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O Griesbeck

Publications and source records attributed to O Griesbeck.

9 recordsLinked to original sources
PubMed2001

Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications.

Yellow mutants of the green fluorescent protein (YFP) are crucial constituents of genetically encoded indicators of signal transduction and fusions to monitor protein-protein interactions. However, previous YFPs show excessive pH sensitivity, chloride interference, poor photostability, or poor expression at 37 degrees C. Protein evolution in Escherichia coli has produced a new YFP named Citrine, in which the mutation Q69M confers a much lower pK(a) (5.7) than for previous YFPs, indifference to chloride, twice the photostability of previous YFPs, and much better expression at 37 degrees C and in organelles. The halide resistance is explained by a 2.2-A x-ray crystal structure of Citrine, showing that the methionine side chain fills what was once a large halide-binding cavity adjacent to the chromophore. Insertion of calmodulin within Citrine or fusion of cyan fluorescent protein, calmodulin, a calmodulin-binding peptide and Citrine has generated improved calcium indicators. These chimeras can be targeted to multiple cellular locations and have permitted the first single-cell imaging of free [Ca(2+)] in the Golgi. Citrine is superior to all previous YFPs except when pH or halide sensitivity is desired and is particularly advantageous within genetically encoded fluorescent indicators of physiological signals.

Amino Acid Substitution↗
PubMed1999

Dynamic and quantitative Ca2+ measurements using improved cameleons.

Cameleons are genetically-encoded fluorescent indicators for Ca2+ based on green fluorescent protein variants and calmodulin (CaM). Because cameleons can be targeted genetically and imaged by one- or two-photon excitation microscopy, they offer great promise for monitoring Ca2+ in whole organisms, tissues, organelles, and submicroscopic environments in which measurements were previously impossible. However, the original cameleons suffered from significant pH interference, and their Ca2+-buffering and cross-reactivity with endogenous CaM signaling pathways was uncharacterized. We have now greatly reduced the pH-sensitivity of the cameleons by introducing mutations V68L and Q69K into the acceptor yellow green fluorescent protein. The resulting new cameleons permit Ca2+ measurements despite significant cytosolic acidification. When Ca2+ is elevated, the CaM and CaM-binding peptide fused together in a cameleon predominantly interact with each other rather than with free CaM and CaM-dependent enzymes. Therefore, if cameleons are overexpressed, the primary effect is likely to be the unavoidable increase in Ca2+ buffering rather than specific perturbation of CaM-dependent signaling.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Neurotrophin release by neurotrophins: implications for activity-dependent neuronal plasticity.

Neurotrophins, secreted in an activity-dependent manner, are thought to be involved in the activity-dependent refinement of synaptic connections. Here we demonstrate that in hippocampal neurons and the rat pheochromocytoma cell line PC12 application of exogenous neurotrophins induces secretion of neurotrophins, an effect that is mediated by the activation of tyrosine kinase neurotrophin receptors (Trks). Like activity-dependent secretion of neurotrophins, neurotrophin-induced neurotrophin secretion requires mobilization of calcium from intracellular stores. Because neurotrophins are likely to be released from both dendrites and axons, neurotrophin-induced neurotrophin release represents a potential positive feedback mechanism, contributing to the reinforcement and stabilization of synaptic connections.

Animals↗
PubMed1997

Rapid gene transfer into cultured hippocampal neurons and acute hippocampal slices using adenoviral vectors.

Primary cultures of hippocampal neurons were infected with an adenovirus coding for beta-galactosidase. Expression could be detected as early as 4 h after infection and steadily increased to high levels at 24 h without evidence for a functional impairment of the infected neurons. Similarly, adenovirus-mediated gene transfer into acute hippocampal slices was detectable 4 h after infection and could be localized to discrete areas of the CA1 region by microinjection of the virus stock solution. Infected slices were still suitable for electrophysiological experiments.

Adenoviridae↗
PubMed1996

Virus-mediated gene transfer into hippocampal CA1 region restores long-term potentiation in brain-derived neurotrophic factor mutant mice.

Long-term potentiation (LTP) has been shown to be impaired in mice deficient in the brain-derived neurotrophic factor (BDNF) gene, as well as in a number of other knockout animals. Despite its power the gene-targeting approach is always fraught with the danger of looking at the cumulative direct and indirect effects of the absence of a particular gene rather than its immediate function. The re-expression of a specific gene at a selective time point and at a specific site in gene-defective mutants presents a potent procedure to overcome this limitation and to evaluate the causal relationship between the absence of a particular gene and the impairment of a function in gene-defective animals. Here we demonstrate that the re-expression of the BDNF gene in the CA1 region almost completely restores the severely impaired LTP in hippocampal slices of BDNF-deficient mice. The results therefore provide strong evidence for the direct involvement of BDNF in the process of LTP.

Adenoviridae↗
PubMed1996

The involvement of brain-derived neurotrophic factor in hippocampal long-term potentiation revealed by gene targeting experiments.

Brain-derived neurotrophic factor (BDNF) is a member of the NGF gene family, which has been shown to influence the survival and differentiation of specific classes of neurons in vitro and in vivo. The possibility that neurotrophins are also involved in processes of neuronal plasticity has only recently begun to receive attention. To determine whether BDNF has a function in processes like long-term potentiation (LTP), we produced a strain of mice with a deletion in the coding sequence of the BDNF-gene. We then used hippocampal slices from these mice to investigate whether LTP is affected by this mutation. Mutant mice showed significantly weaker LTP in the CA1 region. The magnitude of the potentiation as well as the percentage of cases in which LTP could be induced successfully was clearly reduced whereas important pharmacological and morphological control parameters in the hippocampus of these animals were unaffected. Adenoviral vectors were used to re-express BDNF in acute slices of BDNF-knock-out mice. In most cases LTP could be rescued with this approach. These results suggest that BDNF has an important functional role in the expression of LTP in the hippocampus.

Animals↗
PubMed1995

Expression of neurotrophins in skeletal muscle: quantitative comparison and significance for motoneuron survival and maintenance of function.

Neurotrophins play a crucial role in the regulation of survival and maintenance of specific functions of various populations of neurons. Brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin 4/5 (NT-4) have been shown to support motoneuron survival during embryonic development and, after birth, to protect motoneurons from degeneration after nerve lesion. We have compared the levels of these neurotrophins in skeletal muscle by quantitative Northern blot analysis, both during embryonic development and postnatally. We localized the sites of expression of these neurotrophins by in situ hybridisation and analysed the expression of trkB in the spinal cord by in situ hybridisation and immunohistochemistry. NT-3 is most abundantly expressed both during embryonic development and in the postnatal phase, followed by NT-4. The levels of BDNF are very low, in particular after birth. After nerve lesion, NT-3 mRNA essentially remained unchanged, whereas NT-4 mRNA rapidly decreased. The slow increase in BDNF expression seems to be essentially due to the expression in Schwann cells rather than skeletal muscle, demonstrated by in situ hybridisation. Our data indicate that motoneurons can receive trophic support from several members of the neurotrophin gene family during the period of naturally occurring cell death. Postnatally, the predominant ligand acting via trkB on motoneurons is NT-4, whereas BDNF expression seems to play a role mainly after nerve lesion.

Animals↗
PubMedDate not supplied

Are there differences between the secretion characteristics of NGF and BDNF? Implications for the modulatory role of neurotrophins in activity-dependent neuronal plasticity.

In previous experiments the activity-dependent secretion of nerve growth factor (NGF) from native hippocampal slices and from NGF-cDNA transfected hippocampal neurons showed unusual characteristics [Blochl and Thoenen (1995) Eur J Neurosci 7:1220-1228; (1996) Mol Cell Neurosci 7:173-190]. In both hippocampal slices and cultured hippocampal neurons the activity-dependent secretion proved to be independent of extracellular calcium, but dependent on the release of calcium from intracellular stores. Under different experimental conditions, Goodman et al. [(1996) Mol Cell Neurosci 7:222-238] reported that the high potassium-mediated secretion of brain-derived neurotrophic factor (BDNF) from hippocampal cultures was dependent on extracellular calcium. Mowla et al. [(1997) Proc 27th Annu Meet Soc Neurosci New Orleans 875.10] reported on even further-reaching differences between NGF and BDNF secretion, namely, that in hippocampal neurons and in pituitary cell lines NGF was secreted exclusively according to the constitutive pathway, whereas BDNF was exclusively sorted according to the activity-dependent regulated pathway. In view of the crucial importance of such potential differences between the processing, sorting, and secretory mechanisms of different neurotrophins for their modulatory roles in activity-dependent neuronal plasticity, a thorough analysis under comparable experimental conditions was mandatory. We demonstrate that in native hippocampal slices and adenoviral-transduced hippocampal neurons there are no differences between NGF and BDNF with respect to the subcellular distribution and mechanism of secretion; that the activity-dependent secretion of both NGF and BDNF is dependent on intact intracellular calcium stores; and that the differences between our own observations and those of Goodman et al. (ibid.) regarding the dependence on extracellular calcium do not reflect differences between NGF and BDNF sorting and secretion, but reflect the differing experimental conditions used.

Animals↗