingulate Cortex (ACC), Dorso-medial Striatum (DMS), Anterior Pretectal Nucleus (APN), Retrosplenial Cortex (RSC), and Superior Colliculus (SC). This suggests that these regions may also be important in the neural circuit for conscious vision. Here, to understand which brain areas are critical, I used viral labeling and ablation methods to target the subset of neurons within V1 that project to specific brain areas. Ablation of V1 neurons that project to ACC can lead to complete blindness. Death of V1 neurons that project to DMS and SC can lead to partial visuo-behavioral defects, while those that project to RSC and APN leaves behavior largely unaffected. These results suggest that ACC, DMS, and SC may play a more significant role in modulating conscious vision, but the strength of defect within each group varies. To attain more robust effects, I am in the process of dual target ablations where I simultaneously eliminate the subsets of V1 neurons that project to two of these three brain regions. Overall, we have begun to elucidate the neural circuit downstream of V1 in conscious vision.
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Jennifer Garrison, PhD , Buck Institute for Research on Aging, Novato, CA
The Role of nep-2 in C. elegans Egg-Laying Circuit
Abstract:
Neuropeptide signaling in neural circuits regulates a wide range of physiological and behavioral aspects of an organism. In C. elegans, the physical connectome has been mapped but the wireless neuropeptide network and the mechanism by which their signaling leads to behavior are less well-studied. The egg-laying circuit in C. elegans is regulated by both neuropeptide and neurotransmitter signaling. Hermaphrodite Specific Neurons (HSNs), one component of this circuitry, release serotonin and the neuropeptide NLP-3 to initiate and modulate the temporal pattern of egg-laying. In this study, we looked at nep-2, a neuropeptidase gene. Previous studies in the Garrison Lab have shown elevated NLP-3 peptide levels in nep-2 null mutants, suggesting that NEP-2 modulates NLP-3 levels in the egg-laying circuit. The goal of this study is to characterize the role of nep-2 in the egg-laying circuit. In collaboration with other lab members, I characterized the distribution of eggs in the uterus of a nep-2 mutant through live imaging and found them to be hyperactive egg-laying. To study the localization of NEP-2, I will construct a transgenic line that acts both as a translational and transcriptional reporter of the nep-2 gene. By studying nep-2 expression and the pathways it plays a role in, we make progress in fully understanding how signaling controls the egg-laying circuit, which is a promising model for studying more complex neural circuits and neuropeptide signaling biology.
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Spencer Diamond , IGI
From Stable Cyanobacterial Communities Determined Heterotrophic Bacteria Phyla Composition Remains Similar Independent of Nitrogen Concentration
Abstract:
Found globally, cyanobacteria have the ability to fix atmospheric gases into sugars and bioavailable nitrogen compounds respectively, resulting in immense contributions to primary productivity and biogeochemical cycling. Cyanobacteria support and live in complex microbial communities where metabolic exchange dynamics take place among their heterotrophic partners. Hence, cyanobacteria communities serve as a model microbial community to study, and as they participate in aquatic symbiosis, it is very likely there are certain microbes that work in tandem with cyanobacteria. The nitrogen fixing process is a metabolic process of particular interest since there are certain strains such as Anabaena sp. PCC7120 that can form heterocysts, specialized cells for nitrogen fixation, under low nitrogen conditions and their role as a nitrogen provider can predominantly alter bacterial community composition. While the associations between cyanobacterial community dynamics are largely understood, there is a significant gap in our understanding of the specific genes, pathways, and community interactions which veil the community behavior principles underlying nitrogen fixation. In this study, we endeavor to show how cyanobacterial community composition changes in response to a gradient of different concentrations of nitrogen and uncover a mechanistic understanding (ecologically and molecularly) of the cyanobacterial community biology that mediates this metabolic process. Here we show that there is no clear difference in bacteria phyla across different nitrogen concentrations after approximately one month of passaging and growth. This main result reveals that for the parameters of this experiment, nitrogen concentration is not statistically significant in altering community composition. Overall, this research can help to identify shifts in community dynamics and -omics; elucidate novel genes, pathways, species interactions, and co-occurring microbial species; and provide a new framework for studying nitrogen fixation, since the application of metagenomics and metatranscriptomics will illuminate beyond bacterial population abundance and size to study interactions of bacteria and cyanobacteria communities. In addition to a quantitative understanding of microbial community features, this research has a potential application of creating microbial genetic engineering tools to improve human health and the worldwide environment.
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Craig Miller , Department of Molecular & Cell Biology
Genetic Manipulation of Tooth Formation Competency in the Threespine Stickleback
Abstract:
Organ formation competency is highly regulated during development. Fish teeth serve as a tractable model system to explore organ formation competency due to their simplicity, iterated nature, and known genetic requirements. Here we use stickleback fish to characterize developmental rules governing endogenous and ectopic tooth formation competency. We find that endogenous tooth development is severely inhibited by Dkk2, a known modulator of Wnt signaling. Overexpressing Dkk2 from 5 to 17 days post-fertilization led to comparable tooth recovery rates as the control group, overexpression at 4 to 17 days post-fertilization led to decreased recovery rates as well as ceratobranchial fusion. This suggests that interfering with Wnt signaling causes defects in pouch formation during pharyngeal arch segmentation, which secondarily affects tooth number. However, tooth development is able to rebound in some capacity, suggesting that the potential to create teeth persists even when the underlying bone is reduced. This study additionally details the fine-scale distribution of facial teeth under Eda overexpression, showing that ectopic teeth are concentrated along the infraorbital and ventral dentary regions. This is also where neuromasts are found, a type of external epithelial sensory organ in fish. Given the tight spatial relationship observed, we test and find support for a hypothesis whereby facial teeth may differentiate at the expense of neuromasts. Finally, we document the expression of other genes known to mark developing teeth, including Edar, Pitx2, Troy, and Relt during and after tooth formation on the face. These genes all show transcript localization along the facial lateral line, in some cases marking wider regions than just the ectopic tooth organs alone. Together, our data show that tooth formation competency is a robust, highly localized phenomenon that is recalcitrant to permanent cessation via disruptions in Wnt signaling, while demonstrating a spatially-specific capability to be activated near a subset of neuromasts externally.