The development of nonviral gene delivery vehicles for therapeutic applications requires methods capable of quantifying the association between the genes and their carrier counterparts. Here we investigate the potential of fluorescence cross-correlation spectroscopy (FCCS) to characterize and optimize the assembly of nonviral cationic liposome (CL)-DNA complexes based on a CL formulation consisting of the cationic lipid DOTAP and zwitterionic lipid DOPC. We use a DNA plasmid for lipoplex loading encoding the Oct4 gene, critically involved in reprogramming somatic cells into induced pluripotent stem cells. We demonstrate that FCCS is able to quantitatively determine the extent of the association between DNA and the liposomes and assess its loading capacity. We also establish that the cationic lipid fraction, being proportional to the liposome membrane charge density, as well as charge ratio between the CLs and anionic DNA play an important role in the degree of interaction between the liposomes and DNA.
The pathogenesis of human Menkes and Wilson diseases depends on alterations in copper transport. Some reports suggest that intracellular traffic of copper might be regulated by kinase-mediated phosphorylation. However, there is no evidence showing the influence of kinase-related processes in coupled ATP hydrolysis/copper transport cycles. Here, we show that cyclic AMP-dependent protein kinase (PKA) regulates Ccc2p, the yeast Cu(I)-ATPase, with PKA-mediated phosphorylation of a conserved serine (Ser258) being crucial for catalysis. Long-range intramolecular communication between Ser258 and Asp627 (at the catalytic site) modulates the key pumping event: the conversion of the high-energy to the low-energy phosphorylated intermediate associated with copper release.
Plasma membrane Ca 2þ -ATPase is involved in the fine-tuned regulation of intracellular Ca 2þ . In this study, the presence of Ca 2þ -ATPase in caveolae from kidney basolateral membranes was investigated. With the use of a discontinuous sucrose gradient, we show that Ca 2þ -ATPase is exclusively located and fully active in caveolin-containing microdomains. Treatment with methyl-b-cyclodextrin -a cholesterol chelatorleads to a spreading of both caveolin and completely inactive Ca 2þ -ATPase toward high-density fractions. These data support the view that Ca 2þ fluxes mediated by Ca 2þ -ATPase in kidney epithelial cells occur only in caveolae, being strictly dependent on the integrity of these microdomains.
The aim of this study was to investigate (a) whether Ca 2؉ /calmodulin-dependent protein kinase II (CaM kinase II) participates in the regulation of plasma membrane Ca 2؉ -ATPase and (b) its possible cross-talk with other kinase-mediated modulatory pathways of the pump. Using isolated innervated membranes of the electrocytes from Electrophorus electricus L., we found that stimulation of endogenous protein kinase A (PKA) strongly phosphorylated membrane-bound CaM kinase II with simultaneous substantial activation of the Ca 2؉ pump (Ϸ2-fold). The addition of cAMP (5-50 pM), forskolin (10 nM), or cholera toxin (10 or 100 nM) stimulated both CaM kinase II phosphorylation and Ca 2؉ -ATPase activity, whereas these activation processes were cancelled by an inhibitor of the PKA ␣-catalytic subunit. When CaM kinase II was blocked by its specific inhibitor KN-93, the Ca 2؉ -ATPase activity decreased to the levels measured in the absence of calmodulin; the unusually high Ca 2؉ affinity dropped 2-fold; and the PKA-mediated stimulation of Ca 2؉ -ATPase was no longer seen. Hydroxylamine-resistant phosphorylation of the Ca 2؉ -ATPase strongly increased when the PKA pathway was activated, and this phosphorylation was suppressed by inhibition of CaM kinase II. We conclude that CaM kinase II is an intermediate in a complex regulatory network of the electrocyte Ca 2؉ pump, which also involves calmodulin and PKA.
The dynamics of two different fluorescent dyes labelling DNA and cationic liposomes is monitored through Fluorescence Cross‐Correlation Spectroscopy (FCCS). Measuring the extent of correlation between the flickering of the two dyes as they diffuse in and out of overlapping confocal volumes, the association of DNA and liposomes to form lipoplexes is quantified. This was achieved analysing the correlation with non 1:1 stoichiometric sample description.
Further details can be found in the article by Ana I. Gómez‐Varela, Ricardo Gaspar, Adelaide Miranda, Juliane L. Assis, Rafael H.F. Valverde, Marcelo Einicker‐Lamas, Bruno F. B. Silva, and Pieter A.A. De Beule (https://onlinelibrary.wiley.com/doi/10.1002/jbio.202000200).
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