scholarly journals Immunogold Electron Microscopic Demonstration of Distinct Submembranous Localization of the Activated γPKC Depending on the Stimulation

2007 ◽  
Vol 56 (3) ◽  
pp. 253-265 ◽  
Author(s):  
Miho Oyasu ◽  
Mineko Fujimiya ◽  
Kaori Kashiwagi ◽  
Shiho Ohmori ◽  
Hirotsugu Imaeda ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Confocal Laser ◽  
Immunogold Electron Microscopy ◽  
Subcellular Targeting ◽  
Electron Microscopic ◽  
Proper Distance

We examined the precise intracellular translocation of γ subtype of protein kinase C (γPKC) after various extracellular stimuli using confocal laser-scanning fluorescent microscopy (CLSM) and immunogold electron microscopy. By CLSM, treatment with 12- O-tetradecanoylphorbol-13-acetate (TPA) resulted in a slow and irreversible accumulation of green fluorescent protein (GFP)-tagged γPKC (γPKC–GFP) on the plasma membrane. In contrast, treatment with Ca2+ ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of γPKC–GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that γPKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of γPKC–GFP on the plasma membrane, Ca2+ ionophore and NMDA rapidly translocated γPKC–GFP to the plasma membrane and then restricted γPKC–GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca2+ influx alone induced the association of γPKC with the plasma membrane for only a moment and then located this enzyme at a proper distance in a touch-and-go manner, whereas diacylglycerol or TPA tightly anchored this enzyme on the plasma membrane. The distinct subcellular targeting of γPKC in response to various stimuli suggests a novel mechanism for PKC activation.

scholarly journals Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunofluorescence Microscopy and Biochemical Analysis

Crystals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 874
Author(s):  
Zachery Oestreicher ◽  
Carmen Valverde-Tercedor ◽  
Eric Mumper ◽  
Lumarie Pérez-Guzmán ◽  
Nadia N. Casillas-Ituarte ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Western Blot ◽  
Laser Scanning ◽  
Catalytic Domain ◽  
Amino Acid Sequences ◽  
Laser Scanning Microscopy ◽  
Protein Sequencing ◽  
Confocal Laser ◽  
Immunogold Electron Microscopy ◽  
Magnetospirillum Magneticum

Magnetotactic bacteria (MTB) biomineralize intracellular magnetite (Fe3O4) crystals surrounded by a magnetosome membrane (MM). The MM contains membrane-specific proteins that control Fe3O4 mineralization in MTB. Previous studies have demonstrated that Mms13 is a critical protein within the MM. Mms13 can be isolated from the MM fraction of Magnetospirillum magneticum AMB-1 and a Mms13 homolog, MamC, has been shown to control the size and shape of magnetite nanocrystals synthesized in-vitro. The objective of this study was to use several independent methods to definitively determine the localization of native Mms13 in M. magneticum AMB-1. Using Mms13-immunogold labeling and transmission electron microscopy (TEM), we found that Mms13 is localized to the magnetosome chain of M. magneticum AMB-1 cells. Mms13 was detected in direct contact with magnetite crystals or within the MM. Immunofluorescence detection of Mms13 in M. magneticum AMB-1 cells by confocal laser scanning microscopy (CLSM) showed Mms13 localization along the length of the magnetosome chain. Proteins contained within the MM were resolved by SDS-PAGE for Western blot analysis and LC-MS/MS (liquid chromatography with tandem mass spectrometry) protein sequencing. Using Anti-Mms13 antibody, a protein band with a molecular mass of ~14 kDa was detected in the MM fraction only. This polypeptide was digested with trypsin, sequenced by LC-MS/MS and identified as magnetosome protein Mms13. Peptides corresponding to the protein’s putative MM domain and catalytic domain were both identified by LC-MS/MS. Our results (Immunogold TEM, Immunofluorescence CLSM, Western blot, LC-MS/MS), combined with results from previous studies, demonstrate that Mms13 and homolog proteins MamC and Mam12, are localized to the magnetosome chain in MTB belonging to the class Alphaproteobacteria. Because of their shared localization in the MM and highly conserved amino acid sequences, it is likely that MamC, Mam12, and Mms13 share similar roles in the biomineralization of Fe3O4 nanocrystals.

scholarly journals Glycosylphosphatidyl Inositol-anchored Proteins and fyn Kinase Assemble in Noncaveolar Plasma Membrane Microdomains Defined by Reggie-1 and -2

2001 ◽  
Vol 12 (10) ◽  
pp. 3031-3045 ◽  
Author(s):  
Claudia A.O. Stuermer ◽  
Dirk M. Lang ◽  
Friederike Kirsch ◽  
Marianne Wiechers ◽  
Sören-Oliver Deininger ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Laser Scanning ◽  
Jurkat Cells ◽  
Membrane Microdomains ◽  
Confocal Laser ◽  
Immunogold Electron Microscopy ◽  
Cross Linking ◽  
Fyn Kinase ◽  
Glycosylphosphatidyl Inositol ◽  
Plasma Membrane Microdomains

Using confocal laser scanning and double immunogold electron microscopy, we demonstrate that reggie-1 and -2 are colocalized in ≤0.1-μm plasma membrane microdomains of neurons and astrocytes. In astrocytes, reggie-1 and -2 do not occur in caveolae but clearly outside these structures. Microscopy and coimmunoprecipitation show that reggie-1 and -2 are associated with fyn kinase and with the glycosylphosphatidyl inositol-anchored proteins Thy-1 and F3 that, when activated by antibody cross-linking, selectively copatch with reggie. Jurkat cells, after cross-linking of Thy-1 or GM1 (with the use of cholera toxin), exhibit substantial colocalization of reggie-1 and -2 with Thy-1, GM1, the T-cell receptor complex and fyn. This, and the accumulation of reggie proteins in detergent-resistant membrane fractions containing F3, Thy-1, and fyn imparts to reggie-1 and -2 properties of raft-associated proteins. It also suggests that reggie-1 and -2 participate in the formation of signal transduction centers. In addition, we find reggie-1 and -2 in endolysosomes. In Jurkat cells, reggie-1 and -2 together with fyn and Thy-1 increase in endolysosomes concurrent with a decrease at the plasma membrane. Thus, reggie-1 and -2 define raft-related microdomain signaling centers in neurons and T cells, and the protein complex involved in signaling becomes subject to degradation.

scholarly journals Charged residues in the M2 region of α-hENaC play a role in channel conductance

2000 ◽  
Vol 278 (2) ◽  
pp. C277-C291 ◽  
Author(s):  
Anne Lynn B. Langloh ◽  
Bakhrom Berdiev ◽  
Hong-Long Ji ◽  
Kent Keyser ◽  
Bruce A. Stanton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Single Channel ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Channel Conductance ◽  
Α Subunit ◽  
Whole Cell

The epithelial Na+channel (ENaC) is a low-conductance channel that is highly selective for Na+ and Li+ over K+ and impermeable to anions. The molecular basis underlying these conduction properties is not well known. Previous studies with the ENaC subunits demonstrated that the M2 region of α-ENaC is critical to channel function. Here we examine the effects of reversing the negative charges of highly conserved amino acids in α-subunit human ENaC (α-hENaC) M1 and M2 domains. Whole cell and single-channel current measurements indicated that the M2 mutations E568R, E571R, and D575R significantly decreased channel conductance but did not affect Na+:K+permeability. We observed no functional perturbations from the M1 mutation E108R. Whole cell amiloride-sensitive current recorded from oocytes injected with the M2 α-hENaC mutants along with wild-type (wt) β- and γ-hENaC was low (46–93 nA) compared with the wt channel (1–3 μA). To determine whether this reduced macroscopic current resulted from a decreased number of mutant channels at the plasma membrane, we coexpressed mutant α-hENaC subunits with green fluorescent protein-tagged β- and γ-subunits. Confocal laser scanning microscopy of oocytes demonstrated that plasma membrane localization of the mutant channels was the same as that of wt. These experiments demonstrate that acidic residues in the second transmembrane domain of α-hENaC affect ion permeation and are thus critical components of the conductive pore of ENaC.

scholarly journals Insulin-sensitive targeting of the GLUT4 glucose transporter in L6 myoblasts is conferred by its COOH-terminal cytoplasmic tail.

1995 ◽  
Vol 129 (3) ◽  
pp. 641-658 ◽  
Author(s):  
P M Haney ◽  
M A Levy ◽  
M S Strube ◽  
M Mueckler
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Glucose Transporter ◽  
Cytoplasmic Tail ◽  
Structural Basis ◽  
Immunogold Electron Microscopy ◽  
Subcellular Targeting ◽  
Intracellular Targeting ◽  
Necessary And Sufficient

The GLUT4 glucose transporter appears to be targeted to a unique insulin-sensitive intracellular membrane compartment in fat and muscle cells. Insulin stimulates glucose transport in these cell types by mediating the partial redistribution of GLUT4 from this intracellular compartment to the plasma membrane. The structural basis for the unique targeting behavior of GLUT4 was investigated in the insulin-sensitive L6 myoblast cell line. Analysis of immunogold-labeled cells of independent clonal lines by electron microscopy indicated that 51-53% of GLUT1 was present in the plasma membrane in the basal state. Insulin did not significantly affect this distribution. In contrast, only 4.2-6.1% of GLUT4 was present in the plasma membrane of basal L6 cells and insulin increased this percentage by 3.7-6.1-fold. Under basal conditions and after insulin treatment, GLUT4 was detected in tubulovesicular structures, often clustered near Golgi stacks, and in endosome-like vesicles. Analysis of 25 chimeric transporters consisting of reciprocal domains of GLUT1 and GLUT4 by confocal immunofluorescence microscopy indicated that only the final 25 amino acids of the COOH-terminal cytoplasmic tail of GLUT4 were both necessary and sufficient for the targeting pattern observed for GLUT4. A dileucine motif present in the COOH-terminal tail of GLUT4 was found to be necessary, but not sufficient, for intracellular targeting. Contrary to previous studies, the NH2 terminus of GLUT4 did not affect the subcellular distribution of chimeras. Analysis of a chimera containing the COOH-terminal tail of GLUT4 by immunogold electron microscopy indicated that its subcellular distribution in basal cells was very similar to that of wild-type GLUT4 and that its content in the plasma membrane increased 6.8-10.5-fold in the presence of insulin. Furthermore, only the chimera containing the COOH terminus of GLUT4 enhanced insulin responsive 2-deoxyglucose uptake. GLUT1 and two other chimeras lacking the COOH terminus of GLUT4 were studied by immunogold electron microscopy and did not demonstrate insulin-mediated changes in subcellular distribution. The NH2-terminal cytoplasmic tail of GLUT4 did not confer intracellular sequestration and did not cause altered subcellular distribution in the presence of insulin. Intracellular targeting of one chimera to non-insulin-sensitive compartments was also observed. We conclude that the COOH terminus of GLUT4 is both necessary and sufficient to confer insulin-sensitive subcellular targeting of chimeric glucose transporters in L6 myoblasts.

scholarly journals Pro-Apoptotic Apoptosis Protease–Activating Factor 1 (Apaf-1) Has a Cytoplasmic Localization Distinct from Bcl-2 or Bcl-XL

2000 ◽  
Vol 149 (3) ◽  
pp. 623-634 ◽  
Author(s):  
George Hausmann ◽  
Lorraine A. O'Reilly ◽  
Rosemary van Driel ◽  
Jennifer G. Beaumont ◽  
Andreas Strasser ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cytochrome C ◽  
Laser Scanning ◽  
Subcellular Fractionation ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Immunogold Electron Microscopy ◽  
Cytoplasmic Localization ◽  
In Vitro Activation

How Bcl-2 and its pro-survival relatives prevent activation of the caspases that mediate apoptosis is unknown, but they appear to act through the caspase activator apoptosis protease–activating factor 1 (Apaf-1). According to the apoptosome model, the Bcl-2–like proteins preclude Apaf-1 activity by sequestering the protein. To explore Apaf-1 function and to test this model, we generated monoclonal antibodies to Apaf-1 and used them to determine its localization within diverse cells by subcellular fractionation and confocal laser scanning microscopy. Whereas Bcl-2 and Bcl-xL were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocalize with them, even when these pro-survival proteins were overexpressed or after apoptosis was induced. Immunogold electron microscopy confirmed that Apaf-1 was dispersed in the cytoplasm and not on mitochondria or other organelles. After the death stimuli, Bcl-2 and Bcl-xL precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of larger Apaf-1 complexes, which are steps that presage apoptosis. However, neither Bcl-2 nor Bcl-xL could prevent the in vitro activation of Apaf-1 induced by the addition of exogenous cytochrome c. Hence, rather than sequestering Apaf-1 as proposed by the apoptosome model, Bcl-2–like proteins probably regulate Apaf-1 indirectly by controlling upstream events critical for its activation.

scholarly journals Investigation of Spin Coating Cerium-Doped Hydroxyapatite Thin Films with Antifungal Properties

Coatings ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 464
Author(s):  
Simona Liliana Iconaru ◽  
Mihai Valentin Predoi ◽  
Patrick Chapon ◽  
Sofia Gaiaschi ◽  
Krzysztof Rokosz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Photoelectron Spectroscopy ◽  
Spin Coating ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
X Ray ◽  
Antifungal Properties ◽  
Scanning Electron

In this study, the cerium-doped hydroxyapatite (Ca10−xCex(PO4)6(OH)2 with xCe = 0.1, 10Ce-HAp) coatings obtained by the spin coating method were presented for the first time. The stability of the 10Ce-HAp suspension particles used in the preparation of coatings was evaluated by ultrasonic studies, transmission electron microscopy (TEM), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The surface morphology of the 10Ce-HAp coating was studied by SEM and atomic force microscopy (AFM) techniques. The obtained 10Ce-HAp coatings were uniform and without cracks or unevenness. Glow discharge optical emission spectroscopy (GDOES) and X-ray photoelectron spectroscopy (XPS) were used for the investigation of fine chemical depth profiling. The antifungal properties of the HAp and 10Ce-HAp suspensions and coatings were assessed using Candida albicans ATCC 10231 (C. albicans) fungal strain. The quantitative antifungal assays demonstrated that both 10Ce-HAp suspensions and coatings exhibited strong antifungal properties and that they successfully inhibited the development and adherence of C. albicans fungal cells for all the tested time intervals. The scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) visualization of the C. albicans fungal cells adherence to the 10Ce-HAp surface also demonstrated their strong inhibitory effects. In addition, the qualitative assays also suggested that the 10Ce-HAp coatings successfully stopped the biofilm formation.

scholarly journals Biosensors Used for Epifluorescence and Confocal Laser Scanning Microscopies to Study Dickeya and Pectobacterium Virulence and Biocontrol

2021 ◽  
Vol 9 (2) ◽  
pp. 295
Author(s):  
Yvann Bourigault ◽  
Andrea Chane ◽  
Corinne Barbey ◽  
Sylwia Jafra ◽  
Robert Czajkowski ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Infected Plant ◽  
Soft Rot ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Cell Physiology ◽  
In Planta ◽  
Reporter System ◽  
Cell Wall Lysis

Promoter-probe vectors carrying fluorescent protein-reporter genes are powerful tools used to study microbial ecology, epidemiology, and etiology. In addition, they provide direct visual evidence of molecular interactions related to cell physiology and metabolism. Knowledge and advances carried out thanks to the construction of soft-rot Pectobacteriaceae biosensors, often inoculated in potato Solanum tuberosum, are discussed in this review. Under epifluorescence and confocal laser scanning microscopies, Dickeya and Pectobacterium-tagged strains managed to monitor in situ bacterial viability, microcolony and biofilm formation, and colonization of infected plant organs, as well as disease symptoms, such as cell-wall lysis and their suppression by biocontrol antagonists. The use of dual-colored reporters encoding the first fluorophore expressed from a constitutive promoter as a cell tag, while a second was used as a regulator-based reporter system, was also used to simultaneously visualize bacterial spread and activity. This revealed the chronology of events leading to tuber maceration and quorum-sensing communication, in addition to the disruption of the latter by biocontrol agents. The promising potential of these fluorescent biosensors should make it possible to apprehend other activities, such as subcellular localization of key proteins involved in bacterial virulence in planta, in the near future.

scholarly journals Fig mosaic emaravirus p4 protein is involved in cell-to-cell movement

2013 ◽  
Vol 94 (3) ◽  
pp. 682-686 ◽  
Author(s):  
Kazuya Ishikawa ◽  
Kensaku Maejima ◽  
Ken Komatsu ◽  
Osamu Netsu ◽  
Takuya Keima ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Laser Scanning ◽  
Movement Protein ◽  
Rna Virus ◽  
Cell Movement ◽  
Plant Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Series Of Experiments ◽  
Fig Mosaic Virus

Fig mosaic virus (FMV), a member of the newly formed genus Emaravirus, is a segmented negative-strand RNA virus. Each of the six genomic FMV segments contains a single ORF: that of RNA4 encodes the protein p4. FMV-p4 is presumed to be the movement protein (MP) of the virus; however, direct experimental evidence for this is lacking. We assessed the intercellular distribution of FMV-p4 in plant cells by confocal laser scanning microscopy and we found that FMV-p4 was localized to plasmodesmata and to the plasma membrane accompanied by tubule-like structures. A series of experiments designed to examine the movement functions revealed that FMV-p4 has the capacity to complement viral cell-to-cell movement, prompt GFP diffusion between cells, and spread by itself to neighbouring cells. Altogether, our findings demonstrated that FMV-p4 shares several properties with other viral MPs and plays an important role in cell-to-cell movement.

scholarly journals Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites

1998 ◽  
Vol 330 (2) ◽  
pp. 853-860 ◽  
Author(s):  
N. J. Silvia MORENO ◽  
Li ZHONG ◽  
Hong-Gang LU ◽  
Wanderley DE SOUZA ◽  
Marlene BENCHIMOL
Keyword(s):  
Plasma Membrane ◽  
Toxoplasma Gondii ◽  
Laser Scanning ◽  
Membrane Surface ◽  
Surface Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Cytoplasmic Ph ◽  
Bafilomycin A1

Cytoplasmic pH (pHi) regulation was studied in Toxoplasma gondii tachyzoites by using the fluorescent dye 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Their mean baseline pHi (7.07±0.06; n = 5) was not significantly affected in the absence of extracellular Na+, K+ or HCO3- but was significantly decreased in a dose-dependent manner by low concentrations of N,Nʹ-dicyclohexylcarbodi-imide (DCCD), N-ethylmaleimide (NEM) or bafilomycin A1. Bafilomycin A1 also inhibited the recovery of tachyzoite pHi after an acid load with sodium propionate. Similar concentrations of DCCD, NEM and bafilomycin A1 produced depolarization of the plasma membrane potential as measured with bis-(1,3-diethylthiobarbituric)trimethineoxonol (bisoxonol), and DCCD prevented the hyperpolarization that accompanies acid extrusion after the addition of propionate, in agreement with the electrogenic nature of this pump. Confocal laser scanning microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the vacuolar (V)-H+-ATPase of T. gondii tachyzoites is also located in the plasma membrane. Surface localization of the V-H+-ATPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against V-H+-ATPases. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of these intracellular parasites and with an important role of this enzyme in the regulation of pHi homoeostasis in these cells.

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