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A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

University of Ottawa

We present gravitational field-flow fractionation and hydrodynamic chromatography of colloids eluting through 18 μm microchannels. Using video microscopy and mesoscopic simulations, we investigate the average retention ratio of colloids with both a large specific weight and neutral buoyancy. We consider the entire range of colloid sizes, including particles that barely fit in the microchannel and nanoscopic particles. Ideal theory predicts four operational modes, from hydrodynamic chromatography to Faxén-mode field-flow fractionation. We experimentally demonstrate, for the first time, the existence of the Faxén-mode field-flow fractionation and the transition from hydrodynamic chromatography to normal-mode field-flow fractionation. Furthermore, video microscopy and simulations show that the retention ratios are largely reduced above the steric-inversion point, causing the variation of the retention ratio in the steric- and Faxén-mode regimes to be suppressed due to increased drag. We demonstrate that theory can accurately predict retention ratios if hydrodynamic interactions with the microchannel walls (wall drag) are added to the ideal theory. Rather than limiting the applicability, these effects allow the microfluidic channel size to be tuned to ensure high selectivity. Our findings indicate that particle velocimetry methods must account for the wall-induced lag when determining flow rates in highly confining systems.

Field-flow fractionation and hydrodynamic chromatography on a microfluidic chip.

hMSL2 (male-specific lethal 2, human) is a RING finger protein with ubiquitin ligase activity. Although it has been shown to target histone H2B at lysine 34 and p53 at lysine 351, suggesting roles in transcription regulation and apoptosis, its function in these and other processes remains poorly defined. To further characterize this protein, we have disrupted the Msl2 gene in chicken DT40 cells. Msl2(-/-) cells are viable, with minor growth defects. Biochemical analysis of the chromatin in these cells revealed aberrations in the levels of several histone modifications involved in DNA damage response pathways. DNA repair assays show that both Msl2(-/-) chicken cells and hMSL2-depleted human cells have defects in non-homologous end joining (NHEJ) repair. DNA damage assays also demonstrate that both Msl2 and hMSL2 proteins are modified and stabilized shortly after induction of DNA damage. Moreover, hMSL2 mediates modification, presumably ubiquitylation, of a key DNA repair mediator 53BP1 at lysine 1690. Similarly, hMSL1 and hMOF (males absent on the first) are modified in the presence of hMSL2 shortly after DNA damage. These data identify a novel role for Msl2/hMSL2 in the cellular response to DNA damage. The kinetics of its stabilization suggests a function early in the NHEJ repair pathway. Moreover, Msl2 plays a role in maintaining normal histone modification profiles, which may also contribute to the DNA damage response.

Msl2 is a novel component of the vertebrate DNA damage response.

Physical forces arising in the cellular microenvironment have been hypothesized to play a major role in governing cell function. Moreover, it is thought that gene regulation may be sensitive to nuclear deformations taking place in response to extracellular forces over short and long timescales. Although nuclear responses to mechanical stimuli over long timescales are relatively well studied, the short-term responses are poorly understood. Therefore, to characterize the short-term instantaneous deformation of the nucleus in a mechanically dynamic environment, we exposed MDCK epithelial monolayers to varying mechanical strain fields. The results reveal that nuclei deform anisotropically in response to substrate strain, specifically, the minor nuclear axis is significantly more deformable than the major axis. We show that upon microtubule depolymerization, nuclear deformation anisotropy completely disappears. Moreover, the removal of actin causes a significant increase in nuclear deformation along the minor axis and a corresponding increase in mechanical anisotropy. The results demonstrate that the nucleus deforms in a manner that is very much dependent on the direction of strain and the characteristics of the strain field. Actin and microtubules also appear to play distinct roles in controlling the anisotropic deformation of the nucleus in response to mechanical forces that arise in the cellular microenvironment.

Lukasz Andrzejewski

Centre for Interdisciplinary NanoPhysics,
Department of Physics,
Centre for Interdisciplinary NanoPhysics, Department of Physics

University of Ottawa

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Lukasz Andrzejewski

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Centre for Interdisciplinary NanoPhysics,Department of Physics,Centre for Interdisciplinary NanoPhysics, Department of Physics

University of Ottawa

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Centre for Interdisciplinary NanoPhysics,
Department of Physics,
Centre for Interdisciplinary NanoPhysics, Department of Physics