Biomedical subjects

Tobias Meyer

Publications and source records attributed to Tobias Meyer.

At least 19 recordsLinked to original sources

PubMed2026

CDC7 and APC/CCdh1 gate distinct routes to initiate DNA replication.

Precise temporal control of DNA replication initiation is essential for faithful cell division, yet there are conflicting prevailing models for how cells trigger origin firing. Here, we demonstrate that human origins are not fired at fixed thresholds of cell cycle regulators E2F, APC/CCdh1, CDK2/1, and CDC7. Instead, origin firing is triggered at a tunable CDK2/1 threshold that is gated by either CDC7 or APC/CCdh1 activity. CDC7 phosphorylates MCM helicase, enabling Cyclin E-CDK2 to trigger origin firing at low CDK2/1 activity, independent of APC/CCdh1 inactivation. In contrast, APC/CCdh1 inactivation enables Cyclin A-CDK1 to phosphorylate MCM at distinct CDK2/1-specific sites and trigger origin firing, independent of CDC7 activity. Strikingly, this requires much higher CDK2/1 activity, which CDK2 cannot normally reach alone. Thus, having two distinct routes necessitates blocking both routes to prevent S phase: either by inhibiting CDC7 or CDK2 while also preventing APC/CCdh1 inactivation or inhibiting CDK1.

Humans↗

PubMed2007

Cyclin A2 regulates nuclear-envelope breakdown and the nuclear accumulation of cyclin B1.

Mitosis is thought to be triggered by the activation of Cdk-cyclin complexes. Here we have used RNA interference (RNAi) to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in the regulation of centrosome separation and nuclear-envelope breakdown (NEB) in HeLa cells. We found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1. However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion and is capable of supporting NEB if rendered constitutively nuclear.

Cell Nucleus↗

PubMed2006

The alliance for cellular signaling plasmid collection: a flexible resource for protein localization studies and signaling pathway analysis.

Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines.

Animals↗

PubMed2006

PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane.

Many signaling, cytoskeletal, and transport proteins have to be localized to the plasma membrane (PM) in order to carry out their function. We surveyed PM-targeting mechanisms by imaging the subcellular localization of 125 fluorescent protein-conjugated Ras, Rab, Arf, and Rho proteins. Out of 48 proteins that were PM-localized, 37 contained clusters of positively charged amino acids. To test whether these polybasic clusters bind negatively charged phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipids, we developed a chemical phosphatase activation method to deplete PM PI(4,5)P2. Unexpectedly, proteins with polybasic clusters dissociated from the PM only when both PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] were depleted, arguing that both lipid second messengers jointly regulate PM targeting.

ADP-Ribosylation Factors↗

PubMed2006

Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels.

To resolve the controversy about messengers regulating KCNQ ion channels during phospholipase C-mediated suppression of current, we designed translocatable enzymes that quickly alter the phosphoinositide composition of the plasma membrane after application of a chemical cue. The KCNQ current falls rapidly to zero when phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 or PI(4,5)P2] is depleted without changing Ca2+, diacylglycerol, or inositol 1,4,5-trisphosphate. Current rises by 30% when PI(4,5)P2 is overproduced and does not change when phosphatidylinositol 3,4,5-trisphosphate is raised. Hence, the depletion of PI(4,5)P2 suffices to suppress current fully, and other second messengers are not needed. Our approach is ideally suited to study biological signaling networks involving membrane phosphoinositides.

Animals↗

PubMed2006

Synergistic control of protein kinase Cgamma activity by ionotropic and metabotropic glutamate receptor inputs in hippocampal neurons.

Conventional protein kinase C (PKC) isoforms are abundant neuronal signaling proteins with important roles in regulating synaptic plasticity and other neuronal processes. Here, we investigate the role of ionotropic and metabotropic glutamate receptor (iGluR and mGluR, respectively) activation on the generation of Ca2+ and diacylglycerol (DAG) signals and the subsequent activation of the neuron-specific PKCgamma isoform in hippocampal neurons. By combining Ca2+ imaging with total internal reflection microscopy analysis of specific biosensors, we show that elevation of both Ca2+ and DAG is necessary for sustained translocation and activation of EGFP (enhanced green fluorescent protein)-PKCgamma. Both DAG production and PKCgamma translocation were localized processes, typically observed within discrete microdomains along the dendritic branches. Markedly, intermediate-strength NMDA receptor (NMDAR) activation or moderate electrical stimulation generated Ca2+ but no DAG signals, whereas mGluR activation generated DAG but no Ca2+ signals. Both receptors were needed for PKCgamma activation. This suggests that a coincidence detection process exists between iGluRs and mGluRs that relies on a molecular coincidence detection process based on the corequirement of Ca2+ and DAG for PKCgamma activation. Nevertheless, the requirement for costimulation with mGluRs could be overcome for maximal NMDAR stimulation through a direct production of DAG via activation of the Ca2+-sensitive PLCdelta (phospholipase Cdelta) isoform. In a second important exception, mGluRs were sufficient for PKCgamma activation in neurons in which Ca2+ stores were loaded by previous electrical activity. Together, the dual activation requirement for PKCgamma provides a plausible molecular interpretation for different synergistic contributions of mGluRs to long-term potentiation and other synaptic plasticity processes.

Animals↗

PubMed2005

Interlinked fast and slow positive feedback loops drive reliable cell decisions.

Positive feedback is a ubiquitous signal transduction motif that allows systems to convert graded inputs into decisive, all-or-none outputs. Here we investigate why the positive feedback switches that regulate polarization of budding yeast, calcium signaling, Xenopus oocyte maturation, and various other processes use multiple interlinked loops rather than single positive feedback loops. Mathematical simulations revealed that linking fast and slow positive feedback loops creates a "dual-time" switch that is both rapidly inducible and resistant to noise in the upstream signaling system.

Animals↗

PubMed2005

Spines and neurite branches function as geometric attractors that enhance protein kinase C action.

Ca2+ and diacylglycerol-regulated protein kinase Cs (PKCs; conventional PKC isoforms, such as PKCgamma) are multifunctional signaling molecules that undergo reversible plasma membrane translocation as part of their mechanism of activation. In this article, we investigate PKCgamma translocation in hippocampal neurons and show that electrical or glutamate stimulation leads to a striking enrichment of PKCgamma in synaptic spines and dendritic branches. Translocation into spines and branches was delayed when compared with the soma plasma membrane, and PKCgamma remained in these structures for a prolonged period after the response in the soma ceased. We have developed a quantitative model for the translocation process by measuring the rate at which PKCgamma crossed the neck of spines, as well as cytosolic and membrane diffusion coefficients of PKCgamma. Our study suggests that neurons make use of a high surface-to-volume ratio of spines and branches to create a geometric attraction process for PKC that imposes a delayed enhancement of PKC action at synapses and in peripheral processes.

Animals↗

PubMed2005

Reversible intracellular translocation of KRas but not HRas in hippocampal neurons regulated by Ca2+/calmodulin.

The Ras/MAPK pathway regulates synaptic plasticity and cell survival in neurons of the central nervous system. Here, we show that KRas, but not HRas, acutely translocates from the plasma membrane (PM) to the Golgi complex and early/recycling endosomes in response to neuronal activity. Translocation is reversible and mediated by the polybasic-prenyl membrane targeting motif of KRas. We provide evidence that KRas translocation occurs through sequestration of the polybasic-prenyl motif by Ca2+/calmodulin (Ca2+/CaM) and subsequent release of KRas from the PM, in a process reminiscent of GDP dissociation inhibitor-mediated membrane recycling of Rab and Rho GTPases. KRas translocation was accompanied by partial intracellular redistribution of its activity. We conclude that the polybasic-prenyl motif acts as a Ca2+/CaM-regulated molecular switch that controls PM concentration of KRas and redistributes its activity to internal sites. Our data thus define a novel signaling mechanism that differentially regulates KRas and HRas localization and activity in neurons.

Animals↗

PubMed2005

STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx.

Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.

Calcium↗

PubMed2005

Protein localization studies in the age of 'Omics'.

As thousands of new genes are identified in genomics efforts, the rush is on to learn something about the functional roles of the proteins encoded by those genes. Clues to protein functions, activation states and protein-protein interactions have been revealed in focused studies of protein localization. With technical breakthroughs such as GFP protein tagging and recombinase cloning systems, large-scale screens of protein localization are now being undertaken.

Aging↗

PubMed2005

A local coupling model and compass parameter for eukaryotic chemotaxis.

Chemotaxis is a cellular sensing mechanism that guides immune cells to sites of infection and leads fibroblasts to sites of injury. Here, we show in migrating primary dendritic cells and fibroblasts that the leading edge is not a uniform signaling entity, but instead consists of independent coupling units in which transient activation of PI3-kinase links to local lamellipod extension and small discrete turns in the direction of migration. These findings led to a model in which global cell polarization is independent from the chemotaxis mechanism. In this model, chemotaxis does not require spatial integration but is instead a stochastic process in which each receptor binding event within the leading edge triggers a local lamellipod extension and a small turn in the direction of migration. We show that this model and a derived "compass parameter" are sufficient to simulate the observed random migration, biased random walk, and persistent chemotactic behaviors of eukaryotic cells.

Animals↗

PubMed2005

An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways.

We made substantial advances in the implementation of a rapamycin-triggered heterodimerization strategy. Using molecular engineering of different targeting and enzymatic fusion constructs and a new rapamycin analog, Rho GTPases were directly activated or inactivated on a timescale of seconds, which was followed by pronounced cell morphological changes. As signaling processes often occur within minutes, such rapid perturbations provide a powerful tool to investigate the role, selectivity and timing of Rho GTPase-mediated signaling processes.

Animals↗

PubMed2004

The neural F-box protein NFB42 mediates the nuclear export of the herpes simplex virus type 1 replication initiator protein (UL9 protein) after viral infection.

The neural F-box 42-kDa protein (NFB42) is a component of the SCF(NFB42) E3 ubiquitin ligase that is expressed in all major areas of the brain; it is not detected in nonneuronal tissues. We previously identified NFB42 as a binding partner for the herpes simplex virus 1 (HSV-1) UL9 protein, the viral replication-initiator, and showed that coexpression of NFB42 and UL9 in human embryonic kidney (293T) cells led to a significant decrease in the level of UL9 protein. We have now found that HSV-1 infection promotes the shuttling of NFB42 between the cytosol and the nucleus in both 293T cells and primary hippocampal neurons, permitting NFB42 to bind to the phosphorylated UL9 protein, which is localized in the nucleus. This interaction mediates the export of the UL9 protein from the nucleus to the cytosol, leading to its ubiquitination and degradation via the 26S proteasome. Because the intranuclear localization of the UL9 protein, along with other viral and cellular factors, is an essential step in viral DNA replication, degradation of the UL9 protein in neurons by means of nuclear export through its specific interaction with NFB42 may prevent active replication and promote neuronal latency of HSV-1.

Cell Cycle Proteins↗

PubMed2004

Probing the precision of the mitotic clock with a live-cell fluorescent biosensor.

Precise timing of mitosis is essential for high-fidelity cell duplication. However, temporal measurements of the mitotic clock have been challenging. Here we present a fluorescent mitosis biosensor that monitors the time between nuclear envelope breakdown (NEB) and re-formation using parallel total internal reflection fluorescence (TIRF) microscopy. By tracking tens to hundreds of mitotic events per experiment, we found that the mitotic clock of unsynchronized rat basophilic leukemia cells has a marked precision with 80% of cells completing mitosis in 32 +/- 6 min. This assay further allowed us to observe delays in mitotic timing at Taxol concentrations 100 times lower than previous minimal effective doses, explaining why Taxol is clinically active at low concentrations. Inactivation of the spindle checkpoint by targeting the regulator Mad2 with RNAi consistently shortened mitosis, providing direct evidence that the internal mitotic timing mechanism is much faster in cells that lack the checkpoint.

Animals↗

PubMed2004

A critical intramolecular interaction for protein kinase Cepsilon translocation.

Disruption of intramolecular interactions, translocation from one intracellular compartment to another, and binding to isozyme-specific anchoring proteins termed RACKs, accompany protein kinase C (PKC) activation. We hypothesized that in inactive epsilonPKC, the RACK-binding site is engaged in an intramolecular interaction with a sequence resembling its RACK, termed psiepsilonRACK. An amino acid difference between the psiepsilonRACK sequence in epsilonPKC and its homologous sequence in epsilonRACK constitutes a change from a polar non-charged amino acid (asparagine) in epsilonRACK to a polar charged amino acid (aspartate) in epsilonPKC. Here we show that mutating the aspartate to asparagine in epsilonPKC increased intramolecular interaction as indicated by increased resistance to proteolysis, and slower hormone- or PMA-induced translocation in cells. Substituting aspartate for a non-polar amino acid (alanine) resulted in binding to epsilonRACK without activators, in vitro, and increased translocation rate upon activation in cells. Mathematical modeling suggests that translocation is at least a two-step process. Together our data suggest that intramolecular interaction between the psiepsilonRACK site and RACK-binding site within epsilonPKC is critical and rate limiting in the process of PKC translocation.

Amino Acid Substitution↗

PubMed2004

Simplified analogs of bryostatin with anticancer activity display greater potency for translocation of PKCdelta-GFP.

Structurally simplified analogs of bryostatin 1, a marine natural product in clinical trials for the treatment of cancer, have been shown to be up to 50 times more potent than bryostatin 1 at inducing the translocation of PKCdelta-GFP from the cytosol of rat basophilic leukemia (RBL) cells. The end distribution of the protein is similar for all three compounds, despite a significant difference in translocation kinetics. The potency of the compounds for inducing the translocation response appears to be only qualitatively related to their binding affinity for PKC, highlighting the importance of using binding affinity in conjunction with real-time measurements of protein localization for the pharmacological profiling of biologically active agents.

Animals↗

PubMed2004

Function oriented synthesis: the design, synthesis, PKC binding and translocation activity of a new bryostatin analog.

Bryostatin 1 represents a novel and potent therapeutic lead with a unique activity profile. Its natural and synthetic availability is severely limited. Function oriented synthesis provides a means to address this supply problem through the design of synthetically more accessible simplified structures that at the same time incorporate improved functional activity. Pharmacophore searching and a new computer aided visualization of a possible binding mode are combined with an understanding of function and knowledge of synthesis to design and prepare a new and simplified compound with bryostatin-like function in biological systems. This new compound is a potent ligand for protein kinase C in vitro (K(i) = 8.0 nM). More significantly, the described molecule retains the functional ability to translocate a PKCdelta-GFP fusion protein in RBL cells. The extent of protein translocation and the sub-cellular localization induced by this new compound is similar to that seen in response to bryostatin 1, indicating that the new molecule retains the functional activity of the natural product but is simpler and can be synthesized in a practical fashion.

Animals↗