Tension, Free Space, and Cell Damage in a Microfluidic Wound Healing Assay

We use a novel, microfluidics-based technique to deconstruct the classical wound healing scratch assay, decoupling the contribution of free space and cell damage on the migratory dynamics of an epithelial sheet. This method utilizes multiple laminar flows to selectively cleave cells enzymatically, and allows us to present a 'damage free' denudation. We therefore isolate the influence of free space on the onset of sheet migration. First, we observe denudation directly to measure the retraction in the cell sheet that occurs after cell-cell contact is broken, providing direct and quantitative evidence of strong tension within the sheet. We further probe the mechanical integrity of the sheet without denudation, instead using laminar flows to selectively inactivate actomyosin contractility. In both cases, retraction is observed over many cell diameters. We then extend this method and complement the enzymatic denudation with analogies to wounding, including gradients in signals associated with cell damage, such as reactive oxygen species, suspected to play a role in the induction of movement after wounding. These chemical factors are evaluated in combination with the enzymatic cleavage of cells, and are assessed for their influence on the collective migration of a non-abrasively denuded epithelial sheet. We conclude that free space alone is sufficient to induce movement, but this movement is predominantly limited to the leading edge, leaving cells further from the edge less able to move towards the wound. Surprisingly, when coupled with a gradient in ROS to simulate the chemical effects of abrasion however, motility was not restored, but further inhibited.Massachusetts Institute of Technology. Presidential FellowshipNational Institutes of Health (U.S.). Biotechnology Training FellowshipSingapore-MIT Alliance for Research and TechnologyMassachusetts Institute of Biotechnology Training GrantMassachusetts Institute of Technology (Open-source Funding

Myelin-associated proteins block the migration of olfactory ensheathing cells: an in vitro study using single cell tracking and traction force microscopy

Newly generated olfactory receptor axons grow from the peripheral to the central nervous system aided by olfactory ensheathing cells (OECs). Thus, OEC transplantation has emerged as a promising therapy for spinal cord injuries and for other neural diseases. However, these cells do not present a uniform population, but, instead, a functionally heterogeneous population that exhibits a variety of responses including adhesion, repulsion and crossover during cell-cell and cell-matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. Here, we demonstrated that rodent OECs express all the components of the Nogo Receptor complex and that their migration is blocked by Myelin. Next, we used cell tracking and traction force microscopy to analyze OEC migration and its mechanical properties over Myelin. Our data relate the absence of traction force of OEC with lower migratory capacity, which correlates with changes in the F-Actin cytoskeleton and focal adhesion distribution. Lastly, OEC traction force and migratory capacity is enhanced after cell incubation with the Nogo Receptor inhibitor NEP1-40

Epithelial bridges maintain tissue integrity during collective cell migration

10.1038/nmat3814Nature Materials13187-96NMAA

Analysis of baseline and cisplatin-inducible gene expression in Fanconi anemia cells using oligonucleotide-based microarrays

<p>Abstract</p> <p>Background</p> <p>Patients with Fanconi anemia (FA) suffer from multiple defects, most notably of the hematological compartment (bone marrow failure), and susceptibility to cancer. Cells from FA patients show increased spontaneous chromosomal damage, which is aggravated by exposure to low concentrations of DNA cross-linking agents such as mitomycin C or cisplatin. Five of the identified FA proteins form a nuclear core complex. However, the molecular function of these proteins remains obscure.</p> <p>Methods</p> <p>Oligonucleotide microarrays were used to compare the expression of approximately 12,000 genes from FA cells with matched controls. Expression profiles were studied in lymphoblastoid cell lines derived from three different FA patients, one from the FA-A and two from the FA-C complementation groups. The isogenic control cell lines were obtained by either transfecting the cells with vectors expressing the complementing cDNAs or by using a spontaneous revertant cell line derived from the same patient. In addition, we analyzed expression profiles from two cell line couples at several time points after a 1-hour pulse treatment with a discriminating dose of cisplatin.</p> <p>Results</p> <p>Analysis of the expression profiles showed differences in expression of a number of genes, many of which have unknown function or are difficult to relate to the FA defect. However, from a selected number of proteins involved in cell cycle regulation, DNA repair and chromatin structure, Western blot analysis showed that p21^{<it>waf</it>1/<it>Cip</it>1}was significantly upregulated after low dose cisplatin treatment in FA cells specifically (as well as being expressed at elevated levels in untreated FA cells).</p> <p>Conclusions</p> <p>The observed increase in expression of p21^{<it>waf</it>1/<it>Cip</it>1}after treatment of FA cells with crosslinkers suggests that the sustained elevated levels of p21^{<it>waf</it>1/<it>Cip</it>1}in untreated FA cells detected by Western blot analysis likely reflect increased spontaneous damage in these cells.</p