Christof Meisinger

Insertion of Hydrophobic Membrane Proteins into the Inner Mitochondrial Membrane—A Guided Tour

Only a few mitochondrial proteins are encoded by the organellar genome. The majority of mitochondrial proteins are nuclear encoded and thus have to be transported into the organelle from the cytosol. Within the mitochondrion proteins have to be sorted into one of the four sub-compartments: the outer or inner membranes, the intermembrane space or the matrix. These processes are mediated by complex protein machineries within the different compartments that act alone or in concert with each other. The translocation machinery of the outer membrane is formed by a multi-subunit protein complex (TOM complex), that is built up by signal receptors and the general import pore (GIP). The inner membrane houses two multi-subunit protein complexes that each handles special subsets of mitochondrial proteins on their way to their final destination. According to their primary function these two complexes have been termed the pre-sequence translocase (or TIM23 complex) and the protein insertion complex (or TIM22 complex). The identification of components of these complexes and the analysis of the molecular mechanisms underlying their function are currently an exciting and fast developing field of molecular cell biology.

Expression of fibroblast growth factor-2 and fibroblast growth factor receptor 1 messenger RNAs in spinal ganglia and sciatic nerve: regulation after peripheral nerve lesion.

In order to determine functional roles of basic fibroblast growth factor (FGF-2) in the peripheral nervous system we have analysed the expression of FGF-2 and FGF receptor 1 (FGFR1) in spinal ganglia and the sciatic nerve under normal conditions and after nerve crush using RNAse protection assay and in situ hybridization. In intact spinal ganglia, both FGF-2 and FGFR1 messenger RNAs are expressed, albeit at different levels. In situ hybridization identifies satellite cells as the source of FGF-2 and sensory neurons as the source of FGFR1 suggesting a paracrine mode of action of FGF-2 on sensory neurons. One day after crush lesion FGF-2 is significantly up-regulated in sensory ganglia L4-L6. Highest levels are found at day 7; control levels are approached after 28 days. FGFR1 messenger RNA, which is strongly expressed in intact spinal ganglia, displays no significant change after lesion. In the intact sciatic nerve, FGFR1 messenger RNA is detected at higher levels than FGF-2 messenger RNA. After injury, both transcripts display a time-dependent up-regulation in both the proximal and distal nerve stump. Schwann cells, as a putative source of the sciatic nerve-derived FGF-2, express both FGF-2 and FGFR1 messenger RNAs in vitro. The FGFR1 transcript level is increased in the presence of forskolin. FGF-2 does not affect expression of FGFR1 messenger RNA but stimulates its own expression. These results show that during peripheral nerve regeneration FGF-2 is up-regulated in both the crushed nerve and the respective spinal ganglia suggesting a possible physiological function of FGF-2 during the regeneration process.

In vivo expression and localization of the fibroblast growth factor system in the intact and lesioned rat peripheral nerve and spinal ganglia

Basic fibroblast growth factor (FGF‐2) is involved in several cellular processes of the nervous system during development, maintenance, and regeneration. In the central nervous system, FGF‐2 has been shown to be expressed in neurons and glial cells, depending on the developmental stage and brain area. In the present study, a comprehensive analysis was performed of the cellular distribution of the transcripts of FGF‐2 and of the FGF high‐affinity receptors (R) 1–4 in intact and lesioned sciatic nerve and spinal ganglia. In the adult rat sciatic nerve FGF‐2, FGFR1–3 were expressed at low levels as revealed by reverse transcriptase‐polymerase chain reaction (RT‐PCR). Sciatic nerve crush resulted in an increase of these transcript levels. FGFR4 expression was not detected in the intact and crushed nerve as revealed by RT‐PCR and RNase protection assay. In situ hybridization using riboprobes for FGF‐2, FGFR1–3 displayed staining in diverse cell types. Immunocytochemical staining of consecutive sections with cell markers for myelin, macrophages, and neurons revealed colocalization of the transcripts with Schwann cells and macrophages. In addition to FGF‐2 and FGFR1, the transcripts of FGFR2–4 were expressed in neurons of spinal ganglia. Crush lesion of the sciatic nerve resulted in no alterations of the FGFR1–4 transcripts, whereas FGF‐2 and FGFR3 mRNAs were up‐regulated in spinal ganglia. The expression of FGFRs and FGF‐2 in Schwann cells and macrophages at the lesion site of the sciatic nerve and in sensory neurons suggests that FGF‐2 is involved in specific functions of these cells during regeneration. J. Comp. Neurol. 434:342–357, 2001.

Differential Regulation of Fibroblast Growth Factor (FGF)‐2 and FGF Receptor 1 mRNAs and FGF‐2 Isoforms in Spinal Ganglia and Sciatic Nerve After Peripheral Nerve Lesion

Abstract: To study the functional role of endogenous basic fibroblast growth factor‐2 (FGF‐2) during degeneration and regeneration of the sensory system, we have determined the expression and regulation of FGF‐2 and FGF receptor (FGFR)‐1 mRNAs in spinal ganglia and sciatic nerve during experimental transection and crush injury of the sciatic nerve. In contrast to levels of the FGFR‐1 transcript, which is not altered, the level of FGF‐2 mRNA is dramatically up‐regulated in spinal ganglia after injury. In the proximal and distal nerve stumps both transcript levels are significantly elevated, albeit at different time points. The FGF‐2 isoforms are differently up‐regulated in spinal ganglia and sciatic nerve following peripheral nerve lesion. The differential response of FGF‐2 mRNA and protein and of FGFR‐1 mRNA in spinal ganglia and sciatic nerve after lesion is suggestive of different physiological functions: a local reaction at the lesion site where axonal regrowth occurs and a trophic reaction for the degenerating/regenerating sensory neurons.

Mitochondrial outer membrane proteome of Trypanosoma brucei reveals novel factors required to maintain mitochondrial morphology

Trypanosoma brucei is a unicellular parasite that causes devastating diseases in humans and animals. It diverged from most other eukaryotes very early in evolution and, as a consequence, has an unusual mitochondrial biology. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the parasite. The outer mitochondrial membrane defines the boundary of the organelle. Its properties are therefore key for understanding how the cytosol and mitochondria communicate and how the organelle is integrated into the metabolism of the whole cell. We have purified the mitochondrial outer membrane of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal outer membrane proteome consists of 82 proteins, two-thirds of which have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins, 33 of which are specific to trypanosomatids, remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and for the first time identified factors that control mitochondrial shape in T. brucei.

In Vivo and in Vitro Effect of Glucocorticoids on Fibroblast Growth Factor (FGF)-2 and FGF Receptor 1 Expression

In order to clarify the physiological function of fibroblast growth factor (FGF-2) in the adrenal medulla the regulation of FGF-2 and FGF receptor 1 (FGFR1) was studied in vitro and in vivo in response to glucocorticoids. To assess the effects of glucocorticoids, in vivo extracts of adrenal medulla and adrenal cortex were analyzed by RNase protection assay and Western blot analysis. PC12 cells were chosen as a model system to study the effects of glucocorticoids in vitro. In PC12 cells, dexamethasone (DEX) was found to stimulate dramatically the expression of both FGF-2 mRNA and protein. Western blot analysis revealed that exclusively the 21-kDa FGF-2 isoform was enhanced. In contrast to the FGF-2 mRNA level FGFR1 was not affected by treatment with glucocorticoids. In vivo FGF-2 mRNA level and 21-kDa FGF-2 isoform level are significantly enhanced in the adrenal medulla 24 h after DEX injection. In vivo application of DEX leads to an increase of the medullary and cortical FGFR1 transcript levels. Glucocorticoid effects on FGF-2 expression were not found in adrenal cortex, heart, skeletal muscle, and kidney, respectively, in vivo and in L6 rat myoblasts in vitro. In addition to adrenal medullary cells glucocorticoids elevated the FGF-2 mRNA and protein level also in vivo in the brain and in vitro in immortalized Schwann cells. The present results suggest that the 21-kDa FGF-2 isoform mediates a physiological function specific for neuronal tissue which is modulated by glucocorticoids.

Regulation of nerve growth factor and its low-affinity receptor (p75NTR) during myogenic differentiation.

In our preceding report, we have shown that nerve growth factor (NGF) and its low‐affinity receptor (p75NTR) are expressed in C2C12 myoblasts and downregulated during myogenic differentiation. Furthermore, NGF affects myogenic differentiation and cell growth via p75NTR and downregulation of p75NTR is essential for myogenic differentiation (Seidl et al., 1998). Here we show that NGF and p75NTR are regulated by mechanisms preceding terminal differentiation in myogenic cells. These mechanisms include cell‐density phenomena such as cell‐cell contact as well as signaling of basic fibroblast growth factor (FGF‐2) and its receptor (FGFR1). Downregulation of NGF and p75NTR occurred as a consequence of increasing cell density, an important trigger for the onset of myogenic differentiation. FGF‐2 and FGFR1 were shown to be present in C2C12 cells and exogenous FGF‐2 induced NGF and p75NTR expression, implying that FGF/FGFR signaling is an upstream regulator of the NGF/p75NTR system. The fact that FGF‐2 could suspend yet not abolish density‐induced downregulation indicates that cell‐cell contact counteracts the FGF effect and ultimately terminates NGF/p75NTR signaling. This evidence, together with the observation that p75NTR expression is suppressed in muscle progenitors, which constitutively express adenovirus E1A proteins and thus lack the competence of myogenic differentiation, underline the important role for the NGF/p75NTR system in the interplay of multiple factors and biological systems that balance myogenic differentiation at the appropriate spatial and temporal level. J. Cell. Psysiol. 176:22–31, 1998.

Fibroblast growth factor receptor 1 in the adrenal gland and PC12 cells: developmental expression and regulation by extrinsic molecules.

In the present study we have analyzed the expression of fibroblast growth factor receptor 1 (FGFR-1) mRNA in the developing and adult rat adrenal gland and in PC12 cells under different culture conditions. For this purpose a sensitive ribonuclease protection assay using 33P-labelled riboprobes was established. 33P-labelled riboprobes show a high resolution and are relatively easy to handle. FGFR-1 mRNA was found to be present in the postnatal and adult adrenal gland. In the cortex high levels of FGFR-1 mRNA were detected at postnatal day (P) 1 and P8, during the third week the mRNA levels declined, and reached low levels during adulthood. PC12 cells also contained detectable amounts of FGFR-1 mRNA. With the exception of NGF, however, the different treatment procedures did not affect FGFR-1 mRNA levels. The expression pattern of the FGFR-1 transcript matches that of the expression of FGF-2 and of the mitotic activity in the developing and adult cortex. This supports the idea that FGF-2 might act as an autocrine mitogen for adrenocortical cells. In the medulla FGFR-1 mRNA levels were low at the first 3 postnatal weeks and increased towards the adult. In accordance with the developing expression pattern of FGF-2 in the medulla and in vitro effects of this protein on chromaffin and PC12 cells an autocrine/paracrine role as a maintenance and differentiation factor for chromaffin cells is conceivable.

Over-expression of the 18 kD and 21/23 kD fibroblast growth factor-2 isoforms in PC12 cells and Schwann cells results in altered cell morphology and growth

Basic fibroblast growth factor (FGF-2) occurs in different isoforms which represent alternative translation products from a single mRNA. The question of whether the presence of multiple FGF-2 isoforms has physiological implications is compelling but unresolved so far. However, it has been shown recently that the FGF-2 isoforms are differentially regulated in sensory ganglia and peripheral nerve following nerve injury and, moreover, in the adrenal medulla during postnatal development and after hormonal stimuli suggesting that the isoforms may serve different physiological functions. To investigate isoform-specific effects we have established immortalized Schwann cells and PC12 cells stably over-expressing the 18 kD and the HMW isoforms. We found that the over-expression of the different isoforms alters morphology and growth of the Schwann cells. PC12 cells over-expressing the 18 kD FGF-2 were found to differentiate towards the neuronal phenotype whereas over-expression of the HMW isoforms resulted in a stabilization of the endocrine phenotype. Taken together, these data corroborate the idea of FGF-2 isoform-specific functions.

LOCALIZATION, DIFFERENTIAL EXPRESSION AND RETROGRADE AXONAL TRANSPORT SUGGEST PHYSIOLOGICAL ROLE OF FGF-2 IN SPINAL AUTONOMIC NEURONS OF THE RAT

Fibroblast growth factor‐2 (FGF‐2) has marked pharmacological neurotrophic effects on lesioned spinal autonomic neurons following target removal of the adrenal medulla, yet expression and axonal transport in autonomic neurons remain to be shown. We show here FGF‐2 and FGF receptor type 1 (FGFR1) protein and mRNA expression in preganglionic intermediolateral neurons of the rat thoracic spinal cord. While immunoreactivity of both FGF‐2 and FGFR1 co‐localize to intermediolateral neurons, mRNA transcripts of FGFR1, but not of FGF‐2, are detectable in intermediolateral preparations by RNase protection analysis, suggesting protein translocation in viva Unilateral microinjection of 125iodinated FGF‐2 into the adrenal medulla (a major target of intermediolateral neurons) results in significant accumulation of specific radioactivity in thoracic spinal cord tissue, including the intermediolateral neurons, and the ipsilateral splanchnic nerve. Emulsion autoradiography demonstrated labelling over ipsilateral intermediolateral neurons only. Neuronal co‐localization of FGF‐2/FGFR1 protein, differential mRNA expression, specific retrograde axonal transport and the known neurotrophic actions in vivo, strongly suggest unique physiological roles of FGF‐2 in the autonomic nervous system.