scholarly journals A novel mammalian myosin I from rat with an SH3 domain localizes to Con A-inducible, F-actin-rich structures at cell-cell contacts.

1995 ◽  
Vol 129 (3) ◽  
pp. 819-830 ◽  
Author(s):  
H E Stöffler ◽  
C Ruppert ◽  
J Reinhard ◽  
M Bähler
Keyword(s):  
Light Chain ◽  
Sh3 Domain ◽  
Actin Binding ◽  
Cell Contact ◽  
Tail Domain ◽  
Con A ◽  
Cell Contacts ◽  
Head Domain ◽  
Myosin I ◽  
Cell Cell

In an effort to determine diversity and function of mammalian myosin I molecules, we report here the cloning and characterization of myr 3 (third unconventional myosin from rat), a novel mammalian myosin I from rat tissues that is related to myosin I molecules from protozoa. Like the protozoan myosin I molecules, myr 3 consists of a myosin head domain, a single light chain binding motif, and a tail region that includes a COOH-terminal SH3 domain. However, myr 3 lacks the regulatory phosphorylation site present in the head domain of protozoan myosin I molecules. Evidence was obtained that the COOH terminus of the tail domain is involved in regulating F-actin binding activity of the NH2-terminal head domain. The light chain of myr 3 was identified as the Ca(2+)-binding protein calmodulin. Northern blot and immunoblot analyses revealed that myr 3 is expressed in many tissues and cell lines. Immunofluorescence studies with anti-myr 3 antibodies in NRK cells demonstrated that myr 3 is localized in the cytoplasm and in elongated structures at regions of cell-cell contact. These elongated structures contained F-actin and alpha-actinin but were devoid of vinculin. Incubation of NRK cells with Con A stimulated the formation of myr 3-containing structures along cell-cell contacts. These results suggest for myr 3 a function mediated by cell-cell contact.

scholarly journals Synthetic lethality screen identifies a novel yeast myosin I gene (MYO5): myosin I proteins are required for polarization of the actin cytoskeleton.

1996 ◽  
Vol 133 (6) ◽  
pp. 1277-1291 ◽  
Author(s):  
H V Goodson ◽  
B L Anderson ◽  
H M Warrick ◽  
L A Pon ◽  
J A Spudich
Keyword(s):  
Actin Cytoskeleton ◽  
Synthetic Lethality ◽  
Critical Role ◽  
Neck Region ◽  
Actin Binding ◽  
Cell Physiology ◽  
Deletion Mutants ◽  
Tail Domain ◽  
Amino Terminal ◽  
Myosin I

The organization of the actin cytoskeleton plays a critical role in cell physiology in motile and nonmotile organisms. Nonetheless, the function of the actin based motor molecules, members of the myosin superfamily, is not well understood. Deletion of MYO3, a yeast gene encoding a "classic" myosin I, has no detectable phenotype. We used a synthetic lethality screen to uncover genes whose functions might overlap with those of MYO3 and identified a second yeast myosin 1 gene, MYO5. MYO5 shows 86 and 62% identity to MYO3 across the motor and non-motor regions. Both genes contain an amino terminal motor domain, a neck region containing two IQ motifs, and a tail domain consisting of a positively charged region, a proline-rich region containing sequences implicated in ATP-insensitive actin binding, and an SH3 domain. Although myo5 deletion mutants have no detectable phenotype, yeast strains deleted for both MYO3 and MYO5 have severe defects in growth and actin cytoskeletal organization. Double deletion mutants also display phenotypes associated with actin disorganization including accumulation of intracellular membranes and vesicles, cell rounding, random bud site selection, sensitivity to high osmotic strength, and low pH as well as defects in chitin and cell wall deposition, invertase secretion, and fluid phase endocytosis. Indirect immunofluorescence studies using epitope-tagged Myo5p indicate that Myo5p is localized at actin patches. These results indicate that MYO3 and MYO5 encode classical myosin I proteins with overlapping functions and suggest a role for Myo3p and Myo5p in organization of the actin cytoskeleton of Saccharomyces cerevisiae.

scholarly journals Identification, characterization and cloning of myr 1, a mammalian myosin-I.

1993 ◽  
Vol 120 (6) ◽  
pp. 1393-1403 ◽  
Author(s):  
C Ruppert ◽  
R Kroschewski ◽  
M Bähler
Keyword(s):  
Amino Acid ◽  
Light Chain ◽  
Brush Border ◽  
Binding Sites ◽  
Neuronal Development ◽  
Amino Acid Sequences ◽  
Dependent Manner ◽  
Tail Domain ◽  
Myosin I ◽  
Splice Forms

We have identified, characterized and cloned a novel mammalian myosin-I motor-molecule, called myr 1 (myosin-I from rat). Myr 1 exists in three alternative splice forms: myr 1a, myr 1b, and myr 1c. These splice forms differ in their numbers of putative calmodulin/light chain binding sites. Myr 1a-c were selectively released by ATP, bound in a nucleotide-dependent manner to F-actin and exhibited amino acid sequences characteristic of myosin-I motor domains. In addition to the motor domain, they contained a regulatory domain with up to six putative calmodulin/light chain binding sites and a tail domain. The tail domain exhibited 47% amino acid sequence identity to the brush border myosin-I tail domain, demonstrating that myr 1 is related to the only other mammalian myosin-I motor molecule that has been characterized so far. In contrast to brush border myosin-I which is expressed in mature enterocytes, myr 1 splice forms were differentially expressed in all tested tissues. Therefore, myr 1 is the first mammalian myosin-I motor molecule with a widespread tissue distribution in neonatal and adult tissues. The myr 1a splice form was preferentially expressed in neuronal tissues. Its expression was developmentally regulated during rat forebrain ontogeny and subcellular fractionation revealed an enrichment in purified growth cone particles, data consistent with a role for myr 1a in neuronal development.

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.

CD43 Interacts With Moesin and Ezrin and Regulates Its Redistribution to the Uropods of T Lymphocytes at the Cell-Cell Contacts

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4632-4644 ◽  
Author(s):  
Juan M. Serrador ◽  
Marta Nieto ◽  
José L. Alonso-Lebrero ◽  
Miguel A. del Pozo ◽  
Javier Calvo ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Binding Proteins ◽  
Cell Aggregation ◽  
Actin Binding ◽  
Cell Aggregates ◽  
Actin Binding Proteins ◽  
Cell Contacts ◽  
T Lymphoblasts ◽  
Cell Cell

Abstract Chemokines as well as the signaling through the adhesion molecules intercellular adhesion molecule (ICAM)-3 and CD43 are able to induce in T lymphocytes their switching from a spherical to a polarized motile morphology, with the formation of a uropod at the rear of the cell. We investigated here the role of CD43 in the regulation of T-cell polarity, CD43-cytoskeletal interactions, and lymphocyte aggregation. Pro-activatory anti-CD43 monoclonal antibody (MoAb) induced polarization of T lymphocytes with redistribution of CD43 to the uropod and the CCR2 chemokine receptor to the leading edge of the cell. Immunofluorescence analysis showed that all three ezrin-radixin-moesin (ERM) actin-binding proteins localized in the uropod of both human T lymphoblasts stimulated with anti-CD43 MoAb and tumor-infiltrating T lymphocytes. Radixin localized at the uropod neck, whereas ezrin and moesin colocalized with CD43 in the uropod. Biochemical analyses showed that ezrin and moesin coimmunoprecipitated with CD43 in T lymphoblasts. Furthermore, in these cells, the CD43-associated moesin increased after stimulation through CD43. The interaction of moesin and ezrin with CD43 was specifically mediated by the cytoplasmic domain of CD43, as shown by precipitation of both ERM proteins with a GST-fusion protein containing the CD43 cytoplasmic tail. Videomicroscopy analysis of homotypic cell aggregation induced through CD43 showed that cellular uropods mediate cell-cell contacts and lymphocyte recruitment. Immunofluorescence microscopy performed in parallel showed that uropods enriched in CD43 and moesin localized at the cell-cell contact areas of cell aggregates. The polarization and homotypic cell aggregation induced through CD43 was prevented by butanedione monoxime, indicating the involvement of myosin cytoskeleton in these phenomena. Altogether, these data indicate that CD43 plays an important regulatory role in remodeling T-cell morphology, likely through its interaction with actin-binding proteins ezrin and moesin. In addition, the redistribution of CD43 to the uropod region of migrating lymphocytes and during the formation of cell aggregates together with the enhancing effect of anti-CD43 antibodies on lymphocyte cell recruitment suggest that CD43 plays a key role in the regulation of cell-cell interactions during lymphocyte traffic.

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.

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.

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