Plasma membrane organization and dynamics is probe and cell line dependent

It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2+sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membraneversussecretory granules). In this study we established a PC12 cell line “stably expressing” the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as thetrans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca2+-dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (∼60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (∼85–90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expressionversusstable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca2+sensor for exocytosis in endocrine cells.

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A Region Within the Third Extracellular Loop of Rat AQP6 Precludes Its Trafficking to Plasma Membrane in a Heterologous Cell Line.

10.21203/rs.3.rs-147613/v1 ◽

2021 ◽

Author(s):

David Soler ◽

Thomas Kowatz ◽

Andrew Sloan ◽

Thomas McCormick ◽

Kevin Cooper ◽

...

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Membrane Protein ◽

Cell Line ◽

Membrane Trafficking ◽

Extracellular Loop ◽

Reporter System ◽

The Third ◽

Heterologous Cell ◽

Retention Signal

Abstract The inability to over-express AQP6 in the plasma membrane of heterologous cells has hampered efforts to further characterize the function of this aquaglyceroporin membrane protein at atomic detail. Using the AGR reporter system we have identified a region within loop C of AQP6 that is responsible for severely hampering its plasma membrane localization. Serine substitution corroborated that amino acids present within AQP6194-213 of AQP6 loop C contribute to intracellular retention. This intracellular retention signal may preclude proper plasma membrane trafficking and severely curtail expression of AQP6 in heterologous cells.

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Identification of a novel member of the Rab8 family from the rat basophilic leukaemia cell line, RBL.2H3

Journal of Cell Science ◽

10.1242/jcs.109.6.1265 ◽

1996 ◽

Vol 109 (6) ◽

pp. 1265-1274 ◽

Cited By ~ 4

Author(s):

J. Armstrong ◽

N. Thompson ◽

J.H. Squire ◽

J. Smith ◽

B. Hayes ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Line ◽

Northern Blot Analysis ◽

Intracellular Localization ◽

Transient Transfection ◽

The Novel ◽

Leukaemia Cell ◽

Marked Contrast ◽

Epitope Tag ◽

Leukaemia Cell Line

We describe the cloning of a cDNA from the rat basophilic leukaemia cell line (RBL.2H3) encoding a novel member of the Rab family of small GTP binding proteins. The novel clone, which we call Rab8b, is most highly related to the Rab8 family with substantial divergence in the variable C-terminal domain. Northern blot analysis reveals highest levels of expression of Rab8b in the spleen, testis and brain, which is in marked contrast to the tissue distribution of Rab8. The Rab8b cDNA was modified to introduce a c-myc epitope tag at the extreme N terminus of the protein, and transient transfection studies were performed to analyse the intracellular localization of Rab8b by confocal microscopy. Transient expression of the c-myc/Rab8b fusion protein in both PC12 and RBL.2H3 cells shows staining of both the plasma membrane and ill-defined vesicular structures, and in the case of RBL.2H3 cells appears to induce striking outgrowths of the plasma membrane.

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Microgravity-Induced Cell-to-Cell Junctional Contacts Are Counteracted by Antioxidant Compounds in TCam-2 Seminoma Cells

Applied Sciences ◽

10.3390/app10228289 ◽

2020 ◽

Vol 10 (22) ◽

pp. 8289

Author(s):

Angela Catizone ◽

Caterina Morabito ◽

Marcella Cammarota ◽

Chiara Schiraldi ◽

Katia Corano Scheri ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Line ◽

Simulated Microgravity ◽

Cell Metabolism ◽

Ultrastructural Level ◽

Antioxidant Compounds ◽

Signal Molecules ◽

Beta Catenin ◽

Autophagy Induction ◽

A Cell

The direct impact of microgravity exposure on male germ cells, as well as on their malignant counterparts, has not been largely studied. In previous works, we reported our findings on a cell line derived from a human seminoma lesion (TCam-2 cell line) showing that acute exposure to simulated microgravity altered microtubule orientation, induced autophagy, and modified cell metabolism stimulating ROS production. Moreover, we demonstrated that the antioxidant administration prevented both TCam-2 microgravity-induced microtubule disorientation and autophagy induction. Herein, expanding previous investigations, we report that simulated microgravity exposure for 24 h induced the appearance, at an ultrastructural level, of cell-to-cell junctional contacts that were not detectable in cells grown at 1 g. In line with this result, pan-cadherin immunofluorescence analyzed by confocal microscopy, revealed the clustering of this marker at the plasma membrane level on microgravity exposed TCam-2 cells. The upregulation of cadherin was confirmed by Western blot analyses. Furthermore, we demonstrated that the microgravity-induced ROS increase was responsible for the distribution of cadherin nearby the plasma membrane, together with beta-catenin since the administration of antioxidants prevented this microgravity-dependent phenomenon. These results shed new light on the microgravity-induced modifications of the cell adhesive behavior and highlight the role of ROS as microgravity activated signal molecules.

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Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation

Blood ◽

10.1182/blood.v85.3.804.bloodjournal853804 ◽

1995 ◽

Vol 85 (3) ◽

pp. 804-811 ◽

Cited By ~ 68

Author(s):

TG Gabig ◽

CD Crean ◽

PL Mantel ◽

R Rosli

Keyword(s):

Plasma Membrane ◽

Nadph Oxidase ◽

Cell Line ◽

Cell Lines ◽

Wild Type ◽

Free System ◽

Hl60 Cells ◽

Cell Free System ◽

Nadph Oxidase Activation ◽

O2 Production

Studies of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in a cell-free system showed that the low molecular-weight guanosine triphosphatase (GTPase) Rac was required, and that Rap1a may participate in activation of the catalytic complex. Full-length posttranslationally modified Rac2 was active, whereas only the 1–166 truncated form of Rap1a was functional in the cell-free system, and thus, clarification of the function of Rap1a and Rac2 in intact human phagocytes is needed to provide further insight into their roles as signal transducers from plasma membrane receptors. In the present studies, oligonucleotide-directed mutagenesis was used to introduce a series of mutations into human rap1a or rac2 in the mammalian expression vector pSR alpha neo. HL60 cells transfected with wild-type or mutated rac2 or rap1a cDNA constructs and control HL60 cells transfected with the pSR alpha neo vector containing no inserted cDNA were selected in G418-containing media, then subclones were isolated. Compared with the parent HL60 cells, each of the stable transfected cell lines differentiated similarly into neutrophil-like cells and expressed comparable levels of NADPH oxidase components p47- phox, p67-phox and gp91-phox. The differentiated vector control cell line produced O2. in response to receptor stimulation at rates that were not significantly different from parent HL60 cells. O2-. production by differentiated cell lines expressing mutated N17 Rap1a or N17 Rac2 dominant-negative proteins was inhibited, whereas O2-. production by the subline overexpressing wild-type Rap1a was increased by fourfold. O2-. production by the differentiated cell line expressing GTPase-defective V12 Rap1a was also significantly inhibited, a finding that is consistent with a requirement for cycling between guanosine diphosphate- and GTP-bound forms of Rap1a for continuous NADPH oxidase activation in intact neutrophils. A model is proposed in which Rac2 mediates assembly of the p47 and p67 oxidase components on the cytosolic face of the plasma membrane via cytoskeletal reorganization, whereas Rap1a functions downstream as the final activation switch involving direct physical interaction with the transmembrane flavocytochrome component of the NADPH oxidase.

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