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ABSTRACT: Calcium (Ca(2+)) signaling in neurons is mediated by plethora of calcium binding proteins with many of them belonging to the Calmodulin family of calcium sensors. Many studies have shown that the subcellular localization of neuronal EF-hand Ca(2+)-sensors is crucial for their cellular function. To overcome the resolution limit of classical fluorescence and confocal microscopy various imaging techniques have been developed recently that improve the resolution by an order of magnitude in all dimensions. This new microscope techniques make co-localization studies of Ca(2+)-binding proteins more reliable and help to get insights into the macromolecular organization of intracellular structures and signaling pathways beyond the diffraction limit of visible light.
Methods in molecular biology (Clifton, N.J.) 01/2013; 963:147-69.
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ABSTRACT: Glycolytic oscillations of intact yeast cells of the strain Saccharomyces carlsbergensis were investigated at both the levels of cell populations and of individual cells. Individual cells showed glycolytic oscillations even at very low cell densities (e.g. 1.0[Formula: see text]10(5) cells/ml). By contrast, the collective behaviour on the population level was cell density-dependent: at high cell densities it is oscillatory, but below the threshold density of 1.0[Formula: see text]10(6) cells/ml the collective dynamics becomes quiescent. We demonstrate that the transition in the collective dynamics is caused by the desynchronisation of the oscillations of individual cells. This is characteristic for a Kuramoto transition. Spatially resolved measurements at low cell densities revealed that even cells that adhere to their neighbours oscillated with their own, independent frequencies and phases.
PLoS ONE 01/2012; 7(9):e43276. · 4.09 Impact Factor
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ABSTRACT: Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes.
PLoS ONE 01/2011; 6(2):e15820. · 4.09 Impact Factor
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ABSTRACT: F1-ATPase is an enzyme acting as a rotary nano-motor. During catalysis subunits of this enzyme complex rotate relative to other parts of the enzyme. Here we demonstrate that the combination of two input stimuli causes stop of motor rotation. Application of either individual stimulus did not significantly influence motor motion. These findings may contribute to the development of logic gates using single biological motor molecules.
Journal of Nanobiotechnology 06/2009; 7:3. · 5.09 Impact Factor
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Anna Fejtova,
Daria Davydova,
Ferdinand Bischof,
Vesna Lazarevic,
Wilko D Altrock,
Stefano Romorini,
Cornelia Schöne, Werner Zuschratter,
Michael R Kreutz,
Craig C Garner,
Noam E Ziv,
Eckart D Gundelfinger
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ABSTRACT: Bassoon and the related protein Piccolo are core components of the presynaptic cytomatrix at the active zone of neurotransmitter release. They are transported on Golgi-derived membranous organelles, called Piccolo-Bassoon transport vesicles (PTVs), from the neuronal soma to distal axonal locations, where they participate in assembling new synapses. Despite their net anterograde transport, PTVs move in both directions within the axon. How PTVs are linked to retrograde motors and the functional significance of their bidirectional transport are unclear. In this study, we report the direct interaction of Bassoon with dynein light chains (DLCs) DLC1 and DLC2, which potentially link PTVs to dynein and myosin V motor complexes. We demonstrate that Bassoon functions as a cargo adapter for retrograde transport and that disruption of the Bassoon-DLC interactions leads to impaired trafficking of Bassoon in neurons and affects the distribution of Bassoon and Piccolo among synapses. These findings reveal a novel function for Bassoon in trafficking and synaptic delivery of active zone material.
The Journal of Cell Biology 05/2009; 185(2):341-55. · 10.26 Impact Factor
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ABSTRACT: Mice lacking functional presynaptic active zone protein Bassoon are characterized by an enlarged cerebral cortex and an altered cortical activation pattern. This morphological and functional phenotype is associated with defined metabolic distortions as detected by a metabonomic approach using high-field (14.1 T) high-resolution 1H-nuclear magnetic resonance spectroscopy (MRS) in conjunction with statistical pattern recognition. Within the cortex but not in the cerebellum, concentrations of N-acetyl aspartate, glutamine, and glutamate are significantly reduced, whereas the majority of all other detectable low molecular metabolites are unchanged. The reduction of the neuron-specific metabolite N-acetyl aspartate in the cortex coincides with a significant decrease in neuronal density in cortical layer V. Comparing the neuron with glia cell densities across the cortex reveals cortex layer-dependent alterations in the ratio between both cell types. Whereas the ratio shifts significantly toward neurons in the cortical input layers IV, the ratio is reversed in cortical layer V. Consequently, the previously observed altered neuronal activation pattern in the cortex is reflected not only in defined cytoarchitectural anomalies but also in metabolic disturbances in the glutamine-glutamate and N-acetyl aspartate metabolism.
Cerebral Cortex 05/2008; 18(4):890-7. · 6.54 Impact Factor
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ABSTRACT: Neuronal synapses are highly specialized structures for communication between nerve cells. Knowledge about their molecular organization and dynamics is still incomplete. The large multidomain protein Bassoon plays a major role in scaffolding and organizing the cytomatrix at the active zone of neurotransmitter release in presynaptic boutons. Utilizing immunofluorescence techniques, we show that Bassoon is essential for corecruitment of its synaptic interaction partners, C-terminal binding protein 1/brefeldin A-dependent ADP-ribosylation substrate and CAZ-associated structural protein, into protein complexes upon heterologous expression in COS-7 cells. A combination of Foerster's resonance energy transfer and fluorescence lifetime imaging microscopy in the time domain was adopted to investigate the potential for the association of these proteins in the same complexes. A direct physical association between Bassoon and CtBP1 could also be observed at synapses of living hippocampal neurons. Simultaneous analysis of fluorescence decays of the donor and the acceptor probes along with their decay-associated spectra allowed a clear discrimination of energy transfer.
Biophysical Journal 03/2008; 94(4):1483-96. · 3.65 Impact Factor
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Daniela C Dieterich,
Anna Karpova,
Marina Mikhaylova,
Irina Zdobnova,
Imbritt König,
Marco Landwehr,
Martin Kreutz,
Karl-Heinz Smalla,
Karin Richter,
Peter Landgraf,
Carsten Reissner,
Tobias M Boeckers, Werner Zuschratter,
Christina Spilker,
Constanze I Seidenbecher,
Craig C Garner,
Eckart D Gundelfinger,
Michael R Kreutz
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ABSTRACT: NMDA (N-methyl-D-aspartate) receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacob's nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacob's nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacob's extranuclear localization by competing with the binding of Importin-alpha to Jacob's nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB) shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor-induced cellular degeneration.
PLoS Biology 03/2008; 6(2):e34. · 11.45 Impact Factor
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ABSTRACT: In this work, fluorescence lifetime imaging microscopy in the time domain was used to study the fluorescence dynamics of ECFP and of the ratiometric chloride sensor Clomeleon along neuronal development. The multiexponential analysis of fluorophores combined with the study of the contributions of the individual lifetimes (decay-associated spectra) was used to discriminate the presence of energy transfer from other excited state reactions. A characteristic change of sign of the pre-exponential factors of lifetimes from positive to negative near the acceptor emission maxima was observed in presence of energy transfer. By fluorescence lifetime imaging microscopy, we could show that the individual conformations of CFP display differential quenching properties depending on their microenvironment. Suitability of Clomeleon as an optical indicator to obtain a direct readout of the intracellular chloride concentrations in living cells was verified by steady-state and time-resolved spectroscopy. The simultaneous study of the photophysical properties of Clomeleon, the calcium indicator Cameleon, and ECFP with neuronal development provided a kinetic model for the mechanism when competitive quenching effects as well as energy transfer occur in the same molecule. Simultaneous analysis of donor and acceptor kinetics was necessary to discriminate Försters resonance energy transfer along neuronal development due to the different cellular effects involved.
Biophysical Journal 04/2007; 92(6):2237-54. · 3.65 Impact Factor
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ABSTRACT: We report the investigation of Foerster's Resonance Energy Transfer dynamics in GFP based tandem constructs in living T-cells using a combination of Fluorescence Lifetime Imaging Microscopy (FLIM) and Fluorescence Lifetime Micro-Spectroscopy (FLMS) at picosecond time resolution and nanometer spectral resolution. The involvement of multiple lifetimes of CFP in energy transfer was analyzed by plotting pre-exponential factors of individual lifetimes along the wavelength resulting in the Decay Associated Spectra (DAS). A change in the amplitude of pre-exponential factors from positive to negative at the acceptor emission maxima was used as a confirmation of FRET in the multiexponential lifetime analysis.
Optics Express 01/2007; 14(25):12217-29. · 3.59 Impact Factor
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ABSTRACT: In this paper we describe the ''Multiple Image Stack Browser Suite'', a software system for handling very large, multidimensional
image data. We present some of its distinguished properties such as the extensibility by using plug-in mechanisms. In detail
we will discuss some results of searching optimized pixel based strategies for aligning several fluorescence images acquired
by confocal microscopes.
11/2006: pages 355-360;
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ABSTRACT: Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles, little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking. We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins, or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking of distinct cytoplasmic scaffolding proteins.
Journal of Biological Chemistry 04/2006; 281(9):6038-47. · 4.77 Impact Factor
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ABSTRACT: Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called
active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation
and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic
active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles,
little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking.
We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins,
or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region
of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding
of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period
of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix
proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking
of distinct cytoplasmic scaffolding proteins.
Journal of Biological Chemistry 03/2006; 281(9):6038-6047. · 4.77 Impact Factor
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Constanze I Seidenbecher,
Marco Landwehr,
Karl-Heinz Smalla,
Martin Kreutz,
Daniela C Dieterich, Werner Zuschratter,
Carsten Reissner,
James A Hammarback,
Tobias M Böckers,
Eckart D Gundelfinger,
Michael R Kreutz
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ABSTRACT: Caldendrin is a neuronal Ca(2+)-sensor protein (NCS), which represents the closest homologue of calmodulin (CaM) in nerve cells. It is tightly associated with the somato-dendritic cytoskeleton of neurons and highly enriched in the postsynaptic cytomatrix. Here, we report that caldendrin specifically associates with the microtubule cytoskeleton via an interaction with light chain 3 (LC3), a microtubule component with sequence homology to the GABAA receptor-associated protein (GABARAP), which is, like LC3, probably involved in cellular transport processes. Interestingly, two binding sites exist in LC3 for caldendrin from which only one exhibits a strict Ca(2+)-dependency for the interaction to take place but both require the presence of the first two EF-hands of caldendrin. CaM, however, is not capable of binding to LC3 at both sites despite its high degree of primary structure similarity with caldendrin. Computer modelling suggests that this might be explained by an altered distribution of surface charges at the first two EF-hands rendering each molecule, in principle, specific for a discrete set of binding partners. These findings provide molecular evidence that NCS can transduce signals to a specific target interaction irrespective of Ca(2+)-concentrations and CaM-levels.
Journal of Molecular Biology 03/2004; 336(4):957-70. · 4.00 Impact Factor
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ABSTRACT: Exocytosis of neurotransmitter from synaptic vesicles is restricted to specialized sites of the presynaptic plasma membrane called active zones. A complex cytomatrix of proteins exclusively assembled at active zones, the CAZ, is thought to form a molecular scaffold that organizes neurotransmitter release sites. Here, we have analyzed synaptic targeting and cytomatrix association of Bassoon, a major scaffolding protein of the CAZ. By combining immunocytochemistry and transfection of cultured hippocampal neurons, we show that the central portion of Bassoon is crucially involved in synaptic targeting and CAZ association. An N-terminal region harbors a distinct capacity for N-myristoylation-dependent targeting to synaptic vesicle clusters, but is not incorporated into the CAZ. Our data provide the first experimental evidence for the existence of distinct functional regions in Bassoon and suggest that a centrally located CAZ targeting function may be complemented by an N-terminal capacity for targeting to membrane-bounded synaptic organelles.
Molecular and Cellular Neuroscience 07/2003; 23(2):279-91. · 3.66 Impact Factor
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Tobias M Boeckers,
Michael R Kreutz,
Carsten Winter, Werner Zuschratter,
Karl-Heinz Smalla,
Lydia Sanmarti-Vila,
Heike Wex,
Kristina Langnaese,
Juergen Bockmann,
Craig C Garner,
Eckart D Gundelfinger
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ABSTRACT: The postsynaptic density (PSD) is crucially involved in the structural and functional organization of the postsynaptic neu-rotransmitter reception apparatus. Using antisera against rat brain synaptic junctional protein preparations, we isolated cDNAs coding for proline-rich synapse-associated protein-1 (ProSAP1), a PDZ-domain protein. This protein was found to be identical to the recently described cortactin-binding protein-1 (CortBP1). Homology screening identified a related protein, ProSAP2. Specific antisera raised against a C-terminal fusion construct and a central part of ProSAP1 detect a cluster of immunoreactive bands of 180 kDa in the particulate fraction of rat brain homogenates that copurify with the PSD fraction. Transcripts and immunoreactivity are widely distributed in the brain and are upregulated during the period of synapse forma-tion in the brain. In addition, two short N-terminal insertions are detected; they are differentially regulated during brain develop-ment. Confocal microscopy of hippocampal neurons showed that ProSAP1 is predominantly localized in synapses, and im-munoelectron microscopy in situ revealed a strong association with PSDs of hippocampal excitatory synapses. The accumu-lation of ProSAP1 at synaptic structures was analyzed in the developing cerebral cortex. During early postnatal develop-ment, strong immunoreactivity is detectable in neurites and somata, whereas from postnatal day 10 (P10) onward a punc-tate staining is observed. At the ultrastructural level, the immu-noreactivity accumulates at developing PSDs starting from P8. Both interaction with the actin-binding protein cortactin and early appearance at postsynaptic sites suggest that ProSAP1/ CortBP1 may be involved in the assembly of the PSD during neuronal differentiation. Key words: rat brain; synapse; postsynaptic density (PSD); PDZ domain; synaptogenesis; actin-based cytoskeleton; devel-opment; synamon
Journal of Neuroscience 08/2000; 19(15):6506-6518. · 7.11 Impact Factor
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01/1999
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ABSTRACT: GLYCOCONJUGATES are known to be concentrated in plasma membranes, especially in synaptic junctions, where they subserve various functions in neural connectivity. Here we report the cellular distribution of a new monoclonal antibody recognizing (fucogalactosyl) sequences in carbohydrate structures. The most pronounced immunoreactivity was found in fibrous astrocytes, in many parts of the brain and with lower density in various neuronal elements. This points to the expression of identical carbohydrate sequences on molecules within certain glial and neuronal elements. Previous intracerebral injections of the antibody interfered with long term memory formation. Therefore, functions mediated by corresponding glycoproteins in neurons and glia cells or even neuron-glial interactions, might be relevant for information-processing.
(C) Lippincott-Raven Publishers.
Neuroreport 07/1991; 2(8). · 1.66 Impact Factor
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Daniela C Dieterich,
Anna Karpova,
Marina Mikhaylova,
Irina Zdobnova,
Imbritt König,
Marco Landwehr,
Martin Kreutz,
Karl-Heinz Smalla,
Karin Richter,
Peter Landgraf,
Carsten Reissner,
Tobias M Boeckers, Werner Zuschratter,
Christina Spilker,
Constanze I Seidenbecher,
Craig C Garner,
Eckart D Gundelfinger,
Michael R Kreutz
