Biology subjects

Reynolds, P. M.

Publications and source records attributed to Reynolds, P. M..

4 recordsLinked to original sources

bioRxiv2022

Regulation of Cardiomyocyte Adhesion and Mechanosignalling Through Distinct Nanoscale Behaviour of Integrin Ligands Mimicking Healthy or Fibrotic ECM

The stiffness of the cardiovascular environment changes during ageing and in disease and contributes to disease incidence and progression. Changing collagen expression and crosslinking regulate the rigidity of the cardiac ECM. Additionally, basal lamina glycoproteins, especially laminin and fibronectin regulate cardiomyocyte adhesion formation, mechanics and mechano-signalling. Laminin is abundant in the healthy heart, but fibronectin is increasingly expressed in the fibrotic heart. ECM receptors are co-regulated with the changing ECM. Due to differences in integrin dynamics, clustering, and downstream adhesion formation this is expected to ultimately influence cardiomyocyte mechanosignalling; however details remain elusive. Here we sought to investigate how different cardiomyocyte ligand/integrin combinations are affecting adhesion formation, traction forces and mechanosignalling, using a combination of uniformly coated surfaces with defined stiffness, PDMS nanopillars, micropatterning and specifically designed bionanoarrays for precise ligand presentation. Thereby we find that neonatal rat cardiomyocytes (which express both laminin and fibronectin binding integrins) adhesion nanoscale organisation, signalling and traction force generation are strongly dependent on the integrin/ligand combination. Together our data indicates that the presence of fibronectin in combination with the enhanced stiffness in fibrotic areas will strongly impact on the cardiomyocyte behaviour and influence disease progression.

cell biology↗

bioRxiv2020

Nanotopography controls single-molecule mobility to determine overall cell fate

The addition of nanoscale distortion to ordered nanotopographies consistently determines an osteogenic fate in stem cells. Although disordered and ordered nanopit arrays have identical surface areas, array symmetry has opposite effects on cell fate. We aimed to understand how cells sense disorder at the nanoscale. We observed effects in the early formation of cell and focal adhesions that controlled long-term cell fate. Disordered nanopits consistently yielded larger focal adhesions at a faster rate, prompting us to investigate this at the molecular scale. Super-resolution microscopy revealed that the nanopits did not act as nucleation points, as previously thought. Rather, nanopit arrays altered the plasma membrane and acted as barriers that changed molecular diffusion. The local areas corralled by four nanopits were the smallest structures that exerted diverging effects between ordered and disordered arrays. Heterogeneity in the local area on disordered arrays increased the proportion of fastest and slowest diffusing molecules. This resulted in higher quantity, more frequent formation and clustered arrangement of nascent adhesions, i.e., the modular units on which focal adhesions are built. This work presents a new pathway to exploit nanoscale sensing to dictate cell fate.

bioengineering↗

bioRxiv2018

Predicting phenotype using morphological cell responses to nanotopography

Cells respond in complex ways to topographies, making it challenging to identify a direct relationship between surface topography and cell response. A key problem is the lack of informative representations of topographical parameters that translate directly into biological properties. Here, we present a platform to relate the effects of nanotopography on morphology to function. This platform utilizes the morphome, a multivariate dataset containing single cell measures of focal adhesions, the cytoskeleton, and chromatin. We demonstrate that nanotopography-induced changes in cell phenotype are uniquely encoded by the morphome. The morphome was used to create a Bayesian linear regression model that robustly predicted changes in bone, cartilage, muscle and fibrous tissue gene expression induced by nanotopography. Furthermore, the morphome effectively predicted nanotopography-induced phenotype within a complex co-culture microenvironment. Thus, the morphome enables the cell function-oriented exploration of new topographies, with potential applications in the development of novel surface-patterned biomaterials for tissue implants.

bioengineering↗

bioRxiv2018

Controlling fluid flow to improve cell seeding uniformity

Standard methods for seeding monolayer cell cultures in a multiwell plate or dish do not uniformly distribute cells on the surface. With traditional methods, users find aggregation around the circumference, in the centre, or a combination of the two. This variation is introduced due to the macro scale flow of the cell seeding suspension, and movement of the dish before cells can settle and attach to the surface. Reproducibility between labs, users, and experiments is hampered by this variability in cell seeding. We present a simple method for uniform and user-independent cell seeding using an easily produced uniform cell seeder (UCS) device. This allows precise control of cell density in a reproducible manner. By containing the cell seeding suspension in a defined volume above the culture surface with the UCS, fluctuations in cell density are minimised. Seeding accuracy, as defined by the actual cell density versus the target seeding density is improved dramatically across users with various levels of expertise. We go on to demonstrate the impact of local variation in cell density on the lineage commitment of human embryonic stem cells (hESCs) towards pancreatic endoderm (PE). Variations in the differentiation profile of cells across a culture well closely mirror variations in cell density introduced by seeding method - with the UCS correcting variations in differentiation efficiency. The UCS device provides a simple and reproducible method for uniform seeding across multiple culture systems.

cell biology↗