scholarly journals Myoblasts and macrophages share molecular components that contribute to cell–cell fusion

2008 ◽  
Vol 180 (5) ◽  
pp. 1005-1019 ◽  
Author(s):  
Kostandin V. Pajcini ◽  
Jason H. Pomerantz ◽  
Ozan Alkan ◽  
Regis Doyonnas ◽  
Helen M. Blau
Keyword(s):  
Cell Fusion ◽  
Cell Formation ◽  
Cell Types ◽  
Giant Cells ◽  
Nucleotide Exchange ◽  
Multinucleated Cells ◽  
Guanine Nucleotide Exchange ◽  
Distinct Cell ◽  
Molecular Components ◽  
Cell Cell

Cell–cell fusion is critical to the normal development of certain tissues, yet the nature and degree of conservation of the underlying molecular components remains largely unknown. Here we show that the two guanine-nucleotide exchange factors Brag2 and Dock180 have evolutionarily conserved functions in the fusion of mammalian myoblasts. Their effects on muscle cell formation are distinct and are a result of the activation of the GTPases ARF6 and Rac, respectively. Inhibition of ARF6 activity results in a lack of physical association between paxillin and β1-integrin, and disruption of paxillin transport to sites of focal adhesion. We show that fusion machinery is conserved among distinct cell types because Dock180 deficiency prevented fusion of macrophages and the formation of multinucleated giant cells. Our results are the first to demonstrate a role for a single protein in the fusion of two different cell types, and provide novel mechanistic insight into the function of GEFs in the morphological maturation of multinucleated cells.

Macrocyst development in Dictyostelium discoideum. II. Mating-type-specific cell fusion and acquisition of fusion-competence

1983 ◽  
Vol 60 (1) ◽  
pp. 157-168
Author(s):  
Y. Saga ◽  
H. Okada ◽  
K. Yanagisawa
Keyword(s):  
Dictyostelium Discoideum ◽  
Cell Fusion ◽  
Mating Type ◽  
Cell Formation ◽  
Giant Cells ◽  
Specific Cell ◽  
Restrictive Temperature ◽  
Opposite Mating Type ◽  
Multinucleated Cells ◽  
Giant Cell Formation

The early events of macrocyst development in Dictyostelium discoideum have been investigated using a new culturing system. When cells of opposite mating-types, HM1 and NC4, are cultured together at the appropriate temperature in the dark, giant cells appear, ingest the surrounding amoebae, and develop into macrocysts. Although these giant cells have been assumed to be the products of the fusion of opposite mating-type cells, no experimental evidence to prove this assumption has been obtained using such mixed-culture systems. In order to avoid the complexities involved in mixed-culturing, we have developed a new system involving the separate culture, and later mixing, of opposite mating-type cells. This has enabled us to obtain direct evidence that giant cells are produced by fusion between opposite mating-type cells. Cell fusion occurs immediately after mixing and is completed within 30 min. As a number of cells fuse simultaneously, giant cells produced by this method are very large multinucleated cells, and not binucleated zygotes. Using this system we also discovered the following facts related to giant cell formation: (1) cells can acquire their fusion competence without the presence of cells of the opposite mating-type; (2) only HM1 cells require darkness to acquire their fusion competence; (3) the restrictive temperature, 25 degrees C, inhibits the induction of fusion competence in HM1 cells, but not in NC4 cells.

Effects of temperature on reptilian and other cells

Development ◽  
1966 ◽  
Vol 16 (3) ◽  
pp. 455-467
Author(s):  
N. G. Stephenson
Keyword(s):  
Blood Cells ◽  
Cell Types ◽  
Giant Cells ◽  
Hanging Drop ◽  
Culture Studies ◽  
Multinucleated Cells ◽  
Zootoca Vivipara ◽  
Lower Vertebrates ◽  
Initial Culture ◽  
Mononuclear Blood Cells

Only rarely have reptilian cells been used in cell culture studies. Lewis & Lewis (1925, 1926) investigated the transformation of mononuclear blood cells into macrophages, epithelioid cells and giant multinucleated cells in hanging drop cultures of lower vertebrates, including those of reptiles. Cohen (1926) used hanging-drop cultures of turtle blood to examine the formation of giant cells, because experiments could be carried out at room temperature. He recorded the activity of giant cells for 26 days after initial culture. Chang (1934) examined selachian blood in handing-drop cultures of whole blood and for comparison made similar studies of other vertebrates, including a snake. Studies with cell types other than blood cells have been more neglected. Chlopin (1925), studying liver cells of a number of embryonic and adult vertebrates, used a young specimen of Zootoca vivipara, and explanted a liver piece to a hanging drop preparation of rabbit plasma (1–2 months at 22–24 °C).

scholarly journals Reciprocal expression of 17α-hydroxylase-C17,20-lyase and aromatase cytochrome P450 during bovine trophoblast differentiation: a two-cell system drives placental oestrogen synthesis

Reproduction ◽  
2006 ◽  
Vol 131 (4) ◽  
pp. 669-679 ◽  
Author(s):  
G Schuler ◽  
G R Özalp ◽  
B Hoffmann ◽  
N Harada ◽  
P Browne ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Cell Types ◽  
Staining Intensity ◽  
Giant Cells ◽  
Cell System ◽  
Cellular Level ◽  
Specific Staining ◽  
Cytoplasmic Staining ◽  
Distinct Cell ◽  
Trophoblast Giant Cells

No definitive information is yet available on the steroidogenic capacity of the two morphologically distinct cell types forming the bovine trophoblast, the uninucleated trophoblast cells (UTCs) and the trophoblast giant cells (TGCs). Hence, in order to localise 17α-hydroxylase-C17,20-lyase (P450c17) on a cellular level and to monitor its expression as a function of gestational age, placentomes from pregnant (days 80–284; n = 19), prepartal (days 273–282; 24–36 h prior to the onset of labour; n = 3) and parturient cows (n = 5) were immunostained for P450c17 using an antiserum against the recombinant bovine enzyme. At all stages investigated, P450c17 was exclusively found in the UTCs of chorionic villi (CV), where staining was ubiquitous between days 80 and 160, but was largely restricted to primary CV and the branching sites of secondary CV between days 160 and 240. Thereafter, a distinct ubiquitous staining reoccurred in the UTCs of all CV in late pregnant, prepartal and parturient animals. Using an antiserum against human aromatase cytochrome P450 (P450arom), specific cytoplasmic staining was observed in TGCs. In placentomes from pregnant cows, staining intensity was higher in mature compared with immature TGCs and was more pronounced in the trophoblast covering big stem villi compared with the trophoblast at other sites of the villous tree. In placentomes of a parturient cow, specific staining was only found in mature TGCs that survived the normal, but substantial, prepartal decline in TGC numbers. These results clearly showed that bovine UTCs and TGCs exhibit different steroidogenic capacities, constituting a ‘two-cell’ organisation for oestrogen synthesis. P450c17 expression appears to be quickly down-regulated and P450arom is up-regulated when UTCs enter the TGC differentiation pathway.

scholarly journals Functional Analysis of Hepatitis C Virus Envelope Proteins, Using a Cell-Cell Fusion Assay

2006 ◽  
Vol 80 (4) ◽  
pp. 1817-1825 ◽  
Author(s):  
Mariko Kobayashi ◽  
Michael C. Bennett ◽  
Theodore Bercot ◽  
Ila R. Singh
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Fusion ◽  
Viral Entry ◽  
Antiviral Agents ◽  
Cell Types ◽  
Envelope Proteins ◽  
Virus Envelope ◽  
Host Cell Membrane ◽  
Cell Cell

ABSTRACT Hepatitis C virus (HCV) envelope proteins mediate the entry of virus into cells by binding to cellular receptors, resulting in fusion of the viral membrane with the host cell membrane and permitting the viral genome to enter the cytoplasm. We report the development of a robust and reproducible cell-cell fusion assay using envelope proteins from commonly occurring genotypes of HCV. The assay scored HCV envelope protein-mediated fusion by the production of fluorescent green syncytia and allowed us to elucidate many aspects of HCV fusion, including the pH of fusion, cell types that permit viral entry, and the conformation of envelope proteins essential for fusion. We found that fusion could be specifically inhibited by anti-HCV antibodies and by at least one peptide. We also generated a number of insertional mutations in the envelope proteins and tested nine of these using the fusion assay. We demonstrate that this fusion assay is a powerful tool for understanding the mechanism of HCV-mediated fusion, elucidating mutant function, and testing antiviral agents.

scholarly journals DRhoGEF2 and Diaphanous Regulate Contractile Force during Segmental Groove Morphogenesis in the Drosophila Embryo

2008 ◽  
Vol 19 (5) ◽  
pp. 1883-1892 ◽  
Author(s):  
Shai Mulinari ◽  
Mojgan Padash Barmchi ◽  
Udo Häcker
Keyword(s):  
Cell Shape ◽  
Cell Formation ◽  
Small Gtpases ◽  
Cell Junctions ◽  
Drosophila Embryo ◽  
Cell Cortex ◽  
Nucleotide Exchange ◽  
Apical Constriction ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

Morphogenesis of the Drosophila embryo is associated with dynamic rearrangement of the actin cytoskeleton mediated by small GTPases of the Rho family. These GTPases act as molecular switches that are activated by guanine nucleotide exchange factors. One of these factors, DRhoGEF2, plays an important role in the constriction of actin filaments during pole cell formation, blastoderm cellularization, and invagination of the germ layers. Here, we show that DRhoGEF2 is equally important during morphogenesis of segmental grooves, which become apparent as tissue infoldings during mid-embryogenesis. Examination of DRhoGEF2-mutant embryos indicates a role for DRhoGEF2 in the control of cell shape changes during segmental groove morphogenesis. Overexpression of DRhoGEF2 in the ectoderm recruits myosin II to the cell cortex and induces cell contraction. At groove regression, DRhoGEF2 is enriched in cells posterior to the groove that undergo apical constriction, indicating that groove regression is an active process. We further show that the Formin Diaphanous is required for groove formation and strengthens cell junctions in the epidermis. Morphological analysis suggests that Dia regulates cell shape in a way distinct from DRhoGEF2. We propose that DRhoGEF2 acts through Rho1 to regulate acto-myosin constriction but not Diaphanous-mediated F-actin nucleation during segmental groove morphogenesis.

scholarly journals A role for Gic1 and Gic2 in Cdc42 polarization

2018 ◽  
Author(s):  
Christine N. Daniels ◽  
Trevin R. Zyla ◽  
Daniel J. Lew
Keyword(s):  
Feedback Loop ◽  
Cell Types ◽  
Effector Proteins ◽  
Yeast Cells ◽  
Cell Cortex ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Rho Family ◽  
Cytoskeletal Elements

AbstractThe conserved Rho-family GTPase Cdc42 is a master regulator of polarity establishment in many cell types. Cdc42 becomes activated and concentrated in a region of the cell cortex, and recruits a variety of effector proteins to that site. In turn, many effectors participate in regulation of cytoskeletal elements in order to remodel the cytoskeleton in a polarized manner. The budding yeast Saccharomyces cerevisiae has served as a tractable model system for studies of cell polarity. In yeast cells, Cdc42 polarization involves a positive feedback loop in which effectors called p21-activated kinases (PAKs) act to recruit a Cdc42-directed guanine nucleotide exchange factor (GEF), generating more GTP-Cdc42 in areas that already have GTP-Cdc42. The GTPase-interacting components (GICs) Gic1 and Gic2 are also Cdc42 effectors, and have been implicated in regulation of the actin and septin cytoskeleton. However, we report that cells lacking GICs are primarily defective in polarizing Cdc42 itself, suggesting that they act upstream as well as downstream of Cdc42 in yeast. Our findings suggest that feedback pathways involving GTPase effectors may be more prevalent than had been appreciated.

Headless henipaviral receptor binding glycoproteins reveal key post-receptor binding contributions of the globular head and head/stalk interface in fusion promotion

2021 ◽  
Author(s):  
Yao Yu Yeo ◽  
David W. Buchholz ◽  
Amandine Gamble ◽  
Mason Jager ◽  
Hector C. Aguilar
Keyword(s):  
Cell Fusion ◽  
Receptor Binding ◽  
Viral Entry ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Wild Type ◽  
Emerging Viruses ◽  
Multinucleated Cells ◽  
Head Domain ◽  
Cell Cell

Cedar virus (CedV) is a nonpathogenic member of the Henipavirus (HNV) genus of emerging viruses, which includes the deadly Nipah (NiV) and Hendra (HeV) viruses. CedV forms syncytia, a hallmark of henipaviral and paramyxoviral infections and pathogenicity. However, the intrinsic fusogenic capacity of CedV relative to NiV or HeV remains unquantified. HNV entry is mediated by concerted interactions between the attachment (G) and fusion (F) glycoproteins. Upon receptor binding by the HNV G head domain, a fusion-activating G stalk region is exposed and triggers F to undergo a conformational cascade that leads to viral entry or cell-cell fusion. Here, we first demonstrated quantitatively that CedV is inherently significantly less fusogenic than NiV at equivalent G and F cell surface expression levels. We then generated and tested six headless CedV G mutants of distinct stalk C-terminal lengths, surprisingly revealing highly hyperfusogenic cell-cell fusion phenotypes 3 to 4-fold greater than wild-type CedV levels. Additionally, similarly to NiV, a headless HeV G mutant yielded a less pronounced hyperfusogenic phenotype compared to wild-type HeV. Further, coimmunoprecipitation and cell-cell fusion assays revealed heterotypic NiV/CedV functional G/F bidentate interactions, as well as evidence of HNV G head domain involvement beyond receptor binding or G stalk exposure. All evidence points to the G head/stalk junction being key to modulating HNV fusogenicity, supporting the notion that head domains play several distinct and central roles in modulating stalk domain fusion promotion. Further, this study exemplifies how CedV may help elucidate important mechanistic underpinnings of HNV entry and pathogenicity. IMPORTANCE The Henipavirus genus in the Paramyxoviridae family includes the zoonotic Nipah (NiV) and Hendra (HeV) viruses. NiV and HeV infections often cause fatal encephalitis and pneumonia, but no vaccines or therapeutics are currently approved for human use. Upon viral entry, Henipavirus infections yield the formation of multinucleated cells (syncytia). Viral entry and cell-cell fusion are mediated by the attachment (G) and fusion (F) glycoproteins. Cedar virus (CedV), a nonpathogenic henipavirus, may be a useful tool to gain knowledge on henipaviral pathogenicity. Here, using homotypic and heterotypic full-length and headless CedV, NiV, and HeV G/F combinations, we discovered that CedV G/F are significantly less fusogenic than NiV or HeV G/F, and that the G head/stalk junction is key to modulating cell-cell fusion, refining the mechanism of henipaviral membrane fusion events. Our study exemplifies how CedV may be a useful tool to elucidate broader mechanistic understanding for the important henipaviruses.

scholarly journals GGAPs, a New Family of Bifunctional GTP-Binding and GTPase-Activating Proteins

2003 ◽  
Vol 23 (7) ◽  
pp. 2476-2488 ◽  
Author(s):  
Chunzhi Xia ◽  
Wenbin Ma ◽  
Lewis Joe Stafford ◽  
Chengyu Liu ◽  
Liming Gong ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Intramolecular Interaction ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
New Family ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Gtpase Activating Proteins ◽  
Gtp Binding ◽  
Distinct Cell

ABSTRACT G proteins are molecular switches that control a wide variety of physiological functions, including neurotransmission, transcriptional activation, cell migration, cell growth. and proliferation. The ability of GTPases to participate in signaling events is determined by the ratio of GTP-bound to GDP-bound forms in the cell. All known GTPases exist in an inactive (GDP-bound) and an active (GTP-bound) conformation, which are catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs), respectively. In this study, we identified and characterized a new family of bifunctional GTP-binding and GTPase-activating proteins, named GGAP. GGAPs contain an N-terminal Ras homology domain, called the G domain, followed by a pleckstrin homology (PH) domain, a C-terminal GAP domain, and a tandem ankyrin (ANK) repeat domain. Expression analysis indicates that this new family of proteins has distinct cell localization, tissue distribution, and even message sizes. GTPase assays demonstrate that GGAPs have high GTPase activity through direct intramolecular interaction of the N-terminal G domain and the C-terminal GAP domain. In the absence of the GAP domain, the N-terminal G domain has very low activity, suggesting a new model of GGAP protein regulation via intramolecular interaction like the multidomain protein kinases. Overexpression of GGAPs leads to changes in cell morphology and activation of gene transcription.

scholarly journals An Elmo–Dock complex locally controls Rho GTPases and actin remodeling during cadherin-mediated adhesion

2014 ◽  
Vol 207 (5) ◽  
pp. 577-587 ◽  
Author(s):  
Christopher P. Toret ◽  
Caitlin Collins ◽  
W. James Nelson
Keyword(s):  
Cell Adhesion ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Cell Contact ◽  
Nucleotide Exchange ◽  
Cell Contacts ◽  
Guanine Nucleotide Exchange ◽  
A Genome ◽  
Dependent Cell ◽  
Cell Cell

Cell–cell contact formation is a dynamic process requiring the coordination of cadherin-based cell–cell adhesion and integrin-based cell migration. A genome-wide RNA interference screen for proteins required specifically for cadherin-dependent cell–cell adhesion identified an Elmo–Dock complex. This was unexpected as Elmo–Dock complexes act downstream of integrin signaling as Rac guanine-nucleotide exchange factors. In this paper, we show that Elmo2 recruits Dock1 to initial cell–cell contacts in Madin–Darby canine kidney cells. At cell–cell contacts, both Elmo2 and Dock1 are essential for the rapid recruitment and spreading of E-cadherin, actin reorganization, localized Rac and Rho GTPase activities, and the development of strong cell–cell adhesion. Upon completion of cell–cell adhesion, Elmo2 and Dock1 no longer localize to cell–cell contacts and are not required subsequently for the maintenance of cell–cell adhesion. These studies show that Elmo–Dock complexes are involved in both integrin- and cadherin-based adhesions, which may help to coordinate the transition of cells from migration to strong cell–cell adhesion.

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