Rho family GTPase Rnd2 interacts and co-localizes with MgcRacGAP in male germ cells

The male-germ-cell Rac GTPase-activating protein gene (MgcRacGAP) was initially described as a human RhoGAP gene highly expressed in male germ cells at spermatocyte stage, but exhibits significant levels of expression in most cell types. In somatic cells, MgcRacGAP protein was found to both concentrate in the midzone/midbody and be required for cytokinesis. As a RhoGAP, MgcRacGAP has been proposed to down-regulate RhoA, which is localized to the cleavage furrow and midbody during cytokinesis. Due to embryonic lethality in MgcRacGAP-null mutant mice and to the lack of an in vitro model of spermatogenesis, nothing is known regarding the role and mode of action of MgcRacGAP in male germ cells. We have analysed the expression, subcellular localization and molecular interactions of MgcRacGAP in male germ cells. Whereas MgcRacGAP was found only in spermatocytes and early spermatids, the widespread RhoGTPases RhoA, Rac1 and Cdc42 (which are, to various extents, in vitro substrates for MgcRacGAP activity) were, surprisingly, not detected at these stages. In contrast, Rnd2, a Rho family GTPase-deficient G-protein was found to be co-expressed with MgcRacGAP in spermatocytes and spermatids. MgcRacGAP was detected in the midzone of meiotic cells, but also, unexpectedly, in the Golgi-derived pro-acrosomal vesicle, co-localizing with Rnd2. In addition, a stable Rnd2–MgcRacGAP molecular complex could be evidenced by glutathione S-transferase pull-down and co-immunoprecipitation experiments. We conclude that Rnd2 is a probable physiological partner of MgcRacGAP in male germ cells and we propose that MgcRacGAP, and, quite possibly, other RhoGAPs, may participate in signalling pathways involving Rnd family proteins.

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208 CYTOLYTIC ANALYSIS AND NUCLEAR TRANSFER OF hCD46-TRANSGENIC PORCINE EMBRYONIC GERM CELLS TO DEVELOP AND IN VITRO MODEL OF XENOTRANSPLANTATION

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab208 ◽

2006 ◽

Vol 18 (2) ◽

pp. 212

Author(s):

J. Y. Won ◽

K. S. Ahn ◽

S. Y. Heo ◽

J. H. Kang ◽

H. Shim

Keyword(s):

Human Serum ◽

Germ Cells ◽

Nuclear Transfer ◽

In Vitro Model ◽

Regulatory Protein ◽

Cell Types ◽

Human Complement ◽

Transgenic Pigs ◽

Vitro Model

Pigs are considered the most likely source of organs for xenotransplantation due to their anatomical and physiological similarities to humans. Production of transgenic pigs including addition of human complement-regulatory protein genes and deletion of alpha-1,3-galactosyl transferase gene may overcome hyperacute rejection (HAR), the first and currently the most critical immunological hurdle in the development of xenogeneic organs for human transplantation. However, even after resolving HAR in pig-to-human xenotransplantation, a series of other transgenic pigs may be required to alleviate subsequent acute and chronic rejection and incompatibility of porcine proteins to human counterparts. The production of transgenic pigs is not only labor-intensive, time-consuming, and costly, but also the usefulness of such pigs in transplantation to humans is unpredictable. For these reasons, development of a reliable in vitro procedure to pre-evaluate effectiveness of the transgenic approach would be beneficial. This study was preformed to establish an in vitro model of xenotransplantation using porcine embryonic germ (EG) cells, undifferentiated stem cells derived from culture of primordial germ cells. Porcine EG cells were maintained in feeder-free state in DMEM containing 15% (v/v) fetal bovine serum and 1000 units/mL leukemia inhibitory factor. Human complement down-regulator hCD46 (also known as MCP, membrane cofactor protein) gene under the regulation of cytomegalovirus promoter was introduced into porcine EG cells. Transfected cells were selected by antibiotic treatment and confirmed by PCR. To test the resistance of hCD46-transgenic EG cells to human xenoreactive natural antibody and complement, EG cells were cultured for 1.5 days in DMEM containing 15% (v/v) normal human serum. The treatment with human serum did not affect the survival of hCD46-transgenic EG cells, whereas with the same treatment approximately one half of non-transfected EG cells failed to survive (P < 0.01). Transgenic EG cells presumably capable of overcoming HAR were used as nuclear donors for subsequent transfer of nuclei into enucleated oocytes. Among 110 reconstituted oocytes, 19 (17.3%) developed to the blastocyst stage. Analysis of individual nuclear transfer embryos by PCR indicated that 89.5% (17/19) of embryos contained transgene hCD46. The PCR-negative embryos might be due to an incomplete antibiotic selection of cells after transfection. Overall, the results of the present study demonstrate that the cell culture-based model of xenotransplantation may validate the usefulness of particular transgenic pigs prior to actual production. Further experiments on differentiation of transgenic EG cells into various cell types, cytolytic analysis of such cells to assess efficiency of xenotransplantation, and subsequent production and transfer of transgenic clone embryos to recipients may provide a useful new procedure to accelerate xenotransplantation research.

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A Central Role for the Armadillo Protein Plakoglobin in the Autoimmune Disease Pemphigus Vulgaris

The Journal of Cell Biology ◽

10.1083/jcb.153.4.823 ◽

2001 ◽

Vol 153 (4) ◽

pp. 823-834 ◽

Cited By ~ 130

Author(s):

Reto Caldelari ◽

Alain de Bruin ◽

Dominique Baumann ◽

Maja M. Suter ◽

Christiane Bierkamp ◽

...

Keyword(s):

Steric Hindrance ◽

In Vitro Model ◽

Molecular Mechanisms ◽

Pemphigus Vulgaris ◽

Cell Types ◽

Linear Distribution ◽

Igg Binding ◽

Vitro Model ◽

First Time

In pemphigus vulgaris (PV), autoantibody binding to desmoglein (Dsg) 3 induces loss of intercellular adhesion in skin and mucous membranes. Two hypotheses are currently favored to explain the underlying molecular mechanisms: (a) disruption of adhesion through steric hindrance, and (b) interference of desmosomal cadherin-bound antibody with intracellular events, which we speculated to involve plakoglobin. To investigate the second hypothesis we established keratinocyte cultures from plakoglobin knockout (PG−/−) embryos and PG+/+ control mice. Although both cell types exhibited desmosomal cadherin-mediated adhesion during calcium-induced differentiation and bound PV immunoglobin (IgG) at their cell surface, only PG+/+ keratinocytes responded with keratin retraction and loss of adhesion. When full-length plakoglobin was reintroduced into PG−/− cells, responsiveness to PV IgG was restored. Moreover, in these cells like in PG+/+ keratinocytes, PV IgG binding severely affected the linear distribution of plakoglobin at the plasma membrane. Taken together, the establishment of an in vitro model using PG+/+ and PG−/− keratinocytes allowed us (a) to exclude the steric hindrance only hypothesis, and (b) to demonstrate for the first time that plakoglobin plays a central role in PV, a finding that will provide a novel direction for investigations of the molecular mechanisms leading to PV, and on the function of plakoglobin in differentiating keratinocytes.

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CELLULAR IMMUNITY IN VITRO

Journal of Experimental Medicine ◽

10.1084/jem.133.6.1377 ◽

1971 ◽

Vol 133 (6) ◽

pp. 1377-1389 ◽

Cited By ~ 80

Author(s):

Harvey B. Simon ◽

John N. Sheagren

Keyword(s):

Cellular Immunity ◽

In Vitro Model ◽

Specific Antigen ◽

Cell Types ◽

Intracellular Bacteria ◽

Migration Inhibition ◽

Vitro Model ◽

And Control ◽

Macrophage Migration Inhibition

An in vitro model of cellular immunity in the guinea pig was established. Animals were immunized with tubercle bacilli, bovine gamma globulin, or picrylated human serum albumin in complete Freund's adjuvant. Oil-induced peritoneal exudates from immune and control animals were cultured overnight with and without specific antigen. The cultures were washed and the macrophage monolayers were infected with Listeria monocytogenes. At intervals the monolayers were lysed and the numbers of viable intracellular bacteria were quantitated by pour plate cultures. Random monolayers were also evaluated in sequence by visually counting the intracellular bacteria on Gram-stained plates. Both methods demonstrated that the macrophages from immune animals had markedly enhanced listericidal activity when the peritoneal exudates were cultured with antigen before infection. Macrophage migration inhibition was also demonstrated under these conditions. The experiments reported here describe an in vitro model of cellular immunity which will allow separation and recombination of cell types and direct assay of cell products in efforts to elucidate further the mechanisms of the immunologically mediated enhancement of macrophage bactericidal capacity.

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Pluripotent stem cells of the mouse as a potential in vitro model for mammalian germ cells. Sister chromatid exchanges induced by MMC and ENU in undifferentiated cell lines compared to differentiated cell lines

Mutation Research/Genetic Toxicology and Environmental Mutagenesis ◽

10.1016/s1383-5718(99)00090-x ◽

1999 ◽

Vol 444 (1) ◽

pp. 97-102 ◽

Cited By ~ 6

Author(s):

Susanne Bremer ◽

Richard Vogel

Keyword(s):

Stem Cells ◽

Cell Lines ◽

Germ Cells ◽

Pluripotent Stem Cells ◽

In Vitro Model ◽

Sister Chromatid Exchanges ◽

Undifferentiated Cell ◽

Vitro Model ◽

Chromatid Exchanges

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Efficient differentiation of human primordial germ cells through geometric control reveals a key role for NODAL signaling

10.1101/2021.08.04.455129 ◽

2021 ◽

Author(s):

Kyoung Jo ◽

Seth Teague ◽

Bohan Chen ◽

Hina Aftab Khan ◽

Emily Freeburne ◽

...

Keyword(s):

Germ Cells ◽

In Vitro Model ◽

Primordial Germ Cells ◽

Bmp Signaling ◽

Critical Parameters ◽

Nodal Signaling ◽

Relative Timing ◽

Vitro Model ◽

First Time

Human primordial germ cells (hPGCs) form around the time of implantation and are the precursors of eggs and sperm. Many aspects of hPGC specification remain poorly understood. Here we show that micropatterned human pluripotent stem cells (hPSCs) treated with BMP4 give rise to hPGC-like cells (hPGCLC) and use these as a quantitatively reproducible and simple in vitro model to interrogate this important developmental event. We characterize micropatterned hPSCs up to 96h for the first time and show that hPGCLC populations are stable and continue to mature. By perturbing signaling during hPGCLC differentiation, we identify a previously unappreciated role for NODAL signaling and find that the relative timing and duration of BMP and NODAL signaling are critical parameters controlling the number of hPGCLCs. We formulate a mathematical model for a network of cross-repressive fates driven by NODAL and BMP signaling which predicts the measured fate patterns after signaling perturbations. Finally, we show that hPSC colony size dictates the efficiency of hPGCLC specification, which led us to dramatically improve the efficiency of hPGCLC differentiation over current protocols.

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