Impaired neural development in a zebrafish model for Lowe syndrome

Lowe syndrome, which is characterized by defects in the central nervous system, eyes and kidneys, is caused by mutation of the phosphoinositide 5-phosphatase OCRL1. The mechanisms by which loss of OCRL1 leads to the phenotypic manifestations of Lowe syndrome are currently unclear, in part, owing to the lack of an animal model that recapitulates the disease phenotype. Here, we describe a zebrafish model for Lowe syndrome using stable and transient suppression of OCRL1 expression. Deficiency of OCRL1, which is enriched in the brain, leads to neurological defects similar to those reported in Lowe syndrome patients, namely increased susceptibility to heat-induced seizures and cystic brain lesions. In OCRL1-deficient embryos, Akt signalling is reduced and there is both increased apoptosis and reduced proliferation, most strikingly in the neural tissue. Rescue experiments indicate that catalytic activity and binding to the vesicle coat protein clathrin are essential for OCRL1 function in these processes. Our results indicate a novel role for OCRL1 in neural development, and support a model whereby dysregulation of phosphoinositide metabolism and clathrin-mediated membrane traffic leads to the neurological symptoms of Lowe syndrome

Affinity Two-Phase Partitioning of Liposomes and Membranes

Affinity two-phase partitioning based on an immunoaffinity sandwich approach for the rapid and selective purification of membranes is presented in this thesis. The method was developed by studying different parameters governing the affinity partitioning of model membranes. To this end, biotinylated liposomes were used. They specifically distributed to the bottom phase of a poly (ethylene glycol)/dextran two-phase system through interactions with the affinity ligand, NeutrAvidin coupled to dextran. Restrictions of the liposomal biotin-NeutrAvidin affinity interaction in the two-phase system were analysed. The immunoaffinity sandwich approach exploits the very strong interaction between NeutrAvidin and biotin and the introduction of specific antibodies (a primary and a biotinylated secondary antibody) makes the method highly selective. As an example, caveolae from different sources were purified by affinity-two-phase partitioning, yielding a material of the same or better purity as when purified by standard procedures. The same approach, employing other selective primary antibodies, should facilitate the purification of other membrane fractions. The use of biotinylated secondary antibodies in the immunoaffinity sandwich approach obviates the need of biotinylating each primary antibody for each application, facilitating the general applicability of the method. A miniaturised version of affinity two-phase partitioning in levitated drops (< 1 µl) was investigated using biotinylated liposomes as model material and NeutrAvidin-dextran as affinity ligand. Several factors affecting the affinity partitioning in a miniature system, including control of evaporation, and addition of minute amounts of material to the drop via special dispensers, were investigated. Phase separation was followed visually by microscopy and phase extraction was performed by specifically made micropipettes. Biotinylated liposomes were partitioned to the PEG-rich phase in the absence of the NeutrAvidin ligand and to the dextran-rich phase in its presence, similarly to their partition in larger systems. This miniaturised technique would allow the separation of membranes from single cells for analysis as well as being suitable for screening of separation conditions

Isolation of Escherichia coli inner membranes by metal affinity two-phase partitioning

As reduction of sample complexity is a central issue in membrane proteomic research, the need for new pre-fractionation methods is significant. Here we present a method for fast and efficient enrichment of Escherichia coli inner membranes expressing a His-tagged integral membrane L-fucose-proton symporter (FucP). An enriched inner membrane fraction was obtained from a crude membrane mixture using affinity two-phase partitioning in combination with nickel-nitrilotri acetic acid (Ni-NTA) immobilized on agarose beads. Due to interaction between the beads and FucP, inner membranes were selectively partitioned to the bottom phase of a polymer/polymer aqueous two-phase system consisting of poly(ethylene glycol) (PEG) and dextran. The partitioning of membranes was monitored by assaying the activity of an inner membrane marker protein and measuring the total protein content in both phases. The enrichment of inner membrane proteins in the dextran phase was also investigated by proteomic methodology, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), trypsin digestion and liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS). Using a high level of significance (99.95%) in the subsequent database search, 36 proteins assigned to the inner membrane were identified in the bottom phase, compared to 29 when using the standard sucrose gradient centrifugation method for inner membrane isolation. Furthermore, metal affinity two-phase partitioning was up to 10 times faster than sucrose gradient centrifugation. The separation conditions in these model experiments provide a basis for the selective isolation of E. coli membranes expressing His-tagged proteins and can therefore facilitate research on such membrane proteomes. (c) 2006 Elsevier B.V. All rights reserved

Affinity partitioning of biotinylated mixed liposomes: Effect of charge on biotin-NeutrAvidin interaction

The partitioning behaviour of biotinylated mixed liposomes in aqueous poly(ethylene glycol)/dextran two-phase systems containing NeutrAvidin-dextran suggests that the biotin-NeutrAvidin affinity interaction is charge dependent. Biotinylated phosphatidylcholine liposomes with a low negative surface charge distributed in the NeutrAvidin-containing bottom phase at neutral pH, but the introduction of additional negative charges by including phosphatidylserine or the surfactant sodium dodecylsulfate in the liposomes caused them to distribute in the poly(ethylene glycol)-rich top phase instead. By gradually lowering the pH of the affinity two-phase system below the isoelectric point (6.3) of NeutrAvidin, negatively charged phosphatidylserine/phosphatidylcholine liposomes increasingly were attracted by NeutrAvidin to the bottom phase. It is suggested that acidic amino acids present at the rim of the biotin-binding pocket of NeutrAvidin may interact electrostatically with charged residues of the closely apposed liposome surface affecting the affinity interaction

Purification of caveolae by affinity two-phase partitioning using biotinylated antibodies and NeutrAvidin-dextran

A new concept for affinity two-phase partitioning was tested. The partitioning was based on the interaction of target membranes with a primary antibody which, in turn, interacted with a biotinylated secondary antibody and NeutrAvidin-dextran in a poly(ethylene glycol)/dextran two-phase system. Caveolae selectively redistributed from the top phase to the NeutrAvidin-dextran-containing bottom phase by employing anti-caveolin as the primary antibody. This immunoaffinity approach was more selective than the established sucrose gradient centrifugation method and resulted in highly purified caveolae from Triton X-100-treated liver and lung plasma membranes. The same approach, employing other selective primary antibodies, should facilitate the purification also of other membrane fractions. (C) 2004 Elsevier Inc. All rights reserved

Affinity partitioning for membrane purification exploiting the biotin-NeutrAvidin interaction - Model study of mixed liposomes and membranes

Biotinylated negatively charged liposomes as well as membranes were affinity partitioned in an aqueous poly(ethylene glycol)-dextran two-phase system using NeutrAvidin conjugated to dextran as affinity ligand. Both liposomes and membranes redistributed from top to bottom phase upon addition of NeutrAvidin-dextran. The presence of 35-60 mM Li2SO4 was necessary both to force the components into the top phase without ligand and for ligand-dependent redistribution into the bottom phase. Attaching biotin via a hexanamidohexanoyl spacer and an increased density of biotin or NeutrAvidin enhanced the affinity separation. The separation conditions in these model experiments provide a basis for affinity partitioning of membranes using other affinity ligands

TbG63, a golgin involved in Golgi architecture in Trypanosoma brucei

10.1242/jcs.014324Journal of Cell Science12191538-1546JNCS

Measurement of phosphoinositides in the zebrafish Danio rerio

Phosphoinositides represent a minor fraction of the total glycerolipids in cells. Despite the fact that phosphoinositides are present in small quantities, they have crucial roles during cell signaling and in regulating numerous intracellular processes. Measuring changes in the levels of different phosphoinositides in animals is difficult, but it is essential in order to define the important functions of specific members of the phosphoinositide family. Here we detail procedures for measuring phosphoinositides in 2-days-postfertilization (2-d.p.f.) embryos in zebrafish (Danio rerio). Both in vivo radiolabeling (using [(32)P]orthophosphate) followed by thin-layer or high-performance liquid chromatography (TLC or HPLC) analysis and specific in vitro phosphorylation assays (using [(32)P]γATP) permit the quantitative measurement of phosphoinositides. Normalization of both measurements can be achieved by the determination of total lipid phosphate in embryos. All the techniques described are relatively inexpensive and accessible to most laboratories with an interest in studying the effect of gene manipulation on phosphoinositide metabolism in zebrafish. All the procedures described herein will take up to 10 working days.</p