Kaiserslautern - Fachbereich Biologie
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Synapses are the fundamental structures that regulate the functionality of the neural circuit. The ability of the synapse to modulate its structure and function at a fast rate due to various sensory inputs provides the strength to the nervous system to incorporate new adaptations and behaviors in the animal. The synapses are very dynamic throughout the life of the animal starting from early development. Continuous events of formation and elimination of synapse, activation and inhibition of synaptic function are observed in almost all synapses. These processes occur at a high speed and require controlled cellular mechanisms. Imbalance in these processes results in defective nervous system and has been reported in many neurological disorders. Thus, it is important to understand the mechanisms that regulate process of synapse development maintenance and function.
Kinases and phosphatases are the key regulators of cellular mechanisms. Understanding the function of these molecules in the neuron will shed light on the molecular mechanisms of synaptic plasticity. Using Drosophila melanogaster larval neuromuscular junction as a model, Bulat et al. (2014) performed a large RNAi based screen targeting kinome and phosphatome of Drosophila to identify the essential kinases and phosphatases and found Myeloid leukemia factor-1 adaptor molecule (Madm) and Protein phosphatase 4 (PP4) as novel regulators of synapse development and maintenance. The function of these molecules in the nervous system has not been reported and hence I investigated on the role of Madm and PP4 in the regulation of synapse development, maintenance and function.
Myeloid leukemia factor-1 adaptor molecule (Madm), a ubiquitously expressing psuedokinase essentially functions to regulate synaptic growth, stability and function. Using a combination of genetic and high throughput imaging, I could demonstrate that Madm functions to regulate the synaptic growth and stability from the presynapse and synaptic organization form the postsynapse. Also, I could demonstrate that Madm functions in association with mTOR pathway to regulate synapse growth acting downstream of 4E-BP. In addition, using electrophysiology, we could demonstrate that Madm is essential for the basic synaptic transmission with an additive function of retrograde synaptic potentiation. In summary, I could demonstrate that Madm is a novel regulator of synaptic development, maintenance and function.
Protein phosphatase 4 (PP4), a ubiquitously expressing protein phosphatase is involved in the regulation of multiple aspects of the nervous system. I could demonstrate that PP4 is essential for the development of nervous system and the metamorphosis. Using genetics and imaging analysis, I could demonstrate that loss of PP4 results in the abnormal morphology of cell organelles. In addition, I could show that loss of PP4 results in defective brain development with poorly developed structures.
Altogether, in this study, I could demonstrate the importance of novel molecules, a pesudokinase Madm and protein phosphatases PP4 in the nervous system to regulate distinct aspects of the neuron.
More than ten years ago, ER-ANT1 was shown to act as an ATP/ADP antiporter and to exist in the endoplasmic reticulum (ER) of higher plants. Because structurally different transporters generally mediate energy provision to the ER, the physiological function of ER-ANT1 was not directly evident.
Interestingly, mutant plants lacking ER-ANT1 exhibit a photorespiratory phenotype. Although many research efforts were undertaken, the possible connection between the transporter and photorespiration also remained elusive. Here, a forward genetic approach was used to decipher the role of ER-ANT1 in the plant context and its association to photorespiration.
This strategy identified that additional absence of a putative HAD-type phosphatase partially restored the photorespiratory phenotype. Localisation studies revealed that the corresponding protein is targeted to the chloroplast. Moreover, biochemical analyses demonstrate that the HAD-type phosphatase is specific for pyridoxal phosphate. These observations, together with transcriptional and metabolic data of corresponding single (ER-ANT1) and double (ER-ANT1, phosphatase) loss-of-function mutant plants revealed an unexpected connection of ER-ANT1 to vitamin B6 metabolism.
Finally, a scenario is proposed, which explains how ER-ANT1 may influence B6 vitamer phosphorylation, by this affects photorespiration and causes several other physiological alterations observed in the corresponding loss-of-function mutant plants.
Biological clocks exist across all life forms and serve to coordinate organismal physiology with periodic environmental changes. The underlying mechanism of these clocks is predominantly based on cellular transcription-translation feedback loops in which clock proteins mediate the periodic expression of numerous genes. However, recent studies point to the existence of a conserved timekeeping mechanism independent of cellular transcription and translation, but based on cellular metabolism. These metabolic clocks were concluded based upon the observation of circadian and ultradian oscillations in the level of hyperoxidized peroxiredoxin proteins. Peroxiredoxins are enzymes found almost ubiquitously throughout life. Originally identified as H2O2 scavengers, recent studies show that peroxiredoxins can transfer oxidation to, and thereby regulate, a wide range of cellular proteins. Thus, it is conceivable that peroxiredoxins, using H2O2 as the primary signaling molecule, have the potential to integrate and coordinate much of cellular physiology and behavior with metabolic changes. Nonetheless, it remained unclear if peroxiredoxins are passive reporters of metabolic clock activity or active determinants of cellular timekeeping. Budding yeast possess an ultradian metabolic clock termed the Yeast Metabolic Cycle (YMC). The most obvious feature of the YMC is a high amplitude oscillation in oxygen consumption. Like circadian clocks, the YMC temporally compartmentalizes cellular processes (e.g. metabolism) and coordinates cellular programs such as gene expression and cell division. The YMC also exhibits oscillations in the level of hyperoxidized peroxiredoxin proteins.
In this study, I used the YMC clock model to investigate the role of peroxiredoxins in cellular timekeeping, as well as the coordination of cell division with the metabolic clock. I observed that cytosolic 2-Cys peroxiredoxins are essential for robust metabolic clock function. I provide direct evidence for oscillations in cytosolic H2O2 levels, as well as cyclical changes in oxidation state of a peroxiredoxin and a model peroxiredoxin target protein during the YMC. I noted two distinct metabolic states during the YMC: low oxygen consumption (LOC) and high oxygen consumption (HOC). I demonstrate that thiol-disulfide oxidation and reduction are necessary for switching between LOC and HOC. Specifically, a thiol reductant promotes switching to HOC, whilst a thiol oxidant prevents switching to HOC, forcing cells to remain in LOC. Transient peroxiredoxin inactivation triggered rapid and premature switching from LOC to HOC. Furthermore, I show that cell division is normally synchronized with the YMC and that deletion of typical 2-Cys peroxiredoxins leads to complete uncoupling of cell division from metabolic cycling. Moreover, metabolic oscillations are crucial for regulating cell cycle entry and exit. Intriguingly, switching to HOC is crucial for initiating cell cycle entry whilst switching to LOC is crucial for cell cycle completion and exit. Consequently, forcing cells to remain in HOC by application of a thiol reductant leads to multiple rounds of cell cycle entry despite failure to complete the preceding cell cycle. On the other hand, forcing cells to remain in LOC by treating with a thiol oxidant prevents initiation of cell cycle entry.
In conclusion, I propose that peroxiredoxins – by controlling metabolic cycles, which are in turn crucial for regulating the progression through cell cycle – play a central role in the coordination of cellular metabolism with cell division. This proposition, thus, positions peroxiredoxins as active players in the cellular timekeeping mechanism.
The transfer of substrates between to enzymes within a biosynthesis pathway is an effective way to synthesize the specific product and a good way to avoid metabolic interference. This process is called metabolic channeling and it describes the (in-)direct transfer of an intermediate molecule between the active sites of two enzymes. By forming multi-enzyme cascades the efficiency of product formation and the flux is elevated and intermediate products are transferred and converted in a correct manner by the enzymes.
During tetrapyrrole biosynthesis several substrate transfer events occur and are prerequisite for an optimal pigment synthesis. In this project the metabolic channeling process during the pink pigment phycoerythrobilin (PEB) was investigated. The responsible ferredoxin-dependent bilin reductases (FDBR) for PEB formation are PebA and PebB. During the pigment synthesis the intermediate molecule 15,16-dihydrobiliverdin (DHBV) is formed and transferred from PebA to PebB. While in earlier studies a metabolic channeling of DHBV was postulated, this work revealed new insights into the requirements of this protein-protein interaction. It became clear, that the most important requirement for the PebA/PebB interaction is based on the affinity to their substrate/product DHBV. The already high affinity of both enzymes to each other is enhanced in the presence of DHBV in the binding pocket of PebA which leads to a rapid transfer to the subsequent enzyme PebB. DHBV is a labile molecule and needs to be rapidly channeled in order to get correctly further reduced to PEB. Fluorescence titration experiments and transfer assays confirmed the enhancement effect of DHBV for its own transfer.
More insights became clear by creating an active fusion protein of PebA and PebB and comparing its reaction mechanism with standard FDBRs. This fusion protein was able to convert biliverdin IXα (BV IXα) to PEB similar to the PebS activity, which also can convert BV IXα via DHBV to PEB as a single enzyme. The product and intermediate of the reaction were identified via HPLC and UV-Vis spectroscopy.
The results of this work revealed that PebA and PebB interact via a proximity channeling process where the intermediate DHBV plays an important role for the interaction. It also highlights the importance of substrate channeling in the synthesis of PEB to optimize the flux of intermediates through this metabolic pathway.
Substrate channeling is a widespread mechanism in metabolic pathways to avoid decomposition of unstable intermediates, competing reactions, and to accelerate catalytic turnover. During the biosynthesis of light-harvesting phycobilins in cyanobacteria, two members of the ferredoxin-dependent bilin reductases are involved in the reduction of the open-chain tetrapyrrole biliverdin IXα to the pink pigment phycoerythrobilin. The first reaction is catalyzed by 15,16-dihydrobiliverdin:ferredoxin oxidoreductase and produces the unstable intermediate 15,16-dihydrobiliverdin (DHBV). This intermediate is subsequently converted by phycoerythrobilin:ferredoxin oxidoreductase to the final product phycoerythrobilin. Although substrate channeling has been postulated already a decade ago, detailed experimental evidence was missing. Using a new on-column assay employing immobilized enzyme in combination with UV-Vis and fluorescence spectroscopy revealed that both enzymes transiently interact and that transfer of the intermediate is facilitated by a significantly higher binding affinity of DHBV toward phycoerythrobilin:ferredoxin oxidoreductase. Concluding from the presented data, the intermediate DHBV is transferred via proximity channeling.
The scaffolding protein family Fe65, composed of Fe65, Fe65L1, and Fe65L2, was identified as an interaction partner of the amyloid precursor protein (APP), which plays a key function in Alzheimer’s disease. All three Fe65 family members possess three highly conserved interaction domains, forming complexes with diverse binding partners that can be assigned to different cellular functions, such as transactivation of genes in the nucleus, modulation of calcium homeostasis and lipid metabolism, and regulation of the actin cytoskeleton. In this article, we rule out putative new intracellular signaling mechanisms of the APP-interacting protein Fe65 in the regulation of actin cytoskeleton dynamics in the context of various neuronal functions, such as cell migration, neurite outgrowth, and synaptic plasticity.
Compared to our current knowledge of neuronal excitation, little is known about the development and maturation of inhibitory circuits. Recent studies show that inhibitory circuits develop and mature in a similar way like excitatory circuit. One such similarity is the development through excitation, irrespective of its inhibitory nature. Here in this current study, I used the inhibitory projection between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) as a model system to unravel some aspects of the development of inhibitory synapses. In LSO neurons of the rat auditory brainstem, glycine receptor-mediated responses change from depolarizing to hyperpolarizing during the first two postnatal weeks (Kandler and Friauf 1995, J. Neurosci. 15:6890-6904). The depolarizing effect of glycine is due to a high intracellular chloride concentration ([Cl-]i), which induces a reversal potential of glycine (EGly) more positive than the resting membrane potential (Vrest). In older LSO neurons, the hyperpolarizing effect is due to a low [Cl-]i (Ehrlich et al., 1999, J. Physiol. 520:121-137). Aim of the present study was to elucidate the molecular mechanism behind Clhomeostasis in LSO neurons which determines polarity of glycine response. To do so, the role and developmental expression of Cl-cotransporters, such as NKCC1 and KCC2 were investigated. Molecular biological and gramicidin perforated patchclamp experiments revealed, the role of KCC2 as an outward Cl-cotransporter in mature LSO neurons (Balakrishnan et al., 2003, J Neurosci. 23:4134-4145). But, NKCC1 does not appear to be involved in accumulating chloride in immature LSO neurons. Further experiments, indicated the role of GABA and glycine transporters (GAT1 and GLYT2) in accumulating Cl- in immature LSO neurons. Finally, the experiments with hypothyroid animals suggest the possible role of thyroid hormone in the maturation of inhibitory synapse. Altogether, this thesis addressed the molecular mechanism underlying the Cl- regulation in LSO neurons and deciphered it to some extent.
Herbivory is discussed as a key agent in maintaining dynamics and stability of tropical forested ecosystems. Accordingly increasing attention has been paid to the factors that structure tropical herbivore communities. The aim of this study was (1) to describe diversity, density, distribution and host range of the phasmid community (Phasmatodea) of a moist neotropical forest in Panamá, and (2) to experimentally assess bottom-up and top-down factors that may regulate populations of the phasmid Metriophasma diocles. The phasmid community of Barro Colorado Island was poor in species and low in density. Phasmids mainly occurred along forest edges and restricted host ranges of phasmid species reflected the successional status of their host plants. Only M. diocles that fed on early and late successional plants occurred regularly in the forest understory. A long generation time with a comparably low fecundity converted into a low biotic potential of M. diocles. However, modeled potential population density increased exponentially and exceeded the realized densities of this species already after one generation indicating that control factors continuously affect M. diocles natural populations. Egg hatching failure decreased potential population growth by 10 % but was of no marked effect at larger temporal scale. Interspecific differences in defensive physical and chemical leaf traits of M. diocles host plants, amongst them leaf toughness the supposedly most effective anti-herbivore defense, seemed not to affect adult female preference and nymph performance. Alternatively to these defenses, I suggest that the pattern of differential preference and performance may be based on interspecific differences in qualitative toxic compounds or in nutritive quality of leaves. The significant rejection of leaf tissue with a low artificial increase of natural phenol contents by nymphs indicated a qualitative defensive pathway in Piper evolution. In M. diocles, oviposition may not be linked to nymph performance, because the evolutionary prediction of a relation between female adult preference and nymph performance was missing. Consequently, the recruitment of nymphs into the reproductive adult phase may be crucially affected by differential performance of nymphs. Neonate M. diocles nymphs suffered strong predation pressure when exposed to natural levels of predation. Concluding from significantly increased predation-related mortality at night, I argue that arthropods may be the main predators of this nocturnal herbivore. Migratory behavior of nymphs seemed not to reflect predation avoidance. Instead, I provided first evidence that host plant quality may trigger off-plant migration. In conclusion, I suggest that predation pressure with its direct effects on nymph survival may be a stronger factor regulating M. diocles populations, compared to direct and indirect effects of host plant quality, particularly because slow growth and off-host migration both may feed back into an increase of predation related mortality.
On a route from whole genome duplication to aneuploidy and cancer: consequences and adaptations
(2022)
Whole genome duplication (WGD) is commonly accepted as an intermediate state between healthy cells and aneuploid cancer cells. Usually, cells after WGD get removed from the replicating pool by p53-dependent cell cycle arrest or apoptosis. Cells, which are able to bypass these mechanisms exhibit chromosomal instability (CIN) and DNA damage, promoting the formation of highly aneuploid karyotypes. In general, WGD favors several detrimental consequences such as increased drug resistance, transformation and metastasis formation. Therefore, it is of special interest to investigate the limiting factors and consequences of tetraploid proliferation as well as the adaptations to WGD. In the past it has been difficult to study the consequences of such large-scale genomic changes and how cells adapt to tetraploidy in order to survive. Our lab established protocols to generate tetraploids as well as isolated post-tetraploid/aneuploid single cells clones derived from euploid parental cell lines after induction of cytokinesis failure. This system enables to study the consequences and adaptations of WGD in newly generated tetraploid cells and evolved post-tetraploid clones in comparison to their isogenic parental cell line.
Using newly generated tetraploids from HCT116 cells, we identified USP28 and SPINT2 as novel factors limiting the proliferation after WGD. Using mass spectrometry and immunoprecipitation, we revealed an interaction between USP28 and NuMA1 upon WGD, which affects centrosome coalescence of supernumerary centrosomes, an important process that enhances survival of tetraploids. Furthermore, we validated the occurrence of DNA damage in tetraploid cells and found that USP28 depletion diminished the DNA damage dependent checkpoint activation. SPINT2 influences the proliferation after WGD by regulating the transcription of CDKN1A via histone acetylation. Following proliferating tetraploid cells, we confirmed the activation of the DNA damage response (DDR) by immunoblotting and microscopic approaches. Furthermore, we show that the DDR in the arising post-tetraploid clones is reduced. Further experiments verified the appearance of severe mitotic aberrations, replication stress and accumulation of reactive oxygen species in newly generated tetraploids as well as in the aneuploid cancer cells contributing to the occurrence of DNA damage. Using various drug treatments, we observed an increased dependency on the spindle assembly checkpoint in aneuploid cancer cells compared to their diploid parental cell line. Additionally, siRNA knock down experiments revealed the kinesin motor protein KIF18A as an essential protein in aneuploid cells.
Taken together, the results point out cellular consequences of proliferation after tetraploidization as well as the cellular adaptations needed to cope with the increased amount of DNA.
Loss of USP28 and SPINT2 expression promotes cancer cell survival after whole genome doubling
(2021)
Background
Whole genome doubling is a frequent event during cancer evolution and shapes the cancer genome due to the occurrence of chromosomal instability. Yet, erroneously arising human tetraploid cells usually do not proliferate due to p53 activation that leads to CDKN1A expression, cell cycle arrest, senescence and/or apoptosis.
Methods
To uncover the barriers that block the proliferation of tetraploids, we performed a RNAi mediated genome-wide screen in a human colorectal cancer cell line (HCT116).
Results
We identified 140 genes whose depletion improved the survival of tetraploid cells and characterized in depth two of them: SPINT2 and USP28. We found that SPINT2 is a general regulator of CDKN1A transcription via histone acetylation. Using mass spectrometry and immunoprecipitation, we found that USP28 interacts with NuMA1 and affects centrosome clustering. Tetraploid cells accumulate DNA damage and loss of USP28 reduces checkpoint activation, thus facilitating their proliferation.
Conclusions
Our results indicate three aspects that contribute to the survival of tetraploid cells: (i) increased mitogenic signaling and reduced expression of cell cycle inhibitors, (ii) the ability to establish functional bipolar spindles and (iii) reduced DNA damage signaling.