Quantifying myosin light chain phosphorylation in single adherent cells with automated fluorescence microscopy

<p>Abstract</p> <p>Background</p> <p>In anchorage dependent cells, myosin generated contractile forces affect events closely associated with adhesion such as the formation of stress fibers and focal adhesions, and temporally distal events such as entry of the cell into S-phase. As occurs in many signaling pathways, a phosphorylation reaction (in this case, phosphorylation of myosin light chain) is directly responsible for cell response. Western blotting has been useful in measuring intracellular phosphorylation events, but cells are lysed in the process of sample preparation for western blotting, and spatial information such as morphology, localization of the phosphorylated species, and the distribution of individual cell responses across the population is lost. We report here a reliable automated microscopy method for quantitative measurement of myosin light chain phosphorylation in adherent cells. This method allows us to concurrently examine cell morphology, cell-cell contact, and myosin light chain diphosphorylation in vascular smooth muscle cells.</p> <p>Results</p> <p>Paraformaldehyde fixation and Triton X-100 permeabilization preserved cell morphology and myosin light chain phosphorylation better than the alternative fixation/permeabilization methods tested. We utilized automated microscopy methods to acquire three color images, determine cell spread area, and quantify the intensity of staining within each cell with anti-phospho-MLC antibody. Our results indicate that A10 rat aortic smooth muscle cells exhibit a re producible non-Gaussian distribution of MLC phosphorylation across a population of unsynchronized genetically identical cells. Adding an inhibitor of Rho kinase, Y27632, or plating cells on a low density of fibronectin, reduced phospho-myosin light chain signal as expected. On the other hand, adding calyculin A, an activator of contractility, increased myosin light chain phosphorylation. The IC₅₀for myosin light chain phosphorylation using Y27632 was determined to be 2.1 ± 0.6 micrometers. We observed a positive linear relationship between cell area and myosin light chain diphosphorylation, which is consistent with what has been reported in the literature using other methods.</p> <p>Conclusion</p> <p>Our results show that using proper specimen fixation techniques and background subtraction methods, imaging cytometry can be used to reliably measure relative myosin light chain phosphorylation in individual adherent cells. Importantly, the ability to make this measurement in adherent cells allows for simultaneous measurement of and correlation with other parameters of cellular topography such as morphology and cell-cell proximity. This assay has potential application in screening for drug development.</p

In thrombin stimulated human platelets Citalopram, Promethazine, Risperidone, and Ziprasidone, but not Diazepam, may exert their pharmacological effects also through intercalation in membrane phospholipids in a receptor-independent manner

Intercalation of drugs in the platelet membrane affects phospholipid-requiring enzymatic processes according to the drugs’ intercalation capability. We investigated effects of Promethazine, Citalopram, Ziprasidone, Risperidone, and Diazepam on phospholipase A2 (PLA2) and polyphosphoinositide (PPI) metabolism in thrombin-stimulated human platelets. We also examined effects of the drugs on monolayers of glycerophospholipids using the Langmuir technique. Diazepam did not influence PLA2 activity, had no effects on PPI cycle, and caused no change in mean molecular area of phospholipid monolayers. The remaining psychotropic drugs affected these parameters in different ways and levels of potency suggesting that they act by being intercalated between the molecules of adjacent membrane phospholipids, thus causing changes in substrate availability for phospholipid-hydrolyzing enzymes (PLA2 and Phospholipase C). We show that several psychotropic drugs can also have other cellular effects than receptor antagonism. These effects may be implicated in the psychotropic effects of the drugs and/or their side effects

Emerging medical and engineering strategies for the prevention of long-term indwelling catheter blockage

Urinary catheters have been used on an intermittent or indwelling basis for centuries, in order to relieve urinary retention and incontinence. Nevertheless, the use of urinary catheters in the clinical setting is fraught with complication, the most common of which is the development of nosocomial urinary tract infections, known as catheter-associated urinary tract infections. Infections of this nature are not only significant owing to their high incidence rate and subsequent economic burden but also to the severe medical consecutions that result. A range of techniques have been employed in recent years, utilising various technologies in attempts to counteract the perilous medical cascade following catheter blockage. This review will focus on the current advancement (within the last 10 years) in prevention of encrustation and blockage of long-term indwelling catheters both from engineering and medical perspectives, with particular emphasis on the importance of stimuli-responsive systems.</p