an-optogenetic-approach-for-assessing-formation-neuronal-connections

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Nanyang Technological University

We developed a new fluorescence assay for sterol oxidation and used it to study the relationship between free radical-induced sterol oxidation and membrane sterol lateral organization. This assay used dehydroergosterol (DHE) as both a membrane probe and a membrane component. Sterol oxidation was induced by a free radical generator, AAPH (2,2'-azobis(2-amidinopropane)dihydrochloride). Using this new assay, we found that, in unilamellar vesicles composed of DHE and 1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC), the initial rate of DHE oxidation induced by AAPH changed with membrane sterol content in an alternating manner, exhibiting a local maximum at 20.3, 22.2, 25.0, 32.3, and 40.0 mol % DHE. These mole fractions correspond to the critical sterol mole fractions C(r) predicted for maximal sterol superlattice formation. In three-component bilayers composed of POPC, cholesterol, and DHE (fixed at 1 and 5 mol %), the initial rate of AAPH-induced DHE oxidation exhibited a biphasic change whenever the total sterol mole fraction, irrespective of the DHE content, was near C(r), indicating that the correlation between sterol oxidation and sterol superlattice formation revealed in this study is not an artifact due to the use of the fluorescent cholesterol analogue DHE. The alternating variation of AAPH-induced sterol oxidation with sterol content also appeared in multicomponent unilamellar vesicles containing bovine brain sphingomyelins (bbSPM), POPC, and DHE. The present work and our previous study on cholesterol oxidase-induced sterol oxidation [Wang et al. (2004) Biochemistry 43, 2159-2166] suggest that sterol oxidation in general, either by reactive oxygen species or by enzymes, may be regulated by the extent of sterol superlattice in the membrane and thus regulated by the membrane sterol content in a fine-tuning manner.

Role of sterol superlattice in free radical-induced sterol oxidation in lipid membranes.

Lipid rafts are microdomains of plasma membranes enriched in cholesterol and sphingolipids in the outer layer. We determined whether kappa opioid receptors (KOR) in human placenta and FLAG (DYKDDDDK)-tagged human KOR (FLAG-hKOR) expressed in Chinese hamster ovary (CHO) cells are localized in lipid rafts and whether changes in cholesterol contents affect hKOR properties and signaling. Lipid rafts were prepared from placenta membranes and CHO cells expressing FLAG-hKOR using the Na2CO3 method and fractionation through a sucrose density gradient. The majority of the KOR in the placenta and FLAG-hKOR in CHO cells, determined by [3H]diprenorphine binding and/or immunoblotting with an anti-FLAG antibody, was present in low-density fractions, coinciding with high levels of caveolin-1 and cholesterol, markers of lipid rafts, which indicated that the KOR is localized in lipid rafts. Pretreatment with 2% methyl beta-cyclodextrin (MCD) reduced cholesterol content by approximately 48% and changed the cells from spindle-shaped to spherical. MCD treatment disrupted lipid rafts, shifted caveolin-1 and FLAG-hKOR to higher density fractions, increased the affinity of (-)-(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488H) for the hKOR, and greatly increased U50,488H-induced [35S]guanosine 5'-O-(3-thio)triphosphate binding and p42/44 mitogen-activated protein kinase phosphorylation. Cholesterol replenishment reversed all the MCD effects. Caveolin-1 immunoprecipitated with Galphai proteins and MCD treatment reduced caveolin-1 associated with Galphai proteins, which may contribute to the enhanced agonist-induced G protein activation. Caveolin-1 also immunoprecipitated with FLAG-hKOR, but MCD treatment had no effect on the association. Thus, the KOR is located in lipid rafts and its localization in the microdomains greatly affects coupling to G proteins.

Localization of the kappa opioid receptor in lipid rafts.

Opioid receptors have been shown to be located in and regulated by lipid rafts/caveolae in caveolin-rich non-neuronal cells. Here, we found that caveolin-1 level was very low in rat brain and undetectable in NG108-15 cells, which endogenously express delta opioid receptors (DOR). Rat caudate putamen (CPu) membranes, NG108-15 cells and CHO cells stably transfected with FLAG-mouse-DOR (CHO-FLAG-mDOR) were homogenized, sonicated in a detergent-free 0.5M Na(2)CO(3) buffer and fractionated through discontinuous or continuous sucrose density gradients. About 70% of opioid receptors in CPu and DOR in both cell lines were present in low-density (5-20% sucrose) membrane domains enriched in cholesterol and ganglioside M1 (GM1), characteristics of lipid rafts in plasma membranes. In both cells, stimulation with permeable or non-permeable full agonists, but not with partial or inverse agonists, for 30min shifted approximately 25% of DORs out of rafts, by a naloxone-reversible and pertussis toxin-insensitive mechanism, which may undergo internalization. Methyl-beta-cyclodextrin (MCD) treatment greatly reduced cholesterol and shifted DOR to higher density fractions and decreased DPDPE affinities. MCD treatment attenuated DPDPE-induced [(35)S]GTPgammaS binding in CPu and NG108-15 cells, but enhanced it in CHO-FLAG-mDOR cells. In CHO-FLAG-mDOR cells, G(alphai) co-immunoprecipitated with caveolin-1, which was shown to inhibit G(alphai/o), and MCD treatment dramatically reduced the association leading to disinhibition. Thus, although localization in rafts and agonist-induced shift of DOR are independent of caveolin-1, lipid rafts sustain DOR-mediated signaling in caveolin-deficient neuronal cells, but appear to inhibit it in caveolin-enriched non-neuronal cells. Cholesterol-dependent association of caveolin-1 with and the resulting inhibition of G proteins may be a contributing factor.

Cholesterol reduction by methyl-beta-cyclodextrin attenuates the delta opioid receptor-mediated signaling in neuronal cells but enhances it in non-neuronal cells.

The mu opioid receptor (MOR) in the rat and mouse caudate putamen (CPu) and thalamus was demonstrated as diffuse and broad bands by Western blot with a polyclonal antibody against a C-terminal peptide of MOR, which were absent in the cerebellum and brains of MOR-knockout mice. The electrophoretic mobility of MOR differed in the two brain regions with median relative molecular masses (Mr's) of 75 kDa (CPu) vs. 66 kDa (thalamus) for the rat, and 74 kDa (CPu) vs. 63 kDa (thalamus) for the mouse, which was due to its differential N-glycosylation. Rat MOR in CPu was found mainly associated with low-density cholesterol- and ganglioside M1 (GM1)-enriched membrane subdomains (lipid rafts), while the MOR in the thalamus was present in rafts and non-rafts without preference. Cholesterol reduction by methyl-beta-cyclodextrin decreased DAGMO-induced [35S]GTPgammaS binding in rat CPu membranes to a greater extent than in the thalamus membranes.

Brain region-specific N-glycosylation and lipid rafts association of the rat mu opioid receptor.

Lipid rafts are small cholesterol- and glycosphingolipid-enriched membrane subdomains. Here we compared the mu opioid receptor (MOR)-lipid rafts relationship in the rat brain, where neurons have non-caveolae rafts, and in CHO cells stably transfected with HA-rat MOR (CHO-HA-rMOR), which are enriched in caveolae. Membranes of rat caudate putamen (CPu) and thalamus or CHO-HA-rMOR cells were homogenized, sonicated in a detergent-free 0.5 M Na(2)CO(3) buffer and fractionated through sucrose density gradients. Western blot and [(3)H]diprenorphine binding showed that approximately 70% of MOR in CHO-HA-rMOR was present in low-density (5-20% sucrose) fractions enriched in cholesterol and/or ganglioside M1 (GM1) (lipid rafts) in plasma membranes, whereas about 70% and 45% of MOR in CPu and thalamus, respectively, were associated with lipid rafts. Incubation with a saturating concentration of etorphine or morphine at 37 degrees C for 30 min failed to change the MOR location in rafts in CHO-HA-rMOR, indicating that the internalized MOR does not move out of rafts, in contrast to the delta opioid receptor. In vivo, rafts association of MOR in CPu and thalamus was not affected significantly in rats implanted with two 75-mg morphine pellets for 72 h. In addition, cholesterol reduction by methyl-beta-cyclodextrin (MCD) disrupted rafts and shifted MOR to higher density fractions in both CHO-HA-rMOR and CPu membranes. However, MCD treatment had opposite impacts on MOR signaling in the two tissues: it attenuated MOR-mediated [(35)S]GTPgammaS binding in CPu but enhanced it in CHO-HA-rMOR.

Agonist treatment did not affect association of mu opioid receptors with lipid rafts and cholesterol reduction had opposite effects on the receptor-mediated signaling in rat brain and CHO cells.

Hexahistidine ((His)(6)) tags are used to purify genetically engineered proteins. Herein, we describe the construction of a 'turn-on' fluorescent probe system that consists of the fluorescence quencher, Dabcyl, conjugated to (His)(6), and fluorescent tetramethylrhodamine conjugated to nitrilotriacetic acid, which, in the presence of Ni(2+), can bind (His)(6). The system is turned off when Dabcyl-(His)(6) is bound to the fluorescent nitrilotriacetic acid derivative. The binding strength of this system was assessed using electrospray ionization mass spectrometry, fluorescence correlation spectroscopy, and fluorescence intensity distribution analysis-polarization. Although there was no significant enhancement in fluorescence after addition of an equimolar amount of ubiquitin, the fluorescence increased from 14% to 40% of its initial intensity when an equimolar amount of (His)(6)-ubiquitin was added. Therefore, this system should be able to specifically recognize (His)(6)-proteins with good resolution and has the additional advantage that a washing step is not required to remove fluorescent probe, that is, not bound to the (His)(6)-protein.

Construction of a 'turn-on' fluorescent probe system for His-tagged proteins.

A specific protein fluorescent labeling method has been used as a tool for bio-imaging in living cells. We developed a novel system of switching "fluorescent turn on" by the recognition of a fluorescent probe to a hexahistidine-tagged (His-tag) protein. The tetramethyl rhodamine bearing three nitrilotriacetic acids, which was used as a fluorescent probe to target a His-tagged protein, formed a reversible complex with the quencher, (Dabcyl)-conjugated oligohistidines, in the homogeneous solution, causing fluorescence of the fluorophore to be quenched. The complex when applied to living cells (COS-7) expressing His-tagged proteins on the cell surface caused the quencher-conjugated oligohistidines to be dissociated from the complex by specific binding of the fluorescent probe to the tagged protein, resulting in the fluorescent emission. The complex that did not participate in the binding event remained in the quenched state to maintain a low level of background fluorescence.

Su-In Yoon

Lee Kong Chian School of Medicine

Nanyang Technological University

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System