Towards an understanding of peroxisome dynamics in mammalian cells
Peroxisomes are ubiquitous subcellular organelles involved in a variety of important metabolic processes. Recently, it became obvious that many of those functions are carried out in co-operation with mitochondria. The essential role of peroxisomes for human health is exemplified by the severe phenot...
Klinische Zytobiologie und Zytopathologie
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|Summary:||Peroxisomes are ubiquitous subcellular organelles involved in a variety of important metabolic processes. Recently, it became obvious that many of those functions are carried out in co-operation with mitochondria. The essential role of peroxisomes for human health is exemplified by the severe phenotype of peroxisomal disorders. Furthermore, peroxisomes are highly dynamic, adjusting their protein content, morphology and number in response to cellular needs. In recent years, peroxisome dynamics and their proper regulation were closely linked to organelle function and thus, human well-being. In line with this, a patient with a lethal defect in mitochondrial and peroxisomal fission was identified. Peroxisome dynamics are regulated by growth and division of the organelle, however, peroxisomes can also arise de novo from the ER under special conditions. In mammalian cells, peroxisomal growth and division follows a well-defined sequence of morphological alterations. Initial membrane elongation is carried out by the key peroxisomal membrane protein Pex11pβ, while final scission into smaller organelles is achieved by the combined action of the membrane adaptors Mff and Fis1 as well as the large GTPase DLP1. Interestingly, the key components of the peroxisomal fission machinery are shared with mitochondria; however the occurrence of peroxisomal fusion analogous to mitochondria remains a matter of debate. Although several external stimuli were identified to alter peroxisome dynamics, detailed information on their reception and transduction onto the peroxisomal level is limited. Thus, the aim of this study was to gain a deeper understanding of the processes contributing to and regulating peroxisome dynamics in mammalian cells. This thesis contains three parts: in the first section, the contribution of peroxisomal fusion, analogous to mitochondria, to organelle dynamics was addressed. In the second part, the regulation of peroxisome dynamics at the organelle itself was investigated by characterizing post-translational mechanisms modulating the action of Pex11pβ, the key mediator of peroxisome elongation/proliferation in mammalian cells. In the final part, different groups of external stimuli were characterized in regard to their capacity to alter peroxisome dynamics in order to study the regulation of peroxisome dynamics on a transcriptional level in mammalian cell culture.
To investigate fusion of mature peroxisomes in mammalian CHO cells, an in vivo fusion assay was established based on hybridoma formation by cell fusion using cell lines stably expressing GFP- or DsRed-derived peroxisomal matrix and membrane markers. Fluorescence microscopy in time course experiments of fixed cells revealed a merge of different peroxisomal markers in fused cells, pointing to a certain degree of peroxisomal fusion. Although subsequent live cell imaging indicated that peroxisomes did not exchange matrix or membrane markers, the existence of transient, vivid interactions between individual peroxisomes was characterized for the first time. Interacting peroxisomes were shown to be tightly associated, accounting for the marker overlay observed in fixed cells. Using computational modelling and mathematical analysis, transient peroxisome interactions were shown to follow a complex, non-random behaviour that has the potential to facilitate the homogenization of the heterogeneous peroxisomal compartment. Pre-treatment with peroxisomal substrates indicated that transient, peroxisomal interactions do not contribute to the exchange of fatty acids or H2O2, but might facilitate the exchange of other peroxisomal metabolites or be part of a signaling system to sense the state and/or distribution of the peroxisomal population in the cell. Furthermore, for the first time, computational analysis provided an explanation why only 15 % of the peroxisome population is engaged in long-range microtubule-dependent movement. Additionally, evidence was provided that mitochondrial fusion proteins do not localize to peroxisomes, indicating that peroxisome dynamics in mammalian cells are regulated in a distinct manner.
To gain insight into the modulation of peroxisome dynamics at the organelle itself, Pex11pβ was characterized biochemically. Differential permeabilization and protease-protection assays in combination with a newly available commercial antibody localized the position of its first transmembrane domain to the amino acid positions 90 – 110. Subsequently, the contribution of the N-terminal domain to the regulation of human Pex11pβ activity was addressed. Deletion of the first 40 amino acids abolished Pex11pβ-membrane elongation, although the essential amphipathic helix within the protein remained intact. Biochemical cross-linking and enrichment of Pex11pβ in time-course experiments linked its homo-dimerization to its activity which was diminished upon N-terminal deletion. In vivo phospho-labelling did not indicate phosphorylation of Pex11pβ in a manner similar to its S. cerevisiae orthologue. Thus, Pex11 proteins in yeast and mammals appear to be regulated in an opposite manner. Furthermore, overexpression of Pex11pβ in peroxisome-deficient patient fibroblasts resulted in its mistargeting to mitochondria where an excessive fragmentation was induced. This further emphasizes that mitochondria, but not the ER, serve a default membrane for peroxisomal membrane proteins in the absence of peroxisomes in mammals. Potential disturbances of mitochondrial function might thus contribute to the clinical severity of peroxisome disorders.
In the final part of this thesis, external stimuli altering peroxisomal dynamics were characterized to establish a more physiological and amenable cell culture model to investigate the transcriptional regulation of peroxisome dynamics in mammalian cells. Application of the neurotoxin 6-OHDA in SH-S5Y5 neuroblastoma cells did not affect peroxisome dynamics, but led to a profound DLP1-dependent fragmentation of mitochondria. Though DLP1 is a shared component of both organelles, mitochondrial and peroxisomal dynamics further appear to be regulated in a distinct manner. Using a variety of compounds inducing cytosolic and mitochondrial oxidative stress, no morphological alterations of peroxisomes were observed. KillerRed-based induction of ROS in different compartments produced similar results in living cells. Thus, other factors besides the induction of oxidative stress, such as e.g. the intracellular or extracellular origin of the signal, alterations of cellular redox-state or yet unidentified signalling pathways might contribute to induce the alterations of peroxisome dynamics observed before. Addition of the glucocorticoid dexamethasone to rat pancreatic AR42J cells resulted in a profound, continuous elongation of peroxisomes. Notably, peroxisomes maintained their tubular morphology even after removal of the stimulus. Continuous peroxisome tubulation might be linked to cell differentiation or a metabolic function. Dexamethasone application induced Pex11β (and Pex11α) on a transcriptional level by a potentially PPARα-independent mechanism. Thus, dexamethasone-induced peroxisome elongation in AR42J cells has a high potential to serve as a more physiological model to study the regulation of peroxisome dynamics in mammalian cells. Future studies using expression profiling after dexamethasone stimulation in order to identify novel components and/or molecular mechanisms regulating peroxisome dynamics have been initiated.|