scholarly journals Embedding of HIV Egress within Cortical F-Actin

Pathogens ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 56
Author(s):  
Anupriya Aggarwal ◽  
Alberto Ospina Stella ◽  
Catherine C. Henry ◽  
Kedar Narayan ◽  
Stuart G. Turville
Keyword(s):  
Focused Ion Beam ◽  
Positive Curvature ◽  
Ion Beam ◽  
Membrane Curvature ◽  
Cell Contact ◽  
Actin Remodeling ◽  
Cell Contacts ◽  
Viral Release ◽  
Viral Mutagenesis ◽  
Cell Cell

F-Actin remodeling is important for the spread of HIV via cell–cell contacts; however, the mechanisms by which HIV corrupts the actin cytoskeleton are poorly understood. Through live cell imaging and focused ion beam scanning electron microscopy (FIB-SEM), we observed F-Actin structures that exhibit strong positive curvature to be enriched for HIV buds. Virion proteomics, gene silencing, and viral mutagenesis supported a Cdc42-IQGAP1-Arp2/3 pathway as the primary intersection of HIV budding, membrane curvature and F-Actin regulation. Whilst HIV egress activated the Cdc42-Arp2/3 filopodial pathway, this came at the expense of cell-free viral release. Importantly, release could be rescued by cell–cell contact, provided Cdc42 and IQGAP1 were present. From these observations, we conclude that a proportion out-going HIV has corrupted a central F-Actin node that enables initial coupling of HIV buds to cortical F-Actin to place HIV at the leading cell edge. Whilst this initially prevents particle release, the maturation of cell–cell contacts signals back to this F-Actin node to enable viral release & subsequent infection of the contacting cell.

scholarly journals HIV corrupts the Cdc42-IQGAP1 axis of actin regulation to promote cell-cell viral spread

2019 ◽  
Author(s):  
Anupriya Aggarwal ◽  
Alberto Ospina Stella ◽  
Catherine Henry ◽  
Kedar Narayan ◽  
Stuart G. Turville
Keyword(s):  
Focused Ion Beam ◽  
Positive Curvature ◽  
Ion Beam ◽  
Membrane Curvature ◽  
Cell Contact ◽  
Cell Contacts ◽  
Viral Spread ◽  
Viral Release ◽  
Viral Mutagenesis ◽  
Cell Cell

AbstractF-Actin remodelling is important for the spread of HIV via cell-cell contacts, yet the mechanisms by which HIV corrupts the actin cytoskeleton are poorly understood. Through live cell imaging and focused ion beam scanning electron microscopy (FIB-SEM), we observed F-Actin structures that exhibit strong positive curvature to be enriched for HIV buds. Virion proteomics, gene silencing, and viral mutagenesis supported a Cdc42-IQGAP1-Arp2/3 pathway as the primary intersection of HIV budding, membrane curvature and F-Actin regulation. Whilst HIV egress activated the Cdc42-Arp2/3 filopodial pathway, this came at the expense of cell-free viral release. Importantly, release could be rescued by cell-cell contact, providing Cdc42 and IQGAP1 were present. From these observations we conclude that out-going HIV has corrupted a central F-Actin node that enables initial coupling of HIV buds to cortical F-Actin to place HIV at the leading cell edge. Whilst this initially prevents particle release, maturation of cell-cell contacts signals back to this F-Actin node to enable viral release & subsequent infection of the contacting cell.

N-cadherin in adult rat cardiomyocytes in culture. II. Spatio-temporal appearance of proteins involved in cell-cell contact and communication. Formation of two distinct N-cadherin/catenin complexes

1996 ◽  
Vol 109 (1) ◽  
pp. 11-20 ◽  
Author(s):  
C.M. Hertig ◽  
S. Butz ◽  
S. Koch ◽  
M. Eppenberger-Eberhardt ◽  
R. Kemler ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Connexin 43 ◽  
Cell Contact ◽  
Beta Catenin ◽  
Rat Cardiomyocytes ◽  
Cell Contacts ◽  
Adult Rat ◽  
Spatio Temporal ◽  
Cell Cell

The spatio-temporal appearance and distribution of proteins forming the intercalated disc were investigated in adult rat cardiomyocytes (ARC). The ‘redifferentiation model’ of ARC involves extensive remodelling of the plasma membrane and of the myofibrillar apparatus. It represents a valuable system to elucidate the formation of cell-cell contact between cardiomyocytes and to assess the mechanisms by which different proteins involved in the cell-cell adhesion process are sorted in a precise manner to the sites of function. Appearance of N-cadherin, the catenins and connexin43 within newly formed adherens and gap junctions was studied. Here first evidence is provided for a formation of two distinct and separable N-cadherin/catenin complexes in cardiomyocytes. Both complexes are composed of N-cadherin and alpha-catenin which bind to either beta-catenin or plakoglobin in a mutually exclusive manner. The two N-cadherin/catenin complexes are assumed to be functionally involved in the formation of cell-cell contacts in ARC; however, the differential appearance and localization of the two types of complexes may also point to a specific role during ARC differentiation. The newly synthesized beta-catenin containing complex is more abundant during the first stages in culture after ARC isolation, while the newly synthesized plakoglobin containing complex progressively accumulates during the morphological changes of ARC. ARC formed a tissue-like pattern in culture whereby the new cell-cell contacts could be dissolved through Ca2+ depletion. Presence of cAMP and replenishment of Ca2+ content in the culture medium not only allowed reformation of cell-cell contacts but also affected the relative protein ratio between the two N-cadherin/catenin complexes, increasing the relative amount of newly synthesized beta-catenin over plakoglobin at a particular stage of ARC differentiation. The clustered N-cadherin/catenin complexes at the plasma membrane appear to be a prerequisite for the following gap junction formation; a temporal sequence of the appearance of adherens junction proteins and of gap junctions forming connexin-43 is suggested.

Robust T-cell signaling triggered on soft polydimethylsiloxane-supported lipid-bilayers

2020 ◽  
Author(s):  
Anna H. Lippert ◽  
Ivan B. Dimov ◽  
Alexander Winkel ◽  
James McColl ◽  
Jane Humphrey ◽  
...  
Keyword(s):  
T Cell ◽  
Lipid Bilayers ◽  
T Cell Activation ◽  
Cell Activation ◽  
Tyrosine Phosphatase ◽  
Supported Lipid Bilayers ◽  
Cell Contact ◽  
Mechanical Resistance ◽  
Cell Contacts ◽  
Cell Cell

AbstractThe T-cell receptor (TCR) is thought to be triggered either by mechano-transduction or local tyrosine phosphatase exclusion at cell-cell contacts. However, the effects of the mechanical properties of activating surfaces have only been tested for late-stage T-cell activation, and phosphatase segregation has mostly been studied on glass-supported lipid bilayers that favor imaging but are orders-of-magnitude stiffer than typical cells. We developed a method for attaching lipid bilayers to polydimethylsiloxane polymer supports, producing ‘soft bilayers’ with physiological levels of mechanical resistance (Young’s modulus of 4 kPa). Comparisons of T-cell behavior on soft and glass-supported bilayers revealed that early calcium signaling is unaffected by substrate rigidity, implying that early steps in TCR triggering are not mechanosensitive. Robust phosphatase exclusion was observed on the soft bilayers, however, suggesting it likely occurs at cell-cell contacts. This work sets the stage for an imaging-based exploration of receptor signaling under conditions closely mimicking physiological cell-cell contact.

scholarly journals ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells.

1989 ◽  
Vol 109 (3) ◽  
pp. 1047-1056 ◽  
Author(s):  
J M Anderson ◽  
C M Van Itallie ◽  
M D Peterson ◽  
B R Stevenson ◽  
E A Carew ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Polyclonal Antibodies ◽  
Epithelial Cell Line ◽  
Cell Contact ◽  
Mrna Levels ◽  
Expression Library ◽  
Cell Contacts ◽  
Protein Levels ◽  
Cell Cell

We previously identified and characterized ZO-1 as a peripheral membrane protein specifically associated with the cytoplasmic surface of tight junctions. Here we describe the identification of partial cDNA sequences encoding rat and human ZO-1 and their use to study the assembly of tight junctions in the Caco-2 human intestinal epithelial cell line. A rat cDNA was isolated from a lambda-gtll expression library by screening with mAbs. Polyclonal antibodies were raised to cDNA-encoded fusion protein; several properties of these antibodies support this cDNA as encoding ZO-1. Expression of ZO-1 mRNA occurs in the rat and Caco-2 cells with a major transcript of approximately 7.5 kb. To disrupt tight junctions and study the subsequent process of assembly, Caco-2 cells were grown in suspension for 48 h in Ca++/Mg++-free spinner medium during which time they lose cell-cell contacts, become round, and by immunofluorescence microscopy show diffuse and speckled localization of ZO-1. Within hours of replating at confluent density in Ca++/Mg++-containing media, attached cells show discrete localization of ZO-1 at cell-cell contacts. Within 2 d, fully confluent monolayers form, and ZO-1 localizes in a continuous gasket-like fashion circumscribing all cells. ZO-1 mRNA levels are highest in cells in spinner culture, and upon replating rapidly fall and plateau at approximately 10% of initial levels after 2-3 wk in culture. ZO-1 protein levels are lowest in contact-free cells and rise five- to eightfold over the same period. In contrast, mRNA levels for sucrase-isomaltase, an apical membrane hydrolase, increase only after a confluent monolayer forms. Thus, in this model of contact-dependent assembly of the tight junction, there is both a rapid assembly beginning upon cell-cell contact, as well as a long-term modulation involving changes in expression of ZO-1 mRNA and protein levels.

scholarly journals N-Cadherin Association with Lipid Rafts Regulates Its Dynamic Assembly at Cell-Cell Junctions in C2C12 Myoblasts

2005 ◽  
Vol 16 (5) ◽  
pp. 2168-2180 ◽  
Author(s):  
Marie Causeret ◽  
Nicolas Taulet ◽  
Franck Comunale ◽  
Cyril Favard ◽  
Cécile Gauthier-Rouvière
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Lipid Rafts ◽  
Membrane Lipid ◽  
Cell Junctions ◽  
Cell Contact ◽  
C2c12 Myoblasts ◽  
Cell Contacts ◽  
Dynamic Assembly ◽  
Cell Cell

Cadherins are homophilic cell-cell adhesion molecules implicated in cell growth, differentiation, and organization into tissues during embryonic development. They accumulate at cell-cell contact sites and act as adhesion-activated signaling receptors. Here, we show that the dynamic assembly of N-cadherin at cell-cell contacts involves lipid rafts. In C2C12 myoblasts, immunofluorescence and biochemical experiments demonstrate that N-cadherin present at cell-cell contacts is colocalized with lipid rafts. Disruption of lipid rafts leads to the inhibition of cell-cell adhesion and disorganization of N-cadherin–dependent cell-cell contacts without modifying the association of N-cadherin with catenins and its availability at the plasma membrane. Fluorescent recovery after photobleaching experiments demonstrate that at the dorsal plasma membrane, lipid rafts are not directly involved in the diffusional mobility of N-cadherin. In contrast, at cell-cell junctions N-cadherin association with lipid rafts allows its stabilization enabling the formation of a functional adhesive complex. We show that lipid rafts, as homophilic interaction and F-actin association, stabilize cadherin-dependent adhesive complexes. Homophilic interactions and F-actin association of N-cadherin are both required for its association to lipid rafts. We thus identify lipid rafts as new regulators of cadherin-mediated cell adhesion.

scholarly journals Cell contacts orient some cell division axes in the Caenorhabditis elegans embryo.

1995 ◽  
Vol 129 (4) ◽  
pp. 1071-1080 ◽  
Author(s):  
B Goldstein
Keyword(s):  
Cell Division ◽  
Caenorhabditis Elegans ◽  
Mitotic Spindle ◽  
Spindle Orientation ◽  
Cell Contact ◽  
Specific Cell ◽  
Cell Contacts ◽  
Established Cell ◽  
A Site ◽  
Cell Cell

Cells of the early Caenorhabditis elegans embryo divide in an invariant pattern. Here I show that the division axes of some early cells (EMS and E) are controlled by specific cell-cell contacts (EMS-P2 or E-P3 contact). Altering the orientation of contact between these cells alters the axis along which the mitotic spindle is established, and hence the orientation of cell division. Contact-dependent mitotic spindle orientation appears to work by establishing a site of the type described by Hyman and White (1987. J. Cell Biol. 105:2123-2135) in the cortex of the responding cell: one centrosome moves toward the site of cell-cell contact during centrosome rotation in both intact embryos and reoriented cell pairs. The effect is especially apparent when two donor cells are placed on one side of the responding cell: both centrosomes are "captured," pulling the nucleus to one side of the cell. No centrosome rotation occurs in the absence of cell-cell contact, nor in nocodazole-treated cell pairs. The results suggest that some of the cortical sites described by Hyman and White are established cell autonomously (in P1, P2, and P3), and some are established by cell-cell contact (in EMS and E). Additional evidence presented here suggests that in the EMS cell, contact-dependent spindle orientation ensures a cleavage plane that will partition developmental information, received by induction, to one of EMS's daughter cells.

Novel cell contact between podocyte microprojections and parietal epithelial cells analyzed by volume electron microscopy

2020 ◽  
Vol 318 (5) ◽  
pp. F1246-F1251
Author(s):  
Christoph Wrede ◽  
Jan Hegermann ◽  
Christian Mühlfeld
Keyword(s):  
Epithelial Cells ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Cell Contact ◽  
Apical Surface ◽  
Electron Microscopic ◽  
Vascular Perfusion ◽  
Scanning Em ◽  
Parietal Epithelial Cells ◽  
Urinary Space

Podocytes are highly specialized cells with a clear cell polarity. It is known that in health and disease, microvilli protrude from the apical surface of the podocytes into the urinary space. As a basis to better understand the podocyte microprojections/microvilli, the present study analyzed their spatial localization, extension, and contact site with parietal epithelial cells (PECs). Using different electron microscopic (EM) techniques, we analyzed renal corpuscles of healthy young adult male C57BL/6 mice fixed by vascular perfusion. Serial block-face scanning EM was used to visualize entire corpuscles, focused ion beam scanning EM was performed to characterize microprojection/microvilli-rich regions at higher magnification, and transmission EM of serial sections was used to analyze the contact zone between podocyte microprojections and PECs. Numerous microprojections originating from the primary processes of podocytes were present in the urinary space in all regions of the corpuscle. They often reached the apical surface of the PEC but did not make junctional contacts. At high resolution, it was observed that the glycocalyx of both cells was in contact. Depending on the distance between podocytes and PECs, these microprojections had a stretched or coiled state. The present study shows that microprojections/microvilli of podocytes are a physiological feature of healthy mouse kidneys and are frequently in contact with the apical surface of PECs, thus spanning the urinary space. It is proposed that podocyte microprojections serve mechanosensory or communicative functions between podocytes and PECs.

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.

scholarly journals Contact-Induced Mitochondrial Polarization Supports HIV-1 Virological Synapse Formation

2014 ◽  
Vol 89 (1) ◽  
pp. 14-24 ◽  
Author(s):  
Elisabetta Groppelli ◽  
Shimona Starling ◽  
Clare Jolly
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Virological Synapse ◽  
Cell Contact ◽  
Target Cells ◽  
Cell Contacts ◽  
Cell Spread ◽  
Hiv 1 ◽  
Cell Cell

ABSTRACTRapid HIV-1 spread between CD4 T lymphocytes occurs at retrovirus-induced immune cell contacts called virological synapses (VS). VS are associated with striking T cell polarization and localized virus budding at the site of contact that facilitates cell-cell spread. In addition to this, spatial clustering of organelles, including mitochondria, to the contact zone has been previously shown. However, whether cell-cell contact specifically induces dynamic T cell remodeling during VS formation and what regulates this process remain unclear. Here, we report that contact between an HIV-1-infected T cell and an uninfected target T cell specifically triggers polarization of mitochondria concomitant with recruitment of the major HIV-1 structural protein Gag to the site of cell-cell contact. Using fixed and live-cell imaging, we show that mitochondrial and Gag polarization in HIV-1-infected T cells occurs within minutes of contact with target T cells, requires the formation of stable cell-cell contacts, and is an active, calcium-dependent process. We also find that perturbation of mitochondrial polarization impairs cell-cell spread of HIV-1 at the VS. Taken together, these data suggest that HIV-1-infected T cells are able to sense and respond to contact with susceptible target cells and undergo dynamic cytoplasmic remodeling to create a synaptic environment that supports efficient HIV-1 VS formation between CD4 T lymphocytes.IMPORTANCEHIV-1 remains one of the major global health challenges of modern times. The capacity of HIV-1 to cause disease depends on the virus's ability to spread between immune cells, most notably CD4 T lymphocytes. Cell-cell transmission is the most efficient way of HIV-1 spread and occurs at the virological synapse (VS). The VS forms at the site of contact between an infected cell and an uninfected cell and is characterized by polarized assembly and budding of virions and clustering of cellular organelles, including mitochondria. Here, we show that cell-cell contact induces rapid recruitment of mitochondria to the contact site and that this supports efficient VS formation and consequently cell-cell spread. Additionally, we observed that cell-cell contact induces a mitochondrion-dependent increase in intracellular calcium, indicative of cellular signaling. Taken together, our data suggest that VS formation is a regulated process and thus a potential target to block HIV-1 cell-cell spread.

scholarly journals Rab35 regulates cadherin-mediated adherens junction formation and myoblast fusion

2013 ◽  
Vol 24 (3) ◽  
pp. 234-245 ◽  
Author(s):  
Sophie Charrasse ◽  
Franck Comunale ◽  
Sylvain De Rossi ◽  
Arnaud Echard ◽  
Cécile Gauthier-Rouvière
Keyword(s):  
Adherens Junction ◽  
Phosphatidyl Inositol ◽  
Myoblast Fusion ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Cell Contact ◽  
Dependent Manner ◽  
Cell Contacts ◽  
Contact Sites ◽  
Cell Cell

Cadherins are homophilic cell–cell adhesion molecules implicated in many fundamental processes, such as morphogenesis, cell growth, and differentiation. They accumulate at cell–cell contact sites and assemble into large macromolecular complexes named adherens junctions (AJs). Cadherin targeting and function are regulated by various cellular processes, many players of which remain to be uncovered. Here we identify the small GTPase Rab35 as a new regulator of cadherin trafficking and stabilization at cell–cell contacts in C2C12 myoblasts and HeLa cells. We find that Rab35 accumulates at cell–cell contacts in a cadherin-dependent manner. Knockdown of Rab35 or expression of a dominant-negative form of Rab35 impaired N- and M-cadherin recruitment to cell–cell contacts, their stabilization at the plasma membrane, and association with p120 catenin and led to their accumulation in transferrin-, clathrin-, and AP-2–positive intracellular vesicles. We also find that Rab35 function is required for PIP5KIγ accumulation at cell–cell contacts and phosphatidyl inositol 4,5-bisphosphate production, which is involved in cadherin stabilization at contact sites. Finally, we show that Rab35 regulates myoblast fusion, a major cellular process under the control of cadherin-dependent signaling. Taken together, these results reveal that Rab35 regulates cadherin-dependent AJ formation and myoblast fusion.

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