Cells expressing GFP-CpnA were washed three times in starvation buffer, placed on glass bottom plates, and imaged using a confocal microscope. This is "Jenny et Marion les copines d'outre mer video pekin express 2021" by marion panis on Vimeo, the home for high quality videos and the people… Arrowheads point to contractile vacuoles. The presence of C2 domains suggests that copines … BMC Cell Biology All additional files are movies in AVI format except for Movie 9, which is MOV format. J Biol Chem. Marken Cop Copine. Please note that we do … GFP-CpnA binding to membranes was abolished by the EGTA indicating that the membrane binding activity of GFP-CpnA to the plasma membrane and intracellular vacuoles in these disrupted cells is calcium-dependent. The 66 kDa band recognized by the CpnA antiserum on Western blots using whole cell samples from wildtype cells was absent from whole cell samples made from cpnA- null cells (Fig. The full length coding sequence of cpnA was amplifed by PCR from the cDNA clone, SLI-395. See Additional Files – Movies 6, 7, 8. C) Successive time-lapse images taken every 2.5 seconds of a single cell from a plate of cells starved for 9.5 hours (arrowhead points to fluorescent dot). Aya was born in Bamako but immigrated to France with her family and grew up in Aulnay-sous-Bois. GFP-CpnA behaved similarly to the native CpnA, with GFP-CpnA nearly absent from the supernatant in the presence of calcium (Fig. Reactions without reverse transcriptase and with primers to cinD mRNA were run as controls. 2003, 165: 563-574. Many lines of evidence indicate that calcium is involved in the differentiation of stalk cells [26]. Nakayama T, Yaoi T, Kuwajima G: Localization and subcellular distribution of N-Copine in mouse brain. In addition, it has been shown that in neutrophils fast calcium waves accompany phagocytosis [32]. Dictyostelium provides an ideal system for studying copine function for several reasons. This small amount of calcium-independent binding was also seen with the Arabidopsis copine protein [7] and a neurally expressed mouse copine, N-copine [6]. However, cells exhibiting this behavior were found more frequently when the cells were starved for longer periods of time. These results indicate the addition of GFP to the N-terminus of CpnA does not disrupt the calcium-dependent membrane binding activity of CpnA (Fig. Tomsig JL, Creutz CE: Biochemical characterization of copine: a ubiquitous Ca 2+-dependent, phospholipid-binding protein. Creutz CE, Tomsig JL, Snyder SL, Gautier M, Skouri F, Beisson J, Cohen J: The copines, a novel class of C2 domain-containing, calcium-dependent phospholipid binding proteins conserved from Paramecium to humans. 10.1101/gad.918101. The bladders transiently form pores with the plasma membrane to expel water [21]. Page TransparencySee More. (AVI 732 KB), Additional File 3: A single cell expressing GFP-CpnA that has been starved for 4 hrs (not shown in Figure 4). Maitra R, Grigoryev DN, Bera TK, Pastan IH, Byungkook L: Cloning, molecular characterization, and expression analysis of copine 8. Not all GFP-CpnA labeled structures contained red beads; this is perhaps due to the fact that some of the structures are contractile vacuoles and that some are endosomes/lysosomes that do not contain beads. Contractile vacuoles in cells expressing GFP-CpnA were labeled with a primary antibody to calmodulin and a TRITC-conjugated secondary antibody and then imaged for TRITC (E, G, I). Dictyostelium discoideum: Molecular Approaches to Cell Biology. Images of fixed cells look similar to images of disrupted cells and the subset of starved live cells that display the transient membrane localization of GFP-CpnA (Fig. Once the cells burst, the GFP-CpnA did not bind to membranes, but diffused into the water and disappeared (Fig. Tomsig JL, Snyder SL, Creutz CE: Identification of targets for calcium signaling through the copine family of proteins. B) Wildtype cells were disrupted with passage through a French Press and all membranes were pelleted in the presence of calcium or EGTA. Ann Rev Biochem. When imaging a large field of starved cells, we noticed a few cells that displayed a very transient membrane localization of GFP-CpnA. GFP-CpnA translocates from the cytosol to membranes multiple times in each cell. In an earlier study, we identified six copine genes in the Dictyostelium discoideum genome and focused our studies on cpnA . However, CpnA and CpnE contain unique additional sequences not found in each of the other five copine genes. These GFP-CpnA labeled structures forming attachments with the plasma membrane could be bladders caught in the act of expelling water. PubMed  10.1016/j.jmb.2003.08.052. Primers were designed to amplify a 400 bp fragment that crossed an intron. J Mol Biol. Looking for in. Therefore, Dictyostelium serves as a good model for studying membrane trafficking and a particularly good model for many of the phagocytic cells found in human tissues, in which copines are highly expressed [13, 14]. By using this website, you agree to our Western blot analysis indicates that CpnA is present throughout the 24-hour development with only slight differences in protein levels (Fig. Previous studies with synaptotagmin, a protein with two C2 domains, have shown that the lipid binding behavior of C2 domains is highly dependent on the type of phospholipids present and whether they are independent or paired with another C2 domain [17]. Accepts Credit Cards. Farbe Alle anzeigen schwarz blau braun grau lila rot beige pink weiß creme gelb. GFP-CpnA transiently binds to the plasma membrane and intracellular vacuoles in a small subset of starved cells. It is not clear whether cells would continue to produce these oscillations of GFP-CpnA localization over a long period of time because we were only able to image cells for a few minutes before the cells became damaged by the laser. 1991, 18: 113-122. For phospholipid binding assays with CpnA-C2A, bovine brain lipids (Sigma, B-1502, 100 mg) were dissolved in 10 ml of buffer (20 mM HEPES, 30 mM KCl, pH 7.4) and sonicated to form liposomes. Location & Hours. A GFP-tagged CpnA also binds to membranes in a calcium-dependent manner in vitro. In these broken cells, the GFP-CpnA was seen labeling the plasma membrane and intracellular vacuoles (Fig. 5C and additional file 8). 2002, 418: 79-85. However, in a small subset of starved cells, GFP-CpnA was observed to bind transiently (typically ~1–10 s) to the plasma membrane and intracellular vacuoles. We then used the cDNA sequences of cpnA and cpnB to search both genomic and cDNA sequence databases for additional copine genes. 2003, 333: 621-639. 10.1091/mbc.E02-05-0259. Aya Danioko, known as Aya Nakamura, is a French pop singer of Malian origin. Aya was born in Bamako but immigrated to France with her family and grew up in Aulnay-sous-Bois. In addition, we would like to thank Jerry Calvin for technical assistance with microscopy, Carl Creutz for helpful discussions on copines, and Scott Daigle for careful reading of the manuscript. Amino acid sequences of CpnA and CpnB were derived from sequencing of full-length cDNA clones. 1994, 337: 43-47. 10.1016/0003-2697(91)90534-Z. Cells expressing GFP-CpnA were incubated with Alexa Fluor-594-labeled yeast for 1 hour, flattened with agarose, fixed, and imaged for both the yeast and GFP-CpnA simultaneously using confocal microscopy. 7F, H, J). The numerous GFP-CpnA labeled structures not identified as contractile vacuoles are most likely part of the endolysosomal pathway, which includes endosomes, lysosomes, and postlysosomes. Aya Nakamura - Copines (slowed TikTok) (Lyrics) pota potaOriginal Video: https://www.youtube.com/watch?v=0KJoT7vVUBI 10.1016/0014-5793(94)80626-8. These membranes include the plasma membrane and intracellular vacuoles. In cells starved for 4–8 hours, membrane localization of GFP-CpnA occurred for ~1–10 seconds, while in cells starved for 8–10 hours, membrane localization sometimes occurred for longer periods of time ranging from ~1–30 seconds. 2A). To label endosomes/lysosomes, cells expressing GFP-CpnA in culture dishes were incubated with 0.02 μm red fluorescent microspheres (Fluospheres, Molecular Probes, F-8786; [41]) diluted 1:100 in HL-5 for 2 hours, washed once, and placed on coverslips for 15 min before fixation. This search yielded multiple cDNA clones with homologies to human copine I. Chères clientes, demain et samedi à Cruseilles c'est la Braderie des Commercants, venez profitez de nos incroyables prix ronds sur … In contrast, most proteins that have multiple C2 domains are involved in membrane trafficking. They lose their shape and adherence, becoming round and eventually bursting if exposed to the light for more than tens of seconds with a fluorescence microscope or frequent scanning over tens of minutes with a laser scanning confocal microscope. Third, Dictyostelium executes a simple 24-hour developmental program to form multicellular fruiting bodies and thus, Dictyostelium provides a simplistic model to study copine function in programmed cell death and development. CAS  GFP-CpnA translocates from the cytosol to membranes and back to the cytosol once. 2003, 74: 379-388. Using the light sensitivity of Dictyostelium to our advantage, we designed an assay to visualize GFP-CpnA in disrupted cells. Levi S, Polyakov MV, Egelhoff TT: Green fluorescent protein (GFP) and epitope tag fusion vectors for Dictyostelium discoideum. 8 (G, H). The overexpression of GFP-CpnA did not result in any easily identifiable phenotypes. The same cells are displayed twice; GFP-CpnA only is shown on the left (A, C, E), while both the red beads and GFP-CpnA are shown to the right (B, D, F). 2004, 378: 1089-1094. 2001, 13: 2225-2240. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( Plasmid. Using a monoclonal antibody to calmodulin, we performed immunofluorescence studies to label contractile vacuoles with rhodamine (TRITC) (Fig. In addition, we hypothesize that the observed transient oscillatory membrane localization of GFP-CpnA in a small subset of starved cells is caused by fast calcium waves and speculate that CpnA may have a role in development, particularly in the differentiation of stalk cells. (AVI 654 KB), Additional File 2: A single cell expressing GFP-CpnA that has been starved for 5.5 hrs (same cell shown in Figure 4B). M I Y U K I みゆき. Many of these interactions between the copine A domains and their target proteins were verified in in vitro pull-down assays. PubMed  The vacuolar structures were heterogeneous in size and found throughout the cytoplasm (Fig. Coming from a family of griots (West African storytellers, praise singers / poets of oral tradition), she is the oldest of five siblings. babies noun: bébés, petites amies: Find more words! Therefore, the Dictyostelium copine genes are diverse in sequence and may carry out distinct functions. 2000, 150: 1013-1026. In Arabidopsis, CPN1-GFP localizes to the plasma membrane. Article  When imaged using fluorescence microscopy, these same vacuoles were outlined in GFP-CpnA (Fig. 1995, 108: 1105-1115. Wildtype Dictyostelium cells were transformed with the plasmid by electroporation and G418 resistant cells were screened for expression of GFP-CpnA by Western blot analysis using antibodies to both CpnA and GFP. J Cell Biol. Alignment of the amino acid sequences of the six copines genes in Dictyostelium. It has also been shown that increasing calcium levels increases the stalk cell to spore cell ratio [29, 25]. 5C). FASEB J. 3C, arrows). CKD wrote the manuscript, created the figures, and designed and supervised all experiments. 2003, 170: 64-72. Aya Danioko, known as Aya Nakamura, is a French pop singer of Malian origin. Preis Bitte wählen bis 10 Euro 10 - 25 Euro 25 - 50 Euro 50 - 100 Euro. Additional File 4: Adjacent cells expressing GFP-CpnA that have been starved for 10 hrs. Over time the cells sustained plasma membrane ruptures, allowing soluble cytoplasmic proteins to diffuse out of the cell and into the water. 1991, 112: 103-110. Google Scholar. Fey P, Gaudet P, Just EM, Merchant SN, Pilcher KE, Kibbe WA, Chisholm RL: dictyBase. Amino acid sequences were aligned using Lasergene's Megalign Clustal V method. mRNA from cpnA was amplified from each stage of development (Fig. The small amount of GFP-CpnA found in the supernatant in the presence of calcium is most likely due to the overexpression of GFP-CpnA. 5B and additional file 7). EN; FR; NL; Shop at Amazon. (AVI 269 KB), Additional File 5: A small aggregate of cells expressing GFP-CpnA that has been starved for 9.5 hrs (one of these cells is shown in Figure 4C). Because we had problems with proteolysis of GFP-CpnA, we also analyzed the pellets and supernatants using a monoclonal antibody to GFP (Fig. CpnA is unique among the Dictyostelium copines in that it contains a C-terminal tail domain following the A domain that is ~50 amino acids longer (Fig. Springer Nature. Wildtype Dictyostelium cells were disrupted with passage through a French Press, unbroken cells were pelleted, and then all membranes were pelleted by high-speed centrifugation in the presence of calcium or EGTA. However, because the C2 domains are in the N-terminal half of the protein, we tested whether GFP-CpnA has similar membrane binding properties to the endogenous CpnA. We used these sequences to identify and predict amino acid sequences for four additional copine genes (cpnC-F) within the Dictyostelium genome. J Cell Biol. Although copines possess two C2 domains, studies with human copines indicate that copines are involved in calcium-dependent signaling pathways and are therefore, more functionally related to the single C2 domain proteins [8, 9]. We used RT-PCR to amplify a small region of cpnA across an intron to control for any contaminating DNA. Cells expressing GFP-CpnA were placed in water for 1.5 minutes, fixed, and imaged using differential interference contrast microscopy (A, C). The nem-4 mutant strains do not exhibit any obvious phenotype [12].