Kinetic disruption of lipid rafts is a mechanosensor for phospholipase D.

The sensing of physical force, mechanosensation, underlies two of five human senses-touch and hearing. How transduction of force in a membrane occurs remains unclear. We asked if a biological membrane could employ kinetic energy to transduce a signal absent tension. Here we show that lipid rafts are dynamic compartments that inactivate the signalling enzyme phospholipase D2 (PLD2) by sequestering the enzyme from its substrate. Mechanical disruption of the lipid rafts activates PLD2 by mixing the enzyme with its substrate to produce the signalling lipid phosphatidic acid (PA). We calculate a latency time of <650 µs for PLD activation by mixing. Our results establish a fast, non-tension mechanism for mechanotransduction where disruption of ordered lipids initiates a mechanosensitive signal for cell growth through mechanical mixing.

Endothelial nuclear lamina is not required for glucocorticoid receptor nuclear import but does affect receptor-mediated transcription activation.

The lamina serves to maintain the nuclear structure and stiffness while acting as a scaffold for heterochromatin and many transcriptional proteins. Its role in endothelial mechanotransduction, specifically how nuclear mechanics impact gene regulation under shear stress, is not fully understood. In this study, we successfully silenced lamin A/C in bovine aortic endothelial cells to determine its role in both glucocorticoid receptor (GR) nuclear translocation and glucocorticoid response element (GRE) transcriptional activation in response to dexamethasone and shear stress. Nuclear translocation of GR, an anti-inflammatory nuclear receptor, in response to dexamethasone or shear stress (5, 10, and 25 dyn/cm(2)) was observed via time-lapse cell imaging and quantified using a Bayesian image analysis algorithm. Transcriptional activity of the GRE promoter was assessed using a dual-luciferase reporter plasmid. We found no dependence on nuclear lamina for GR translocation from the cytoplasm into the nucleus. However, the absence of lamin A/C led to significantly increased expression of luciferase under dexamethasone and shear stress induction as well as changes in histone protein function. PCR results for NF-κB inhibitor alpha (NF-κBIA) and dual specificity phosphatase 1 (DUSP1) genes further supported our luciferase data with increased expression in the absence of lamin. Our results suggest that absence of lamin A/C does not hinder passage of GR into the nucleus, but nuclear lamina is important to properly regulate GRE transcription. Nuclear lamina, rather than histone deacetylase (HDAC), is a more significant mediator of shear stress-induced transcriptional activity, while dexamethasone-initiated transcription is more HDAC dependent. Our findings provide more insights into the molecular pathways involved in nuclear mechanotransduction.

Bayesian image analysis of dexamethasone and shear stress-induced glucocorticoid receptor intracellular movement.

Endothelial cells are continuously exposed to hemodynamic shear stress, which has been shown to induce an array of physiological responses at the cellular and molecular levels. Uniform high shear stress is protective against vascular diseases such as atherosclerosis which preferentially occur at regions of disturbed flow and low shear. The glucocorticoid receptor (GR), a member of the steroid nuclear receptors with anti-inflammatory functions, has been shown to be activated by shear stress. Using a unique expectation-maximization (EM) algorithm based on Bayesian statistics, we have developed an image analysis algorithm to quantitatively assess GR nuclear translocation based on time-lapse images of green fluorescence protein-tagged GR (GFP-GR) under continuous exposure to a shear stress of 10 or 25 dynes/cm(2) as well as to Dexamethasone, a GR agonist. Average fluorescence brightness is generated for nucleus and cytoplasm. Real-time imaging of sheared cells revealed a steady and significant nuclear GFP-GR increase of approximately 20% within 2 h, compared to a rapid 60% increase in Dexamethasone-treated cells within 30 min. Furthermore, we found that that GFP-GR nuclear translocation under shear is not dependent on an intact cytoskeleton. Our image analysis algorithm provides a novel quantitative method to further study shear-sensitive mechanotransduction pathways in endothelial cells.