Among the central constituents of the innate immune system are macrophages, which are known for phagocytosis or ‘eating’ foreign particles or pathogens. Macrophages express several cell-surface proteins including transmembrane and membrane-anchored receptors, which play a vital role in their response to pathogenic stimuli. The plasma membrane is a highly fluid and dynamic environment, which facilitates the diffusion of lipids and proteins within the plane of the membrane. This study aims to measure the lateral diffusion of two types of plasma membrane receptors on macrophages, toll-like receptor II (TLR2) and cluster of differentiation 14 (CD14), to answer three main research questions: 1) Which type of fluorescence-based microscopy techniques is best suited for measuring the lateral diffusion of TLR2 and CD14 on macrophage plasma membrane? 2) Does culturing macrophages on different surface topographies impact the diffusion of TLR2 in the plasma membrane and its pro-inflammatory response, along with morphological changes? 3) Does aging alter the lateral diffusion of TLR2 in the plasma membrane of macrophages? To date, a variety of fluorescence-based methods have been developed to study the dynamics of cell membrane constituents. These techniques are based on either ensemble or single particle measurements. We have used single particle tracking methods to track the mobility of fluorescently labeled membrane receptors on murine bone marrow-derived macrophages. Total internal reflection fluorescence microscopy (TIRF) was used to visualize and capture the dynamics in live cells. Using a custom routine algorithm we detected, localized, and tracked the particles to calculate their diffusion coefficient, extracted from the mean-squared displacement as the most common measure of diffusion. We also measured the diffusion coefficient using an ensemble-based technique known as Raster Image Correlation Spectroscopy (RICS) with a confocal laser-scanning microscope. The use of confocal eliminates the out-of-focus signal and enables measurements that are confined to a narrow plane in the cell. Also, the ability of RICS to separate the slow and immobile fractions of particles makes it possible to detect heterogeneities in diffusion. To our knowledge, this is the first study that has utilized both SPT and RICS to directly compare receptors’ diffusion in different membrane sections. Moreover, this is the first study that has examined the diffusion of receptors on macrophages adhered to different surface topographies, and the first that has investigated the receptors’ diffusion in young and old macrophages. / Thesis / Doctor of Philosophy (PhD) / The immune system is highly dependent on a specialized subset of white blood cells known as macrophages that are capable of clearing damaged and dead cells as well as a wide range of invading micro-organisms. Specific receptor proteins present on the membrane of macrophages are involved in the recognition of particles and subsequent signaling to recruit other immune cells or to promote healing and wound repair. To date, a variety of fluorescence-based microscopy methods have been used to study the dynamics of cell membrane components. The mobility of several membrane receptors in macrophages has been studied using microscopy techniques, which have provided valuable insights into their function. However, there is still insufficient information about the behavior of two key receptors (TLR2 and CD14) that participate in signaling in response to bacterial products. This thesis aims to answer three major questions with regard to receptor mobility (i.e., diffusion) within macrophage membrane: 1) Which type of fluorescence-based microscopy technique is more suitable for measuring the mobility of TLR2 and CD14 receptors on macrophage membranes? 2) What is the impact of different surface topographies on TLR2 diffusion in adhered macrophages, as well as cell shape, and the ability of macrophages to internalize particles? 3) Does aging alter TLR2 mobility in the membrane of macrophages? The following chapters provide detailed answers to these questions. In brief, we have demonstrated that TLR2 and CD14 diffusion measurements in adhered macrophages highly depend on the membrane section chosen. In addition, our results show that micro- and nanostructured surface topographies alter the shape of adhered macrophages and yield higher bacteria internalization, while the diffusion of TLR2 is not changed. When comparing macrophages derived from young and old mice, we find similar diffusion rate of TLR2 in macrophages of the two age groups.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/25233 |
| Date | January 2020 |
| Creators | Makaremi, Sara |
| Contributors | Moran-Mirabal, Jose, Bowdish, Dawn, Biomedical Engineering |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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