[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development by activating 3 receptor tyrosine kinases (RTKs), VEGFR-1, -2, and -3, and by binding to coreceptors such as neuropilin-1 (NRP-1). We investigated how different VEGF-A isoforms, in particular VEGF-A(165)a and VEGF-A(165)b, control the balance between VEGFR-2 recycling, degradation, and signaling. Stimulation of cells with the NRP-1-binding VEGF-A(165)a led to sequential NRP-1-mediated VEGFR-2 recycling through Rab5, Rab4, and Rab11 vesicles. Recycling was accompanied by dephosphorylation of VEGFR-2 between Rab4 and Rab11 vesicles and quantitatively and qualitatively altered signal output. In cells stimulated with VEGF-A(165)b, an isoform unable to bind NRP-1, VEGFR-2 bypassed Rab11 vesicles and was routed to the degradative pathway specified by Rab7 vesicles. Deletion of the GIPC (synectin) binding motif of NRP-1 prevented transition of VEGFR-2 through Rab11 vesicles and attenuated signaling. Coreceptor engagement was specific for VEGFR-2 because EGFR recycled through Rab11 vesicles in the absence of known coreceptors. Our data establish a distinct role of NRP-1 in VEGFR-2 signaling and reveal a general mechanism for the function of coreceptors in modulating RTK signal output.
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of the type V receptor tyrosine kinases VEGFR-1, -2 and -3. In addition, VEGFs interact with co-receptors such as neuropilins, integrins, semaphorins or heparansulfate glycosaminoglycans. Ligand binding dimerises the receptors and activates their intracellular tyrosine kinase domains, resulting in phosphorylation of tyrosine residues acting as docking sites for intracellular signalling molecules. Ligand-induced receptor is internalised and then transported through early, late, and recycling endosomes, and finally degraded by proteasomal or lysosomal pathways. Biological output by VEGF is mediated through distinct receptor/co-receptor complexes and generates signals in all cellular compartments triggering cellular responses such as cell migration, cell proliferation, vessel formation and maturation, as well as changes in vessel fenestration, constriction and permeability. Here we review recent experiments showing how VEGFR-2 is transported through intracellular vesicular compartments specified by Rab family GTPases, and discuss how different VEGF-A isoforms specify intracellular receptor trafficking. We also discuss how the biological consequences of aberrant receptor trafficking bear on the development of vascular disease.
[Show abstract][Hide abstract] ABSTRACT: The introduction of heterologous genetic information, particularly of multiple genes, into mammalian cells is a key technology in contemporary experimental biological research. The coexpression of fluorescently tagged sensors is required to simultaneously analyse multiple parameters in living cells and the coexpression of several proteins is necessary to manipulate cell fate in stem cell biology. Current technologies for multigene expression in mammalian cells are inefficient, inflexible and time-consuming. In this paper we describe MultiLabel, a novel and highly efficient modular plasmid-based eukaryotic expression system. Independent expression vectors are assembled by a Cre/LoxP reaction into a plasmid with multiple expression cassettes. MultiLabel enables rapid construction of multigene expression vectors for the single-step creation of transiently or stably transfected mammalian cells.
[Show abstract][Hide abstract] ABSTRACT: Charcot-Marie-Tooth disease type 4B is caused by mutations in the genes encoding either the lipid phosphatase myotubularin-related protein-2 (MTMR2) or its regulatory binding partner MTMR13/SBF2. Mtmr2 dephosphorylates PI-3-P and PI-3,5-P2 to form phosphatidylinositol and PI-5-P, respectively, while Mtmr13/Sbf2 is an enzymatically inactive member of the myotubularin protein family. We have found altered levels of the critical signalling protein AKT in mouse mutants for Mtmr2 and Mtmr13/Sbf2. Thus, we analysed the influence of Mtmr2 and Mtmr13/Sbf2 on signalling processes. We found that overexpression of Mtmr2 prevents the degradation of the epidermal growth factor receptor (EGFR) and leads to sustained Akt activation whereas Erk activation is not affected. Mtmr13/Sbf2 counteracts the blockage of EGFR degradation without affecting prolonged Akt activation. Our data indicate that Mtmr2 and Mtmr13/Sbf2 play critical roles in the sorting and modulation of cellular signalling which are likely to be disturbed in CMT4B.
Journal of Cellular and Molecular Medicine 11/2009; 15(2):307-15. · 3.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Structural and functional studies of many multiprotein complexes depend on recombinant-protein overexpression. Rapid revision of expression experiments and diversification of the complexes are often crucial for success of these projects; therefore, automation is increasingly indispensable. We introduce Acembl, a versatile and automatable system for protein-complex expression in Escherichia coli that uses recombineering to facilitate multigene assembly and diversification. We demonstrated protein-complex expression using Acembl, including production of the complete prokaryotic holotranslocon.
[Show abstract][Hide abstract] ABSTRACT: Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous disorder. All mendelian patterns of inheritance have been described. We identified a homozygous p.A335V mutation in the MED25 gene in an extended Costa Rican family with autosomal recessively inherited Charcot-Marie-Tooth neuropathy linked to the CMT2B2 locus in chromosome 19q13.3. MED25, also known as ARC92 and ACID1, is a subunit of the human activator-recruited cofactor (ARC), a family of large transcriptional coactivator complexes related to the yeast Mediator. MED25 was identified by virtue of functional association with the activator domains of multiple cellular and viral transcriptional activators. Its exact physiological function in transcriptional regulation remains obscure. The CMT2B2-associated missense amino acid substitution p.A335V is located in a proline-rich region with high affinity for SH3 domains of the Abelson type. The mutation causes a decrease in binding specificity leading to the recognition of a broader range of SH3 domain proteins. Furthermore, Med25 is coordinately expressed with Pmp22 gene dosage and expression in transgenic mice and rats. These results suggest a potential role of this protein in the molecular etiology of CMT2B2 and suggest a potential, more general role of MED25 in gene dosage sensitive peripheral neuropathy pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: The peripheral myelin protein 22 (PMP22) is highly expressed in myelinating Schwann Cells and in neurons of the peripheral nervous system where it accounts for 2–5% of total protein in the myelin sheat. Minor changes in PMP22 gene dosage have profound effects on the development and maintenance of peripheral nerves. This is evident from the genetic disease mechanisms in Charcot–Marie tooth disease type 1 A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) as well as transgenic animals with altered PMP22 gene dosage. Thus, regulation of PMP22 is a crucial aspect in understanding the function of this protein in health and disease. In this study, we describe important regulatory elements of PMP22 by the generation of transgenic mice containing 10 kb of 5′-flanking region of the PMP22 gene, including the two previously identified alternative promoters, fused to a lacZ reporter gene. We show that this part of the PMP22 gene contains the necessary information to mirror the endogenous expression pattern in peripheral nerves during development, regeneration, and in mouse models of demyelination due to genetic lesions. Transgene expression is strongly regulated during myelination, demyelination and remyelination in Schwann cells demonstrating the crucial influence of neuron–Schwann cell interactions in the regulation of PMP22. These results provide the crucial basis for the ongoing further dissection of the elements that direct the temporal and spatial regulation of the PMP22 gene and to elucidate the molecular basis of the master program regulating peripheral nerve myelination.
Journal of Neurochemistry 06/2008; 81:77-77. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Charcot-Marie-Tooth (CMT) disease denotes a large group of genetically heterogeneous hereditary motor and sensory neuropathies and ranks among the most common inherited neurological disorders. Mutations in the Myotubularin-Related Protein-2 (MTMR2) or MTMR13/Set-Binding Factor-2 (SBF2) genes are associated with the autosomal recessive disease subtypes CMT4B1 or CMT4B2. Both forms of CMT share similar features including a demyelinating neuropathy associated with reduced nerve conduction velocity (NCV) and focally folded myelin. Consistent with a common disease mechanism, the homodimeric MTMR2 acts as a phosphoinositide D3-phosphatase with phosphatidylinositol (PtdIns) 3-phosphate and PtdIns 3,5-bisphosphate as substrates while MTMR13/SBF2 is catalytically inactive but can form a tetrameric complex with MTMR2, resulting in a strong increase of the enzymatic activity of complexed MTMR2. To prove that MTMR13/SBF2 is the disease-causing gene in CMT4B2 and to provide a suitable animal model, we have generated Mtmr13/Sbf2-deficient mice. These animals reproduced myelin outfoldings and infoldings in motor and sensory peripheral nerves as the pathological hallmarks of CMT4B2, concomitant with decreased motor performance. The number and complexity of myelin misfoldings increased with age, associated with axonal degeneration, and decreased compound motor action potential amplitude. Prolonged F-wave latency indicated a mild NCV impairment. Loss of Mtmr13/Sbf2 did not affect the levels of its binding partner Mtmr2 and the Mtmr2-binding Dlg1/Sap97 in peripheral nerves. Mice deficient in Mtmr13/Sbf2 together with known Mtmr2-deficient animals will be of major value to unravel the disease mechanism in CMT4B and to elucidate the critical functions of protein complexes that are involved in phosphoinositide-controlled processes in peripheral nerves.
Human Molecular Genetics 01/2008; 16(24):2991-3001. · 6.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The concept of the cell as a collection of multisubunit protein machines is emerging as a cornerstone of modern biology, and molecular-level study of these machines in most cases will require recombinant production. Here, we present and validate a strategy to rapidly produce, permutate, and posttranslationally modify large, eukaryotic multiprotein complexes by using DNA recombination in a process that is fully automatable. Parallel production of 12 protein complex variants within a period of weeks resulted in specimens of sufficient quantity and homogeneity for structural biology applications.
[Show abstract][Hide abstract] ABSTRACT: Elucidation of the molecular basis of protein-interaction networks, in particular in higher eukaryotes, is hampered by insufficient quantities of endogenous multiprotein complexes. Present recombinant expression methods often require considerable investment in both labor and materials before multiprotein expression, and after expression and biochemical analysis these methods do not provide flexibility for expressing an altered multiprotein complex. To meet these demands, we have recently introduced MultiBac, a modular baculovirus-based system specifically designed for eukaryotic multiprotein expression. Here we describe new transfer vectors and a combination of DNA recombination-based methods, which further facilitate the generation of multigene cassettes for protein coexpression (Fig. 1), thus providing a flexible platform for generation of protein expression vectors and their rapid regeneration for revised expression studies. Genes encoding components of a multiprotein complex are inserted into a suite of compatible transfer vectors by homologous recombination. These progenitor constructs are then rapidly joined in the desired combination by Cre-loxP-mediated in vitro plasmid fusion. Protocols for integration of the resulting multigene expression cassettes into the MultiBac baculoviral genome are provided that rely on Tn7 transposition and/or Cre-loxP reaction carried out in vivo in Escherichia coli cells tailored for this purpose. Detailed guidelines for multigene virus generation and amplification, cell culture maintenance and protein production are provided, together with data illustrating the simplicity and remarkable robustness of the present method for multiprotein expression using a composite MultiBac baculoviral vector.
[Show abstract][Hide abstract] ABSTRACT: Over the last 15 years, a number of mutations in a variety of genes have been identified that lead to inherited motor and sensory neuropathies (HMSN), also called Charcot-Marie-Tooth disease (CMT). In this review we will focus on the molecular and cellular mechanisms that cause the Schwann cell pathologies observed in dysmyelinating and demyelinating forms of CMT. In most instances, the underlying gene defects alter primarily myelinating Schwann cells followed by secondary axonal degeneration. The first set of proteins affected by disease-causing mutations includes the myelin components PMP22, P0/MPZ, Cx32/GJB1, and periaxin. A second group contains the regulators of myelin gene transcription EGR2/Krox20 and SOX10. A third group is composed of intracellular Schwann cells proteins that are likely to be involved in the synthesis, transport and degradation of myelin components. These include the myotubularin-related lipid phosphatase MTMR2 and its regulatory binding partner MTMR13/SBF2, SIMPLE, and potentially also dynamin 2. Mutations affecting the mitochondrial fission factor GDAP1 may indicate an important contribution of mitochondria in myelination or myelin maintenance, whereas the functions of other identified genes, including NDRG1, KIAA1985, and the tyrosyl-tRNA synthase YARS, are not yet clear. Mutations in GDAP1, YARS, and the pleckstrin homology domain of dynamin 2 lead to an intermediate form of CMT that is characterized by moderately reduced nerve conduction velocity consistent with minor myelin deficits. Whether these phenotypes originate in Schwann cells or in neurons, or whether both cell types are directly affected, remains a challenging question. However, based on the advances in systematic gene identification in CMT and the analyses of the function and dysfunction of the affected proteins, crucially interconnected pathways in Schwann cells in health and disease have started to emerge. These networks include the control of myelin formation and stability, membrane trafficking, intracellular protein sorting and quality control, and may extend to mitochondrial dynamics and basic protein biosynthesis.
[Show abstract][Hide abstract] ABSTRACT: Mutations in myotubularin-related protein-2 (MTMR2) or MTMR13/set-binding factor-2 (SBF2) genes are responsible for the severe autosomal recessive hereditary neuropathies, Charcot-Marie-Tooth disease (CMT) types 4B1 and 4B2, both characterized by reduced nerve conduction velocities, focally folded myelin sheaths and demyelination. MTMRs form a large family of conserved dual-specific phosphatases with enzymatically active and inactive members. We show that homodimeric active Mtmr2 interacts with homodimeric inactive Sbf2 in a tetrameric complex. This association dramatically increases the enzymatic activity of the complexed Mtmr2 towards phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate. Mtmr2 and Sbf2 are considerably, but not completely, co-localized in the cellular cytoplasm. On membranes of large vesicles formed under hypo-osmotic conditions, Sbf2 favorably competes with Mtmr2 for binding sites. Our data are consistent with a model suggesting that, at a given cellular location, Mtmr2 phosphatase activity is highly regulated, being high in the Mtmr2/Sbf2 complex, moderate if Mtmr2 is not associated with Sbf2 or functionally blocked by competition through Sbf2 for membrane-binding sites.
Human Molecular Genetics 03/2006; 15(4):569-79. · 6.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We review the putative functions and malfunctions of proteins encoded by genes mutated in Charcot-Marie-Tooth disease (CMT; inherited motor and sensory neuropathies) in normal and affected peripheral nerves. Some proteins implicated in demyelinating CMT, peripheral myelin protein 22, protein zero (P0), and connexin32 (Cx32/GJB1) are crucial components of myelin. Periaxin is involved in connecting myelin to the surrounding basal lamina. Early growth response 2 (EGR2) and Sox10 are transcriptional regulators of myelin genes. Mutations in the small integral membrane protein of lysosome/late endosome, the myotubularin-related protein 2 (MTMR2), and MTMR13/set-binding factor 2 are involved in vesicle and membrane transport and the regulation of protein degradation. Pathomechanisms related to alterations of these processes are a widespread phenomenon in demyelinating neuropathies because mutations of myelin components may also affect protein biosynthesis, transport, and/or degradation. Related disease mechanisms are also involved in axonal neuropathies although there is considerably more functional heterogeneity. Some mutations, most notably in P0, GJB1, ganglioside-induced differentiation-associated protein 1 (GDAP1), neurofilament light chain (NF-L), and dynamin 2 (DNM2), can result in demyelinating or axonal neuropathies introducing additional complexity in the pathogenesis. Often, this relates to the intimate connection between Schwann cells and neurons/axons leading to axonal damage even if the mutation-caused defect is Schwann-cell-autonomous. This mechanism is likely for P0 and Cx32 mutations and provides the basis for the unifying hypothesis that also demyelinating neuropathies develop into functional axonopathies. In GDAP1 and DNM2 mutants, both Schwann cells and axons/neurons might be directly affected. NF-L mutants have a primary neuronal defect but also cause demyelination. The major challenge ahead lies in determining the individual contributions by neurons and Schwann cells to the pathology over time and to delineate the detailed molecular functions of the proteins associated with CMT in health and disease.
NeuroMolecular Medicine 02/2006; 8(1-2):217-42. · 3.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Charcot-Marie-Tooth disease (CMT) comprises a family of clinically and genetically very heterogeneous hereditary peripheral neuropathies and is one of the most common inherited neurological disorders. We have generated a mouse model for CMT type 4B1 using embryonic stem cell technology. To this end, we introduced a stop codon into the Mtmr2 locus within exon 9, at the position encoding amino acid 276 of the MTMR2 protein (E276X). Concomitantly, we have deleted the chromosomal region immediately downstream of the stop codon up to within exon 13. The resulting allele closely mimics the mutation found in a Saudi Arabian CMT4B1 patient. Animals homozygous for the mutation showed various degrees of complex myelin infoldings and outfoldings exclusively in peripheral nerves, in agreement with CMT4B1 genetics and pathology. Mainly, paranodal regions of the myelin sheath were affected, with a high degree of quantitative and qualitative variability between individuals. This pathology was progressive with age, and axonal damage was occasionally observed. Distal nerve regions were more affected than proximal parts, in line with the distribution in CMT. However, we found no significant electrophysiological changes, even in aged (16-month-old) mice, suggesting that myelin infoldings and outfoldings per se are not invariably associated with detectable electrophysiological abnormalities. Our animal model provides a basis for future detailed molecular and cellular studies on the underlying disease mechanisms in CMT4B1. Such an analysis will reveal how the disease develops, in particular, the enigmatic myelin infoldings and outfoldings as well as axonal damage, and provide mechanistic insights that may aid in the development of potential therapeutic approaches.
Human Molecular Genetics 01/2006; 14(23):3685-95. · 6.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Charcot-Marie-Tooth disease (CMT) comprises a group of frequent, genetically and clinically heterogenous peripheral neuropathies. Two main CMT forms are distinguished: the demyelinating CMT type 1 (CMT1) and the axonal CMT type 2 (CMT2). Recently, we reported linkage of the axonally pronounced CMT2B2 type to chromosome 19q13.3 (OMIM %605589). Analysis of 53 genes in the critical interval resulted in an A335V mutation in a subunit of the mediator complex associated with RNA polymerase II. This mutation is embedded in a proline-rich motif typical for Abelson-SH3 binding sites. Wildtype and mutant peptides were incubated with AblSH3- and Src-family SH3 protein. While wildtype and mutant CMT2B2 strongly bind Abl-SH3, the mutation results in a drastically increased affinity for Src-family SH3 domains. This indicates a severe loss of specificity in target recognition. A mild myelin impairment in these patients guided us to investigate the CMT2B2 gene expression by qPCR in Pmp22 over-and underexpressing mice. A significant correlation of CMT2B2 expression with Pmp22 expression could be clearly shown-a high Pmp22 expression level resulted in a high CMT2B2 level and vice versa. These results were confirmed in transgenic CMT1 rats. Negative phenotypic and behavioural effects of progesterone treatment in CMT1 rats have been reported recently. We could show that progesterone-treated male CMT1 rats revealed a tremendous increase of the CMT2B2 expression correlated with increased Pmp22 expression. Our data show that the CMT2B2 A335V mutation causes an inherited peripheral neuropathy. Furthermore the tightly linked expression to Pmp22 points to a more general role in peripheral nerve pathogenesis, possibly via transcriptional (mis-) regulation of multiple nerve specific genes in a yet unknown signal transduction cascade. A common late stage feature of inherited peripheral neuropathies is the axonal damage, the CMT2B2 gene could be a crucial player in this context.
Journal of the Peripheral Nervous System 01/2005; 10(Supplement 1):77. · 2.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations in the gene encoding N-myc downstream-regulated gene-1 (NDRG1) lead to truncations of the encoded protein and are associated with an autosomal recessive demyelinating neuropathy--hereditary motor and sensory neuropathy-Lom. NDRG1 protein is highly expressed in peripheral nerve and is localized in the cytoplasm of myelinating Schwann cells, including the paranodes and Schmidt-Lanterman incisures. In contrast, sensory and motor neurons as well as their axons lack NDRG1. NDRG1 mRNA levels in developing and injured adult sciatic nerves parallel those of myelin-related genes, indicating that the expression of NDRG1 in myelinating Schwann cells is regulated by axonal interactions. Oligodendrocytes also express NDRG1, and the subtle CNS deficits of affected patients may result from a lack of NDRG1 in these cells. Our data predict that the loss of NDRG1 leads to a Schwann cell autonomous phenotype resulting in demyelination, with secondary axonal loss.
Neurobiology of Disease 12/2004; 17(2):290-9. · 5.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genetic disease mechanisms in the demyelinating peripheral neuropathies Charcot-Marie-Tooth disease type 1A (CMTA) and hereditary neuropathy with liability to pressure palsies (HNPP) as well as transgenic animals with altered PMP22 gene dosage revealed that alterations in PMP22 gene expression have profound effects on the development and maintenance of peripheral nerves. Consequently, the regulation of PMP22 is a crucial aspect in understanding the function of this protein in health and disease. In this study, we dissected and analyzed different cis-acting elements in the 5'-flanking region of the Pmp22 gene in vivo. We found two separate elements that contribute to different aspects of Pmp22 expression. The first is located 5' distally to promoter 1 and is involved in gene regulation during late phases of myelination in development ["late myelination Schwann cell-specific element" (LMSE)] and in remyelination after injury. The second element was identified upstream of promoter 2 and guides Pmp22 expression in sensory neurons. These results suggest that multiple distinct signaling pathways regulating Pmp22 expression in myelination as well as in neurons converge on distinct segments of the PMP22 promoter region. The underlying molecular mechanisms are likely to be crucially involved in the maintenance of the integrity of myelinated peripheral nerves.
Molecular and Cellular Neuroscience 12/2003; 24(3):803-17. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: E-cadherin is thought to mediate intercellular adhesion in the mammalian epidermis and in hair follicles as the adhesive component of adherens junctions. We have tested this role of E-cadherin directly by conditional gene ablation in the mouse. We show that postnatal loss of E-cadherin in keratinocytes leads to a loss of adherens junctions and altered epidermal differentiation without accompanying signs of inflammation. Overall tissue integrity and desmosomal structures were maintained, but skin hair follicles were progressively lost. Tumors were not observed and beta-catenin levels were not strongly altered in the mutant skin. We conclude that E-cadherin is required for maintaining the adhesive properties of adherens junctions in keratinocytes and proper skin differentiation. Furthermore, continuous hair follicle cycling is dependent on E-cadherin.
The EMBO Journal 12/2003; 22(21):5723-33. · 10.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations in the myotubularin (MTM)-related protein 2 (MTMR2) gene are responsible for the severe autosomal recessive neuropathy Charcot-Marie-Tooth disease type 4B1. MTMR2 belongs to the MTM family of dual-specific phosphatases that use phosphatidylinositol (PI) 3,5-bisphosphate [PI(3,5)P2] and PI 3-phosphate [PI(3)P] as their substrate. Because these substrates are localized in the membrane bilayer, membrane targeting of Mtmr2 is an important regulatory mechanism. In hypoosmotically stressed COS cells with increased levels of PI(3,5)P2, Mtmr2 is bound to the membrane of vacuoles formed under these conditions. Using several mutant forms of Mtmr2, we identified two domains that are necessary for membrane association: (i) A pleckstrin homology-GRAM domain; and (ii) a coiled-coil module. Protein-lipid overlay assays show that the pleckstrin homology-GRAM domain binds to PI(3,5)P2 and PI(5)P, a substrate and a product of the Mtmr2 enzyme, respectively. We also demonstrate that Mtmr2 forms a dimer and that the C-terminal coiled-coil is responsible for homodimerization, in addition to membrane association. Our data indicate that phosphoinositide-protein interactions, as well as protein-protein interactions, are necessary for the correct regulation of MTMR2.
Proceedings of the National Academy of Sciences 11/2003; 100(21):12177-82. · 9.81 Impact Factor