Membrane cholesterol depletion by methyl-beta-cyclodextrin enhances the expression of cardiac differentiation markers.

Cholesterol is a sterol lipid that plays pleiotropic roles in the plasma membrane; it is involved in maintaining membrane fluidity and permeability and the structure of lipid microdomains. Despite its importance, the consequences of membrane cholesterol depletion during cardiac differentiation have not been described. Therefore, we investigated the cellular and molecular mechanisms associated with cholesterol depletion in cultures of chick cardiac cells. We used methyl-beta-cyclodextrin (MCD) to deplete membrane cholesterol and investigate its role in cardiac differentiation by following the expression of several markers including the transcriptional factor Nkx2.5, the myofibrillar protein tropomyosin, the cytoskeletal intermediate filament protein desmin, the caveolar protein caveolin-3, the cadherin/beta-catenin adhesion complex, and the junctional protein connexin 43. Confocal microscopy showed that desmin-positive cells were located more externally in the aggregates in relation to the more internally located caveolin-3-positive cells. Desmin and caveolin-3 were co-localized in filamentous structures in the subsarcolemmal region of well-spread cells outside the aggregates. beta-Catenin was concentrated in regions of cell-cell contact, and tropomyosin in sarcomeric structures. Western blot tests showed that immediately following cholesterol depletion, there was a slight decrease in the expression of caveolin-3 and desmin, and at the same time there was a sharp increase in the expression of cadherin, tropomyosin, Nkx2.5 and connexin 43. Further, we found an increase in the expression of cardiac beta-myosin heavy chain 7, a marker of the cardiac hypertrophic phenotype. These observations suggest that membrane cholesterol plays a significant role in regulating cardiomyocyte differentiation.

Culture of neural cells of the eyestalk of a mangrove crab is optimized on poly-L-ornithine substrate.

Although there is a considerable demand for cell culture protocols from invertebrates for both basic and applied research, few attempts have been made to culture neural cells of crustaceans. We describe an in vitro method that permits the proliferation, growth and characterization of neural cells from the visual system of an adult decapod crustacean. We explain the coating of the culture plates with different adhesive substrates, and the adaptation of the medium to maintain viable neural cells for up to 7 days. Scanning electron microscopy allowed us to monitor the conditioned culture medium to assess cell morphology and cell damage. We quantified cells in the different substrates and performed statistical analyses. Of the most commonly used substrates, poly-L-ornithine was found to be the best for maintaining neural cells for 7 days. We characterized glial cells and neurons, and observed cell proliferation using immunocytochemical reactions with specific markers. This protocol was designed to aid in conducting investigations of adult crustacean neural cells in culture. We believe that an advantage of this method is the potential for adaptation to neural cells from other arthropods and even other groups of invertebrates.