Sucrose Gradient Centrifugation Research Articles

Membrane Vesicles Nucleate Mineralo-organic Nanoparticles and Induce Carbonate Apatite Precipitation in Human Body Fluids

Recent studies indicate that membrane vesicles (MVs) secreted by various cells are associated with human diseases, including arthritis, atherosclerosis, cancer, and chronic kidney disease. The possibility that MVs may induce the formation of mineralo-organic nanoparticles (NPs) and ectopic calcification has not been investigated so far. Here, we isolated MVs ranging in size between 20 and 400 nm from human serum and FBS using ultracentrifugation and sucrose gradient centrifugation. The MV preparations consisted of phospholipid-bound vesicles containing the serum proteins albumin, fetuin-A, and apolipoprotein A1; the mineralization-associated enzyme alkaline phosphatase; and the exosome proteins TNFR1 and CD63. Notably, we observed that MVs induced mineral precipitation following inoculation and incubation in cell culture medium. The mineral precipitates consisted of round, mineralo-organic NPs containing carbonate hydroxyapatite, similar to previous descriptions of the so-called nanobacteria. Annexin V-immunogold staining revealed that the calcium-binding lipid phosphatidylserine (PS) was exposed on the external surface of serum MVs. Treatment of MVs with an anti-PS antibody significantly decreased their mineral seeding activity, suggesting that PS may provide nucleating sites for calcium phosphate deposition on the vesicles. These results indicate that MVs may represent nucleating agents that induce the formation of mineral NPs in body fluids. Given that mineralo-organic NPs represent precursors of calcification in vivo, our results suggest that MVs may initiate ectopic calcification in the human body.

Evidence for the interaction of the human metapneumovirus G and F proteins during virus-like particle formation

BackgroundHuman metapneumovirus (HMPV) is now a major cause of lower respiratory infection in children. Although primary isolation of HMPV has been achieved in several different cell lines, the low level of virus replication and the subsequent recovery of low levels of infectious HMPV have hampered biochemical studies on the virus. These experimental methodologies usually require higher levels of biological material that can be achieved following HMPV infection. In this study we demonstrate that expression of the HMPV F, G and M proteins in mammalian cells leads to HMPV virus-like particles (VLP) formation. This experimental strategy will serve as a model system to allow the process of HMPV virus assembly to be examined.MethodsThe HMPV F, G and M proteins were expressed in mammalian cell lines. Protein cross-linking studies, sucrose gradient centrifugation and in situ imaging was used to examine interactions between the virus proteins. VLP formation was examined using sucrose density gradient centrifugation and electron microscopy analysis.ResultsAnalysis of cells co-expressing the F, G and M proteins demonstrated that these proteins interacted. Furthermore, in cells co-expression the three HMPV proteins the formation VLPs was observed. Image analysis revealed the VLPs had a similar morphology to the filamentous virus morphology that we observed on HMPV-infected cells. The capacity of each protein to initiate VLP formation was examined using a VLP formation assay. Individual expression of each virus protein showed that the G protein was able to form VLPs in the absence of the other virus proteins. Furthermore, co-expression of the G protein with either the M or F proteins facilitated their incorporation into the VLP fraction.ConclusionCo-expression of the F, G and M proteins leads to the formation of VLPs, and that incorporation of the F and M proteins into VLPs is facilitated by their interaction with the G protein. Our data suggests that the G protein plays a central role in VLP formation, and further suggests that the G protein may also play a role in the recruitment of the F and M proteins to sites of virus particle formation during HMPV infection.

Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for Illumina genome sequencing

With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Kruger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis. The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.

Abstract 350: Downregulation of C-terminal Src Kinase (csk) and Csk-binding Protein (cbp) is Associated With Increased Aldosterone-induced C-src Phosphorylation in Shr Vascular Smooth Muscle Cells

c-Src phosphorylation is controlled by the recruitment of enzyme regulators, such as C-terminal Src kinase (CSK) which inhibits Src activity, and interactions with transmembrane adaptors. These complex regulatory mechanisms coordinate activity of c-Src at multiple levels. We previously showed that in aldosterone-stimulated SHR vascular smooth muscle cells (VSMCs), c-Src phosphorylation and its downstream signaling are upregulated. Here we hypothesized that mechanisms underlying vascular c-Src hyperactivation in SHR are related to dysregulated CSK and altered autophosphorylation at Tyr416 and Tyr527 in aldosterone-stimulated SHR cells. Studies were performed in cultured VSMCs from WKY and SHR. C-terminal Src kinase (Csk) cytosol/membrane translocation, Csk-binding protein (CBP), and c-Src phosphorylation were evaluated by western blot. Cholesterol-enriched fractions were obtained by sucrose-gradient centrifugation. Aldosterone (100 nM) induced Tyr527 c-Src phosphorylation (153.5 ± 13.6 %) which locks the kinase in an inactive conformation. This was blunted in SHR cells. Csk is a cytosolic kinase that catalyzes c-Src Tyr527 phosphorylation. ASN (10 uM), a Csk inhibitor, prevented the kinase translocation to the membrane and inhibited Tyr527 c-Src phosphorylation induced by aldosterone in WKY cells. Inhibition of Csk induced an increase in Tyr416 c-Src (180 ± 21%) under basal conditions. In SHR cells, Csk translocation to the membrane was reduced by aldosterone compared with WKY cells. Aldosterone induced an increase in expression (180.3 ± 39 %) and phosphorylation (169 ± 23 %) of the adaptor protein CBP in WKY, but not in SHR cells. Aldosterone stimulation increased Csk trafficking into lipid rafts/caveolae in WKY cells, without affecting the kinase content in the cholesterol-enriched fractions from SHR. Our findings demonstrate that 1) key regulators of c-Src activation by aldosterone, specifically Csk and CBP, are altered in SHR VSMCs; 2) c-Src regulation by aldosterone involves lipid rafts/caveolae. These novel findings suggest that modulation of Csk could be an important strategy to blunt c-Src-dependent aldosterone vascular effects.

Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid

The mature nucleocapsid (NC) of hepatitis B virus containing the relaxed circular (RC) DNA genome can be secreted extracellularly as virions after envelopment with the viral surface proteins or, alternatively, can be disassembled to release RC DNA (i.e., uncoating) into the host cell nucleus to form the covalently closed circular (CCC) DNA, which sustains viral replication and persistence. In contrast, immature NCs containing the viral single-stranded DNA or the pregenomic RNA are incompetent for either envelopment or uncoating. Little is currently known about how mature NCs, and not the immature ones, are specifically selected for these processes. Here, we have carried out a biochemical analysis of the different NC populations upon their separation through sucrose gradient centrifugation. We have found that the maturation of NCs is associated with their destabilization, manifested as increased protease and nuclease sensitivity, altered sedimentation during sucrose gradient centrifugation, and retarded mobility during native agarose gel electrophoresis. Also, three distinct populations of intracellular mature NCs could be differentiated based on these characteristics. Furthermore, mature NCs generated in vitro under cell-free conditions acquired similar properties. These results have thus revealed significant structural changes associated with NC maturation that likely play a role in the selective uncoating of the mature NC for CCC DNA formation and/or its preferential envelopment for virion secretion.

A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification.

BackgroundGarlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors.ResultsIn this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3′-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR.ConclusionsThe expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts.

Expression and assembly of Norwalk virus-like particles in plants using a viral RNA silencing suppressor gene

Binary vector-based transient expression of heterologous proteins in plants is a very attractive strategy due to the short time required for proceeding from planning to expression. However, this expression system is limited by comparatively lower yields due to strong post-transcriptional gene silencing (PTGS) in the host plants. The aim of this study was to optimize a procedure for expression of norovirus virus-like particles (VLPs) in plants using a binary vector with co-expression of a PTGS suppressor to increase the yield of the target protein. The effects of four plant viral PTGS suppressors on protein expression were evaluated using green fluorescent protein (GFP) as a reporter. Constructs for both GFP and PTGS suppressor genes were co-infiltrated in Nicotiana benthamiana plants, and the accumulation of GFP was evaluated. The most effective PTGS suppressor was the 126K protein of Pepper mild mottle virus. Therefore, this suppressor was selected as the norovirus capsid gene co-expression partner for subsequent studies. The construct containing the major (vp1) and minor capsid (vp2) genes with a 3'UTR produced a greater amount of protein than the construct with the major capsid gene alone. Thus, the vp1-vp2-3'UTR and 126K PTGS suppressor constructs were co-infiltrated at middle scale and VLPs were purified by sucrose gradient centrifugation. Proteins of the expected size, specific to the norovirus capsid antibody, were observed by Western blot. VLPs were observed by transmission electron microscopy. It was concluded that protein expression in a binary vector co-expressed with the 126K PTGS suppressor protein enabled superior expression and assembly of norovirus VLPs.

Proteomic characterization of Pseudomonas aeruginosa PAO1 inner membrane

Pseudomonas aeruginosa is a Gram-negative bacterium that can inhabit a wide variety of environments and infect different hosts. Human infections by this microbe are nowadays perceived as a major health problem due notably to its multiresistance. The present data set provides the first description of P. aeruginosa inner membrane proteome. To achieve this, we combined efficient separation of membranes from PAO1 reference strain using discontinuous sucrose gradient centrifugation and MS-based proteomic analysis. A core list of 991 nonredundant proteins was established and analyzed in terms of trans-membrane domains, signal peptide, and lipobox sequence prediction. Furthermore, functional insights into membrane-spanning and membrane-associated protein complexes have been explored. All mass spectrometry data have been deposited in the ProteomeXchange with identifier PXD000107.

Shotgun proteomic analysis of sarcoplasmic reticulum preparations from rabbit skeletal muscle

To obtain a comprehensive understanding of proteins involved in excitation-contraction coupling, a catalog of proteins from sarcoplasmic reticulum (SR) membrane fractions of New Zealand white rabbit skeletal muscle was analyzed by an optimized shotgun proteomic method. Light and heavy SR membrane fractions were obtained by nonlinear sucrose gradient centrifugation and separated by 1DE followed by a highly reproducible, automated LC-MS/MS on the hybrid linear ion trap (LTQ) Orbitrap mass spectrometer. By integrating as low as 1% false discovery rate as one of the features for quality control method, 483 proteins were identified from both of the two independent SR preparations. Proteins involved in calcium release unit complex, including ryanodine receptor 1, dihydropyridine receptor, calmodulin, triadin, junctin, and calsequestrin, were all detected, which offered validation for this protein identification method. Rigorous bioinformatics analysis was performed. Protein pI value, molecular weight range, hydrophobicity index, and transmembrane region were calculated using bioinformatics softwares. Eighty-three proteins were classified as hydrophobic proteins and 175 proteins were recognized as membrane proteins. Based on the proteomic analysis results, we found as the first time that not only transverse tubule but also mitochondrion physically connected to SR. The complete mapping of these proteomes may help in the elucidation of the process of excitation-contraction coupling and excitation-metabolism coupling.

Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Modulates N-Methyl-d-aspartate (NMDA) Receptor-dependent Intracellular Signaling and NMDA-induced Regulation of Postsynaptic Protein Complexes

The lipoprotein receptor LRP1 is essential in neurons of the central nervous system, as was revealed by the analysis of conditional Lrp1-deficient mouse models. The molecular basis of its neuronal functions, however, is still incompletely understood. Here we show by immunocytochemistry, electron microscopy, and postsynaptic density preparation that LRP1 is located postsynaptically. Basal and NMDA-induced phosphorylation of the transcription factor cAMP-response element-binding protein (CREB) as well as NMDA target gene transcription are reduced in LRP1-deficient neurons. In control neurons, NMDA promotes γ-secretase-dependent release of the LRP1 intracellular domain (LRP1-ICD). However, pull-down and chromatin immunoprecipitation (ChIP) assays showed no direct interaction between the LRP1-ICD and either CREB or target gene promoters. On the other hand, NMDA-induced degradation of the postsynaptic scaffold protein PSD-95 was impaired in the absence of LRP1, whereas its ubiquitination was increased, indicating that LRP1 influences the composition of postsynaptic protein complexes. Accordingly, NMDA-induced internalization of the AMPA receptor subunit GluA1 was impaired in LRP1-deficient neurons. These results show a role of LRP1 in the regulation and turnover of synaptic proteins, which may contribute to the reduced dendritic branching and to the neurological phenotype observed in the absence of LRP1.

Folic acid modulates eNOS activity via effects on posttranslational modifications and protein–protein interactions

Folic acid enhances endothelial function and improves outcome in primary prevention of cardiovascular disease. The exact intracellular signalling mechanisms involved remain elusive and were therefore the subject of this study. Particular focus was placed on folic acid-induced changes in posttranslational modifications of endothelial nitric oxide synthase (eNOS). Cultured endothelial cells were exposed to folic acid in the absence or presence of phosphatidylinositol-3' kinase/Akt (PI3K/Akt) inhibitors. The phosphorylation status of eNOS was determined via western blotting. The activities of eNOS and PI3K/Akt were evaluated. The interaction of eNOS with caveolin-1, Heat-Shock Protein 90 and calmodulin was studied using co-immunoprecipitation. Intracellular localisation of eNOS was investigated using sucrose gradient centrifugation and confocal microscopy. Folic acid promoted eNOS dephosphorylation at negative regulatory sites, and increased phosphorylation at positive regulatory sites. Modulation of phosphorylation status was concomitant with increased cGMP concentrations, and PI3K/Akt activity. Inhibition of PI3K/Akt revealed specific roles for this kinase pathway in folic acid-mediated eNOS phosphorylation. Regulatory protein and eNOS protein associations were altered in favour of a positive regulatory effect in the absence of bulk changes in intracellular eNOS localisation. Folic acid-mediated eNOS activation involves the modulation of eNOS phosphorylation status at multiple residues and positive changes in important protein–protein interactions. Such intracellular mechanisms may in part explain improvements in clinical vascular outcome following folic acid treatment.

The 786-0 renal cancer cell-derived exosomes promote angiogenesis by downregulating the expression of hepatocyte cell adhesion molecule

The aims of the current study were to determine whether 786‑0 renal cancer cell‑derived exosomes promote human umbilical vein endothelial cells (HUVECs) to form tubular structures and to uncover the underlying mechanisms associated with this process. Exosomes were extracted and purified using ultrafiltration and sucrose gradient centrifugation and characterized by transmission electron microscopy. Tubular structure formation was observed using the matrigel tubular assay. In addition, an adenovirus vector was used to transfect the hepatocyte cell adhesion molecule (hepaCAM) gene into renal cancer 786‑0 cells. The expression of hepaCAM and vascular endothelial growth factor (VEGF) mRNA and protein was determined by reverse transcription‑polymerase chain reaction and western blot analysis, respectively. Tumor cell‑derived exosomes were observed to significantly increase tubular formation in HUVECs. Following transfection with the hepaCAM gene, VEGF expression in 786‑0 cells was markedly decreased. In HUVECs, exosome treatment increased VEGF mRNA and protein expression, while hepaCAM expression was only decreased at the protein level. In the present study, renal cancer 786‑0 cell‑derived exosomes significantly promoted angiogenesis via upregulation of VEGF expression in HUVECs, which may be induced by the downregulation of hepaCAM.

Abstract 835: hRrp15, a ribosome RNA processing protein, has profound function on nucleoli construction, ribosome biogenesis and cell proliferation.

Abstract Nucleolus is the sub-organelle that controls ribosome biogenesis in cells, and ribosome biogenesis is a highly regulated process that ensures cell growth (increase in biomass) coordinated with cell proliferation. In the nucleolus, ribosomal RNA precursor (47S rRNA) is initially transcribed from rDNA and then subsequently processes into 5.8S, 18S and 28S rRNAs, together with numerous ribosome proteins and nonribosomal factors, to form 60S and 40S pre-ribosomal particles before they are exported to the cytoplasm. A large body of evidence demonstrates that perturbation of rRNA synthesis, process and/or pre-ribosomal factor function impairs ribosome biogenesis and induces cell cycle arrest. This is attributed to nucleolar disruption and diffusion of ribosomal proteins, such as Rpl11 and Rpl5, into the nucleoplasm in which these ribosomal proteins bind to and inhibit MDM2 function, resulting in p53 induction and activation. In this study, we described the identification and characterization of a human protein, hRrp15, that has sequence homology with yeast pre-ribosomal factor, Rrp15p. Previous studies showed that Rrp15p is a ribosome RNA processing protein required for 60S pre-ribosomal particle formation and/or maturation in yeast. Using immunofluorescence and sucrose gradient centrifugation analyses, we demonstrated that hRrp15 was mainly localized at nucleoli in human cells. Depletion of hRrp15 expression disrupted nucleolar structure, inducing diffusion of nucleolar protein, nucleolin, in the nucleus. In contrast to Rrp15p, hRrp15 mainly associated with 60S pre-ribosomal particle but it also presented in 40S pre-ribosomal particle. Consistently, inhibition of hRrp15 expression perturbed nucleolar formation and impaired ribosomal biogenesis in both 60S and 40S pre-ribosomal subunits. In addition, depletion of hRrp15 in human non-transformed cells, RPE1, resulted in the induction of p53 and p21 proteins and cell cycle arrest at G1-G1/S whereas depletion of hRrp15 in human tumor cells, HeLa and MCF7, caused DNA damage response with detectable γ-H2AX and apoptosis. These results indicated that hRrp15 plays a critical role in regulating formation of both 60S and 40S pre-ribosomal particles, ribosome biogenesis, construction of nucleoli, cell growth and cell proliferation in human cells. Hence, hRrp15 may serve as a potential target for cancer therapy. Citation Format: Zhixiong Dong, Changjun Zhu, Wei Jiang. hRrp15, a ribosome RNA processing protein, has profound function on nucleoli construction, ribosome biogenesis and cell proliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 835. doi:10.1158/1538-7445.AM2013-835

Organization of cytochrome P450 system components in the endoplasmic reticulum

P450s are membrane‐bound proteins found in the endoplasmic reticulum (ER). The ER is known to be heterogeneous, containing both ordered and disordered regions. Ordered microdomains are more tightly packed and not readily solubilized with non‐ionic detergents and are called detergent resistant membranes (DRM). The goal of this study is to determine if these microdomains affect P450 localization. The localization of P450 system proteins was examined in rabbit liver microsomes after incubation with 1% Brij 98, followed by sucrose gradient centrifugation. Unlike CYP1A2 (which localizes in the DRM), CYP2E1 proteins localized in non‐DRM and CYP2B4 was found to be equally distributed between both domains. Importantly, for P450 to function, it is necessary for this heme protein to form a complex with its electron donor CPR; however, CPR is found largely in the DRM. Consequently, for CYP2E1 to function, a mechanism for complex formation is required, such as translocation of either CYP2E1 or CPR between the lipid domains, or the interaction of CYP2E1 with the small amount of residual CPR localized in the disordered region, which would limit its activity. These results demonstrate that the components of the P450 system are organized in specific membrane regions, and not randomly distributed throughout in the ER membrane. Supported by ES004344

Decreased nitration of mitochondrial complex I by ROS/RNS scavenging during cardiac ischemia reperfusion injury

Protein nitration is a deleterious post‐translational modification mediated by peroxynitrite during cardiac ischemia reperfusion (I+R) injury. Nitration of mitochondrial complex I (CI) proteins could directly impair mitochondrial function and cardiac recovery. We tested this in 4 groups of guinea pig isolated hearts: time control (TC), I 25 min + R 20 min (IR), IR + 32°C hypothermia only during R (IRH), and IRH + a ROS/RNS scavenging cocktail (IRHS), i.e. MnTBAP, catalase, glutathione, and resveratrol, given only during R. Mitochondria were isolated from hearts at 20 min R, and CI proteins were enriched by sucrose gradient centrifugation. Identity was verified by Western blot with anti‐CI subunit antibodies of NDUFS1/NDUFS9 and purity by Commassie Blue staining on a native gel. Anti‐3‐nitrotyrosine AB was used to detect nitrated CI bands normalized to % density of NDUFS1 in the same blot after stripping. Relative nitration at different kDa's was expressed as % density of TC. Compared to TC (100%), nitrated band density was increased after IR (75 kDa = 156%; 30 kDa = 127%; 25 kDa = 123%; 18 kDa = 128%) and decreased after IRH for 75 kDa (118%) and 25 kDa (102%), and after IRHS for 18 kDa (100%). Orbitrap MS analysis of 30 and 18 kDa bands cut from BB stained gels identified several CI subunits as potential proteins for reduced nitration after IRHS. Residues less nitrated after treatment are potential sites to target for protection.

Methamphetamine and amphetamine influence biophysical properties of dopamine transporter in a phosphorylation – dependent manner

The psychostimulants amphetamine (AMPH) & methamphetamine (METH) increase synaptic dopamine (DA) by competing with DA uptake and stimulating the reverse transport of DA via dopamine transporter (DAT). At maximally effective concentrations, METH releases twice as much [Ca2+]in from internal stores as AMPH. [Ca2+]in responses to both drugs were inhibited by DAT antagonists. We have shown that the intact N‐terminal phosphorylation sites on DAT molecule is required for the enhanced effects of METH on [Ca2+]in mobilization. We used discontinuous sucrose gradient centrifugation and Fluorescence Recovery After Photobleaching (FRAP) to examine the microdomain distribution, diffusion rate (D) and mobile fraction (Mf) of Yellow Fluorescent Protein (YFP)‐tagged DAT (YFP‐DAT), in Chinese Hamster Ovarian cells and mice neurons before and after METH exposure. METH exposure (10μM/10min) increases DAT levels in the raft fraction & slows the lateral mobility of YFP‐DAT at the surface membrane. METH exposure significantly decreased the D and Mf of YFP‐DAT as compared to vehicle control (p < 0.05, n= 15–23 cells). Our ongoing studies examine the hypothesis that METH‐regulation of DAT mobility is due to METH‐induced increase in [Ca2+]in and is dependent on Nphosphorylation of DAT. Funding was provided by DA026947/DA/NIDA and NS071122/NS/NINDS

Cholesterol Dependence of Collagen and Echovirus 1 Trafficking along the Novel α2β1 Integrin Internalization Pathway

We have previously shown that soluble collagen and a human pathogen, echovirus 1 (EV1) cluster α2β1 integrin on the plasma membrane and cause their internalization into cytoplasmic endosomes. Here we show that cholesterol plays a major role not only in the uptake of α2β1 integrin and its ligands but also in the formation of α2 integrin-specific multivesicular bodies (α2-MVBs) and virus infection. EV1 infection and α2β1 integrin internalization were totally halted by low amounts of the cholesterol-aggregating drugs filipin or nystatin. Inhibition of cholesterol synthesis and accumulation of lanosterol after ketoconazole treatment inhibited uptake of collagen, virus and clustered integrin, and prevented formation of multivesicular bodies and virus infection. Loading of lipid starved cells with cholesterol increased infection to some extent but could not completely restore EV1 infection to control levels. Cold Triton X-100 treatment did not solubilize the α2-MVBs suggesting, together with cholesterol labeling, that the cytoplasmic endosomes were enriched in detergent-resistant lipids in contrast to αV integrin labeled control endosomes in the clathrin pathway. Cholesterol aggregation leading to increased ion permeability caused a significant reduction in EV1 uncoating in endosomes as judged by sucrose gradient centrifugation and by neutral red-based uncoating assay. In contrast, the replication step was not dependent on cholesterol in contrast to the reports on several other viruses. In conclusion, our results showed that the integrin internalization pathway is dependent on cholesterol for uptake of collagen, EV1 and integrin, for maturation of endosomal structures and for promoting EV1 uncoating. The results thus provide novel information for developing anti-viral strategies and more insight into collagen and integrin trafficking.

Enrichment of Golgi membranes from HeLa cells by sucrose gradient ultracentrifugation

This is a protocol to extract intact Golgi Membranes from HeLa cells using sucrose gradient centrifugation. This extraction is very useful for several applications including pull-down of Golgi membrane proteins, electron microscopy and reconstitution of protein transport into an isolated system. Protocol adapted from Balch et al. (1984).

Cell-Free Electrophysiological Functional Measurements with Native Membranes of Torpedo Californica Electric Tissue

Objective: Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels which mediate Na+, K+ and Ca2+ conductance. The human muscle subtype α1β1δe is an important target in drug discovery related to anaesthesia or therapeutic approaches of intoxications with organophosphorus compounds. Because of its high degree of homology with the human muscle subtype, αβδγ nAChRs from the Torpedo electric organ are a suitable surrogate. For functional characterisation, cell-free electrophysiological methods based on solid supported membranes (SSM) can be useful in cases where conventional electrophysiology cannot be applied. In this sensor based technique, charge translocation is measured via capacitive coupling of the supporting membrane. To obtain a sufficient current density, multiple parameters have an impact evaluated in this study.Experimental procedures: Crude membranes from frozen electric organ of Torpedo californica were purified with sucrose gradient centrifugation and then adsorbed to SSM varying buffers, average vesicle size and incubation. The activation of nAChR was measured with a SURFE2R workstation allowing automated high throughput measurements. Voltage dependent sodium channels were blocked with ambroxol. Rapid exchange of a non-activating buffer to an activating buffer containing the agonist carbamoylcholine initiated Na+ influx in the membrane vesicles. The results were correlated with the different experimental designs.Results: Depending on the average particle size, zeta potential and incubation, stable and reproducible cholinergic signals were recorded (∼ 1 nA). The use of ambroxol prevented a premature collapse of the sodium gradient. Increasing carbamoylcholine content in the activating solution led to curves with saturation behaviour. However, carbamoylcholine concentrations > 1 mM seemed to induce desensitisation of the receptors.Conclusion: The results presented here demonstrate that functional high-throughput measurements can be performed with native membranes of Torpedo californica electric tissue. Though, maintaining and regeneration of sodium gradient requires sensitive measurements.

Abstract P6-06-03: Caveolin-1: A potential mediator of RhoC GTPase driven Inflammatory breast cancer cell invasion.

Abstract The proposed study focuses on Inflammatory Breast Cancer (IBC), which is one of the most aggressive forms of locally advanced breast cancer. IBC is an understudied disease in terms of identifying various molecular entities that could potentially be responsible for the aggressiveness of this disease. Recent advancements reveal that IBC has a unique molecular profile when compared with non-IBC. Caveolin-1 is found to have opposite expression pattern in IBC versus non-IBC. Our lab has shown that Caveolin-1 is found to be over-expressed in IBC whereas in non-IBC its expression is much reduced compared to normal mammary epithelial cells. Caveolin-1 is known to be associated with specialized lipid rafts in the plasma membrane called ‘caveolae’. ‘Caveolin scaffolding domain (CSD)’ from amino acids 82–101 concentrates variety of signaling molecules such as growth factor receptors, protein kinases, heterotrimeric G proteins and Rho GTPases. Thus it is known as a potential regulator of many signaling pathways. Rho GTPases are members of the Ras superfamily of small GTP binding proteins involved in cytoskeletal rearrangements during cell motility and invasion. Our lab has previously shown that RhoC GTPase is over expressed in IBC and that exogenous expression of RhoC GTPase produces motile and invasive phenotype in human mammary epithelial cells (HMECs) similar to SUM149 IBC cells. Thus the overall objective of this study is to determine if caveolin-1 over expression mediates the RhoC GTPase driven invasive phenotype of IBC via its scaffolding action. The first objective of this study is to elucidate the effect of caveolin-1 over expression on SUM149 IBC cell invasion. This was done by down regulating caveolin-1 to a level that is comparable to human mammary epithelial cells (HMECs) by using si caveolin-1. With the same si caveolin-1 or exogenous CSD synthetic peptide, we performed Matrigel invasion assays followed by cell viability and proliferation assay. Si caveolin-1 did not produce significant changes in cell viability over the period of four days as well as in Ki-67 staining which was used as a proliferation marker. Caveolin-1 down regulation reduced cell invasion by 90% compared to untreated cells. Similarly, exogenous CSD synthetic peptide treatment reduced SUM149 cell invasion significantly. In order to understand caveolin-1 localization in SUM149 cells, we performed immuno gold labeling of caveolin-1 by transmission electron microscopy as well as sucrose gradient centrifugation. Both these methods showed distinct pool of cytoplasmic caveolin-1 in addition to plasma membrane associated caveolin-1. Co-localization of caveolin-1 and RhoC GTPase was studied by co-transfecting cells with GFP-caveolin-1 and RFP-RhoC GTPase. Thus, this data suggests potential role of caveolin-1 over expression in regulating IBC cell invasion. Due to its ability to concentrate variety of signaling molecules, caveolin-1 could act as a principle regulator of several downstream events that make cells motile and invasive through RhoC GTPase activation. Thus studying caveolin-1 localization and effect of its regulation on the signaling pathways could help understand the important molecular mechanisms in IBC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-06-03.