Abstract
The local environment of cells is known to regulate their shape and motility. The vast majority of research to date has focused on analysis of motile behaviour of cells on two-dimensional surfaces. However, cells migrating within three-dimensional extracellular matrices (ECM) represent a more physiologically relevant environment, showing substantially different morphological and cell behaviour. Moreover, tissues in the human body are not uniform in stiffness or structure, presenting additional environmental variability for cells to navigate. In vivo, it is unlikely that cells experience fully two- or three-dimensional environments, but rather several structures of different dimensionalities that all influence the behaviour of the cell, and all interact with each other.Here we investigate the role the local environment plays in regulating the structure and dynamics of single cells imbedded in three-dimensional collagen matrices of increasing stiffness. Increasing ECM stiffness is achieved through non-enzymatic glycation with D-ribose, across three concentrations at 0-, 50- and 200-mM. This represents a powerful technique for studying the short- and long-term dynamics and cell-ECM interactions imposed on one another in a consistent and reproducible way. Our investigations focus on the dynamics of single cells, the speed and location of actin treadmilling with respect to migration behaviour and matrix topography quantification in accordance with that of the ECM stiffness.
Using methods in atomic force microscopy, for ECM characterization, and live super resolution imaging enables us to analyse these three-dimensional structures without changing cell behaviour due to phototoxic effects, while still being able to capture dynamic cell events at high and low temporal resolution within relatively thick specimens in high resolution. Live super-resolution imaging revealed that initial seeding of single Hela cells to be affected directly in a stiffness dependant manner by the local environment. Studies showed matrices of lower stiffness having higher protrusion number and turnover in single cells, with smaller protrusion volume, causing a reduced invasion of the extracellular environment. Conversely in matrices of increasing stiffnesses single cells demonstrated greater protrusion development and life span, with higher numbers of stress fibres and greater reach on its surrounding environment. High temporal resolution imaging data, acquired at 24-hours, enabled the analyse in greater depth of the correlation between protrusion, actin and collagen dynamics respectively to local environments of increasing stiffnesses.
These studies demonstrate a novel and reproducible approach, paving the way for future studies to understanding mechano-regulated mechanisms of single cells in three-dimensional environments at a molecular scale. Shedding light on how the ECM organisation and stiffness plays a role in changing local cytoskeletal dynamics and the effects of cytoskeletal components on effectively navigating its local environment, provides new insight into cell behaviour in healthy and diseased tissue environments.
| Date of Award | 1 May 2023 |
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| Original language | English |
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| Supervisor | Susan Cox (Supervisor) & Madeline Parsons (Supervisor) |