Lucy O'Brien, Postdoctoral Faculty Sponsor
Oligomerization of peripheral membrane proteins provides tunable control of cell surface polarity
2022; 121 (23): 4543-4559
View details for DOI 10.1016/j.bpj.2022.10.035
View details for Web of Science ID 000908349700013
A novel membrane complex is required for docking and regulated exocytosis of lysosome-related organelles in Tetrahymena thermophila.
2022; 18 (5): e1010194
In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon called regulated exocytosis. The molecular bases underlying regulated exocytosis have been extensively described in animals but it is not clear whether similar mechanisms exist in ciliates or their sister lineage, the Apicomplexan parasites, which together belong to the ecologically and medically important superphylum Alveolata. Beginning with a T. thermophila mutant in mucocyst exocytosis, we used a forward genetic approach to uncover MDL1 (Mucocyst Discharge with a LamG domain), a novel gene that is essential for regulated exocytosis of mucocysts. Mdl1p is a 40 kDa membrane glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. This sub-localization of Mdl1p, which occurs prior to docking, underscores a functional asymmetry in mucocysts that is strikingly similar to that of highly polarized secretory organelles in other Alveolates. A mis-sense mutation in the LamG domain results in mucocysts that dock but only undergo inefficient exocytosis. In contrast, complete knockout of MDL1 largely prevents mucocyst docking itself. Mdl1p is physically associated with 9 other proteins, all of them novel and largely restricted to Alveolates, and sedimentation analysis supports the idea that they form a large complex. Analysis of three other members of this putative complex, called MDD (for Mucocyst Docking and Discharge), shows that they also localize to mucocysts. Negative staining of purified MDD complexes revealed distinct particles with a central channel. Our results uncover a novel macromolecular complex whose subunits are conserved within alveolates but not in other lineages, that is essential for regulated exocytosis in T. thermophila.
View details for DOI 10.1371/journal.pgen.1010194
View details for PubMedID 35587496
View details for PubMedCentralID PMC9159632
The Dynamics of P Granule Liquid Droplets Are Regulated by the Caenorhabditis elegans Germline RNA Helicase GLH-1 via Its ATP Hydrolysis Cycle
2020; 215 (2): 421-434
View details for DOI 10.1534/genetics.120.303052
View details for Web of Science ID 000538153800011
The PAR proteins: from molecular circuits to dynamic self-stabilizing cell polarity
2017; 144 (19): 3405-3416
PAR proteins constitute a highly conserved network of scaffolding proteins, adaptors and enzymes that form and stabilize cortical asymmetries in response to diverse inputs. They function throughout development and across the metazoa to regulate cell polarity. In recent years, traditional approaches to identifying and characterizing molecular players and interactions in the PAR network have begun to merge with biophysical, theoretical and computational efforts to understand the network as a pattern-forming biochemical circuit. Here, we summarize recent progress in the field, focusing on recent studies that have characterized the core molecular circuitry, circuit design and spatiotemporal dynamics. We also consider some of the ways in which the PAR network has evolved to polarize cells in different contexts and in response to different cues and functional constraints.
View details for DOI 10.1242/dev.139063
View details for Web of Science ID 000412120700003
View details for PubMedID 28974638
View details for PubMedCentralID PMC5665476
Mechanisms and Disease Associations of Haplotype-Dependent Allele-Specific DNA Methylation
AMERICAN JOURNAL OF HUMAN GENETICS
2016; 98 (5): 934-955
Haplotype-dependent allele-specific methylation (hap-ASM) can impact disease susceptibility, but maps of this phenomenon using stringent criteria in disease-relevant tissues remain sparse. Here we apply array-based and Methyl-Seq approaches to multiple human tissues and cell types, including brain, purified neurons and glia, T lymphocytes, and placenta, and identify 795 hap-ASM differentially methylated regions (DMRs) and 3,082 strong methylation quantitative trait loci (mQTLs), most not previously reported. More than half of these DMRs have cell type-restricted ASM, and among them are 188 hap-ASM DMRs and 933 mQTLs located near GWAS signals for immune and neurological disorders. Targeted bis-seq confirmed hap-ASM in 12/13 loci tested, including CCDC155, CD69, FRMD1, IRF1, KBTBD11, and S100A(∗)-ILF2, associated with immune phenotypes, MYT1L, PTPRN2, CMTM8 and CELF2, associated with neurological disorders, NGFR and HLA-DRB6, associated with both immunological and brain disorders, and ZFP57, a trans-acting regulator of genomic imprinting. Polymorphic CTCF and transcription factor (TF) binding sites were over-represented among hap-ASM DMRs and mQTLs, and analysis of the human data, supplemented by cross-species comparisons to macaques, indicated that CTCF and TF binding likelihood predicts the strength and direction of the allelic methylation asymmetry. These results show that hap-ASM is highly tissue specific; an important trans-acting regulator of genomic imprinting is regulated by this phenomenon; and variation in CTCF and TF binding sites is an underlying mechanism, and maps of hap-ASM and mQTLs reveal regulatory sequences underlying supra- and sub-threshold GWAS peaks in immunological and neurological disorders.
View details for DOI 10.1016/j.ajhg.2016.03.027
View details for Web of Science ID 000375869300011
View details for PubMedID 27153397
View details for PubMedCentralID PMC4863666
Trans effects of chromosome aneuploidies on DNA methylation patterns in human Down syndrome and mouse models
2015; 16: 263
Trisomy 21 causes Down syndrome (DS), but the mechanisms by which the extra chromosome leads to deficient intellectual and immune function are not well understood.Here, we profile CpG methylation in DS and control cerebral and cerebellar cortex of adults and cerebrum of fetuses. We purify neuronal and non-neuronal nuclei and T lymphocytes and find biologically relevant genes with DS-specific methylation (DS-DM) in each of these cell types. Some genes show brain-specific DS-DM, while others show stronger DS-DM in T cells. Both 5-methyl-cytosine and 5-hydroxy-methyl-cytosine contribute to the DS-DM. Thirty percent of genes with DS-DM in adult brain cells also show DS-DM in fetal brains, indicating early onset of these epigenetic changes, and we find early maturation of methylation patterns in DS brain and lymphocytes. Some, but not all, of the DS-DM genes show differential expression. DS-DM preferentially affected CpGs in or near specific transcription factor binding sites (TFBSs), implicating a mechanism involving altered TFBS occupancy. Methyl-seq of brain DNA from mouse models with sub-chromosomal duplications mimicking DS reveals partial but significant overlaps with human DS-DM and shows that multiple chromosome 21 genes contribute to the downstream epigenetic effects.These data point to novel biological mechanisms in DS and have general implications for trans effects of chromosomal duplications and aneuploidies on epigenetic patterning.
View details for DOI 10.1186/s13059-015-0827-6
View details for Web of Science ID 000365321200001
View details for PubMedID 26607552
View details for PubMedCentralID PMC4659173