Isotope coded protein labeling coupled immunoprecipitation (ICPL-IP): a novel approach for quantitative protein complex analysis from native tissue.

High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms--including humans--are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)(1) with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated ß-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method.

A QUICK screen for Lrrk2 interaction partners--leucine-rich repeat kinase 2 is involved in actin cytoskeleton dynamics.

Mutations in human leucine-rich repeat kinase 2 (Lrrk2), a protein of yet unknown function, are linked to Parkinson's disease caused by degeneration of midbrain dopaminergic neurons. The protein comprises several domains including a GTPase and a kinase domain both affected by several pathogenic mutations. To elucidate the molecular interaction network of endogenous Lrrk2 under stoichiometric constraints, we applied QUICK (quantitative immunoprecipitation combined with knockdown) in NIH3T3 cells. The identified interactome reveals actin isoforms as well as actin-associated proteins involved in actin filament assembly, organization, rearrangement, and maintenance, suggesting that the biological function of Lrrk2 is linked to cytoskeletal dynamics. In fact, we demonstrate Lrrk2 de novo binding to F-actin and its ability to modulate its assembly in vitro. When tested in intact cells, knockdown of Lrrk2 causes morphological alterations in NIH3T3 cells. In developing dopaminergic midbrain primary neurons, Lrrk2 knockdown results in shortened neurite processes, indicating a physiological role of Lrrk2 in cytoskeletal organization and dynamics of dopaminergic neurons. Hence, our results demonstrate that molecular interactions as well as the physiological function of Lrrk2 are closely related to the organization of the actin-based cytoskeleton, a crucial feature of neuronal development and neuron function.

Structural and functional protein network analyses predict novel signaling functions for rhodopsin.

Orchestration of signaling, photoreceptor structural integrity, and maintenance needed for mammalian vision remain enigmatic. By integrating three proteomic data sets, literature mining, computational analyses, and structural information, we have generated a multiscale signal transduction network linked to the visual G protein-coupled receptor (GPCR) rhodopsin, the major protein component of rod outer segments. This network was complemented by domain decomposition of protein-protein interactions and then qualified for mutually exclusive or mutually compatible interactions and ternary complex formation using structural data. The resulting information not only offers a comprehensive view of signal transduction induced by this GPCR but also suggests novel signaling routes to cytoskeleton dynamics and vesicular trafficking, predicting an important level of regulation through small GTPases. Further, it demonstrates a specific disease susceptibility of the core visual pathway due to the uniqueness of its components present mainly in the eye. As a comprehensive multiscale network, it can serve as a basis to elucidate the physiological principles of photoreceptor function, identify potential disease-associated genes and proteins, and guide the development of therapies that target specific branches of the signaling pathway.

GDNF family ligands trigger indirect neuroprotective signaling in retinal glial cells.

Apoptotic cell death of photoreceptors is the final event leading to blindness in the heterogeneous group of inherited retinal degenerations. GDNF (glial cell-line-derived neurotrophic factor) was found to rescue photoreceptor function and survival very effectively in an animal model of retinal degeneration (M. Frasson, S. Picaud, T. Leveillard, M. Simonutti, S. Mohand-Said, H. Dreyfus, D. Hicks, and J. Sahel, Investig. Ophthalmol. Vis. Sci. 40:2724-2734, 1999). However, the cellular mechanism of GDNF action remained unresolved. We show here that in porcine retina, GDNF receptors GFRalpha-1 and RET are expressed on retinal Mueller glial cells (RMG) but not on photoreceptors. Additionally, RMG express the receptors for the GDNF family members artemin and neurturin (GFRalpha-2 and GFRalpha-3). We further investigated GDNF-, artemin-, and neurturin-induced signaling in isolated primary RMG and demonstrate three intracellular cascades, which are activated in vitro: MEK/ERK, stress-activated protein kinase (SAPK), and PKB/AKT pathways with different kinetics in dependence on stimulating GFL. We correlate the findings to intact porcine retina, where GDNF induces phosphorylation of ERK in the perinuclear region of RMG located in the inner nuclear layer. GDNF signaling resulted in transcriptional upregulation of FGF-2, which in turn was found to support photoreceptor survival in an in vitro assay. We provide here a detailed model of GDNF-induced signaling in mammalian retina and propose that the GDNF-induced rescue effect on mutated photoreceptors is an indirect effect mediated by retinal Mueller glial cells.

16-BAC/SDS-PAGE analysis of membrane proteins of yeast mitochondria purified by free flow electrophoresis.

Mitochondria are essential organelles in cellular metabolism. These organelles are bounded by two membranes, the outer and inner membrane. Especially the inner membrane comprises a high content of proteins, for example, the protein complexes of the respiratory chain. High-resolution separation and analysis of such membrane proteins, for example, by two-dimensional gel electrophoresis (2-DE), is hampered by their hydrophobicity and tendency for aggregation. Here, we describe the separation of mitochondrial membrane proteins of Saccharomyces cerevisiae by 16-benzyldimethyl-n-hexadecylammonium chloride/sodium dodecyl sulfate polyacrylamide gel electrophoresis (16-BAC/SDS-PAGE). This method enables the separation of membrane proteins owing to the solubilizing power of the ionic detergents 16-BAC and SDS, respectively. Mitochondria were isolated from yeast cultures by differential centrifugation and were further purified by free flow electrophoresis (FFE) in zone-electrophoretic mode (ZE). Subsequently, membrane proteins from ZE-FFE-purified mitochondria were enriched by carbonate extraction and subjected to 16-BAC/SDS-PAGE. The resulting protein spot patterns were visualized by a highly sensitive fluorescence stain with ruthenium-II-bathophenantroline disulfonate chelate (RuBP), and by colloidal Coomassie staining. Proteins were identified by Maldi-Tof mass spectrometry and peptide mass fingerprinting.

Delta-like 1 participates in the specification of ventral midbrain progenitor derived dopaminergic neurons.

Delta-like 1 (Dlk1), a member of the Delta/Notch protein family, is expressed in the mouse ventral midbrain (VM) as early as embryonic day 11.5 (E11.5) followed by exclusive expression in tyrosine 3-monooxygenase (TH) positive neurons from E12.5 onwards. To further elucidate the yet unknown function of Dlk1 in VM neuron development, we investigated the effect of soluble Dlk1 protein as well as the intrinsic Dlk1 function in the course of VM progenitor expansion and dopaminergic (DA) neuron differentiation in vitro. Dlk1 treatment during expansion increased DA progenitor proliferation and the proportion of NR4A2+ neurons expressing TH after differentiation, whereas Dlk1 treatment during the course of DA precursor differentiation did not alter TH+ neuron counts. In contrast, silencing of endogenously expressed Dlk1 prior to DA precursor differentiation partially prevented the expression of DA neuron markers, which was not accompanied with alteration of overall or local proliferation. Due to the latter finding in combination with the absence of Dlk1 negative DA neurons in differentiated cultures, we suggest that Dlk1 expression might have a permissive effect on DA neuron differentiation in vitro. The study presented here is the first publication identifying Dlk1 effects on ventral midbrain-derived DA precursor differentiation.

Purification of Saccharomyces cerevisiae mitochondria by zone electrophoresis in a free flow device.

This chapter describes the isolation of yeast mitochondria by differential centrifugation followed by mitochondrial purification through zone electrophoresis (ZE) using a free flow device (FFE). Starting from a yeast colony, cultures are grown under respiratory conditions to logarithmic phase. Cells are collected, their cell walls enzymatically disintegrated and the resulting spheroplasts are homogenized. Mitochondria are pre-fractionated from this homogenate by differential centrifugation. With the focus on further purification, pre-fractionated mitochondria are subjected to ZE-FFE. In ZE-FFE, mitochondria are transported with the buffer flow through the separation chamber and purified from contaminants by specific deflection through a perpendicularly oriented electric field. The purified mitochondria can be collected by centrifugation and used for further experiments and analysis such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, 2-DE or electron microscopy.

Basic fibroblast and epidermal growth factors stimulate survival in adult porcine photoreceptor cell cultures.

PURPOSE: To investigate the effects of basic fibroblast and epidermal growth factor (FGF2 and EGF, respectively) on the survival and phenotypic expression of photoreceptors isolated from adult mammalian retinas. METHODS: Primary cultures highly enriched in photoreceptors were prepared from adult domestic pig retinas and maintained in chemically defined medium. Cell culture composition was characterized through the use of specific antibody markers of retinal neurons, and neuronal survival was quantified through viability assays as a function of time in the presence or absence of different doses of FGF2 and EGF. Western blot analysis of phosphotyrosine residues was used to monitor activation of FGF2 and EGF signaling pathways. RESULTS: Reproducible survival of adult pig rod and cone photoreceptors was obtained for approximately 2 weeks in vitro, with the continued expression of rod opsin, recoverin, S-antigen, cone arrestin, and neuron-specific enolase. Purity of cultures was routinely more than 95% photoreceptors, with a rod-cone ratio of 2:3.1. Photoreceptor survival was stable for the initial week, decreasing slowly during the second, with rapid cell loss occurring thereafter. In the presence of FGF2 (20 ng/mL), the percentage of photoreceptor survival during the second week in culture was statistically significantly different, at least two times higher than in control experiments. Photoreceptor survival correlated directly with increasing concentrations of FGF2, and also of EGF. Combined treatment with FGF2 and EGF did not induce higher survival than either factor alone. There was no detectable selective loss of rods or cones in the experimental model. Phosphotyrosine immunoblots after stimulation of cultures with FGF2 and EGF revealed time-dependent appearance of multiple immunoreactive bands. CONCLUSIONS: The adult pig photoreceptor culture in the current study exhibits reproducible neuronal survival in vitro and represents a useful novel experimental system for the study of potential neuroprotective effects and signaling pathways of neurotrophic factors such as FGF2 and EGF in fully adult higher mammalian retina.

Fibroblast growth factor receptor (FGFR) and candidate signaling molecule distribution within rat and human retina.

PURPOSE: To map the expression and distribution of FGFR and potential FGFR-related signaling molecules within rat and human retina. METHODS: Sections of postnatal 5 day old and adult rat, and aged human retina, and cell cultures prepared from selected cell populations of young rat retina, were immunolabeled with specific antisera to FGFR (FGFR-1, -2, -3, and -4) or candidate signaling molecules [phospholipase Cg1 (PLCg1), son of sevenless 1 and 2 (SOS1, SOS2), extracellular signal-regulated kinase 1 and 2 (ERK1/2), protein tyrosine phosphatase (SH-PTP2) and SH2-containing protein (Shc)], and with multiple retinal cell-type specific antibodies. Controls were conducted using primary antisera pre-adsorbed with the corresponding immunizing peptide. RESULTS: All FGFR antisera showed strong labeling of inner retina [inner nuclear layer, inner plexiform layer and ganglion cell layer (INL, IPL and GCL respectively)] in rat and human retina, although there were distinct differences in individual patterns. FGFR-3 was particularly intense in ganglion cell bodies and dendrites, and was absent from photoreceptors and bipolar cells in vitro. FGFR-1 and FGFR-4 also labeled the outer nuclear layer (ONL), more intensely in adult than in young tissue, and FGFR-4 was especially prominent within inner segments. FGFR-2 and -3 were only weakly expressed in the ONL, but FGFR-2 showed specific labeling of cone outer segments in human retina. Candidate FGFR-signaling molecules also showed generally higher expression in the inner than outer retina in the different samples. Shc immunolabeling was apparent in the GCL and nascent photoreceptor outer segments in young and adult retina. SOS1 expression was much more intense than SOS2 in the ONL, although the latter showed selective intense staining of a sub-population in the INL and GCL. These ex vivo data were confirmed in cultures prepared from young rat retina. Pure photoreceptor cultures exhibited strong expression of FGFR-1 and -4, and faint expression of FGFR-2 and -3. In mixed inner retinal cultures, anti-FGFR-1 labeled neurons and Müller glia with equal intensity, while the other FGFR antisera showed preferential staining of neurons. FGFR-3 was strongly expressed by ganglion and amacrine cells but not by other types. Signaling molecules showed widespread expression, but of variable intensity, in all cells. All control experiments using corresponding peptide pre-adsorption led to complete removal of immunostaining. CONCLUSIONS: Rat and human retinal cells showed a largely similar, widespread expression of multiple FGFR and candidate FGFR-related signaling molecules. Distinct differences in development, species, cell- and sub-cell type distribution were apparent, suggesting that specific FGFR/FGF ligands and transduction pathways may operate in different cells.

Disruption of intraflagellar protein transport in photoreceptor cilia causes Leber congenital amaurosis in humans and mice.

The mutations that cause Leber congenital amaurosis (LCA) lead to photoreceptor cell death at an early age, causing childhood blindness. To unravel the molecular basis of LCA, we analyzed how mutations in LCA5 affect the connectivity of the encoded protein lebercilin at the interactome level. In photoreceptors, lebercilin is uniquely localized at the cilium that bridges the inner and outer segments. Using a generally applicable affinity proteomics approach, we showed that lebercilin specifically interacted with the intraflagellar transport (IFT) machinery in HEK293T cells. This interaction disappeared when 2 human LCA-associated lebercilin mutations were introduced, implicating a specific disruption of IFT-dependent protein transport, an evolutionarily conserved basic mechanism found in all cilia. Lca5 inactivation in mice led to partial displacement of opsins and light-induced translocation of arrestin from photoreceptor outer segments. This was consistent with a defect in IFT at the connecting cilium, leading to failure of proper outer segment formation and subsequent photoreceptor degeneration. These data suggest that lebercilin functions as an integral element of selective protein transport through photoreceptor cilia and provide a molecular demonstration that disrupted IFT can lead to LCA.

Two-dimensional electrophoresis of membrane proteins.

One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples that applies isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, has a strong bias against membrane proteins. This review describes two-dimensional electrophoretic techniques that can be used to separate membrane proteins. Alternative methods for performing conventional 2-DE are highlighted; these involve replacing the IEF with electrophoresis using cationic detergents, namely 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and cetyl trimethyl ammonium bromide (CTAB), or the anionic detergent SDS. Finally, the separation of native membrane protein complexes through the application of blue and clear native gel electrophoresis (BN/CN-PAGE) is reviewed, as well as the free-flow electrophoresis (FFE) of membranes.

The Parkinson disease causing LRRK2 mutation I2020T is associated with increased kinase activity.

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) have been recently identified in families with autosomal dominant late-onset Parkinson disease (PD). The LRRK2 protein consists of multiple domains and belongs to the Roco family, a novel group of the Ras/GTPase superfamily. Besides the GTPase (Roc) domain, it contains a predicted kinase domain, with homology to MAP kinase kinase kinases. Using cell fractionation and immunofluorescence microscopy, we show that LRRK2 is localized in the cytoplasm and is associated with cellular membrane structures. The purified LRRK2 protein demonstrates autokinase activity. The disease-associated I2020T mutant shows a significant increase in autophosphorylation of approximately 40% in comparison to wild-type protein in vitro. This suggests that the pathology of PD caused by the I2020T mutation is associated with an increase rather than a loss in LRRK2 kinase activity.

Possible involvement of a fibroblast growth factor 9 (FGF9)-FGF receptor-3-mediated pathway in adult pig retinal ganglion cell survival in vitro.

The expression and potential roles of fibroblast growth factors (FGF) and their cognate FGF receptors (FGFR) in adult mammalian retinal ganglion cells (RGC) are poorly known. We show that FGFR-3 and FGFR-4 are especially pronounced on RGC and amacrine cell bodies in adult pig inner retinae both in vivo and in vitro. Western blotting revealed distinct profiles for each receptor. Expression of each FGFR and effects of the preferred ligand for FGFR-3, FGF9, upon RGC survival and neurite outgrowth were examined in primary retinal cell cultures: whereas there was no stimulation of neuritogenesis, RGC survival was promoted in a dose-dependent manner (ED(50) approximately 500 pg/ml, mean maximal increase of 60%) and could be completely blocked by addition of FGF9 neutralising antibody. Experiments with three additional FGF (FGF1, FGF2, and FGF4) showed no stimulation of RGC survival above control levels. Taken together, these data suggest that the ligand-receptor couple FGF9-FGFR-3 may function to promote survival of adult mammalian RGC, and their application might be beneficial in retinal degenerative diseases such as glaucoma.