To enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cancer ...cells confined to droplets. The barcodes are then used to reassemble the genetic profiles of cells from next-generation sequencing data. By using this approach, we sequenced longitudinally collected acute myeloid leukemia (AML) tumor populations from two patients and genotyped up to 62 disease relevant loci across more than 16,000 individual cells. Targeted single-cell sequencing was able to sensitively identify cells harboring pathogenic mutations during complete remission and uncovered complex clonal evolution within AML tumors that was not observable with bulk sequencing. We anticipate that this approach will make feasible the routine analysis of AML heterogeneity, leading to improved stratification and therapy selection for the disease.
Aβ (beta-amyloid peptide) is an important contributor to Alzheimer's disease (AD). We modeled Aβ toxicity in yeast by directing the peptide to the secretory pathway. A genome-wide screen for toxicity ...modifiers identified the yeast homolog of phosphatidylinositol binding clathrin assembly protein (PICALM) and other endocytic factors connected to AD whose relationship to Aβ was previously unknown. The factors identified in yeast modified Aβ toxicity in glutamatergic neurons of Caenorhabditis elegans and in primary rat cortical neurons. In yeast Aβ impaired the endocytic trafficking of a plasma membrane receptor, which was ameliorated by endocytic pathway factors identified in the yeast screen. Thus, links between Aβ, endocytosis, and human AD risk factors can be ascertained with yeast as a model system.
Single cell analysis tools are crucial to understand the role that rare or heterogeneous cancer cells play in tumor progression. To enable the characterization of genetic variation within cancer cell ...populations, we developed a novel approach that barcodes amplified genomic DNA of individual cells confined to microfluidic droplets. The barcodes are used to reassemble the genetic profiles of individual cells from next-generation sequencing data. A key feature of our approach is the “two-step” microfluidic workflow. The microfluidic workflow first encapsulates individual cells in droplets, lyses the cells and prepares the genomic DNA for amplification with proteases. Following this lysate preparation step, the proteases are inactivated and droplets containing the genomes of individual cells are then paired with molecular barcodes and PCR reagents. We demonstrate that the two-step microfluidic approach is superior to workflows without the two-step process for efficient DNA amplification on thousands of individual cells per run with high coverage uniformity and low allelic dropout of targeted genomic loci.
To apply our single-cell sequencing technology to human tumor samples, we developed a targeted panel to partially sequence 23 genes frequently mutated in acute myeloid leukemia (AML) including TP53 , DNMT3A , FLT3 , NPM1 , NRAS , IDH1 and IDH2 . Using this panel, we were able to sensitively assay SNP and indel defined clones within AML samples collected longitudinally at the time of diagnosis, remission and relapse. Our single-cell data indicates that clonal populations inferred from VAFs obtained from bulk sequencing data may not fully resolve the heterogeneity within tumors; moreover, the single-cell nature of our approach enabled the unambiguous colocalization of multiple mutations within subclones not possible with bulk measurements. Collectively, our results show a greater degree of heterogeneity in AML tumor samples than is commonly appreciated with traditional sequencing paradigms and they demonstrate the value of single-cell analysis for AML.
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Eastburn:Mission Bio: Employment, Equity Ownership. Pellegrino:Mission Bio: Employment, Equity Ownership. Sciambi:Mission Bio: Employment, Equity Ownership. Treusch:Mission Bio: Employment, Equity Ownership. Gokhale:Mission Bio: Employment, Equity Ownership. Jacob:Mission Bio: Employment, Equity Ownership. Chen:Mission Bio: Employment, Equity Ownership. Jones:Mission Bio: Employment, Equity Ownership. Takahashi:Symbio Pharmaceuticals: Consultancy.
Signaling pathways enable cells to sense and respond to their environment. Many cellular signaling strategies are conserved from fungi to humans, yet their activity and phenotypic consequences can ...vary extensively among individuals within a species. A systematic assessment of the impact of naturally occurring genetic variation on signaling pathways remains to be conducted. In S. cerevisiae, both response and resistance to stressors that activate signaling pathways differ between diverse isolates. Here, we present a quantitative trait locus (QTL) mapping approach that enables us to identify genetic variants underlying such phenotypic differences across the genetic and phenotypic diversity of S. cerevisiae. Using a Round-robin cross between twelve diverse strains, we identified QTL that influence phenotypes critically dependent on MAPK signaling cascades. Genetic variants under these QTL fall within MAPK signaling networks themselves as well as other interconnected signaling pathways. Finally, we demonstrate how the mapping results from multiple strain background can be leveraged to narrow the search space of causal genetic variants.
Yeast cell biology has yielded fundamental insight into a variety of processes involved in human disease. Treusch et al. (p. 1241, published online 27 October; see the Perspective by McGurk and ...Bonini) have now created a yeast model of the cellular toxicity caused by the beta-Amyloid peptide Aβ 1-42, which is thought to be causal in Alzheimer's disease (AD). An unbiased genome-wide screen for modifiers of toxicity revealed the yeast homolog of PICALM, a confirmed AD risk factor involved in endocytosis. Three additional genes were also identified that appear to impact AD risk, based on their associations with AD onset and pathologic burden. A model of Aβ toxicity in the glutamatergic neurons of nematodes was created and used to confirm the role of the toxicity modifiers. PICALM was also shown to protect rat cortical neurons from toxic Aβ oligomers. Aβ (beta-amyloid peptide) is an important contributor to Alzheimer's disease (AD). We modeled Aβ toxicity in yeast by directing the peptide to the secretory pathway. A genome-wide screen for toxicity modifiers identified the yeast homolog of phosphatidylinositol binding clathrin assembly protein (PICALM) and other endocytic factors connected to AD whose relationship to Aβ was previously unknown. The factors identified in yeast modified Aβ toxicity in glutamatergic neurons of Caenorhabditis elegans and in primary rat cortical neurons. In yeast, Aβ impaired the endocytic trafficking of a plasma membrane receptor, which was ameliorated by endocytic pathway factors identified in the yeast screen. Thus, links between Aβ, endocytosis, and human AD risk factors can be ascertained with yeast as a model system. PUBLICATION ABSTRACT
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2011.
This electronic version was submitted by the student author. The certified thesis is available in the Institute ...Archives and Special Collections.
Cataloged from student submitted PDF version of thesis.
Includes bibliographical references.
Numerous neurodegenerative diseases are pathologically characterized by idiosyncratic protein amyloid inclusions. Not surprisingly amyloid fibrils have long been proposed to be the toxic protein species in these neurodegenerative diseases. However, more recent work has begun to suggest that the formation of ordered inclusions serves a protective role and that soluble oligomers on pathway to amyloid formation cause neuronal death. In that regard, ordered protein inclusions, such as aggresomes, have also been shown to facilitate the asymmetric inheritance of protein damage during the mitoses of cells ranging from E. coli to human stem cells. Yeast prion proteins are another group of proteins capable of adapting an amyloid conformation. The self-templating amyloid fold allows yeast prions to act as non-Mendelian elements of inheritance. We have shown that yeast prion amyloid fibrils, especially upon prion protein overexpression, localize to the IPOD (insoluble protein deposit), an ordered inclusion proximal to the vacuole, and that the majority of the prion amyloid is asymmetrically inherited upon cell division. I used the yeast prion Rnq1 to investigate how amyloid formation contributes to proteotoxicity. Ectopic overexpression of Rnq1 was extremely toxic, but only if the endogenous Rnq1 protein had adopted its amyloid conformation. The Hsp40 co-chaperone Sis1 was able to counteract the Rnq1-induced toxicity when co-overexpressed. In collaboration with Doug Cyr's lab I showed that Sis1-mediated amyloid formation was cytoprotective and that disordered non-amyloid aggregates induced toxicity. These results provide evidence that the formation of ordered inclusions can be cytoprotective. I further characterized Rnq1 toxicity, conducted two genome-ide screens for modifiers and found that Rnq1 induced a G2/M cell cycle arrest. Rnq1 overexpression resulted in the mislocalization of the core spindle pole body component Spc42 to the IPOD and an unduplicated spindle pole body. In mammalian cells aggresomes localize to centrosomes, the mammalian equivalent of the yeast spindle pole body. The finding that a yeast prion can interact with a spindle pole body component represents a new connection between the IPOD and aggresomes. Lastly, I studied a yeast model of Abeta 1-42 toxicity. Accumulation of the amyloid beta peptide is thought to be causal in both sporadic and familial Alzheimer's disease. In collaboration with Kent Matlack I developed a yeast model that expressed Abeta 1-42 in a manner recapitulating mammalian Abeta 1-42 generation and that was amenable to screens for genetic modifiers of Abeta 1-42 toxicity. The screen identified the yeast homolog of PICALM, a known Alzheimer's disease risk factor. I showed that Abeta 1-42 expression resulted in a defect in endocytosis that could be reverted by several of the genetic suppressors. In collaboration with the Caldwell lab, we showed that the genetic modifiers also modulated Abeta 1-42 toxicity in a neuronal setting, C. elegans glutamatergic neurons. Finally, we showed that PICALM could protect primary rat cortical neuron cultures from Abeta oligomer toxicity.
by Sebastian Treusch.
Ph.D.
Many DNA sequence variants influence phenotypes by altering gene expression. Our understanding of these variants is limited by sample sizes of current studies and by measurements of mRNA rather than ...protein abundance. We developed a powerful method for identifying genetic loci that influence protein expression in very large populations of the yeast Saccharomyes cerevisiae. The method measures single-cell protein abundance through the use of green-fluorescent-protein tags. We applied this method to 160 genes and detected many more loci per gene than previous studies. We also observed closer correspondence between loci that influence protein abundance and loci that influence mRNA abundance of a given gene. Most loci cluster at hotspot locations that influence multiple proteins - in some cases, more than half of those examined. The variants that underlie these hotspots have profound effects on the gene regulatory network and provide insights into genetic variation in cell physiology between yeast strains.
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