Plasma Membrane Marker Research Articles

Imaging the Alphavirus Exit Pathway

Alphaviruses are small enveloped RNA viruses with highly organized structures that exclude host cell proteins. They contain an internal nucleocapsid and an external lattice of the viral E2 and E1 transmembrane proteins. Alphaviruses bud from the plasma membrane (PM), but the process and dynamics of alphavirus assembly and budding are poorly understood. Here we generated Sindbis viruses (SINVs) with fluorescent protein labels on the E2 envelope protein and exploited them to characterize virus assembly and budding in living cells. During virus infection, E2 became enriched in localized patches on the PM and in filopodium-like extensions. These E2-labeled patches and extensions contained all of the viral structural proteins. Correlative light and electron microscopy studies established that the patches and extensions colocalized with virus budding structures, while light microscopy showed that they excluded a freely diffusing PM marker protein. Exclusion required the interaction of the E2 protein with the capsid protein, a critical step in virus budding, and was associated with the immobilization of the envelope proteins on the cell surface. Virus infection induced two distinct types of extensions: tubulin-negative extensions that were ∼2 to 4 μm in length and excluded the PM marker, and tubulin-positive extensions that were >10 μm long, contained the PM marker, and could transfer virus particles to noninfected cells. Tubulin-positive extensions were selectively reduced in cells infected with a nonbudding SINV mutant. Together, our data support a model in which alphavirus infection induces reorganization of the PM and cytoskeleton, leading to virus budding from specialized sites. Alphaviruses are important and widely distributed human pathogens for which vaccines and antiviral therapies are urgently needed. These small highly organized viruses bud from the host cell PM. Virus assembly and budding are critical but little understood steps in the alphavirus life cycle. We developed alphaviruses with fluorescent protein tags on one of the viral membrane (envelope) proteins and used a variety of microscopy techniques to follow the envelope protein and a host cell PM protein during budding. We showed that alphavirus infection induced the formation of patches and extensions on the PM where the envelope proteins accumulate. These sites excluded other PM proteins and correlated with virus budding structures. Exclusion of PM proteins required specific interactions of the viral envelope proteins with the internal capsid protein. Together, our data indicate that alphaviruses extensively reorganize the cell surface and cytoskeleton to promote their assembly and budding.

A novel cytoplasmic tail fqqi motif mediates internalization and intracellular trafficking of guanylyl‐cyclase/natriuretic peptide receptor‐A (539.4)

Cardiac hormone, atrial natriuretic peptide (ANP) modulates blood pressure and blood volume by activation of guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA), which produces the second messenger cGMP. The aim of the present study was to examine the role of small sequence motifs in the internalization and intracellular trafficking pathway of NPRA by immunofluorescence confocal microscopy. A chimeric protein of enhanced green fluorescence protein (eGFP) and NPRA (eGFP‐NPRA) was used to study the NPRA trafficking in the intact mouse mesangial cells (MMCs). We have identified a FQQI (Phe790, Gln791, Gln792 and Ile793) motif in the cytoplasmic tail of NPRA sequence and mutated with alanine residues (FQQI/AQQA) and cells were transiently transfected with both constructs. In the ANP‐treated cells, colocalization of wild‐type eGFP‐NPRA with pan‐Cadherin, a plasma membrane marker, indicated that internalization of receptor accounted for 59.2%, 70.0%, 71.8%, and 77.4% at 5, 10, 15, and 30 min, respectively of total fluorescence intensity compared with untreated cells. Colocalization of eGFP‐NPRA with early endosome antigen‐1 (EEA‐1) marker was maximum at 5 min (64.5%), and gradually decreased at 30 min (24.4%). However, the mutation of FQQI/AQQA decreased the receptor internalization by 45% (P< 0.001) and the receptor colocalization into endosome by 58% (P<0.001), respectively, compared with wild type receptor. Our, study suggest that a novel FQQI motif in the cytoplasmic tail mediates the internalization and intracellular trafficking of NPRA, which plays critical roles in hypertension and cardiovascular homeostasis.Grant Funding Source: Supported by the NIH grant (HL57531)

Sweet taste of cell death: role of carbohydrate recognition systems

T he number of cellular and molecular markers of apoptosis described in cytoplasm, nucleus and mitochondria, is increasing yearly. Howe ver, up to recent time, plasma membrane (PM) markers stayed to be less studied [1], and only phosphatidylserine (PS) externalization has been well characterized in that compartment, and already proposed for application in biolo gy studies and medical diagnostics [2]. It should be stressed that PM markers of apoptosis possess some important advantages compared to all other markers of this phenomenon, since they are better accessible to extra-cellular interactions without disturbing cell integrity. Since PM markers of apoptotic cells can be recognized by the phagocytic and neighboring epithelial cells, they are very important for efficient elimination of apoptotic cells from tissues and organs. Cell death was shown to be accompanied by time-dependent sequence of intracellular events, starting with a trigger signal and ending with a complete removal of dying cells by their neighbors and macrophages. It is discussed since when one can consider the cell “dead” or “apoptotic”. The organism senses the dying cells by a change of their surface markers and by a release of particular intercellular molecules [3] also related to changes in plasma membrane. Thus, the PM-derived apoptosis markers seem to be the most “physiological” measure of whether the cell is alive, dying, or already dead (necrotic). Recently, glycoproteins (GPs) integrated in the PM of apoptotic cells, were addressed by the investigators searching for novel cell surface markers of apoptosis. The carbohydrate moiety of plasma membrane GPs, unlike the DNA or proteins, does not need a specific matrix for its synthesis. Besides, unlike 3′→5′ phospodiester bonds in DNA, or peptide bonds in proteins, two carbohydrate molecules can be joined together by eleven different bond combinations providing a huge number of possible oligosaccharides and much higher information potential that is perfectly described by H.-J. Gabius [4]. Carbohydrate binding proteins, the lectins, have demonstrated to be a powerful tool for recognizing different carbohydrate moieties. They were discovered over 100 years ago, and hundreds of lectins with various carbohydrate specificity and different functions were described in plants and animals [5, 6]. A principal function of lectin-carbohydrate interaction is well expressed at the immune response [7, 8], including autoimmunity [9], and cell elimination from the organism [10]. It was hypothesized [11], that such interaction could be an evolutionary ancestor of the antibody-antigen interaction.

Early sequence of events triggered by the interaction ofNeisseria meningitidiswith endothelial cells

Neisseria meningitidis is a bacterium responsible for severe sepsis and meningitis. Following type IV pilus-mediated adhesion to endothelial cells, bacteria proliferating on the cellular surface trigger a potent cellular response that enhances the ability of adhering bacteria to resist the mechanical forces generated by the blood flow. This response is characterized by the formation of numerous 100 nm wide membrane protrusions morphologically related to filopodia. Here, a high-resolution quantitative live-cell fluorescence microscopy procedure was designed and used to study this process. A farnesylated plasma membrane marker was first detected only a few seconds after bacterial contact, rapidly followed by actin cytoskeleton reorganization and bulk cytoplasm accumulation. The bacterial type IV pili-associated minor pilin PilV is necessary for the initiation of this cascade. Plasma membrane composition is a key factor as cholesterol depletion with methyl-β-cyclodextrin completely blocks the initiation of the cellular response. In contrast membrane deformation does not require the actin cytoskeleton. Strikingly, plasma membrane remodelling undermicrocolonies is also independent of common intracellular signalling pathways as cellular ATP depletion is not inhibitory. This study shows that bacteria-induced plasma membrane reorganization is a rapid event driven by a direct cross-talk between type IV pili and the plasma membrane rather than by the activation of an intracellular signalling pathway that would lead to actin remodelling.

Structural and functional alterations in the atrioventricular node and atrioventricular ring tissue in ischaemia-induced heart failure.

Heart failure (HF) causes dysfunction of the atrioventricular node (AVN) - first or second-degree heart block is a risk factor for sudden cardiac death in HF patients. The aim of the study was to determine if HF causes remodelling of the AVN and right atrioventricular ring (RAVR). HF was induced in rats (n=4) by ligation of the proximal left coronary artery, which resulted in a large infarct of the left ventricle. Sham-operated rats (n=4) were used as controls. Eight weeks after surgery, functional experiments were performed and the hearts were frozen. The body weight of HF rats was similar to control rats, but the mean heart weight of HF rats was significantly enlarged. In HF rats compared to controls, the left ventricle was dilated, left ventricular end-diastolic pressure elevated (21.0 ± 0.6 and 5.4 ± 0.2 mm Hg), left ventricular ejection fraction reduced (0.2 ± 0.02 and 0.5 ± 0.02) and left ventricular end-systolic pressure reduced (102 ± 4.2 and 127 ± 3.1 mm Hg). In HF rats, the in vivo and in vitro PR intervals were increased (41% and 20%), as was the Wenckebach cycle length, indicative of AVN dysfunction. The collagen content was significantly increased in the AVN and RAVR indicating fibrosis. Immunolabelling of caveolin3 (cell membrane marker) showed that there was hypertrophy in HF (cell diameter was increased by 63%, 39% in AVN, RAVR). The TUNEL assay showed that the myocytes of the AVN and RAVR in HF undergo apoptotic cell death. Immunolabelling showed that expression of HCN4 was significantly decreased in the AVN and RAVR (43% and 47%) in HF. We conclude that in HF there is remodelling of the AVN and RAVR and this remodelling may explain the AVN dysfunction.

Potential Significance of EMP3 in Patients with Upper Urinary Tract Urothelial Carcinoma: Crosstalk with ErbB2-PI3K-Akt Pathway

Upper urinary tract (pyelocalyceal cavities and ureter) urothelial carcinoma is a relatively rare neoplastic disease. Although diagnosis and treatment of this tumor variant have improved significantly, accurate risk stratification remains a challenge. To identify the putative oncogene involved in urothelial carcinoma progression we performed bioinformatics guided experimental investigation targeting chromosome 19q13. We investigated the effects of EMP3 on cancer cell growth, migration and adhesion in transfection and siRNA experiments in vitro. Crosstalk of integrins or ErbB2 with EMP3 was examined by reverse transcriptase-polymerase chain reaction and immunoblot. The potential involvement of epigenetic alterations of EMP3 in vitro and in vivo was analyzed by methylation specific polymerase chain reaction. To validate clinical relevance we measured EMP3 expression at the mRNA and protein levels in a cohort of 77 patients with upper urinary tract urothelial carcinoma and compared prognostic significance in relation to that of ErbB2 expression. We noted functional crosstalk between ErbB2 and EMP3 in vitro. EMP3 over expression promoted cancer cell proliferation and migration but suppressed cell adhesion in vitro. EMP3 activated the ErbB2-PI3K-AKT pathway to increase cell growth in vitro. In the clinical cohort Kaplan-Meier survival estimates showed that ErbB2 and EMP3 co-expression was the most important indicator of progression-free and metastasis-free survival in patients with upper urinary tract urothelial carcinoma (log rank test p = 0.018 and 0.04, respectively). EMP3 is an important prognostic indicator for selecting patients with upper urinary tract urothelial carcinoma for more intensive therapy. EMP3 is an innovative co-targeting candidate for designing ErbB2 based cancer therapy.

The RNA/DNA‐binding protein PSF relocates to cell membrane and contributes cells' sensitivity to antitumor drug, doxorubicin

Cell surface proteins play an important role in multidrug resistance (MDR). However, the identification involving chemoresistant features for cell surface proteins is a challenge. To identify potential cell membrane markers in hematologic cancer MDR, we used a cell- and antibody-based strategy of subtractive immunization coupled with cell surface comparative screening of leukemia cell lines from sensitive HL60 and resistant HL60/DOX cells. Fifty one antibodies that recognized the cell surface proteins expressed differently between the two cell lines were generated. One of them, the McAb-5D12 not only recognizes its antigen but also block its function. Comparative analysis of immunofluorescence, flow cytometry, and mass spectrum analysis validated that the membrane antigen of McAb-5D12 is a nucleoprotein-polypyrimidine tract binding protein associated splicing factor, PSF. Our results identified that PSF overexpressed on the membrane of sensitive cells compared with resistant cells and its relocation from the nuclear to the cell surface was common in hematological malignancy cell lines and marrow of leukemia patients. Furthermore, we found that cell surface PSF contributed to cell sensitivity by inhibiting cell proliferation. The results represent a novel and potentially useful biomarker for MDR prediction. The strategy enables the correlation of expression levels and functions of cell surface protein with some cell-drug response traits by using antibodies.

Therapy‐induced enrichment of putative lung cancer stem‐like cells

Tumour drug resistance is a major issue in the management of lung cancer patients as almost all lung tumours are either intrinsically resistant or quickly develop acquired resistance to chemotherapeutic drugs. Cancer drug resistance has recently been linked, at least in part, to the existence of cancer stem-like cells (CSLCs), a small sub-population of cells within the tumour that possess stem-like properties. CSLCs are often isolated by fluorescence activated cell sorting (FACS) according to the expression of certain stem-like cell membrane markers. Conflicting results regarding the specificity of particular stem cell surface markers for isolating CSLCs have, however, been recently reported. Therefore, alternative strategies enabling the identification and study of CSLCs should be considered, particularly in tumour types where appropriate stem cell markers are not well established and validated, like in lung cancer. In this article, we review data indicating therapy-selection as a valid approach for putative lung CSLCs enrichment. We believe that this strategy would be determinant for correctly assessing and characterising the sub-populations of CSLCs that are able to survive chemo or radiotherapy regimens and, at the same time, also have the ability to recapitulate and sustain tumour growth. Using therapy-induced enrichment of CSLCs may, therefore, prove to be an extremely useful method for studying CSLCs and provide new clues regarding potential therapeutic targets for their efficient elimination, which will undoubtedly play a decisive role in improving lung cancer patients' survival.

Plasma Membrane Density of GABAB-R1a, GABAB-R1b, GABA-R2 and Trimeric G-proteins in the Course of Postnatal Development of Rat Brain Cortex

With the aim to understand the onset of expression and developmental profile of plasma membrane (PM) content /density of crucial components of GABA(B)-R signaling cascade, GABA(B)-R1a, GABA(B)-R1b, GABA(B)-R2, G(i)1/G(i)2alpha, G(i)3alpha, G(o)alpha, G(z)alpha and Gbeta subunit proteins were determined by quantitative immunoblotting and compared in PM isolated from brain cortex of rats of different ages: between postnatal-day-1 (PD1) and 90 (PD90). PM density of GABA(B)-R1a, GABA(B)-R2, G(i)1/G(i)2alpha, G(i)3alpha, G(o)alpha, G(z)alpha and Gbeta was high already at birth and further development was reflected in parallel decrease of both GABA(B)-R1a and GABA(B)-R2 subunits. The major decrease of GABA(B)-R1a and GABA(B)-R2 occurred between the birth and PD15: to 55 % (R1a, **) and 51 % (R2, **), respectively. Contrarily, PM level of the cognate G-proteins G(i)1/G(i)2alpha, G(i)3alpha, G(o)alpha, G(z)alpha and Gbeta was unchanged in the course of the whole postnatal period of brain cortex development. Maturation of GABA(B)-R cascade was substantially different from ontogenetic profile of prototypical plasma membrane marker, Na, K-ATPase, which was low at birth and further development was reflected in continuous increase of PM density of this enzyme. Major change occurred between the birth and PD25. In adult rats, membrane content of Na, K-ATPase was 3-times higher than around the birth.

Abstract 282: Aromatic Residue-based Tetrapeptide Fqqi Motif Mediates Internalization and Trafficking of Guanylyl-cyclase/natriuretic Peptide Receptor-a in Mouse Mesangial Cells

Atrial natriuretic peptide (ANP) is a cardiac hormone that modulates blood pressure and blood volume by activation of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) and subsequent generation of intracellular second messenger cGMP. The aim of the present study was to examine the role of small sequence motifs in the internalization and trafficking pathway of NPRA by immunofluorescence confocal microscopy. A chimeric protein of enhanced green fluorescence protein (eGFP) and NPRA (eGFP-NPRA) was used to study the dynamics of NPRA trafficking in the intact mouse mesangial cells (MMCs). We have identified a FQQI (Phe 790 , Gln 791 , Gln 792 and Ile 793 ) motif in the carboxyl-terminal domain of NPRA sequence and mutated with alanine residues (FQQI/AQQA) using QuickChange II site-directed mutagenesis kit. Cells were transiently transfected with mutated and wild-type constructs and cultured at 37 0 C in an atmosphere of 5% CO 2 and 95% O 2 . In the ANP-treated cells, colocalization of wild-type eGFP-NPRA with pan-Cadherin, a plasma membrane marker, indicated that internalization of receptor accounted for 59.2%±3.4, 70.0%±4.3, 71.8%±4.3, and 77.4%±3.2 at 5, 10, 15, and 30 min, respectively of total fluorescence intensity compared with controls. Colocalization of eGFP-NPRA with early endosome antigen-1 (EEA-1) marker was maximum at 5 min (64.5%±2.3), and gradually decreased at 30 min (24.4%±3.8). However, the mutation of FQQI/AQQA decreased the receptor internalization by 45% ( P< 0.001) and the receptor colocalization in to endosome by 58% ( P <0.0001), respectively, compared with wild type receptor. After ANP treatment, immunofluorescence analysis showed a marked increase in the accumulation of intracellular cGMP (29-fold) concurrent with receptor internalization compared with controls. The results indicate that ligand-receptor complexes continue to traffic and produce cGMP even after receptor sequestration into endosomes. Our, data demonstrate that FQQI motif plays essential role in the internalization of NPRA, which regulates hypertension and cardiovascular homeostasis.

Concurrent and separate inside‐out transition of platelet apoptosis and activation markers to the platelet surface

The cell plasma membrane is tightly coupled with the vital processes of apoptosis and activation. In the current study, we investigated exposure of the apoptosis marker phosphatidylserine (PS) and activation marker P-selectin (CD62) on the plasma membrane of anucleate platelets. We found that, depending on triggering stimuli, the plasma membrane of human platelets may exist in four states with predominant exposure of (i) PS but not CD62 (75·9±2·8% of total cells), (ii) CD62 but not PS (86·2±1·3%), (iii) both PS and CD62 (89·6±1·0%) or (iv) neither PS nor CD62 (87·9-97·5%), when platelets were treated at optimal conditions with pro-apoptotic BH3 mimetic ABT-737, thrombin, calcium ionophore A23187 or control diluents, respectively. The dynamics of PS exposure induced by ABT-737 is a slow temperature-dependent process requiring 90min treatment at 37°C rather than at room temperature for obtaining high levels of PS exposure. In contrast, thrombin-induced CD62 exposure and A23187-induced PS and CD62 exposure showed fast temperature-independent dynamics. This model of selective and concurrent stimulation of PS and/or CD62 transition to the platelet surface provides an experimental horizon for elucidating the roles of plasma membrane markers of platelet apoptosis and activation in platelet clearance.

New Visible and NIR Highly Photostable Fluorescent Silica Nanoparticles for Laser Scanning Imaging Applications

One way to improve the photophysical and photochemical properties of NIR probes is to incorporate them in nanoparticles (NPs). The characteristics of NP-based imaging agents, such as the ability to carry large payloads of optical probes and multiple targeting groups, together with the possibility to tune their pharmacokinetics, have motivated the development of different systems for use in combined diagnostic and therapeutic (theranostic) applications. The use of silica nanoparticles (SiNP) in nanomedicine, as carriers for drug delivery and imaging agents has increase remarkably in the last decade. On the other hand, it is well known that the use of near infrared (NIR) light (750-1400 nm) in optical bioimaging presents several advantages over lower wavelength light (visible), namely higher tissue penetration, lower interference from autofluorescence in biological specimens, and reduced light scattering. These factors have greatly increased the interest in NIR fluorescent dyes for use in biological imaging. An additional advantage of NIR dyes is that they can be used with inexpensive laser diode excitation sources in laser scanning microscopy.In this communication we evaluate the performance of new fluorescent SiNP in laser scanning microscopy. The particles are labelled with two different perylenediimide (PDI) derivatives, one with emission in the visible region of the spectrum (PDIvis) and the other emitting in the NIR (PDInir). The PDI derivatives were covalently incorporated in the SiNPs during their synthesis by a modified Stöber method. The labelled SiNP were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), fluorescence emission spectroscopy and laser scanning fluorescence confocal microscopy (LSFCM).The excellent photophysical properties and stability of the fluorescently labelled SiNP encouraged us to evaluate their performance in vivo, as markers for LSFCM. The nanoparticles were internalized in HEK293 cells co-stained with plasma membrane (AF594-WGA) and/or nucleus (Hoechst 33258) markers. Confocal microscopy images show that both SiNP-PDInir and SiNP-PDIvis were efficiently internalized in the cells (Figure 1 and 2, respectively). The major difference between the two sets of SiNPs is that while SiNP-PDIvis is excited at 488 nm (argon laser) showing emission in the green region of the spectra, SiNP-PDInir can be excited at 633 nm (using a low power/low cost HeNe laser) with the emission observed in the NIR. The in vivo photostability of the labelled nanoparticles was compared with that of a commercial membrane marker, DiIC18(5), showing much higher photostability during laser scanning, due to the shielding effect of the silica nanoparticles.This work was partially supported by Fundação para a Ciência e a Tecnologia (FCT-Portugal) and COMPETE (FEDER) within projects PTDC/CTM/101627/2008 and PEst-OE/CTM/LA0024/2011. T.R., R.S. and F.F. also thank FCT for Ph.D. (SFRH/BD/64702/2009) and Pos-Doc (SFRH/BPD/71249/2010; SFRH/BPD/64320/2009) grants.

DNA Damage in Mammalian Neural Stem Cells Leads to Astrocytic Differentiation Mediated by BMP2 Signaling through JAK-STAT

SummaryThe consequences of DNA damage generation in mammalian somatic stem cells, including neural stem cells (NSCs), are poorly understood despite their potential relevance for tissue homeostasis. Here, we show that, following ionizing radiation-induced DNA damage, NSCs enter irreversible proliferative arrest with features of cellular senescence. This is characterized by increased cytokine secretion, loss of stem cell markers, and astrocytic differentiation. We demonstrate that BMP2 is necessary to induce expression of the astrocyte marker GFAP in irradiated NSCs via a noncanonical signaling pathway engaging JAK-STAT. This is promoted by ATM and antagonized by p53. Using a SOX2-Cre reporter mouse model for cell-lineage tracing, we demonstrate irradiation-induced NSC differentiation in vivo. Furthermore, glioblastoma assays reveal that irradiation therapy affects the tumorigenic potential of cancer stem cells by ablating self-renewal and inducing astroglial differentiation.

Basic Studies on Radioimmunotargeting of CD133-Positive HCT116 Cancer Stem Cells

As cancer stem cells (CSCs) are postulated to play critical roles in cancer development, including metastasis and recurrence, CSC imaging would provide valuable information for cancer treatment and lead to CSC-targeted therapy. To assess the possibility of in vivo CSC targeting, we conducted basic studies on radioimmunotargeting of cancer cells positive for CD133, a CSC marker recognized in various cancers. Antibodies against CD133 were labeled with ¹²⁵I, and their in vitro cell binding properties were tested. Using the same isotype IgG as a control, in vivo biodistribution of the labeled antibody retaining immunoreactivity was examined in mice bearing an HCT116 xenograft in which a population of the cancer cells expressed CD133. Intratumoral distribution of the labeled antibody was examined and compared to the CD133 expression pattern. The ¹²⁵I-labeled anti-CD133 antibody showed a modest but significantly higher accumulation in the HCT116 xenograft compared to the control IgG. The intratumoral distribution of the labeled antibody mostly overlapped with the CD133 expression, whereas the control IgG was found in the area close to the necrotic tumor center. Our results indicate that noninvasive in vivo targeting of CSCs could be possible with radiolabeled antibodies against cell membrane markers.

Analysis of gamete membrane dynamics during double fertilization of Arabidopsis

Angiosperms have a unique sexual reproduction system called “double fertilization.” One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.Electronic supplementary materialThe online version of this article (doi:10.1007/s10265-012-0528-0) contains supplementary material, which is available to authorized users.

An aryleneethynylene fluorophore for cell membrane staining

The use of an amphiphilic aryleneethynylene fluorophore as a plasma membrane marker in fixed and living mammalian cells and liposome model systems is demonstrated. We show here that the optical properties of the novel dye are almost independent on pH, in the range 5.0–8.0. Spectroscopic characterization performed on unilamellar liposomes ascertained that the fluorescence intensity of the aryleneethynylene fluorophore greatly increases after incorporation in lipidic membranes. Experiments performed on different mammalian cells demonstrated that the novel membrane marker exhibits fast staining and a good photostability that make it a suitable tool for live cell imaging. Importantly, the aryleneethynylene fluorophore was also shown to be a fast and reliable blue membrane marker in classical multicolor immunofluorescence experiments. This study adds new important findings to the recent exploitation of the wide class of aryleneethynylene molecules as luminescent markers for biological investigations.

Study of the Compartmentalization of Type 1 Angiotensin Receptor Using Bioluminescence Resonance Energy Transfer‐based Sensors

Initiation and termination of signaling of the type I angiotensin receptor (AT1‐R) can lead to dynamic changes in its localization in membrane microdomains.We used YFP‐labeled fusion constructs constructs (i.e. raft‐, or non‐raft plasma membrane markers) to analyze the molecular dynamics of AT1‐R trafficking in response to stimulation in bioluminescence resonance energy transfer (BRET) measurements. Our data demonstrate that angiotensin II (AngII) stimulus changes the microdomain localization of wild type or mutant (DRY/AAY or TSTS/A) AT1‐Rs in HEK293 cells. The comparison of the trafficking of AT1‐R upon AngII stimulus to either [Sar1,Ile8]‐ AngII or [Sar1,Ile4,Ile8]‐AngII stimulus revealed different type of molecular dynamics depending on the nature of the ligand of the AT1‐R. The molecular dynamics of AT1‐R also changes upon cholesterol depletion, which suggests that the mechanism is raft‐dependent. The molecular dynamics of the AT1‐R are strikingly differ from those of 5HT‐2C and EGF receptors, which demonstrate the usefulness of the BRET based measurements in the investigation of receptor trafficking in living cell experiments. These studies should provide valuable information about the distribution and molecular dynamics of AT1‐R upon angiotensin stimulus among membrane microdomains.Supported by Hungarian National Grant OTKA NK‐100883, TÁMOP 4.2.1 B‐09/1/KMR‐2010‐0001.

Interrogating Therapeutic Manipulation of the Endocannabinoid System in Human Colon

The endocannabinoid system (ECS) plays a role in colon inflammation and cancer. Human colon explants and a novel gene expression profiling technology, the GenomeLab System (Beckman) have been developed to investigate ECS regulation in human colon. A custom designed GeXP (Beckman) multiplex gene expression assay, the hECSplex, was developed. hECSplex gene expression signatures of EC receptors (CNR1, CNR2, GPR55 and TRPV1), ECS enzymes (NAPE‐PLD, GDE1, DAGLA, DAGLB, FAAH, FAAH2 and PTGS2), inflammatory (IL1B, IL10, IL6, LEP, TNF and SOCS3), signalling pathway (ID1, BCL2, CFL1, BIRC5, TP53, MYC and KRAS), lipid production (SREBF1, ACACA), and plasma‐membrane (OCLN) markers were assayed using 50ng of total RNA from human colon explants treated with either cannabidiol (CBD) or tetrahydrocannabinol (THC). Preliminary results indicate that both therapeutics regulate EC receptor expression, but notably regulate diverse expression of ECS enzymes, signalling targets and inflammatory markers. The GenomeLab System presents new opportunities to interrogate multiple components of the ECS in small colon explant tissue samples in response to novel therapeutics.

Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae, the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P(2) production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

Development of a cell microarray chip for detection of circulating tumor cells

Detection of circulating tumor cells (CTCs) in the peripheral blood of metastatic cancer patients has clinical significance in earlier diagnosis of metastases. In this study, a novel cell microarray chip for accurate and rapid detection of tumor cells from human leukocytes was developed. The chip with 20,944 microchambers (105 μm diameter and 50 μm depth) was made from polystyrene, and the surface was rendered to hydrophilic by means of reactive-ion etching, which led to the formation of mono-layers of leukocytes on the microchambers. As the model of CTCs detection, we spiked human bronchioalveolar carcinoma (H1650) cells into human T lymphoblastoid leukemia (CEM) cells suspension and detected H1650 cells using the chip. A CEM suspension contained with H1650 cells was dispersed on the chip surface, followed by 10 min standing to allow the cells to settle down into the microchambers. About 30 CEM cells were accommodated in each microchamber, over 600,000 CEM cells in total being on a chip. We could detect 1 H1650 cell per 106 CEM cells on the microarray by staining with fluorescence-conjugated antibody (Anti-Cytokeratin) and cell membrane marker (DiD). Thus, this cell microarray chip has highly potential to be a novel tool of accurate and rapid detection of CTCs.