scholarly journals Fluorescent tagging of Plasmodium circumsporozoite protein allows imaging of sporozoite formation but blocks egress from oocysts

2020 ◽  
Author(s):  
Mirko Singer ◽  
Friedrich Frischknecht
Keyword(s):  
Fluorescent Protein ◽  
Surface Protein ◽  
Dominant Negative ◽  
Circumsporozoite Protein ◽  
Negative Function ◽  
Mammalian Liver ◽  
Parasite Replication ◽  
Fluorescent Tagging ◽  
Green Fluorescent ◽  
Major Surface

AbstractThe circumsporozoite protein, CSP is the major surface protein of Plasmodium sporozoites, the form of malaria parasites transmitted by mosquitoes. CSP is involved in sporozoite formation within and egress from oocysts, entry into mosquito salivary glands and mammalian liver as well as migration in the skin. Antibodies against CSP can stop infection prior to the first round of parasite replication in the liver. CSP consists of different domains and is proteolytically cleaved prior to hepatocyte invasion. Part of CSP has been developed into a licensed vaccine against malaria. Yet, how CSP facilitates sporozoite formation, oocyst egress and hepatocyte specific invasion is still not fully understood. Here, we generated a series of parasites expressing full-length versions of CSP as fusion proteins with the green fluorescent protein. This enabled the investigation of sporozoite formation in living oocysts and revealed a dominant negative function of some GFP-CSP fusions during sporozoite egress.

scholarly journals Binding of the Major Phasin, PhaP1, from Ralstonia eutropha H16 to Poly(3-Hydroxybutyrate) Granules

2008 ◽  
Vol 190 (8) ◽  
pp. 2911-2919 ◽  
Author(s):  
Liv Neumann ◽  
Francesco Spinozzi ◽  
Raffaele Sinibaldi ◽  
Franco Rustichelli ◽  
Markus Pötter ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Surface Protein ◽  
Binding Motif ◽  
Rhodococcus Ruber ◽  
Storage Granules ◽  
Granule Surface ◽  
Green Fluorescent ◽  
Major Surface

ABSTRACT The surface of polyhydroxybutyrate (PHB) storage granules in bacteria is covered mainly by proteins referred to as phasins. The layer of phasins stabilizes the granules and prevents coalescence of separated granules in the cytoplasm and nonspecific binding of other proteins to the hydrophobic surfaces of the granules. Phasin PhaP1 Reu is the major surface protein of PHB granules in Ralstonia eutropha H16 and occurs along with three homologues (PhaP2, PhaP3, and PhaP4) that have the capacity to bind to PHB granules but are present at minor levels. All four phasins lack a highly conserved domain but share homologous hydrophobic regions. To identify the region of PhaP1 Reu which is responsible for the binding of the protein to the granules, N-terminal and C-terminal fusions of enhanced green fluorescent protein with PhaP1 Reu or various regions of PhaP1 Reu were generated by recombinant techniques. The fusions were localized in the cells of various recombinant strains by fluorescence microscopy, and their presence in different subcellular protein fractions was determined by immunodetection of blotted proteins. The fusions were also analyzed to determine their capacities to bind to isolated PHB granules in vitro. The results of these studies indicated that unlike the phasin of Rhodococcus ruber, there is no discrete binding motif; instead, several regions of PhaP1 Reu contribute to the binding of this protein to the surface of the granules. The conclusions are supported by the results of a small-angle X-ray scattering analysis of purified PhaP1 Reu , which revealed that PhaP1 Reu is a planar, triangular protein that occurs as trimer. This study provides new insights into the structure of the PHB granule surface, and the results should also have an impact on potential biotechnological applications of phasin fusion proteins and PHB granules in nanobiotechnology.

scholarly journals The malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host

2011 ◽  
Vol 208 (2) ◽  
pp. 341-356 ◽  
Author(s):  
Alida Coppi ◽  
Ramya Natarajan ◽  
Gabriele Pradel ◽  
Brandy L. Bennett ◽  
Eric R. James ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Critical Role ◽  
Domain Architecture ◽  
Surface Protein ◽  
Circumsporozoite Protein ◽  
Mammalian Host ◽  
Multifunctional Protein ◽  
Mosquito Midgut ◽  
Cell Adhesive ◽  
Mammalian Liver

Plasmodium sporozoites make a remarkable journey from the mosquito midgut to the mammalian liver. The sporozoite’s major surface protein, circumsporozoite protein (CSP), is a multifunctional protein required for sporozoite development and likely mediates several steps of this journey. In this study, we show that CSP has two conformational states, an adhesive conformation in which the C-terminal cell-adhesive domain is exposed and a nonadhesive conformation in which the N terminus masks this domain. We demonstrate that the cell-adhesive domain functions in sporozoite development and hepatocyte invasion. Between these two events, the sporozoite must travel from the mosquito midgut to the mammalian liver, and N-terminal masking of the cell-adhesive domain maintains the sporozoite in a migratory state. In the mammalian host, proteolytic cleavage of CSP regulates the switch to an adhesive conformation, and the highly conserved region I plays a critical role in this process. If the CSP domain architecture is altered such that the cell-adhesive domain is constitutively exposed, the majority of sporozoites do not reach their target organs, and in the mammalian host, they initiate a blood stage infection directly from the inoculation site. These data provide structure–function information relevant to malaria vaccine development.

scholarly journals Differential Localization of Rho Gtpases in Live Cells

2001 ◽  
Vol 152 (1) ◽  
pp. 111-126 ◽  
Author(s):  
David Michaelson ◽  
Joseph Silletti ◽  
Gretchen Murphy ◽  
Peter D'Eustachio ◽  
Mark Rush ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Fluorescent Protein ◽  
Essential Feature ◽  
Hypervariable Region ◽  
Dominant Negative ◽  
Live Cells ◽  
Rho Proteins ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
Membrane Compartment ◽  
Green Fluorescent

Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)α. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDIα in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDIα. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDIα binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDIα and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.

scholarly journals Recycling of Golgi-resident Glycosyltransferases through the ER Reveals a Novel Pathway and Provides an Explanation for Nocodazole-induced Golgi Scattering

1998 ◽  
Vol 143 (6) ◽  
pp. 1505-1521 ◽  
Author(s):  
Brian Storrie ◽  
Jamie White ◽  
Sabine Röttger ◽  
Ernst H.K. Stelzer ◽  
Tatsuo Suganuma ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Time Lapse ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Protein Synthesis Inhibitors ◽  
Microtubule Depolymerization ◽  
Golgi Stack ◽  
Gradual Accumulation ◽  
Green Fluorescent ◽  
Er Export

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11–amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme β1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2–GFP and GalT–GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2–VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1pdn) blocking ER export. Sar1pdn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2–VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1pdn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.

scholarly journals Heterologous downregulation of vasopressin type 2 receptor is induced by transferrin

2013 ◽  
Vol 304 (5) ◽  
pp. F553-F564 ◽  
Author(s):  
Richard Bouley ◽  
Paula Nunes ◽  
Billy Andriopoulos ◽  
Margaret McLaughlin ◽  
Matthew J. Webber ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Dominant Negative ◽  
Target Cells ◽  
Intracellular Camp ◽  
Coated Pits ◽  
Parathyroid Hormone Receptor ◽  
Body Fluid Homeostasis ◽  
Green Fluorescent ◽  
G Protein Coupled

Vasopressin (VP) binds to the vasopressin type 2 receptor (V2R) to trigger physiological effects including body fluid homeostasis and blood pressure regulation. Signaling is terminated by receptor downregulation involving clathrin-mediated endocytosis and V2R degradation. We report here that both native and epitope-tagged V2R are internalized from the plasma membrane of LLC-PK1 kidney epithelial cells in the presence of another ligand, transferrin (Tf). The presence of iron-saturated Tf (holo-Tf; 4 h) reduced V2R binding sites at the cell surface by up to 33% while iron-free (apo-Tf) had no effect. However, no change in green fluorescent protein-tagged V2R distribution was observed in the presence of bovine serum albumin, atrial natriuretic peptide, or ANG II. Conversely, holo-Tf did not induce the internalization of another G protein-coupled receptor, the parathyroid hormone receptor. In contrast to the effect of VP, Tf did not increase intracellular cAMP or modify aquaporin-2 distribution in these cells, although addition of VP and Tf together augmented VP-induced V2R internalization. Tf receptor coimmunoprecipitated with V2R, suggesting that they interact closely, which may explain the additive effect of VP and Tf on V2R endocytosis. Furthermore, Tf-induced V2R internalization was abolished in cells expressing a dominant negative dynamin (K44A) mutant, indicating the involvement of clathrin-coated pits. We conclude that Tf can induce heterologous downregulation of the V2R and this might desensitize VP target cells without activating downstream V2R signaling events. It also provides new insights into urine-concentrating defects observed in rat models of hemochromatosis.

scholarly journals The Mycobacterium tuberculosis Phagosome in Human Macrophages Is Isolated from the Host Cell Cytoplasm

2002 ◽  
Vol 70 (10) ◽  
pp. 5800-5807 ◽  
Author(s):  
Daniel L. Clemens ◽  
Bai-Yu Lee ◽  
Marcus A. Horwitz
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Fluorescent Protein ◽  
Medium Size ◽  
Immunogold Electron Microscopy ◽  
Human Macrophages ◽  
Fab Fragments ◽  
Green Fluorescent ◽  
Major Surface ◽  
Phagosomal Membrane

ABSTRACT Knowledge of whether Mycobacterium tuberculosis resides within a relatively impermeable membrane-bound vacuole or is free within the cytoplasm within its host cell is central to an understanding of the immunobiology of this intracellular parasite but is a matter of controversy. To explore this issue, we assessed the accessibility of medium-size protein molecules (Fab fragments of 50,000 Da) to M. tuberculosis within human macrophages. We infected the macrophages with wild-type or green fluorescent protein-expressing M. tuberculosis, microinjected Fab fragments directed against a major surface antigen of M. tuberculosis into the host cell, and assayed the accessibility of the bacteria to the Fab fragments by both immunofluorescence microscopy and immunogold electron microscopy. Whereas microinjected intact immunoglobulin G molecules against cytoplasmic early endosomal antigen 1 readily stained this antigen, microinjected Fab fragments against M. tuberculosis did not stain the bacterium within its phagosome. In contrast, microinjected Fab fragments against Listeria monocytogenes, an intracellular bacterium known to permeabilize its phagosomal membrane, strongly stained this bacterium. Our study shows that M. tuberculosis resides in an isolated phagosome that is relatively impermeable to cytoplasmic constituents.

Smooth muscle cell-specific transcription is regulated by nuclear localization of the myocardin-related transcription factors

2007 ◽  
Vol 292 (2) ◽  
pp. H1170-H1180 ◽  
Author(s):  
Jeremiah S. Hinson ◽  
Matthew D. Medlin ◽  
Kashelle Lockman ◽  
Joan M. Taylor ◽  
Christopher P. Mack
Keyword(s):  
Smooth Muscle ◽  
Transcription Factors ◽  
Nuclear Localization ◽  
Transforming Growth Factor ◽  
Serum Response Factor ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Dominant Negative ◽  
Specific Gene ◽  
Green Fluorescent

On the basis of our previous studies on RhoA signaling in smooth muscle cells (SMC), we hypothesized that RhoA-mediated nuclear translocalization of the myocardin-related transcription factors (MRTFs) was important for regulating SMC phenotype. MRTF-A protein and MRTF-B message were detected in aortic SMC and in many adult mouse organs that contain a large SMC component. Both MRTFs upregulated SMC-specific promoter activity as well as endogenous SM22α expression in multipotential 10T1/2 cells, although to a lesser extent than myocardin. We used enhanced green fluorescent protein (EGFP) fusion proteins to demonstrate that the myocardin factors have dramatically different localization patterns and that the stimulation of SMC-specific transcription by certain RhoA-dependent agonists was likely mediated by increased nuclear translocation of the MRTFs. Importantly, a dominant-negative form of MRTF-A (ΔB1/B2) that traps endogenous MRTFs in the cytoplasm inhibited the SM α-actin, SM22α, and SM myosin heavy chain promoters in SMC and attenuated the effects of sphingosine 1-phosphate and transforming growth factor (TGF)-β on SMC-specific transcription. Our data confirmed the importance of the NH2-terminal RPEL domains for regulating MRTF localization, but our analysis of MRTF-A/myocardin chimeras and myocardin RPEL2 mutations indicated that the myocardin B1/B2 region can override this signal. Gel shift assays demonstrated that myocardin factor activity correlated well with ternary complex formation at the SM α-actin CArGs and that MRTF-serum response factor interactions were partially dependent on CArG sequence. Taken together, our results indicate that the MRTFs regulate SMC-specific gene expression in at least some SMC subtypes and that regulation of MRTF nuclear localization may be important for the effects of selected agonists on SMC phenotype.

scholarly journals Lamin A/C Binding Protein LAP2α Is Required for Nuclear Anchorage of Retinoblastoma Protein

2002 ◽  
Vol 13 (12) ◽  
pp. 4401-4413 ◽  
Author(s):  
Ewa Markiewicz ◽  
Thomas Dechat ◽  
Roland Foisner ◽  
Roy. A Quinlan ◽  
Christopher J. Hutchison
Keyword(s):  
Tissue Culture ◽  
Fluorescent Protein ◽  
Solid Phase ◽  
Retinoblastoma Protein ◽  
Dominant Negative ◽  
Lamin A ◽  
Dependent Manner ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Green Fluorescent

The phosphorylation-dependent anchorage of retinoblastoma protein Rb in the nucleus is essential for its function. We show that its pocket C domain is both necessary and sufficient for nuclear anchorage by transiently expressing green fluorescent protein (GFP) chimeras of Rb fragments in tissue culture cells and by extracting the cells with hypotonic solutions. Solid phase binding assays using glutathioneS-transferase-fusion of Rb pockets A, B, and C revealed a direct association of lamin C exclusively to pocket C. Lamina-associated polypeptide (LAP) 2α, a binding partner of lamins A/C, bound strongly to pocket C and weakly to pocket B. When LAP2α was immunoprecipitated from soluble nuclear fractions, lamins A/C and hypophosphorylated Rb were coprecipitated efficiently. Similarly, immunoprecipitation of expressed GFP-Rb fragments by using anti-GFP antibodies coprecipitated LAP2α, provided that pocket C was present in the GFP chimeras. On redistribution of endogenous lamin A/C and LAP2α into nuclear aggregates by overexpressing dominant negative lamin mutants in tissue culture cells, Rb was also sequestered into these aggregates. In primary skin fibroblasts, LAP2α is expressed in a growth-dependent manner. Anchorage of hypophosphorylated Rb in the nucleus was weakened significantly in the absence of LAP2α. Together, these data suggest that hypophosphorylated Rb is anchored in the nucleus by the interaction of pocket C with LAP2α–lamin A/C complexes.

scholarly journals Golgi-bound Rab34 Is a Novel Member of the Secretory Pathway

2007 ◽  
Vol 18 (12) ◽  
pp. 4762-4771 ◽  
Author(s):  
Neil M. Goldenberg ◽  
Sergio Grinstein ◽  
Mel Silverman
Keyword(s):  
Hela Cells ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Fluid Phase ◽  
Dominant Negative ◽  
Temperature Sensitive ◽  
Golgi Stack ◽  
Green Fluorescent ◽  
Intracellular Vesicle Transport

Golgi-localized Rab34 has been implicated in repositioning lysosomes and activation of macropinocytosis. Using HeLa cells, we undertook a detailed investigation of Rab34 involvement in intracellular vesicle transport. Immunoelectron microscopy and immunocytochemistry confirmed that Rab34 is localized to the Golgi stack and that active Rab34 shifts lysosomes to the cell center. Contrary to a previous report, we found that Rab34 is not concentrated at membrane ruffles and is not involved in fluid-phase uptake. Also, Rab34-induced repositioning of lysosomes does not affect mannose 6-phosphate receptor trafficking. Most strikingly, HeLa cells depleted of Rab34 by transfection with dominant-negative Rab34 or after RNA interference, failed to transport the temperature-sensitive vesicular stomatitis virus G-protein (VSVG) fused to green fluorescent protein (VSVG-GFP) from the Golgi to the plasma membrane. Transfection with mouse Rab34 rescued this defect. Using endogenous major histocompatibility complex class I (MHCI) as a marker, an endoglycosidase H resistance assay showed that endoplasmic reticulum (ER) to medial Golgi traffic remains intact in knockdown cells, indicating that Rab34 specifically functions downstream of the ER. Further, brefeldin A treatment revealed that Rab34 effects intra-Golgi transport, not exit from the trans-Golgi network. Collectively, these results define Rab34 as a novel member of the secretory pathway acting at the Golgi.

scholarly journals Reciprocal Modulation Between Amyloid Precursor Protein and Synaptic Membrane Cholesterol Revealed By Live Cell Imaging

2018 ◽  
Author(s):  
Claire E. DelBove ◽  
Claire E. Strothman ◽  
Roman M. Lazarenko ◽  
Hui Huang ◽  
Charles R. Sanders ◽  
...  
Keyword(s):  
Amyloid Precursor Protein ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Precursor Protein ◽  
Membrane Cholesterol ◽  
Synaptic Membrane ◽  
Surface Distribution ◽  
Cholesterol Levels ◽  
App Trafficking ◽  
Green Fluorescent

SummaryThe amyloid precursor protein (APP) has been extensively studied because of its association with Alzheimer’s disease (AD). However, APP distribution across different subcellular membrane compartments and its function in neurons remains unclear. We generated an APP fusion protein with a pH-sensitive green fluorescent protein at its ectodomain and a pH-insensitive blue fluorescent protein at its cytosolic domain and used it to measure APP’s distribution, subcellular trafficking and cleavage in live neurons. This reporter, closely resembling endogenous APP, revealed only a limited correlation between synaptic activities and APP trafficking. However, the synaptic surface distribution of APP was inversely correlated to membrane cholesterol levels, a phenomenon that involves APP’s cholesterol-binding site. Mutations within this site not only altered surface APP and cholesterol levels in a dominant negative manner, but also increased synaptic vulnerability to moderate membrane cholesterol reduction. Our results reveal reciprocal modulation of APP and membrane cholesterol levels at synaptic boutons.

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