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Articles by Anke Scholz in JoVE
JoVE General
Isolamento e manipulação genética de adultos miócitos cardíacos for Imaging Confocal
Institute for Molecular Cell Biology, Universty of Saarland
Adulto miócitos cardíacos são células primárias que podem ser isoladas de corações de animais e cultivados por vários dias. Dentro deste período a cultura de transferência de gene adenoviral podem ser usadas para expressar biossensores geneticamente codificado (GEBS) ou proteínas de fusão fluorescente. Ambas as abordagens permitem investigações celular por meio de microscopia confocal.
Other articles by Anke Scholz on PubMed
Phosphorylation of Sucrose Synthase at Serine 170: Occurrence and Possible Role As a Signal for Proteolysis
Sequence analysis identified serine 170 (S170) of the maize (Zea mays L.) SUS1 sucrose synthase (SUS) protein as a possible, second phosphorylation site. Maize leaves contained two calcium-dependent protein kinase activities and a calcium-independent kinase activity with characteristics of an sucrose non-fermenting 1 (SNF1)-related protein kinase. Phosphorylation of the novel S170 and the known serine 15 (S15) site by these protein kinases was determined in peptide substrates and detected in SUS1 protein substrates utilizing sequence- and phosphorylation-specific antibodies. We demonstrate phosphorylation of S170 in vitro and in vivo. The calcium-dependent protein kinases phosphorylated both S170 and S15, whereas SNF1-related protein kinase activity was restricted to S15. Calcium-dependent protein-kinase-mediated S170 and S15 phosphorylation kinetics were determined in wild-type and mutant SUS1 substrates. These analyses revealed that kinase specificity for S170 was threefold lower than that for S15, and that phosphorylation of S170 was stimulated by prior phosphorylation at the S15 site. The SUS-binding peptides encoded by early nodulin 40 (ENOD40) specifically antagonized S170 phosphorylation in vitro. A model wherein S170 phosphorylation functions as part of a mechanism targeting SUS for proteasome-mediated degradation is supported by the observations that SUS proteolytic fragments: (i) were detected and possessed relatively high phosphorylated-S170 (pS170) stoichiometry; (ii) were spatially coincident with proteasome activity within developing leaves; and (iii) co-sedimented with proteasome activity. In addition, full-length pS170-SUS protein was less stable than S170-SUS in cultured leaf segments and was stabilized by proteasome inhibition. Post-translational control of SUS protein level through pS170-promoted proteolysis may explain the specific and significant decrease in SUS abundance that accompanies the sink-to-source transition in developing maize leaves.
Remodelling of Ca2+ Handling Organelles in Adult Rat Ventricular Myocytes During Long Term Culture
It is well known that for cardiomyocytes, isolation and culturing induce largely unknown remodelling processes. We analysed changes in the structure of cell compartments with optical techniques such as confocal microscopy and fluorescence redistribution after photobleaching employing adenoviral-mediated transduction of targeted fluorescent proteins and small molecule dyes. We identified characteristic remodelling processes: the T-tubular membrane system was gradually lost by a process referred to as "sequential pinching off", in an outward direction. Mitochondria fell in one of three classes, very small (0.9 microm length), medium long (1.8 microm) or extended shape (3.6 microm) organelles. Over the culturing time mitochondria gradually fused. Bleaching of individual mitochondria revealed association between apparently separated mitochondria by "tunnelling" via sub-resolution organelle-tubes. This tunnelling process was increasing over the culturing time. A gradual loss of the cross-striation arrangement in the endoplasmic/sarcoplasmic reticulum was visualised. Analysis of large populations of Ca(2+) sparks by video-rate confocal 2D-scanning revealed significant albeit small changes of these elementary SR-Ca(2+) release events in adult cardiomyocytes that could be related to changes in SR-Ca(2+) content rather than resting Ca(2+) concentration. In conclusion, primary isolated cardiomyocytes from adult hearts undergo a well-defined, but reproducible subcellular remodelling during optimised long term culture.
Optical Action Potential Screening on Adult Ventricular Myocytes As an Alternative QT-screen
QT-interval screens are increasingly important for cardiac safety on all new medications. So far, investigations rely on animal experiments or cell-based screens solely probing for conductance alterations in heterologously expressed hERG-channels in cell lines allowing for a high degree of automation. Adult cardiomyocytes can not be handled by automated patch-clamp setups. Therefore optical screening of primary isolated ventricular myocytes is regarded as an alternative. Several optical voltage sensors have been reported for ratiometric measurements, but they all influenced the naïve action potential. The aim of the present study was to explore the recording conditions and define settings that allow optical QT-interval screens.
Functional and Morphological Preservation of Adult Ventricular Myocytes in Culture by Sub-micromolar Cytochalasin D Supplement
In cardiac myocytes, cytochalasin D (CytoD) was reported to act as an actin disruptor and mechanical uncoupler. Using confocal and super-resolution STED microscopy, we show that CytoD preserves the actin filament architecture of adult rat ventricular myocytes in culture. Five hundred nanomolar CytoD was the optimal concentration to achieve both preservation of the T-tubular structure during culture periods of 3 days and conservation of major functional characteristics such as action potentials, calcium transients and, importantly, the contractile properties of single myocytes. Therefore, we conclude that the addition of CytoD to the culture of adult cardiac myocytes can indeed be used to generate a solid single-cell model that preserves both morphology and function of freshly isolated cells. Moreover, we reveal a putative link between cytoskeletal and T-tubular remodeling. In the absence of CytoD, we observed a loss of T-tubules that led to significant dyssynchronous Ca(2+)-induced Ca(2+) release (CICR), while in the presence of 0.5 μM CytoD, T-tubules and homogeneous CICR were majorly preserved. Such data suggested a possible link between the actin cytoskeleton, T-tubules and synchronous, reliable excitation-contraction-coupling. Thus, T-tubular re-organization in cell culture sheds some additional light onto similar processes found during many cardiac diseases and might link cytoskeletal alterations to changes in subcellular Ca(2+) signaling revealed under such pathophysiological conditions.
