Using STORM and TIRF in tandem to investigate adherence and migration patterns in stem cells
| dc.contributor.advisor | Van de Vyver, Mari | en_ZA |
| dc.contributor.advisor | Engelbrecht, Lize | en_ZA |
| dc.contributor.advisor | Van Vuuren, Derick | en_ZA |
| dc.contributor.author | Matyesini, Sandisiwe Manyathi-Mankwali | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine: Clinical Pharmacology. | en_ZA |
| dc.date.accessioned | 2021-06-07T08:18:05Z | |
| dc.date.available | 2021-06-07T08:18:05Z | |
| dc.date.issued | 2021-03 | |
| dc.description | Thesis (MMed)--Stellenbosch University, 2021. | en_ZA |
| dc.description.abstract | 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. | en_ZA |
| dc.description.abstract | Agtergrond: Selmigrasie is 'n dinamiese proses in fisiologie wat gebruik maak van pseudopodiam of selverlengings wat assosieer met die ekstrasellulêre matriks (ESM) en selle vorentoe dryf. Die interaksie word met die opbou en afbreek van fokale adhesie (FA) -komplekse bewerkstellig, wat as sentrums dien vir kommunikasie tussen die ESM en die sel. Veranderinge binne die ESM en die belangrikste proteïene daarin, soos kollageen, veroorsaak reaksies deur en van die FA-komplekse. Hier is askorbiensuur-2-fosfaat (AAP) aangevul aangesien dit bekend is dat AAP kollageenneerlegging stimuleer. AAP is 'n stabiele askorbiensuurderivaat wat kollageensintese en selgroei verbeter. Die veranderde rigiditeit van die matriks laat FA-proteïene wat hierdie komplekse vorm, toe om te verander soos nodig om op stimuli te kan reageer. In hierdie studie is die samestelling van belangrike FA- proteïene, integrien β1, talien en vinkulien in kollageenversterkte migrerende selle met verskillende mikroskopietegnieke, insluitend TIRF en STORM ondersoek. Metodes: Die optimale AAP-konsentrasie (0.6mM) wat nie die lewensvatbaarheid van die sel beïnvloed nie, was deur ‘n kristalviolet-dosisresponstoetse bepaal. Selmigrasie met en sonder AAP was om 0h (kontrole) en 8h ondersoek; die gekose tydspunt uit 'n 24h lewendige-sel-waarneemingseksperiment waartydens duidelike pseudopodia in alle groepe sigbaar was. Daarna was 'n immunositochemiese protokol opgestel om die impak van AAP-aanvulling tydens migrasie op talien, vinkulien en integrien- β1 te ondersoek. Proteïenuitdrukking, sowel as medelokalisering van hierdie proteïene was bepaal. TIRF-mikroskopie het voorsien vir die visualisering van die FA komplekse op die vlak van aanhegting, aangesien dit die fokusvlak direk bokant die dekglas kon beeld. Die FA-lengte, breedte en oppervlakte was met behulp van beeldanalise-sagteware (FIJI) bepaal. Laastens wasdie STORM tegniek geoptimaliseer en toegepas op taliengekleurde monsters om vas te stel of fyner strukturele veranderinge tydens migrasie waargeneem kan word.Resultate: Talien en integrien-β1 het saam gegroepeer terwyl die meerderheid vinkulien rondom die kern gesentraliseer het, ongeag van selmigrasie met of sonder AAP-aanvulling. TIRF-assessering van FA in migrasieselle wat deur AAP behandel is, dui op 'n beduidende verskil in FA-kompleksgroottes in vergelyking met SGM-migrerende selle met duidelike en prominente FA-komplekse wat duidelik sigbaar is. Gevolgtrekking: Selmigrasie in AAP aangevulde media was verminder as gevolg van verhoogde FA proteïene wat reageer op die teenwoordigheid van die rigiede ECM. Soortgelyk het die TIRF gegenereerde data digte prominente strukture uitgebeeld, wat al die lig wat nie in fokus was nie uitgeskakel het en die kotras van die FA komplekse verhoog het. Die verbeterde TIRF-resolusie het beklemtoon dat AAP-migrerende selle se FA-komplekse groter was in vergelyking met FA in SGM- migrerende selle. | af_ZA |
| dc.description.version | Masters | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/110541 | |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | Stem cells | en_ZA |
| dc.subject | UCTD | en_ZA |
| dc.subject | Cell migration | en_ZA |
| dc.subject | Extracellular matrix | en_ZA |
| dc.subject | Mesenchymal stem cells | en_ZA |
| dc.title | Using STORM and TIRF in tandem to investigate adherence and migration patterns in stem cells | en_ZA |
| dc.type | Thesis | en_ZA |