Clinical Pharmacology

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https://scholar.sun.ac.za/handle/10019.1/164
This division was known as Pharmacology until 27 June 2013.

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Browsing Clinical Pharmacology by browse.metadata.advisor "Engelbrecht, Lize"

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    Using STORM and TIRF in tandem to investigate adherence and migration patterns in stem cells
    (Stellenbosch : Stellenbosch University, 2021-03) Matyesini, Sandisiwe Manyathi-Mankwali; Van de Vyver, Mari; Engelbrecht, Lize; Van Vuuren, Derick; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine: Clinical Pharmacology.
    Background: Cell migration is a dynamic process in physiology requiring pseudopodia, elongated cell protrusions, to associate with the extracellular matrix (ECM) and propel cells forward. The interaction is established with the assembly and disassembly of focal adhesion (FA) complexes, acting as signalling centres for communication between the ECM and the cell. Alterations within the ECM and its key proteins, such as collagen, elicit responses through the FA complexes. Here, collagen deposition was stimulated in mesenchymal stem cells (MSC) through the addition of ascorbic acid-2-phosphate (AAP). AAP is stable ascorbic acid derivative that enhances collagen synthesis and cell growth. The altered matrix stiffness allowed FA proteins forming these complexes to change as needed in response to stimuli. In this study the elusive assembly of key FA proteins, integrin β1, talin and vinculin were assessed in collagen reinforced migrating cells with various microscopy techniques, including TIRF and STORM. Methods: The optimal AAP concentration (0.6mM) which did not affect cell viability was determined with a crystal violet dose response assay. Cell migration with and without AAP was assessed at (control at 0h) and 8h, a time point selected from a 24h live cell imaging experiment when distinct pseudopodia were visible in all groups. Thereafter, an immunocytochemistry (ICC) protocol was established to investigate the impact of AAP supplementation during migration on talin, vinculin and integrin-β1. Protein expression, as well as co-localisation of these proteins were determined. TIRF microscopy provided a tool for visualization of the FA complexes at the level of attachment as it imaged the focal plane directly above the coverslip. The FA complex length, breadth and area were determined using image analysis software (FIJI). Finally, STORM was optimised and applied on talin stained samples to establish if finer structural changes during migration could be revealed. Results: Talin and integrin-β1 co-occurred while the majority of vinculin centralized around the nucleus irrespective of cell migration with or without AAP supplementation. TIRF assessment of FA in AAP treated migrating cells indicated a significant difference in FA complex sizes compared to SGM migrating cells with distinct and prominent FA complexes clearly visible. Conclusion: Cell migration in AAP supplemented media was reduced due to increased FA proteins responding to the stiff ECM. Similarly, TIRF generated data portrayed dense prominent structures, eliminating all out of focus light and increase the FA complex contrast. The improved TIRF resolution, highlighted that AAP migrating cells FA complexes were bigger in size compared to FA in SGM migrating cells.