scholarly journals An invasive podosome-like structure promotes fusion pore formation during myoblast fusion

2010 ◽  
Vol 191 (5) ◽  
pp. 1013-1027 ◽  
Author(s):  
Kristin L. Sens ◽  
Shiliang Zhang ◽  
Peng Jin ◽  
Rui Duan ◽  
Guofeng Zhang ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Pore Formation ◽  
Actin Polymerization ◽  
Single Channel ◽  
Myoblast Fusion ◽  
Fusion Pore ◽  
Cellular Mechanisms ◽  
Cytoskeletal Remodeling ◽  
Specific Manner ◽  
Cell Type Specific

Recent studies in Drosophila have implicated actin cytoskeletal remodeling in myoblast fusion, but the cellular mechanisms underlying this process remain poorly understood. Here we show that actin polymerization occurs in an asymmetric and cell type–specific manner between a muscle founder cell and a fusion-competent myoblast (FCM). In the FCM, a dense F-actin–enriched focus forms at the site of fusion, whereas a thin sheath of F-actin is induced along the apposing founder cell membrane. The FCM-specific actin focus invades the apposing founder cell with multiple finger-like protrusions, leading to the formation of a single-channel macro fusion pore between the two muscle cells. Two actin nucleation–promoting factors of the Arp2/3 complex, WASP and Scar, are required for the formation of the F-actin foci, whereas WASP but not Scar promotes efficient foci invasion. Our studies uncover a novel invasive podosome-like structure (PLS) in a developing tissue and reveal a previously unrecognized function of PLSs in facilitating cell membrane juxtaposition and fusion.

scholarly journals Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion.

1994 ◽  
Vol 127 (6) ◽  
pp. 1885-1894 ◽  
Author(s):  
J Zimmerberg ◽  
R Blumenthal ◽  
D P Sarkar ◽  
M Curran ◽  
S J Morris
Keyword(s):  
Cell Membrane ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Pore Formation ◽  
Fusion Pore ◽  
Restricted Movement ◽  
Influenza Hemagglutinin ◽  
Simultaneous Measurements ◽  
Cell Membrane Capacitance ◽  
Total Fusion

The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane capacitance, next by a flux of fluorescent lipid, and finally by flux of aqueous fluorescent dye. Fusion pore conductance increased by small steps. A retardation of lipid and aqueous dyes occurred during fusion pore fluctuations. The flux of aqueous dye depended on the size of the molecule. The lack of movement of aqueous dyes while total fusion pore conductance increased suggests that initial HA-triggered fusion events are characterized by the opening of multiple small pores: the formation of a "sieve".

scholarly journals The block to membrane fusion differs with the site of ligand insertion in modified retroviral envelope proteins

2008 ◽  
Vol 89 (4) ◽  
pp. 1049-1058 ◽  
Author(s):  
Byoung Y. Ryu ◽  
Tatiana Zavorotinskaya ◽  
Bernadette Trentin ◽  
Lorraine M. Albritton
Keyword(s):  
Membrane Fusion ◽  
Pore Formation ◽  
Leukemia Virus ◽  
Point Mutations ◽  
Retroviral Vectors ◽  
Membrane Lipid ◽  
Fusion Pore ◽  
Subsequent Step ◽  
Cell Type Specific ◽  
Proline Rich Region

Efforts to achieve cell type-specific transduction of retroviral vectors for gene therapy have centred on modification of the envelope protein (Env). Typically, addition of a ligand to Env gives binding to the new or target receptor, but little or no infection, and affects the subunit association of the modified Env. We previously discovered two point mutations that increase targeted infection by over 1000-fold when added to an Env modified by N-terminal insertion of the receptor-binding domain from amphotropic murine leukemia virus Env. Here, we asked whether these mutations would similarly increase transduction by Env modified with a clinically relevant ligand, human interleukin-13 (IL-13L). Addition of the point mutations stabilized the weak subunit association observed in some IL-13L-modified Env proteins, but infection via the target IL-13 receptor still did not occur. Fluorescence-based cell–cell fusion assays and studies with a membrane-curving agent revealed that defects in membrane fusion differed with the site of ligand insertion. When IL-13 was inserted into the N terminus of Env, membrane fusion was blocked prior to membrane-lipid mixing, regardless of whether flanking flexible linkers were added. Unexpectedly, insertion of IL-13 in the proline-rich region showed evidence of initiation of fusion and fusion-peptide exposure, but fusion was blocked at a subsequent step prior to fusion-pore formation. Thus, the site of ligand insertion influenced initiation of membrane fusion and its progression. These observations suggest that a novel site for ligand insertion must be identified before clinically useful targeted transduction will be achieved.

Are second messengers crucial for opening the pore associated with P2X7receptor?

2005 ◽  
Vol 288 (2) ◽  
pp. C260-C271 ◽  
Author(s):  
R. X. Faria ◽  
F. P. DeFarias ◽  
Luiz Anastácio Alves
Keyword(s):  
Cell Membrane ◽  
Pore Formation ◽  
Single Channel ◽  
Second Messengers ◽  
Second Messenger ◽  
Fluorescent Imaging ◽  
Cationic Channels ◽  
Intracellular Ca ◽  
Single Channel Currents ◽  
Channel Currents

Stimulation of the P2X7receptor by ATP induces cell membrane depolarization, increase in intracellular Ca2+concentration, and, in most cases, permeabilization of the cell membrane to molecules up to 900 Da. After the activation of P2X7, at least two phenomena occur: the opening of low-conductance (8 pS) cationic channels and pore formation. At least two conflicting hypotheses have been postulated to reconcile these findings: 1) the P2X7pore is formed as a result of gradual permeability increase (dilation) of cationic channels, and 2) the P2X7pore represents a distinct channel, possibly activated by a second messenger and not directly by extracellular nucleotides. In this study, we investigated whether second messengers are necessary to open the pore associated with the P2X7receptor in cells that expressed the pore activity by using the patch-clamp technique in whole cell and cell-attached configurations in conjunction with fluorescent imaging. In peritoneal macrophages and 2BH4 cells, we detected permeabilization and single-channel currents in the cell-attached configuration when ATP was applied outside the membrane patch in a condition in which oxidized ATP and Lucifer yellow were maintained within the pipette. Our data support Ca2+as a second messenger associated with pore formation because the permeabilization depended on the presence of intracellular Ca2+and was blocked by BAPTA-AM. In addition, MAPK inhibitors (SB-203580 and PD-98059) blocked the permeabilization and single-channel currents in these cells. Together our data indicate that the P2X7pore depends on second messengers such as Ca2+and MAP kinases.

scholarly journals Evidence for Pore Formation in Host Cell Membranes by ESX-1-Secreted ESAT-6 and Its Role in Mycobacterium marinum Escape from the Vacuole

2008 ◽  
Vol 76 (12) ◽  
pp. 5478-5487 ◽  
Author(s):  
Jennifer Smith ◽  
Joanna Manoranjan ◽  
Miao Pan ◽  
Amro Bohsali ◽  
Junjie Xu ◽  
...  
Keyword(s):  
Host Cell ◽  
Pore Formation ◽  
Actin Polymerization ◽  
Cell Membranes ◽  
Critical Role ◽  
Mycobacterial Infection ◽  
Cellular Mechanisms ◽  
Direct Role ◽  
Membrane Pores ◽  
Host Cell Membranes

ABSTRACT The ESX-1 secretion system plays a critical role in the virulence of M. tuberculosis and M. marinum, but the precise molecular and cellular mechanisms are not clearly defined. Virulent M. marinum is able to escape from the Mycobacterium-containing vacuole (MCV) into the host cell cytosol, polymerize actin, and spread from cell to cell. In this study, we have examined nine M. marinum ESX-1 mutants and the wild type by using fluorescence and electron microscopy detecting MCV membranes and actin polymerization. We conclude that ESX-1 plays an essential role in M. marinum escape from the MCV. We also show that the ESX-1 mutants acquire the ability to polymerize actin after being artificially delivered into the macrophage cytosol by hypotonic shock treatment, indicating that ESX-1 is not directly involved in initiation of actin polymerization. We provide evidence that M. marinum induces membrane pores ∼4.5 nm in diameter, and this activity correlates with ESAT-6 secretion. Importantly, purified ESAT-6, but not the other ESX-1-secreted proteins, is able to cause dose-dependent pore formation in host cell membranes. These results suggest that ESAT-6 secreted by M. marinum ESX-1 could play a direct role in producing pores in MCV membranes, facilitating M. marinum escape from the vacuole and cell-to-cell spread. Our study provides new insight into the mechanism by which ESX-1 secretion and ESAT-6 enhance the virulence of mycobacterial infection.

Cell Membrane-Coated Electrospun Fibers Enhance Keratinocyte Growth through Cell-Type Specific Interactions

2021 ◽  
Author(s):  
Wai Hon Chooi ◽  
Quanbin Dong ◽  
Jeremy Zhi Yan Low ◽  
Clement Yuen ◽  
Jiah Shin Chin ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Electrospun Fibers ◽  
Specific Interactions ◽  
Cell Type ◽  
Cell Type Specific

scholarly journals Ca2+ Entry, Oxidative Stress, Ceramide and Suicidal Erythrocyte Death Following Diosgenin Treatment

2016 ◽  
Vol 39 (4) ◽  
pp. 1626-1637 ◽  
Author(s):  
Morena Mischitelli ◽  
Mohamed Jemaà ◽  
Mustafa Almasry ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Haemoglobin Concentration ◽  
Annexin V ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Surface Signaling

Background/Aims: The bioactive steroid sapogenin diosgenin is considered for a wide variety of applications including treatment of malignancy. The substance counteracts tumor growth in part by stimulating apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether diosgenin induces eryptosis and, if so, to decipher cellular mechanisms involved. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified by determination of haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to diosgenin significantly increased the percentage of annexin-V-binding cells (≥ 5 µM), significantly decreased forward scatter (15 µM), significantly increased Fluo3-fluorescence (≥ 10 µM), significantly increased DCF fluorescence (15 µM), significantly increased ceramide abundance (15 µM) and significantly increased hemolysis (15 µM). The effect of diosgenin (15 µM) on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Diosgenin stimulates eryptosis with erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca2+ entry, oxidative stress and ceramide.

scholarly journals Actin Polymerization in Macrophages in Response to Oxidized LDL and Apoptotic Cells: Role of 12/15-Lipoxygenase and Phosphoinositide 3-Kinase

2003 ◽  
Vol 14 (10) ◽  
pp. 4196-4206 ◽  
Author(s):  
Yury I. Miller ◽  
Dorothy S. Worrall ◽  
Colin D. Funk ◽  
James R. Feramisco ◽  
Joseph L. Witztum
Keyword(s):  
Cell Membrane ◽  
Actin Polymerization ◽  
Oxidized Ldl ◽  
Low Density Lipoprotein ◽  
Specific Inhibitor ◽  
Density Lipoprotein ◽  
Oxidation Products ◽  
Apoptotic Cells ◽  
Lipid Oxidation Products ◽  
Phosphoinositide 3 Kinase

Formation of filamentous F-actin drives many cellular processes, including phagocytosis and cell spreading. We have recently reported that mouse macrophage 12/15-lipoxygenase (12/15-LO) activity promotes F-actin formation in filopodia during phagocytosis of apoptotic cells. Oxidized low-density lipoprotein (OxLDL) also stimulates robust F-actin formation and spreading of macrophages. However, unlike apoptotic cells, OxLDL did not cause specific translocation of 12/15-LO to the cell membrane, neither in macrophages nor in GFP-15LO–transfected COS-7 cells. Moreover, inhibition of 12/15-LO activity in macrophages by a specific inhibitor or by 12/15-LO gene disruption did not affect OxLDL-induced actin polymerization. Among LDL modifications modeling OxLDL, LDL modified by incubation with 15LO-overexpressing fibroblasts was as active in eliciting F-actin response as was OxLDL. This LDL modification is well known to produce minimally modified LDL (mmLDL), which is bioactive and carries lipid oxidation products similar to those produced by 12/15-LO catalysis. MmLDL activated phosphoinositide 3-kinase (PI3K), and PI3K inhibitors abolished mmLDL-induced macrophage spreading. We hypothesize that OxLDL and mmLDL may contribute oxidized lipids to the macrophage cell membrane and thereby mimic intracellular 12/15-LO activity, which leads to uncontrolled actin polymerization and dramatic cytoskeletal changes in macrophages.

scholarly journals Rel Induces Interferon Regulatory Factor 4 (IRF-4) Expression in Lymphocytes

2000 ◽  
Vol 191 (8) ◽  
pp. 1281-1292 ◽  
Author(s):  
Raelene J. Grumont ◽  
Steve Gerondakis
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Nuclear Factor Κb ◽  
Wild Type ◽  
Cell Type ◽  
Regulatory Factor ◽  
Specific Manner ◽  
A Cell ◽  
Cell Type Specific ◽  
Interferon Regulatory Factor 4

In lymphocytes, the Rel transcription factor is essential in establishing a pattern of gene expression that promotes cell proliferation, survival, and differentiation. Here we show that mitogen-induced expression of interferon (IFN) regulatory factor 4 (IRF-4), a lymphoid-specific member of the IFN family of transcription factors, is Rel dependent. Consistent with IRF-4 functioning as a repressor of IFN-induced gene expression, the absence of IRF-4 expression in c-rel−/− B cells coincided with a greater sensitivity of these cells to the antiproliferative activity of IFNs. In turn, enforced expression of an IRF-4 transgene restored IFN modulated c-rel−/− B cell proliferation to that of wild-type cells. This cross-regulation between two different signaling pathways represents a novel mechanism that Rel/nuclear factor κB can repress the transcription of IFN-regulated genes in a cell type–specific manner.

scholarly journals The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface.

1995 ◽  
Vol 106 (5) ◽  
pp. 783-802 ◽  
Author(s):  
G B Melikyan ◽  
W D Niles ◽  
F S Cohen
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Pore Formation ◽  
Surface Proteins ◽  
Fusion Pore ◽  
Planar Bilayer ◽  
Bilayer Membranes ◽  
Pore Evolution ◽  
Fusion Pores ◽  
Kinetics Of

Time-resolved admittance measurements were used to follow formation of individual fusion pores connecting influenza virus hemagglutinin (HA)-expressing cells to planar bilayer membranes. By measuring in-phase, out-of-phase, and dc components of currents, pore conductances were resolved with millisecond time resolution. Fusion pores developed in stages, from small pores flickering open and closed, to small successful pores that remained open until enlarging their lumens to sizes greater than those of viral nucleocapsids. The kinetics of fusion and the properties of fusion pores were studied as functions of density of the fusion protein HA. The consequences of treating cell surfaces with proteases that do not affect HA were also investigated. Fusion kinetics were described by waiting time distributions from triggering fusion, by lowering pH, to the moment of pore formation. The kinetics of pore formation became faster as the density of active HA was made greater or when cell surface proteins were extensively cleaved with proteases. In accord with this faster kinetics, the intervals between transient pore openings within the flickering stage were shorter for higher HA density and more extensive cell surface treatment. Whereas the kinetics of fusion depended on HA density, the lifetimes of open fusion pores were independent of HA density. However, the lifetimes of open pores were affected by the proteolytic treatment of the cells. Faster fusion kinetics correlated with shorter pore openings. We conclude that the density of fusion protein strongly affects the kinetics of fusion pore formation, but that once formed, pore evolution is not under control of fusion proteins but rather under the influence of mechanical forces, such as membrane bending and tension.

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