27 research outputs found
Viscoelastic response of cells and the role of actin cytoskeletal remodelling.
PhDThe mechanical properties of living cells provide useful information on cellular structure and function. In the present study a micropipette aspiration technique was developed to investigate the viscoelastic parameters of isolated articular chondrocytes. The Standard Linear Solid (SLS) and the Boltzmann Standard Linear Solid (BSLS) models were used to compute the instantaneous and equilibrium moduli and viscosity based on the response to an aspiration pressure of 7 cm of water.
The modulus and viscosity of the chondrocytes increased with decreasing pressure rate. For example, the median equilibrium moduli obtained using the BSLS model increased from 0.19 kPa at 5.48 cmH2O/s to 0.62 kPa at 0.35 cmH2O/s. Cell deformation during micropipette aspiration was associated with an increase in cell volume and remodelling of the cortical actin visualised using GFP-actin. Interestingly, GFP-actin transfection inhibited the increase in cell moduli observed at the slower aspiration rate. Thus actin remodelling appears to be necessary for the pressure rate-dependent behaviour. A hypothesis is proposed explaining the role of actin remodelling and interaction with the membrane in regulating cell mechanics.
Further studies investigated a mechanical injury model of cartilage explants which resulted in significant increases in all three viscoelastic parameters. Treatment with IL-1β also increased the instantaneous moduli of cells treated in explants but there was no difference in equilibrium moduli or viscosity. IL-1β treatment in monolayer had no effect on cell mechanics suggesting that previously reported changes in actin associated with IL-1β may be lost during cell isolation or trypsinisation. Separate studies demonstrated increases in chondrocyte moduli and viscosity during passage indicating changes in cell structure-function associated with de-differentiation in monolayer.
In conclusion, this study has developed an optimised micropipette aspiration technique which was successfully used to quantify chondrocyte viscoelastic behaviour and to elucidate the underlying role of actin dynamics and response to pathological stimuli and in vitro culture.EPSR
Bardet-Biedl syndrome proteins control cilia length through regulation of actin polymerisation.
Primary cilia are cellular appendages important for signal transduction and sensing the environment. Bardet-Biedl syndrome proteins form a complex that is important for several cytoskeleton-related processes such as ciliogenesis, cell migration and division. However, the mechanisms by which BBS proteins may regulate the cytoskeleton remain unclear. We discovered that Bbs4 and Bbs6 deficient renal medullary cells display a characteristic behaviour comprising poor migration, adhesion and division with an inability to form lamellipodial and filopodial extensions. Moreover, fewer mutant cells were ciliated (48% ± 6 for wild-type cells vs 23% ± 7 for Bbs4 null cells; P-value < 0.0001) and their cilia were shorter (2.55 μm ± 0.41 for wild-type cells vs 2.16 μm ± 0.23 for Bbs4 null cells; P-value < 0.0001). Whilst the microtubular cytoskeleton and cortical actin were intact, actin stress fibre formation was severely disrupted, forming abnormal apical stress fibre aggregates. Furthermore, we observed over-abundant focal adhesions in Bbs4, Bbs6 and Bbs8-deficient cells. In view of these findings and the role of RhoA in regulation of actin filament polymerisation, we showed that RhoA-GTP levels were highly upregulated in the absence of Bbs proteins. Upon treatment of Bbs4-deficient cells with chemical inhibitors of RhoA, we were able to restore cilia length and number as well as the integrity of the actin cytoskeleton. Together these findings indicate that Bbs proteins play a central role in the regulation of the actin cytoskeleton and control cilia length through alteration of RhoA levels
Viscoelastic Cell Mechanics and actin remodelling are dependent on the rate of applied pressure
Background: living cells are subjected to external and internal mechanical stresses. The effects of these stresses on the deformation and subsequent biological response of the cells remains unclear. This study tested the hypothesis that the rate at which pressure (or stress) is applied influence the viscoelastic properties of the cell associated with differences in the dynamics of the actin cytoskeleton.Principal finding: micropipette aspiration was used to determine the instantaneous and equilibrium moduli and the viscosity of isolated chondrocytes based on the standard linear solid (SLS) model and a variation of this incorporating Boltzmann superposition. Cells were visualised for 180 seconds following aspiration to 7 cmH2O at 0.35, 0.70 and 5.48 cmH2O/sec. Cell recovery was then examined for a further 180 seconds once the pressure had been removed. Reducing the rate of application of pressure reduced the levels of cell deformation and recovery associated with a significant increase in modulus and viscosity. Using GFP transfection and confocal microscopy, we show that chondrocyte deformation involves distortion, disassembly and subsequent reassembly of the cortical actin cytoskeleton. At faster pressure rates, cell deformation produced an increase in cell volume associated with membrane bleb formation. GFP-actin transfection inhibited the pressure rate dependent variation in cell mechanics indicating that this behaviour is regulated by GFP-sensitive actin dynamics.Conclusion: we suggest that slower rates of aspiration pressure enable greater levels of cortical actin distortion. This is partially inhibited by GFP or faster aspiration rates leading to membrane bleb formation and an increase in cell volume. Thus the rate of application of pressure regulates the viscoelastic mechanical properties of living cells through pressure rate sensitive differences in actin dynamics. Therefore cells appear softer when aspirated at a faster rate in contrast to what is expected of a normal viscoelastic materia
Stem cell differentiation increases membrane-actin adhesion regulating cell blebability, migration and mechanics
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/K. S. is funded by an EPSRC PhD studentship. S.T. is funded by an EU Marie Curie Intra European Fellowship (GENOMICDIFF)
The rate of hypo-osmotic challenge influences regulatory volume decrease (RVD) and mechanical properties of articular chondrocytes
Zhao Wang is funded on a China Scholarships PhD Studentship. Jerome Irianto was supported on a project grant from The Wellcome Trust (ref no. 084717)
Characterization of biomechanical properties of cells through dielectrophoresis-based cell stretching and actin cytoskeleton modeling
Cell deformation is associated with actin distortion, disassembly and remodelling.
<p>a) Representative confocal images showing characteristic actin organisation in a chondrocyte cultured in monolayer and transfected with GFP-actin (green). F-actin has been co-labelled with alexa-phalloidin (red). Selected images from time series showing GFP-actin distortion and dynamics during micropipette aspiration applied at a) 0.35 and b) 5.48 cmH<sub>2</sub>O/sec. Arrow indicates breakdown/fluidization of the actin cortex. Arrowhead shows initiation of a membrane bleb. All scale bars represent 10 microns. The corresponding temporal changes in GFP actin intensity at the leading edge (ROI 1) and the rear edge (ROI 2) are shown for a cell aspirated at d) 0.35 and e) 5.48 cmH<sub>2</sub>O/sec.</p
Pressure rate influences cellular mechanical properties estimated using the SLS and BSLS models.
<p>Plots show median and quartile values for instantaneous modulus (a,b), equilibrium modulus (c,d) and viscosity (e,f) estimated using the SLS model (a,c,e) and BSLS model (b,d,f) for cells aspirated at 0.35, 0.70 and 5.48 cmH<sub>2</sub>O/sec. Statistically significant differences (p<0.05) are indicated between the ramp rates (*) and between the models (+).</p
Viscoelastic cell deformation can be modelled using the SLS and BSLS models.
<p>Representative examples of cell aspiration data fitted using a) the SLS model and b) the BSLS model. Cells were aspirated at 0.35, 0.70 and 5.48 cmH<sub>2</sub>O/sec.</p
GFP-actin transfection inhibits pressure-rate sensitive changes in cell mechanics.
<p>The BSLS model was used to estimate the mechanical properties of transfected and non-transfected chondrocytes aspirated at two different pressure rates of 0.35 and 5.48 cmH<sub>2</sub>O/sec. Media and quartile values are shown for a) instantaneous modulus b) equilibrium modulus and c) viscosity. Statistically significant difference exist between transfected and non-transfected chondrocytes at 0.35 cmH<sub>2</sub>O/sec (*, p<0.05). For non transfected cells the moduli and viscosity were significantly greater at the slower aspiration rate (p<0.05).</p