Intracellular Amplification for Applications in Single-cell DNA Sequencing

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Description
Single-cell DNA sequencing (scDNA-seq) can identify genetic differencesbetween individual cells and has broad applications in studying biology. For example, because scDNA-seq preserves haplotypes, it enables the addition of information about the fitness of different combinations of mutations into studies that…

Single-cell DNA sequencing (scDNA-seq) can identify genetic differencesbetween individual cells and has broad applications in studying biology. For example, because scDNA-seq preserves haplotypes, it enables the addition of information about the fitness of different combinations of mutations into studies that quantify the fitness of individual mutations. However, it requires separating cells manually or using machinery, which is time-consuming and costly as every cell requires a separate reaction. Thus, most studies are limited to a few hundred cells, and scaling up is expensive and challenging. This problem also makes it difficult to multiplex samples or to study multiple sample types in the same experiment. To solve these problems, I introduce a novel method for sequencing DNA in heterogeneous cell populations by using the cell itself as a container for sequencing reactions, eliminating the need to isolate individual cells. The method involves diffusing DNA polymerase and barcoded primers into intact cells and amplifying its DNA Intracellularly. To ensure that DNA from each cell can be uniquely identified, I use combinatorial barcoding, which assigns a specific barcode to each cell using a unique combination of non-unique nucleotide block sequences. This allows for the pooling of cells, making the method multiplexable and enabling the analysis of dozens of samples containing thousands of cells. The method is flexible and allows for targeted sequencing of a region of interest and whole genome sequencing. I optimize the method for various organisms and applications so it can be made accessible to a wide range of research groups.
Date Created
2024
Agent

A Study of the Cockroach Gut Flagellates in Genus Lophomonas with Single Cell Approaches

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Description
Lophomonas is a genus of flagellated parabasalids that exist as commensal symbionts in the hindguts of a variety of pest cockroaches. The genus contains two species: Lophomonas blattarum and Lophomonas striata. The two species differ by way of bacterial ectosymbionts…

Lophomonas is a genus of flagellated parabasalids that exist as commensal symbionts in the hindguts of a variety of pest cockroaches. The genus contains two species: Lophomonas blattarum and Lophomonas striata. The two species differ by way of bacterial ectosymbionts that attach to the outside of L. striata, giving rise to a striated and spindle-shaped appearance. As the attachment of bacterial symbionts prohibits L. striata from taking up large food particles in the same manner as L. blattarum, it is likely the two species differ in which metabolic genes they possess. Here, a comparison of transcriptomes between the two Lophomonas species show slight differences between the species. Metagenomic analysis of L. striata also presents the possibility of L. striata ectosymbionts as belonging to the genus Parabacteroides.
Date Created
2023
Agent

Mechanism of the FO Motor in the F-ATP Synthase

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Description
The FOF1 ATP synthase is responsible for generating the majority of adenosine triphosphate (ATP) in almost all organisms on Earth. A major unresolved question is the mechanism of the FO motor that converts the transmembrane flow of protons into rotation…

The FOF1 ATP synthase is responsible for generating the majority of adenosine triphosphate (ATP) in almost all organisms on Earth. A major unresolved question is the mechanism of the FO motor that converts the transmembrane flow of protons into rotation that drives ATP synthesis. Using single-molecule gold nanorod experiments, rotation of individual FOF1 were observed to measure transient dwells (TDs). TDs occur when the FO momentarily halts the ATP hydrolysis rotation by the F1-ATPase. The work presented here showed increasing TDs with decreasing pH, with calculated pKa values of 5.6 and 7.5 for wild-type (WT) Escherichia coli (E. coli) subunit-a proton input and output half-channels, respectively. This is consistent with the conclusion that the periplasmic proton half-channel is more easily protonated than the cytoplasmic half-channel. Mutation in one proton half-channel affected the pKa values of both half-channels, suggesting that protons flow through the FO motor via the Grotthuss mechanism. The data revealed that 36° stepping of the E. coli FO subunit-c ring during ATP synthesis consists of an 11° step caused by proton translocations between subunit-a and the c-ring, and a 25° step caused by the electrostatic interaction between the unprotonated c-subunit and the aR210 residue in subunit-a. The occurrence of TDs fit to the sum of three Gaussian curves, which suggested that the asymmetry between the FO and F1 motors play a role in the mechanism behind the FOF1 rotation. Replacing the inner (N-terminal) helix of E. coli c10-ring with sequences derived from c8 to c17-ring sequences showed expression and full assembly of FOF1. Decrease in anticipated c-ring size resulted in increased ATP synthesis activity, while increase in c-ring size resulted in decreased ATP synthesis activity, loss of Δψ-dependence to synthesize ATP, decreased ATP hydrolysis activity, and decreased ACMA quenching activity. Low levels of ATP synthesis by the c12 and c15-ring chimeras are consistent with the role of the asymmetry between the FO and F1 motors that affects ATP synthesis rotation. Lack of a major trend in succinate-dependent growth rates of the chimeric E. coli suggest cellular mechanisms that compensates for the c-ring modification.
Date Created
2023
Agent

Understanding the Heterogeneity in Gene Regulatory Responses to Misfolded Protein Toxicity

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Description
Protein misfolding is a problem faced by all organisms, but the reasons behind misfolded protein toxicity are largely unknown. It is difficult to pinpoint one exact mechanism as the effects of misfolded proteins can be widespread and variable between cells.…

Protein misfolding is a problem faced by all organisms, but the reasons behind misfolded protein toxicity are largely unknown. It is difficult to pinpoint one exact mechanism as the effects of misfolded proteins can be widespread and variable between cells. To better understand their impacts, here I explore the consequences of misfolded proteins and if they affect all cells equally or affect some cells more than others. To investigate cell subpopulations, I built and optimized a cutting-edge single-cell RNA sequencing platform (scRNAseq) for yeast. By using scRNAseq, I can study the expression variability of many genes (i.e. how the transcriptomes of single cells differ from one another). To induce misfolding and study how single cells deal with this stress, I use engineered strains with varying degrees of an orthogonal misfolded protein. When I computationally cluster the cells expressing misfolded proteins by their sequenced transcriptomes, I see more cells with the severely misfolded protein in subpopulations undergoing canonical stress responses. For example, I see these cells tend to overexpress chaperones, and upregulate mitochondrial biogenesis and transmembrane transport. Both of these are hallmarks of the “Generalized” or “Environmental Stress Response” (ESR) in yeast. Interestingly, I do not see all components of the ESR upregulated in all cells, which may suggest that the massive transcriptional changes characteristic of the ESR are an artifact of having defined the ESR in bulk studies. Instead, I see some cells activate chaperones, while others activate respiration in response to stress. Another intriguing finding is that growth supporting proteins, such as ribosomes, have particularly heterogeneous expression levels in cells expressing misfolded proteins. This suggests that these cells potentially reallocate their metabolic functions at the expense of growth but not all cells respond the same. In sum, by using my novel single-cell approach, I have gleaned new insights about how cells respond to stress. which can help me better understand diseased cells. These results also teach how cells contend with mutation, which commonly causes protein misfolding and is the raw material of evolution. My results are the first to explore single-cell transcriptional responses to protein misfolding and suggest that the toxicity from misfolded proteins may affect some cells’ transcriptomes differently than others.
Date Created
2023
Agent

How do Endosymbionts Evade the Endocytic Cycle: The Story of Planococcus Citri Mealybugs

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Description
Planococcus citri mealybugs are an incredibly unique species due to their nested endosymbiosis, in which Moranella endobia resides within Tremblaya princeps. These endosymbionts work together with their host to provide nutritional support throughout its life cycle and onto future offspring.…

Planococcus citri mealybugs are an incredibly unique species due to their nested endosymbiosis, in which Moranella endobia resides within Tremblaya princeps. These endosymbionts work together with their host to provide nutritional support throughout its life cycle and onto future offspring. Though what makes these endosymbionts even more interesting is that when viewed form a cell biological perspective, it becomes evident that they should have been exocytosed out of the host millions of years ago. One of the three membranes that surrounds Tremblaya, particularly the outermost vacuolar membrane, acts as the endosomal compartment around the bacteriome. In a traditional case of the endocytic cycle, the contents within the vacuole would be marked by the GTPase proteins Rab5 and Rab7 respectively until the fate of lysosomal digestion occurred. Though what is unique about the vacuolar membrane that surrounds Tremblaya is two things: first is that that membrane is lost and regained upon maternal transmission and secondly the endosymbionts within the membrane are not reaching their lysosomal fate, rather they are being passed down onto future generations. How these endosymbionts can redirect the endocytic pathway can possibly be explained by one of these four mechanisms: 1. Rabex-5 fails to recruit to the membrane of the early endosome, 2. Interruption of Rab7 activation by inhibiting membrane translocation of Ccz1, 3. Failure of the HOPS complex to bind to the late endosome, 4. Inhibition of translocation of ORP1L to the late endosome. Though the four mechanisms outlined above are very clearly regarding a cell biological process, they are not easily testable in a real-world setting. Thus, to adjust and account for this, the early and late endosomal marker proteins (Rab5 and Rab7) were used as the proteins of interest throughout immunofluorescence and western blot experimentation. These experiments revealed significant difficulties in working with commercially made antibodies but more importantly provided insight as to how is best to go forth with this research. In addition to this, qPCR experimentation and Rab7 epitope analysis did reveal that Rab5 and Rab7 are in fact key players in understanding how these endosymbionts are able to evade the endocytic cycle.
Date Created
2023
Agent

A Spatial Proteome of Paramecium tetraurelia

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Description
I studied the evolution and cell biology of Paramecium tetraurelia—a model ciliate with over 40,000 distinct protein-coding genes resulting from as many as three ancient whole-genome duplication events. I was interested in the functional diversification of these gene duplicates at…

I studied the evolution and cell biology of Paramecium tetraurelia—a model ciliate with over 40,000 distinct protein-coding genes resulting from as many as three ancient whole-genome duplication events. I was interested in the functional diversification of these gene duplicates at the level of protein localization, but the commonly used tools to study this were tedious. I instead applied a protein-correlation profiling approach to this system by way of generating a dozen sub-cellular fractions with different protein constituents due to the density of their resident organelle and then assayed these fractions using quantitative mass spectrometry. Each protein’s unique abundance profile provided evidence for its subcellular localization, and I used both supervised and unsupervised classification algorithms to cluster proteins together based on the similarity of these profiles to several hundred “marker proteins” which I manually curated. After expanding the protein inventory for numerous organelles by as many as a thousand proteins, I determined many features not previously understood or appreciated such as mosaic biochemical pathways, evidence for differential sorting mechanisms, and the abnormal evolutionary patterns of the mitochondrial proteome of ciliates. I developed a simple bioinformatic tool to probe spatial proteomics datasets more easily for proteins of interest. I demonstrate its applicability using a handful of well-characterized proteins in the budding yeast Saccharomyces cerevisiae as well as interesting proteins in less well-studied model systems like P. tetraurelia and the apicomplexan Toxoplasma gondii to both recapitulate known interactions and discover new ones. Finally, I look for large-scale evidence of gene duplicates relocalizing to new cellular compartments in P. tetraurelia and S. cerevisiae using this new dataset and a previously generated one, respectively. I find thousands of pairs of duplicates which are differentially identified and display evidence for subcellular divergence, and this seems to be largely decoupled from large changes in protein sequence but are instead associated with indels in their N-terminal peptide. These findings support the use of high-throughput proteomic techniques to determine evidence of functional divergence of gene duplicates. Taken together, this works provides a deep characterization of one of the largest unicellular proteomes in nature.
Date Created
2022
Agent

Profiling of Indel Phases in Coding Regions

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Description
Advances in sequencing technology have generated an enormous amount of data over the past decade. Equally advanced computational methods are needed to conduct comparative and functional genomic studies on these datasets, in particular tools that appropriately interpret indels within an…

Advances in sequencing technology have generated an enormous amount of data over the past decade. Equally advanced computational methods are needed to conduct comparative and functional genomic studies on these datasets, in particular tools that appropriately interpret indels within an evolutionary framework. The evolutionary history of indels is complex and often involves repetitive genomic regions, which makes identification, alignment, and annotation difficult. While previous studies have found that indel lengths in both deoxyribonucleic acid and proteins obey a power law, probabilistic models for indel evolution have rarely been explored due to their computational complexity. In my research, I first explore an application of an expectation-maximization algorithm for maximum-likelihood training of a codon substitution model. I demonstrate the training accuracy of the expectation-maximization on my substitution model. Then I apply this algorithm on a published 90 pairwise species dataset and find a negative correlation between the branch length and non-synonymous selection coefficient. Second, I develop a post-alignment fixation method to profile each indel event into three different phases according to its codon position. Because current codon-aware models can only identify the indels by placing the gaps between codons and lead to the misalignment of the sequences. I find that the mouse-rat species pair is under purifying selection by looking at the proportion difference of the indel phases. I also demonstrate the power of my sliding-window method by comparing the post-aligned and original gap positions. Third, I create an indel-phase moore machine including the indel rates of three phases, length distributions, and codon substitution models. Then I design a gillespie simulation that is capable of generating true sequence alignments. Next I develop an importance sampling method within the expectation-maximization algorithm that can successfully train the indel-phase model and infer accurate parameter estimates from alignments. Finally, I extend the indel phase analysis to the 90 pairwise species dataset across three alignment methods, including Mafft+sw method developed in chapter 3, coati-sampling methods applied in chapter 4, and coati-max method. Also I explore a non-linear relationship between the dN/dS and Zn/(Zn+Zs) ratio across 90 species pairs.
Date Created
2022
Agent

Obstacle Induced Branching in Neurospora crass and Rhizopus oryzae

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Description
The branching dynamics and navigation of filamentous fungi that have an apical vesical crescent (AVC) are poorly understood. Here, Rhizopus oryzae (Mucoromycota), which has an AVC, is compared to Neurospora crassa (Ascomycota), which has a Spitzenkörper (Spk), as they navigated…

The branching dynamics and navigation of filamentous fungi that have an apical vesical crescent (AVC) are poorly understood. Here, Rhizopus oryzae (Mucoromycota), which has an AVC, is compared to Neurospora crassa (Ascomycota), which has a Spitzenkörper (Spk), as they navigated microfluidic maze environments varying in pattern. The different maze patterns (diamonds, squares, and chevrons) presented increasing angles of impact, and degrees of obstruction. This investigation addressed questions regarding advantages or disadvantages that a Spk or AVC may provide in hyphal growth. All branching phenomena were compared to the regular branching of unobstructed growth to determine obstacle induced branching. Neurospora crassa generated more branches per impact amongst all three maze types and was unable to complete the chevron maze types. Rhizopus oryzae generated less branches per impact but was able to complete every maze type. The greatest difference in branch formation was seen in the chevron maze design where N. crassa generated a greater number than R. oryzae. Neurospora crassa exhibited a hyperbranching response in the chevron mazes not seen in R. oryzae. Closer inspection of the hyperbranching events revealed that they were composed of initial branching events followed by secondary and tertiary branching events. The directional memory of N. crassa was also observed, and was a characteristic of R. oryzae. While the branching dynamics and navigation of N. crassa and R. oryzae were different, and N. crassa exhibited branching and navigational phenomenon that R. oryzae did not, R. oryzae seemingly had the advantage with its use of an AVC over N. crassa, as it was able to complete every maze type, which N. crassa was unable to do.
Date Created
2021
Agent

Evolutionary History of Eukaryotic Oxysterol Binding Proteins

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Description

Cells have mechanisms in place to maintain the specific lipid composition of distinct organelles including vesicular transport by the endomembrane system and non-vesicular lipid transport by lipid transport proteins. Oxysterol Binding Proteins (OSBPs) are a family of lipid transport proteins…

Cells have mechanisms in place to maintain the specific lipid composition of distinct organelles including vesicular transport by the endomembrane system and non-vesicular lipid transport by lipid transport proteins. Oxysterol Binding Proteins (OSBPs) are a family of lipid transport proteins that transfer lipids at various membrane contact sites (MCSs). OSBPs have been extensively investigated in human and yeast cells where twelve have been identified in Homo sapiens and seven in Saccharomyces cerevisiae. The evolutionary relationship between these well-characterized OSBPs is still unclear. Reconstructed OSBP phylogenies revealed that the ancestral Saccharomycotinan had four OSBPs, the ancestral Holomycotan had five OSBPs, the ancestral Holozoan had six OSBPs, the ancestral Opisthokont had three OSBPs, and the ancestral Eukaroyte had three OSBPs. Our analysis identified three clades of ancient OSBPs not present in animals or fungi.

Date Created
2022-05
Agent

TSPO Function and Phylogeny in Eukaryotes

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Description

TSPO was discovered in 1977 and it’s function is still currently unknown. Significant research has suggested that TSPO functions in steroidogenesis to import cholesterol from the mitochondrial outer membrane (MOM) to the mitochondrial inner membrane (MIM) where it is converted…

TSPO was discovered in 1977 and it’s function is still currently unknown. Significant research has suggested that TSPO functions in steroidogenesis to import cholesterol from the mitochondrial outer membrane (MOM) to the mitochondrial inner membrane (MIM) where it is converted into steroids. There were two indications that this is TSPOs main function: its elevated levels in steroidogenic tissue and its primary location in the MOM. There is evidence of TSPO binding cholesterol with high affinity, however there is not currently evidence of TSPO transporting cholesterol. STAR, ACBD1, and ACBD3 are proteins thought to be associated with TSPO and steroidogenesis. However, the distribution of these proteins in various eukaryotes show little similarity suggesting that TSPO functions independently. The function of TSPO in steroid synthesis has been called into question because a well-cited research paper claimed that TSPO knockdown resulted in embryonic lethal mice, however there was no evidence presented from their study and this experiment did not produce the same results when repeated in later studies. There are also studies that show TSPO may not be involved in regulation of sterols, but instead may regulate cell stress. The elevated levels of TSPO during inflammation suggest a role for TSPO in cellular stress. Binding interactions with porphyrins and heme also support that TSPO may modulate stress levels. We used the phylogeny of TSPO in order to gain greater insight into the evolutionary function of TSPO. NCBI BLAST searches revealed that TSPO was present in bacteria and had a widespread but patchy distribution in a small set of eukaryotes. From these initial results, we were prompted to search a larger set of eukaryotes for TSPO. All of the prokaryotic and eukaryotic TSPO sequences were used to create a phylogenetic tree that would provide greater insight into the evolution and function of TSPO. If TSPO was from a common ancestor, it is probable that its function is related to sterol regulation whereas if gained in eukaryotes by horizontal gene transfer from bacteria its function is related to stress regulation. The phylogenetic tree was most consistent with an ancestral origin of TSPO with an evolutionary function related to steroid synthesis regulation. However, there is not sufficient research to confirm the function of TSPO.

Date Created
2021-12
Agent
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