5 forskare från skotska universitet,däribland Centre for Regenerative Medicine, Institute for Regeneration and Repair, Edinburgh bioQuarter, har idag fått sina studier om cellers förmåga att reagera och förändras vid olika typer av stimuli publicerad.Studien visar på cellers anpassning vid regenerativa processer,dvs återskapning av skadade celler som i denna studie tar upp konsekvens därav som kan leda till cancerutveckling.Forskarna har använt sig av HoloMonitor tämligen flitigt ser man i studien som är betitlad :
The Hippo pathway drives the cellular response to hydrostatic pressure
15 June 2022
Abstract
Cells need to rapidly and precisely react to multiple mechanical and chemical stimuli in order to ensure precise context-dependent responses. This requires dynamic cellular signalling events that ensure homeostasis and plasticity when needed. A less well-understood process is cellular response to elevated interstitial fluid pressure, where the cell senses and responds to changes in extracellular hydrostatic pressure. Here, using quantitative label-free digital holographic imaging, combined with genome editing, biochemical assays and confocal imaging, we analyse the temporal cellular response to hydrostatic pressure. Upon elevated cyclic hydrostatic pressure, the cell responds by rapid, dramatic and reversible changes in cellular volume. We show that YAP and TAZ, the co-transcriptional regulators of the Hippo signalling pathway, control cell volume and that cells without YAP and TAZ have lower plasma membrane tension. We present direct evidence that YAP/TAZ drive the cellular response to hydrostatic pressure, a process that is at least partly mediated via clathrin-dependent endocytosis. Additionally, upon elevated oscillating hydrostatic pressure, YAP/TAZ are activated and induce TEAD-mediated transcription and expression of cellular components involved in dynamic regulation of cell volume and extracellular matrix. This cellular response confers a feedback loop that allows the cell to robustly respond to changes in interstitial fluid pressure.
The Hippo pathway controls development and facilitates regenerative processes through regulating its transcriptional co-activators YAP and TAZ, and can cause cancer if the pathway is not tightly regulated (Moroishi et al, 2015a; Fulford et al, 2018; Davis & Tapon, 2019; Rognoni & Walko, 2019; Salem & Hansen, 2019; Zanconato et al, 2019; Thompson, 2020). The Hippo pathway contains an upstream serine/threonine kinase module and a downstream transcriptional effector module, consisting of YAP and TAZ (encoded, respectively, by YAP1 and WWTR1) and their cognate transcription factors (Hansen et al, 2015a; Fulford et al, 2018). YAP/TAZ are regulated by LATS1/2-mediated inhibitory phosphorylation on five (YAP) or four (TAZ) serine residues (Huang et al, 2005; Zhao et al, 2007; Liu et al, 2010). Upon relief from this inhibitory phosphorylation, YAP and TAZ localize to the nucleus to exert their co-transcriptional activity (Huang et al, 2005; Zhao et al, 2007; Liu et al, 2010). In solid tumours, high YAP/TAZ activity in general increase the risk of metastasis (Steinhardt et al, 2008; Lamar et al, 2012), impede cancer treatment and confer poor prognosis (Moroishi et al, 2015a; Rognoni & Walko, 2019; Salem & Hansen, 2019; Zanconato et al, 2019; Thompson, 2020). However, distinct core Hippo pathway components are mutated only in a subset of cancers, and the underlying reasons as to why YAP/TAZ are predominantly nuclear in solid tumours are not fully understood (Moroishi et al, 2015a; Fulford et al, 2018; Rognoni & Walko, 2019; Salem & Hansen, 2019; Zanconato et al, 2019; Thompson, 2020).
Under Results och Methods får vi bekräftelse på forskarnas användande av HoloMonitor (DHM).
Digital holographic microscopy (DHM)
Aforementioned microfluidic set up was coupled to a Digital Holographic Microscope (Phi LAB, Holomonitor M4) to investigate the effect of hydrostatic pressure on cell response in real time. Time-lapse imaging was acquired for 60 s at a rate of 1 Hz. Then, cells were segmented with the Hstudio Tracking software (Otsu’s thresholding) to extract for each cell in the field of view and for each time point quantitative cellular parameters. In particular, cell area and mean optical thickness were used to calculate the cellular volume considering a mean cellular index of 1.38 and a mean media refractive index of 1.34. A Matlab script was written to automate calculation of average cell volume at steady state and percentage average change in cell volume in response to hydrostatic pressure.
YAP/TAZ regulate cell volume
To establish the macroscopic cellular response to hydrostatic pressure, we took advantage of live cell digital holographic imaging (DHM). DHM is a technique that allows for quantitative label-free cellular imaging with single-cell resolution.
Cells respond to oscillating hydrostatic pressure by YAP/TAZ-TEAD-dependent rapid volume changes
As the cellular consequences of increased interstitial fluid pressure are not well established (Heldin et al, 2004; Myers et al, 2007; Li et al, 2020), we sought to determine if the force exerted by hydrostatic pressure regulates cell size. To this end, we established a workflow that allows us to analyse the dynamic cellular response to elevated hydrostatic pressure in real time with a temporal resolution of seconds. In our system, the hydrostatic pressure is controlled by a microfluidic pump coupled to closed cell culture chambers, where cells are imaged using digital holographic microscopy (Marquet et al, 2005). We are consequently able to dictate the precise and temporal hydrostatic pressure experienced by cells, driven by the extra- and intracellular pressure differences, in a physiologically relevant manner while imaging the cells without labelling and at single-cell resolution.
- Cell volume of WT and Y/T DKO clones at steady state measured using DHM. Each dot represents a single cell. Error bars represent mean ± 95% CI. Kruskal–Wallis test with Dunn’s post-hoc. Data from three independent experiments. ***P < 0.001 and P = 0.0917 (Y/T DKO #1 vs. #2).
- Comparison of cell volume of WT and LATS1/2 DKO clones at steady state obtained using DHM. Each dot represents a single cell. Data from three independent experiments. Error bars represent mean ± 95% CI. Kruskal–Wallis test with Dunn’s post-hoc. **P = 0.0018 (WT vs. LATS1/2 DKO #1), ***P < 0.001 (WT vs. LATS1/2 DKO #2) and P = 0.0557 (LATS1/2 DKO #1 vs. #2).
- Comparison of cell volume of WT and MST1/2 DKO at steady state obtained using DHM. Each dot represents a single cell. Data from three independent experiments. Error bars represent mean ± 95% CI. Mann–Whitney U test. P = 0.2731.
- HEK293A NF2 KO cells response to 100 mbar cyclic hydrostatic pressure. Each dot represents a single cell and error bars represent mean ± 95% CI. Data pooled from three independent experiments. Mann–Whitney U test. P = 0.4072.
- Comparison of cell volume of WT and TEAD KO clones at steady state obtained using DHM. Each dot represents a single cell. Data from three independent experiments. Error bars represent mean ± 95% CI. Kruskal–Wallis test with Dunn’s post-hoc. P > 0.9999 for all comparisons.
- Maxima and minima are identified to calculate average percentage change in cell volume in response to hydrostatic pressure using measurements obtained by DHM.
- Representative WT and Y/T DKO single-cell volume change in response to hydrostatic pressure recorded by DHM.
- WT and Y/T DKO cell volume were measured at steady state and the average change in cell volume was quantified using the DHM as in previous experiments; these are labelled (−). Cells were then subjected to cyclic hydrostatic pressure for 2 h and their cell volume was quantified to determine whether prior exposure to hydrostatic pressure (“pre-conditioning”) would change their cell volume response; these are labelled (+). Each dot represents a single cell and error bars represent 95% CI. Graphs include data obtained from four independent experiments. Kruskal–Wallis with Dunn’s post-hoc. ns = P > 0.9999.
- The average percentage change in cell volume was quantified under the same experimental conditions as in “O”. The average change in cell volume in response to cyclic hydrostatic pressure of cells with no previous exposure to hydrostatic pressure (labelled as (−)) was quantified using DHM and compared to those with prior exposure to hydrostatic pressure (labelled (+)). Each dot represents a single cell and error bars represent 95% CI. Graphs include data obtained from four independent experiments. Kruskal–Wallis with Dunn’s post-hoc. **P = 0.006 (WT), P = 0.4173 (Y/T DKO) and ns = P > 0.9999.
Det finns fler hänvisningar till forskarnas användande av HoloMonitor = DHM.
quantitative single-cell measurements är ett exempel på vad HoloMonitor förmår.
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