Thesis (PH. D.)--Michigan State University. Genetics, 2008. / Title from PDF t.p. (viewed on Sept. 2, 2009) Includes bibliographical references. Also issued in print.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 82-90).

No description available.

No description available.

The lysosome, a multi-copy organelle, was chosen as an example to study intraorganelle dynamics. Lysosomal contents and membrane proteins were shown to intermix rapidly in fused mammalian cells, with a t_½ of ~30 min. Lysosomal content intermixing, shown by a sensitive invertase-lysosome/[¹⁴C]-sucrose-lysosome pairing assay, was inhibited greatly by ATP inhibitors and partially by cytochalasin D. Lysosomal membrane protein intermixing was shown by the transfer of LAMP-2, a mouse specific lysosomal membrane antigen, from mouse lysosomes to hamster sucrosomes, sucrose-swollen lysosomes. Lysosomal membrane protein intermixing was also shown by the co-localization of LIMP I, a rat specific lysosomal membrane antigen, and LAMP-1, a mouse specific lysosomal membrane antigen. Co-localization was assessed by both double immunofluorescent staining and double immunogold labeling of thin cryosections. Both lysosomal content and membrane protein intermixing were inhibited by nocodazole, a microtubule disruptor. In fused cells, lysosomes remained small, punctate and scattered throughout the cytoplasm. In comparison to lysosomes, the prelysosomal compartment (PLC), a single copy organelle which is related to the lysosome, congregated together to form an extended PLC complex associated with clustered nuclei. The intermixing of both resident and transient Golgi membrane proteins was studied in fused cells. Resident Golgi membrane protein intermixing was slow, with a t_½ of ~ 1.75 h; it was concomitant with the congregation of the Golgi units. In comparison, the transient Golgi membrane protein was transported much faster from Golgi units to the other Golgi units, with the t_½ ≤ 15 min. Transient Golgi membrane protein transport occurred between separate Golgi units. These results are consistent with two different pathways for resident and transient Golgi membrane protein transport: a slow, lateral diffusion along the Golgi connections transport pathway for resident Golgi membrane proteins; and a rapid, transient protein selective, vesicle-mediated transport pathway for transient Golgi membrane proteins. / Ph. D.

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Trichomonas vaginalis is a human pathogen that affects annually approximately 258 million people worldwide. This parasite possesses organelles of mitochondrial origin called hydrogenosomes, which generate ATP under anaerobic conditions. The identification of the protein content at the subcellular level may provide new targets for antiparasitic drugs developments as well as it contributes for our understanding of the organelles function and evolution. The availability of protocols for organelles purification and the complete genome sequence allow the study of the organellar proteomes using mass spectrometry and bioinformatics, providing a powerful strategy that combine cell biology and proteomics. In our research, we used several approaches to identify the protein composition in hydrogenosomes and mitosomes. We performed transcriptomic and proteomic analysis to investigate the molecular responses of Trichomonas vaginalis upon iron availability. Furthermore, the changes in the proteome during the development of metronidazole resistance were also studied. The organelles separated by differential and Optiprep-sucrose gradient centrifugation were analyzed with nano- RP-HPLC/MALDI-TOF/TOF. We also used Triton X-114 phase partitioning to separate membrane proteins and iTRAQ technique to label the peptides...

This thesis describes the use of antibodies and immunofluorescence for subcellular localization of proteins. The key objective is the creation of an open-source atlas with information on the subcellular location of every human protein. Knowledge of the spatial distribution and the precise location of a protein within a cell is important for its functional characterization, and describing the human proteome in terms of compartment proteomes is important to decipher cellular organization and function.   Immunofluorescence and confocal microscopy of cultured cells were used for high-resolution detection of proteins on a high-throughput scale. Critical to immunofluorescence results are sample preparation and specific antibodies. Antibody staining of cells requires fixation and permeabilization, both of which can result in loss or redistribution of proteins and masking of epitopes. A high-throughput approach demands a standardized protocol suitable for the majority of proteins across cellular compartments. Paper I presents an evaluation of sample preparation techniques from which such a single fixation and permeabilization protocol was optimized. Paper II describes the results from applying this protocol to 4000 human proteins in three cell lines of different origin.   Paper III presents a strategy for application-specific antibody validation. Antibodies are the key reagents in immunofluorescence, but all antibodies have potential for off-target binding and should be validated thoroughly. Antibody performance varies across sample types and applications due to the competition present and the effect of the sample preparation on antigen accessibility. In this paper application-specific validation for immunofluorescence was conducted using colocalization with fluorescently tagged protein in transgenic cell lines. / <p>QC 20160509</p>

Compartmentalization allows for the spatial organization of cellular components and is crucial for numerous biological functions. One recently uncovered strategy for intracellular compartmentalization is phase separation via the de-mixing of biomacromolecules. Membraneless organelles, also referred to as biomolecular condensates, are compartments formed by phase separation and create distinct environments that are essential to cellular processes ranging from cell signaling to gene expression. Biomolecular condensates offer several advantages – for example, dynamic restructuring of internal constituents and diffusion of cellular components into/out of compartments – that make them suitable for applications in biocatalysis or pharmaceutical production. However, the ability to independently engineer the formation and disassembly of condensates in vivo remains a challenge. Here, concepts from polymer science have been used to understand parameters that govern intracellular phase separation. Many biomolecular condensates exhibit physical properties that are similar to complex coacervates as both are liquid-like phase separated mixtures formed via associative phase separation, frequently with oppositely charged polyelectrolytes. We utilize the physical phenomenon of complex coacervation and principles underlying the formation of liquid-like biological condensates to identify design parameters for engineering synthetic, phase separated organelles in E. coli. In this dissertation, we employed a library of cationic charge variants derived from superfolder green fluorescent protein (sfGFP) to elucidate the effects of overall cationic charge on intracellular phase separation. We first investigated the complex coacervation of engineered proteins with biological polyelectrolytes to determine predictive design rules for protein phase separation and translated these design rules in vivo to engineer bacterial condensates. Characterization of the coacervate-like properties and macromolecular composition revealed that these condensates can undergo dynamic restructuring and exhibit biomolecular specificity. To facilitate the engineering of active supercharged proteins, we also developed short, cationic peptide tags, ranging from 6-27 amino acids in length, that can be appended onto any protein of interest to promote intracellular phase separation. We find that overall charge generally determines protein phase behavior and observe the formation and disassembly of condensates near the physiological phase boundary. Interestingly, we find that small modifications in charge density can tune the interaction strength between associating biomacromolecules and thus tune condensate stability. We demonstrate the use of these protein design parameters and cationic peptide tags to sequester catalytic enzymes and manipulate the intracellular localization of multiple proteins. These studies pave the way to building synthetic, functional organelles.

The human cell is complex, with countless processes ongoing in parallel in specialized compartments, the organelles. Cells can be studied in vitro by using immortalized cell lines that represent cells in vivo to a varying degree. Gene expression varies between cell types and an average cell line expresses around 10,000-12,000 genes, as measured with RNA sequencing. These genes encode the cell’s proteome; the full set of proteins that perform functions in the cell. In paper I we show that RNA sequencing is a necessary tool for studying the proteome of the human cell. By studying the proteome, and proteins’ localization in the cell, information can be assembled on how the cell functions. Image-based methods allow for detailed spatial resolution of protein localization as well as enable the study of temporal events. Visualization of a protein can be accomplished by using either a cell line that is transfected to express the protein with a fluorescent tag, or by targeting the protein with an affinity reagent such as an antibody. In paper II we present subcellular data for a majority of the human proteins, showing that there is a high degree of complexity in regard to where proteins localize in the cell. Cellular energy is generated in the mitochondria, an important organelle that is also active in many other different functions. Today approximately only a third of the estimated mitochondrial proteome has been validated experimentally, indicating that there is much more to understand with regard to the functions of the mitochondria. In paper III we explore the mitochondrial proteome, based on the results of paper II. We also present a method for sublocalizing proteins to subcompartments that can be performed in a high-throughput manner. To conclude, this thesis shows that transcriptomics is a useful tool for proteome-wide subcellular localization, and presents high-resolution spatial distribution data for the human cell with a deeper analysis of the mitochondrial proteome. / <p>QC 20170512</p>