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Book ; Online ; Thesis: The behavior of DASPMI in living cells

Ramadass, Radhan [Verfasser]

spectrally and spatially resolved fluorescence lifetime imaging

2008

Author's details	von Radhan Ramadass
Keywords	Biowissenschaften, Biologie ; Life Science, Biology
Subject code	sg570
Language	English
Document type	Book ; Online ; Thesis
Database	Digital theses on the web

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Article ; Online: Integration of multiple imaging platforms to uncover cardiovascular defects in adult zebrafish.

Bensimon-Brito, Anabela / Boezio, Giulia L M / Cardeira-da-Silva, João / Wietelmann, Astrid / Ramkumar, Srinath / Lundegaard, Pia R / Helker, Christian S M / Ramadass, Radhan / Piesker, Janett / Nauerth, Arno / Mueller, Clemens / Stainier, Didier Y R

Cardiovascular research

2021 Volume 118, Issue 12, Page(s) 2665–2687

Abstract: Aims: Mammalian models have been instrumental in investigating adult heart function and human disease. However, electrophysiological differences with human hearts and high costs motivate the need for non-mammalian models. The zebrafish is a well- ... ...

Abstract	Aims: Mammalian models have been instrumental in investigating adult heart function and human disease. However, electrophysiological differences with human hearts and high costs motivate the need for non-mammalian models. The zebrafish is a well-established genetic model to study cardiovascular development and function; however, analysis of cardiovascular phenotypes in adult specimens is particularly challenging as they are opaque. Methods and results: Here, we optimized and combined multiple imaging techniques including echocardiography, magnetic resonance imaging, and micro-computed tomography to identify and analyse cardiovascular phenotypes in adult zebrafish. Using alk5a/tgfbr1a mutants as a case study, we observed morphological and functional cardiovascular defects that were undetected with conventional approaches. Correlation analysis of multiple parameters revealed an association between haemodynamic defects and structural alterations of the heart, as observed clinically. Conclusion: We report a new, comprehensive, and sensitive platform to identify otherwise indiscernible cardiovascular phenotypes in adult zebrafish.
MeSH term(s)	Animals ; Cardiovascular System ; Echocardiography ; Heart ; Humans ; Mammals ; X-Ray Microtomography ; Zebrafish/genetics
Language	English
Publishing date	2021-10-05
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	80340-6
ISSN	1755-3245 ; 0008-6363
ISSN (online)	1755-3245
ISSN	0008-6363
DOI	10.1093/cvr/cvab310
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Article ; Online: Stimulation of glycolysis promotes cardiomyocyte proliferation after injury in adult zebrafish.

Fukuda, Ryuichi / Marín-Juez, Rubén / El-Sammak, Hadil / Beisaw, Arica / Ramadass, Radhan / Kuenne, Carsten / Guenther, Stefan / Konzer, Anne / Bhagwat, Aditya M / Graumann, Johannes / Stainier, Didier Yr

EMBO reports

2020 Volume 21, Issue 8, Page(s) e49752

Abstract: Cardiac metabolism plays a crucial role in producing sufficient energy to sustain cardiac function. However, the role of metabolism in different aspects of cardiomyocyte regeneration remains unclear. Working with the adult zebrafish heart regeneration ... ...

Abstract	Cardiac metabolism plays a crucial role in producing sufficient energy to sustain cardiac function. However, the role of metabolism in different aspects of cardiomyocyte regeneration remains unclear. Working with the adult zebrafish heart regeneration model, we first find an increase in the levels of mRNAs encoding enzymes regulating glucose and pyruvate metabolism, including pyruvate kinase M1/2 (Pkm) and pyruvate dehydrogenase kinases (Pdks), especially in tissues bordering the damaged area. We further find that impaired glycolysis decreases the number of proliferating cardiomyocytes following injury. These observations are supported by analyses using loss-of-function models for the metabolic regulators Pkma2 and peroxisome proliferator-activated receptor gamma coactivator 1 alpha. Cardiomyocyte-specific loss- and gain-of-function manipulations of pyruvate metabolism using Pdk3 as well as a catalytic subunit of the pyruvate dehydrogenase complex (PDC) reveal its importance in cardiomyocyte dedifferentiation and proliferation after injury. Furthermore, we find that PDK activity can modulate cell cycle progression and protrusive activity in mammalian cardiomyocytes in culture. Our findings reveal new roles for cardiac metabolism and the PDK-PDC axis in cardiomyocyte behavior following cardiac injury.
MeSH term(s)	Animals ; Cell Proliferation ; Glycolysis ; Myocytes, Cardiac/metabolism ; Protein Serine-Threonine Kinases/genetics ; Protein Serine-Threonine Kinases/metabolism ; Zebrafish/metabolism
Chemical Substances	Protein Serine-Threonine Kinases (EC 2.7.11.1)
Language	English
Publishing date	2020-07-09
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2020896-0
ISSN	1469-3178 ; 1469-221X
ISSN (online)	1469-3178
ISSN	1469-221X
DOI	10.15252/embr.201949752
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Article ; Online: Real-time 3D visualization of cellular rearrangements during cardiac valve formation.

Pestel, Jenny / Ramadass, Radhan / Gauvrit, Sebastien / Helker, Christian / Herzog, Wiebke / Stainier, Didier Y R

Development (Cambridge, England)

2016 Volume 143, Issue 12, Page(s) 2217–2227

Abstract: During cardiac valve development, the single-layered endocardial sheet at the atrioventricular canal (AVC) is remodeled into multilayered immature valve leaflets. Most of our knowledge about this process comes from examining fixed samples that do not ... ...

Abstract	During cardiac valve development, the single-layered endocardial sheet at the atrioventricular canal (AVC) is remodeled into multilayered immature valve leaflets. Most of our knowledge about this process comes from examining fixed samples that do not allow a real-time appreciation of the intricacies of valve formation. Here, we exploit non-invasive in vivo imaging techniques to identify the dynamic cell behaviors that lead to the formation of the immature valve leaflets. We find that in zebrafish, the valve leaflets consist of two sets of endocardial cells at the luminal and abluminal side, which we refer to as luminal cells (LCs) and abluminal cells (ALCs), respectively. By analyzing cellular rearrangements during valve formation, we observed that the LCs and ALCs originate from the atrium and ventricle, respectively. Furthermore, we utilized Wnt/β-catenin and Notch signaling reporter lines to distinguish between the LCs and ALCs, and also found that cardiac contractility and/or blood flow is necessary for the endocardial expression of these signaling reporters. Thus, our 3D analyses of cardiac valve formation in zebrafish provide fundamental insights into the cellular rearrangements underlying this process.
MeSH term(s)	Animals ; Cell Movement ; Coronary Circulation ; Endocardium/cytology ; Endocardium/embryology ; Gene Expression Regulation, Developmental ; Heart Atria/cytology ; Heart Atria/embryology ; Heart Valves/cytology ; Heart Valves/embryology ; Heart Ventricles/cytology ; Heart Ventricles/embryology ; Imaging, Three-Dimensional ; Mutation/genetics ; Myocardial Contraction ; Organogenesis/genetics ; Receptors, Notch/metabolism ; Wnt Signaling Pathway ; Zebrafish
Chemical Substances	Receptors, Notch
Language	English
Publishing date	2016--15
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	90607-4
ISSN	1477-9129 ; 0950-1991
ISSN (online)	1477-9129
ISSN	0950-1991
DOI	10.1242/dev.133272
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Article ; Online: How DASPMI reveals mitochondrial membrane potential: fluorescence decay kinetics and steady-state anisotropy in living cells.

Ramadass, Radhan / Bereiter-Hahn, Jürgen

Biophysical journal

2008 Volume 95, Issue 8, Page(s) 4068–4076

Abstract: Spectroscopic responses of the potentiometric probe 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were investigated in living cells by means of a time- and space-correlated single photon counting technique. Spatially resolved ... ...

Abstract	Spectroscopic responses of the potentiometric probe 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were investigated in living cells by means of a time- and space-correlated single photon counting technique. Spatially resolved fluorescence decays from single mitochondria or only a very few organelles of XTH2 cells exhibited three-exponential decay kinetics. Based on DASPMI photophysics in a variety of solvents, these lifetimes were attributed to the fluorescence from the locally excited state, intramolecular charge transfer state, and twisted intramolecular charge transfer state. A considerable variation in lifetimes among mitochondria of different morphologies and within single cells was evident, corresponding to high physiological variations within single cells. Considerable shortening of the short lifetime component (tau(1)) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents. Under these conditions tau(2) and tau(3) increased with decreasing contribution. Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence. Increased DASPMI fluorescence under conditions that elevate the mitochondrial membrane potential has been attributed to uptake according to Nernst distributions, delocalization of pi-electrons, quenching processes of the methyl pyridinium moiety, and restricted torsional dynamics at the mitochondrial inner membrane. Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential. The direct influence of the local electric field on the transition dipole moment of the probe and its torsional dynamics monitor changes in mitochondrial energy status within living cells.
MeSH term(s)	Animals ; Anisotropy ; Cell Line ; Cell Survival/drug effects ; Fluorescence ; Kinetics ; Membrane Potential, Mitochondrial/drug effects ; Mitochondrial Membranes/drug effects ; Pyridinium Compounds/pharmacology ; Time Factors ; Xenopus
Chemical Substances	Pyridinium Compounds ; 2-(4-(dimethylamino)styryl)-1-methylpyridinium (2156-29-8)
Language	English
Publishing date	2008-07-18
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	218078-9
ISSN	1542-0086 ; 0006-3495
ISSN (online)	1542-0086
ISSN	0006-3495
DOI	10.1529/biophysj.108.135079
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Article ; Online: Actin binding GFP allows 4D in vivo imaging of myofilament dynamics in the zebrafish heart and the identification of Erbb2 signaling as a remodeling factor of myofibril architecture.

Reischauer, Sven / Arnaout, Rima / Ramadass, Radhan / Stainier, Didier Y R

Circulation research

2014 Volume 115, Issue 10, Page(s) 845–856

Abstract: Rationale: Dilated cardiomyopathy is a leading cause of congestive heart failure and a debilitating complication of antineoplastic therapies. Despite disparate causes for dilated cardiomyopathy, maladaptive cardiac remodeling and decreased systolic ... ...

Abstract	Rationale: Dilated cardiomyopathy is a leading cause of congestive heart failure and a debilitating complication of antineoplastic therapies. Despite disparate causes for dilated cardiomyopathy, maladaptive cardiac remodeling and decreased systolic function are common clinical consequences, begging an investigation of in vivo contractile dynamics in development and disease, one that has been impossible to date. Objective: To image myocardial contractile filament dynamics in vivo and to assess potential causes of dilated cardiomyopathy in antineoplastic therapies targeting the epidermal growth factor receptor Erbb2. Methods and results: We generated a transgenic zebrafish line expressing an actin-binding green fluorescent protein in cardiomyocytes, allowing an in vivo imaging of myofilaments. Analysis of this line revealed architectural differences in myofibrils of the distinct cardiomyocyte subtypes. We used this model to investigate the effects of Erbb2 signaling on myofibrillar organization because drugs targeting ERBB2 (HER2/NEU) signaling, a mainstay of breast cancer chemotherapy, cause dilated cardiomyopathy in many patients. High-resolution in vivo imaging revealed that Erbb2 signaling regulates a switch between a dense apical network of filamentous myofibrils and the assembly of basally localized myofibrils in ventricular cardiomyocytes. Conclusions: Using this novel line, we compiled a reference for myofibrillar microarchitecture among myocardial subtypes in vivo and at different developmental stages, establishing this model as a tool to analyze in vivo cardiomyocyte contractility and remodeling for a broad range of cardiovascular questions. Furthermore, we applied this model to study Erbb2 signaling in cardiomyopathy. We show a direct link between Erbb2 activity and remodeling of myofibrils, revealing an unexpected mechanism with potentially important implications for prevention and treatment of cardiomyopathy.
MeSH term(s)	Actins/metabolism ; Amino Acid Sequence ; Animals ; Animals, Genetically Modified ; Cells, Cultured ; Green Fluorescent Proteins/metabolism ; Heart/anatomy & histology ; Heart/physiology ; Imaging, Three-Dimensional/methods ; Molecular Sequence Data ; Myofibrils/chemistry ; Myofibrils/metabolism ; Protein Binding/physiology ; Receptor, ErbB-2/analysis ; Receptor, ErbB-2/genetics ; Receptor, ErbB-2/metabolism ; Signal Transduction/physiology ; Time Factors ; Zebrafish
Chemical Substances	Actins ; Green Fluorescent Proteins (147336-22-9) ; ERBB2 protein, human (EC 2.7.10.1) ; Receptor, ErbB-2 (EC 2.7.10.1)
Language	English
Publishing date	2014-09-16
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	80100-8
ISSN	1524-4571 ; 0009-7330 ; 0931-6876
ISSN (online)	1524-4571
ISSN	0009-7330 ; 0931-6876
DOI	10.1161/CIRCRESAHA.115.304356
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Article: Photophysical properties of DASPMI as revealed by spectrally resolved fluorescence decays.

Ramadass, Radhan / Bereiter-Hahn, Jürgen

The journal of physical chemistry. B

2007 Volume 111, Issue 26, Page(s) 7681–7690

Abstract: Photophysical properties of 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) in various solvents were investigated using time- and space-correlated single photon counting. DASPMI is known to selectively stain mitochondria in living cells.1, ... ...

Abstract	Photophysical properties of 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) in various solvents were investigated using time- and space-correlated single photon counting. DASPMI is known to selectively stain mitochondria in living cells.1,2 The uptake and fluorescence intensity of DASPMI in mitochondria is a dynamic measure of membrane potential. Hence, an endeavor has been made to elucidate the mechanism of DASPMI fluorescence by obtaining spectrally resolved fluorescence decays in different solvents. A biexponential decay model was sufficient to globally describe the wavelength-dependent fluorescence in ethanol and chloroform. While in glycerol, a three-exponential decay model was necessary for global analysis. In the polar low-viscous solvent water, a monoexponential decay model fitted the decay data. The sensitivity of DASPMI to solvent viscosity was analyzed using various proportions of glycerol-ethanol mixtures. The lifetimes were found to increase with increasing solvent viscosity. The negative amplitudes of the short lifetime component found in chloroform and glycerol at the longer wavelengths validated the formation of new excited-state species from the initially excited state. Time-resolved emission spectra in chloroform and glycerol showed a biphasic increase of spectral width and emission maxima. The spectral width had an initial fast increase within 150 ps and a near constant thereafter. A three-state model of generalized scheme, on the basis of successive formation of locally excited state (LE), intramolecular charge transfer state (ICT), and twisted intramolecular charge transfer (TICT) state, has been proposed to explain the excited-state kinetics. The presumed role of solvation dynamics of ICT and TICT states leading to the asymmetrical broadening and structureless fluorescence has been substantiated by the decomposition of time-resolved emission spectra in chloroform, glycerol, and ethanol/glycerol mixtures.
MeSH term(s)	Photochemistry ; Pyridinium Compounds/chemistry ; Spectrometry, Fluorescence
Chemical Substances	Pyridinium Compounds ; 2-(4-(dimethylamino)styryl)-1-methylpyridinium (2156-29-8)
Language	English
Publishing date	2007-07-05
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1520-6106
ISSN	1520-6106
DOI	10.1021/jp070378k
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Article ; Online: Mechanical Forces Regulate Cardiomyocyte Myofilament Maturation via the VCL-SSH1-CFL Axis.

Fukuda, Ryuichi / Gunawan, Felix / Ramadass, Radhan / Beisaw, Arica / Konzer, Anne / Mullapudi, Sri Teja / Gentile, Alessandra / Maischein, Hans-Martin / Graumann, Johannes / Stainier, Didier Y R

Developmental cell

2019 Volume 51, Issue 1, Page(s) 62–77.e5

Abstract: Mechanical forces regulate cell behavior and tissue morphogenesis. During cardiac development, mechanical stimuli from the heartbeat are required for cardiomyocyte maturation, but the underlying molecular mechanisms remain unclear. Here, we first show ... ...

Abstract	Mechanical forces regulate cell behavior and tissue morphogenesis. During cardiac development, mechanical stimuli from the heartbeat are required for cardiomyocyte maturation, but the underlying molecular mechanisms remain unclear. Here, we first show that the forces of the contracting heart regulate the localization and activation of the cytoskeletal protein vinculin (VCL), which we find to be essential for myofilament maturation. To further analyze the role of VCL in this process, we examined its interactome in contracting versus non-contracting cardiomyocytes and, in addition to several known interactors, including actin regulators, identified the slingshot protein phosphatase SSH1. We show how VCL recruits SSH1 and its effector, the actin depolymerizing factor cofilin (CFL), to regulate F-actin rearrangement and promote cardiomyocyte myofilament maturation. Overall, our results reveal that mechanical forces generated by cardiac contractility regulate cardiomyocyte maturation through the VCL-SSH1-CFL axis, providing further insight into how mechanical forces are transmitted intracellularly to regulate myofilament maturation.
MeSH term(s)	Actin Cytoskeleton/metabolism ; Actin Depolymerizing Factors/metabolism ; Actins/metabolism ; Aminobenzoates/pharmacology ; Animals ; Cofilin 1/metabolism ; Gene Expression Regulation, Developmental ; Heart/embryology ; Microfilament Proteins/metabolism ; Myocardium/metabolism ; Myocytes, Cardiac/metabolism ; Myofibrils/metabolism ; Phosphoprotein Phosphatases/metabolism ; Sodium-Calcium Exchanger/metabolism ; Vinculin/metabolism ; Zebrafish
Chemical Substances	Actin Depolymerizing Factors ; Actins ; Aminobenzoates ; Cofilin 1 ; Microfilament Proteins ; Sodium-Calcium Exchanger ; sodium-calcium exchanger 1 ; tricaine (02591PHL19) ; Vinculin (125361-02-6) ; Phosphoprotein Phosphatases (EC 3.1.3.16)
Language	English
Publishing date	2019-09-05
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2054967-2
ISSN	1878-1551 ; 1534-5807
ISSN (online)	1878-1551
ISSN	1534-5807
DOI	10.1016/j.devcel.2019.08.006
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Article ; Online: Focal adhesions are essential to drive zebrafish heart valve morphogenesis.

Gunawan, Felix / Gentile, Alessandra / Fukuda, Ryuichi / Tsedeke, Ayele Taddese / Jiménez-Amilburu, Vanesa / Ramadass, Radhan / Iida, Atsuo / Sehara-Fujisawa, Atsuko / Stainier, Didier Y R

The Journal of cell biology

2019 Volume 218, Issue 3, Page(s) 1039–1054

Abstract: Elucidating the morphogenetic events that shape vertebrate heart valves, complex structures that prevent retrograde blood flow, is critical to understanding valvular development and aberrations. Here, we used the zebrafish atrioventricular (AV) valve to ... ...

Abstract	Elucidating the morphogenetic events that shape vertebrate heart valves, complex structures that prevent retrograde blood flow, is critical to understanding valvular development and aberrations. Here, we used the zebrafish atrioventricular (AV) valve to investigate these events in real time and at single-cell resolution. We report the initial events of collective migration of AV endocardial cells (ECs) into the extracellular matrix (ECM), and their subsequent rearrangements to form the leaflets. We functionally characterize integrin-based focal adhesions (FAs), critical mediators of cell-ECM interactions, during valve morphogenesis. Using transgenes to block FA signaling specifically in AV ECs as well as loss-of-function approaches, we show that FA signaling mediated by Integrin α5β1 and Talin1 promotes AV EC migration and overall shaping of the valve leaflets. Altogether, our investigation reveals the critical processes driving cardiac valve morphogenesis in vivo and establishes the zebrafish AV valve as a vertebrate model to study FA-regulated tissue morphogenesis.
MeSH term(s)	Animals ; Cell Movement ; Endocardium/embryology ; Extracellular Matrix/genetics ; Extracellular Matrix/metabolism ; Focal Adhesions/genetics ; Focal Adhesions/metabolism ; Heart Valves/embryology ; Integrin alpha5beta1/genetics ; Integrin alpha5beta1/metabolism ; Organogenesis ; Signal Transduction ; Talin/genetics ; Talin/metabolism ; Zebrafish/embryology ; Zebrafish/genetics ; Zebrafish Proteins/genetics ; Zebrafish Proteins/metabolism
Chemical Substances	Integrin alpha5beta1 ; Talin ; Zebrafish Proteins ; tln1 protein, zebrafish
Language	English
Publishing date	2019-01-11
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	218154-x
ISSN	1540-8140 ; 0021-9525
ISSN (online)	1540-8140
ISSN	0021-9525
DOI	10.1083/jcb.201807175
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Article ; Online: In vivo

Uribe, Veronica / Ramadass, Radhan / Dogra, Deepika / Rasouli, S Javad / Gunawan, Felix / Nakajima, Hiroyuki / Chiba, Ayano / Reischauer, Sven / Mochizuki, Naoki / Stainier, Didier Y R

Development (Cambridge, England)

2018 Volume 145, Issue 14

Abstract: Cardiomyocyte proliferation is crucial for cardiac growth, patterning and regeneration; however, few studies have investigated the behavior of dividing ... ...

Abstract	Cardiomyocyte proliferation is crucial for cardiac growth, patterning and regeneration; however, few studies have investigated the behavior of dividing cardiomyocytes
MeSH term(s)	Animals ; Cell Division ; Cell Proliferation ; Cell Shape ; Cell Size ; Gene Expression Regulation, Developmental ; Heart/growth & development ; Ligands ; Myocytes, Cardiac/cytology ; Myocytes, Cardiac/metabolism ; Organogenesis ; Sarcomeres/metabolism ; Signal Transduction ; Transforming Growth Factor beta/metabolism ; Zebrafish/genetics ; Zebrafish/growth & development ; Zebrafish Proteins/metabolism
Chemical Substances	Ligands ; Transforming Growth Factor beta ; Zebrafish Proteins
Language	English
Publishing date	2018-07-30
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	90607-4
ISSN	1477-9129 ; 0950-1991
ISSN (online)	1477-9129
ISSN	0950-1991
DOI	10.1242/dev.164194
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