title: The role of Protein Kinase D signaling and the thermal microenvironment on single cell migration
creator: Khachaturyan, Galina
subject: 500
subject: 500 Natural sciences and mathematics
subject: 570
subject: 570 Life sciences
description: Cell migration plays an essential role in development and homeostasis. This extraordinarily complex process, which is dynamically regulated by a coordinated interplay between the extracellular environment and intracellular signaling pathways, can also cause the emergence of disease states when dysregulated. A prominent example is cancer metastasis, which is the  leading cause of cancer-related mortality. Despite an increasing body of literature focused on cell migration, many aspects remain unclear. In the current work, two complementary systematic in vitro cell migration studies were performed to gain insights into both healthy immune cell migration and pathological cancer cell migration.     Temperature-dependent neutrophil migration was analyzed using differentiated HL-60 cells as a model cell line. Neutrophils are the first cells to be activated during inflammation and subsequently migrate toward an injured tissue or infection site. This response is dependent on both biochemical signaling and the extracellular environment, one aspect of which includes increased temperature in the tissues surrounding the inflammation site. The results of this study reveal a positive correlation between the average speed of randomly migrating cells and temperatures from 30 ˚C to 42 ˚C. Higher temperatures also induce a concomitant increase in cell detachment. Further analysis of the migration data showed that persistence time is higher at low temperatures, while migration persistence length remains constant throughout the temperature range. Coupled with the increased speed observed at elevated temperatures, the obtained results indicate the ability of neutrophils to adapt their migration characteristics to the temperature in order to maintain relatively constant persistence length. As temperature gradients exist on both cellular and tissue scales, the ability of the HL-60 cells to sense and react to the presence of temperature gradients, a process known as thermotaxis, was also investigated. Towards this aim, a two-dimensional temperature gradient chamber was developed. In a gradient with the range of 27-43 ˚C both positive and negative thermotaxis was observed with cells migrating both to the high and the low temperature sources. To date, thermotaxis in neutrophil differentiated HL-60 cells has never been reported.    Next, an in-depth in vitro migration study was carried out on Panc1 pancreatic cancer cells to elucidate the role of Protein Kinase D (PKD) in cancer cell migration. CRISPR-mediated knockouts of PKD1, 2, and 3 in Panc1 cells were used to investigate isoform-specific effects on cancer cell invasion-related motility. The data presented in this project reveals that absence of each PKD isoform plays a significant role in cell speed and migration persistence. To investigate the pancreatic cancer PKD isoform-dependent contact guidance, a panel of more complex PDMS-based substrates containing microgrooves of different heights and widths  was utilized. These experiments further demonstrate pancreatic cancer cell shape, speed, and contact guidance dependence on the presence of different PKD isoforms. Based on these findings, in combination with previous works showing that contact guidance is affected by cell viscoelasticity, mechanical properties of each PKD isoform knockout cell line were also measured using a laser-based optical cell stretcher. These experiments showed a significant increase in Panc1 cell deformability in the absence of PKD1, demonstrating for the first time PKD involvement in regulation of cellular mechanical properties. Finally, these basic  mechanical properties were correlated to MMP - independent confined migration, which is required for cells to metastasize in vivo.
date: 2020
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/26531/1/Thesis_Galina%20Khachaturyan.pdf
identifier: DOI:10.11588/heidok.00026531
identifier: urn:nbn:de:bsz:16-heidok-265317
identifier:   Khachaturyan, Galina  (2020) The role of Protein Kinase D signaling and the thermal microenvironment on single cell migration.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/26531/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng