A Study of Nanomaterials and Endocytic Marked Compartments in Cells using Image Correlation Spectroscopy Analysis

• Author / Creator

  Shearer, Lindsay J

• Nanomaterials have gained interest due to their unique physical and chemical phenomena that is only observed for materials in this size regime. In the biohealth sector, gold nanoparticles are good platforms for use in curative treatments and detection of cancer and disease. However, their effectiveness depends on how well they are internalized and destined to travel in cells. One pathway gold nanoparticles use to travel in cells is clathrin mediated endocytosis. In this pathway, gold nanoparticles are encapsulated in membrane vesicles that undergo fusions and maturations with four intracellular vesicles (endosomes) before being directed to the lysosome for degradation. There are four proteins which “mark” the pathway; Rab5 (Early Endosome), Rab11 (Recycling Endosome), Rab7 (Late Endosome), and LAMP-1 (Lysosome). In this work, we first studied the distribution and co-localization of the four proteins which “mark” the pathway to determine the extent to which these markers are unique for specific endosomes or co-exist with other markers on endosomes. This study was done for C2C12 and A549 cells to determine if the distribution and co-localization of markers was cell type specific. We determined that markers are not unique for specific endosomes and markers can co-exist in the same endosome, the distribution and co-localization of markers in cells is cell type specific and the distribution of ternary complexes varies with cell type. We then studied the time dependent internalization and uptake of phospholipid coated gold nanoparticles and the extent of co-localization of phospholipid coated gold nanoparticles with markers in C2C12 and A549 cells. C2C12 and A549 cells are able to continuously internalize phospholipid coated gold nanoparticles with time, but to different extents. It was also observed that the ability for cells to withhold phospholipid coated gold nanoparticles in a nanoparticle free extracellular environment depends on cell type. We also found that binary and ternary complex formation in A549 and C2C12 cells varied. A fraction of binary complexes consisting of one marker and phospholipid coated gold nanoparticles may also include ternary complexes in both cell types. This work was consistent with results obtained in Chapter 3. As we were able to effectively study the endocytic pathway and uptake of phospholipid coated gold nanoparticles in two different cell types, we chose to study α-synuclein (α-syn) in SH-SY5Y cells. α-syn is a protein associated with Parkinson’s disease; until recently, oligomeric forms of the protein are understood to be toxic and are the current focus of study for scientists for detection and curative methods. Thus, we applied the same logic and methods in Chapters 3 and 4 to study oligomeric forms of α-synuclein. We studied the time dependent uptake of oligomers, distribution and extent of co-localization of markers, extent of co-localization of oligomers, and the extent of co-localization of oligomers with markers in SH-SY5Y cells. The oligomers chosen for this study were monomers, as well as engineered dimers and tetramers. We found each oligomer was able to passively enter cells and each oligomer was observed to also undergo clathrin mediated endocytosis. Each oligomer had shown to be internalized and taken up in cells in a time dependent manner; however, the degree of aggregation of monomers decreases the most with time. Real-time observation of monomers, dimers, and tetramers of α-synuclein in SH-SY5Y cells was also studied. This was the first time real-time observation was to be undertaken for this study; we determined an experiment consisting of 50 images is sufficient to estimate the diffusion time, diffusion coefficient, and temporal auto correlation amplitude. We also observed that the diffusion time of oligomers in cells was proportional to the size of the oligomer. In this work, membrane trafficking and possible roles of endocytic proteins was studied along with the endocytic uptake of two nanomaterial systems of interest for potential therapeutic and diagnostic applications. This is the first ICS analysis of endocytic marker and gold nanoparticle association and distribution and the first successful quantitative three color co-localization performed in-vitro in C2C12 and A549 cells, using Image Correlation Spectroscopy analysis. The work presented in this thesis significantly contributes to the field of nanotechnology and cell biology. The work in this thesis was accomplished using Image Correlation Spectroscopy analysis of images obtained using a laser scanning confocal microscope.

• Subjects / Keywords

  • Petersen
  • Image Correlation Spectroscopy
  • Shearer

• Graduation date

  Spring 2016

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3Z31P48W

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.