Targeting of the myosin-I myr 3 to intercellular adherens type junctions induced by dominant active Cdc42 in HeLa cells

1998 ◽  
Vol 111 (18) ◽  
pp. 2779-2788 ◽  
Author(s):  
H.E. Stoffler ◽  
U. Honnert ◽  
C.A. Bauer ◽  
D. Hofer ◽  
H. Schwarz ◽  
...  
Keyword(s):  
Hela Cells ◽  
Adherens Junction ◽  
Intercellular Junctions ◽  
Distinct Type ◽  
Beta Catenin ◽  
Tail Domain ◽  
Filamentous Actin ◽  
Myosin I ◽  
Myosin Motor Domain ◽  
Constitutively Active

Myr 3, a member of the myosin-I family from rat, is shown in this study to be localized at adherens-type intercellular junctions in epithelial and nonepithelial tissues. Formation of intercellular junctions and the accompanying recruitment of myr 3 to these junctions involves signaling by the Rho subfamily of small GTP-binding proteins. This conclusion is based on studies with HtTA-1 HeLa cells that were induced by overexpression of constitutively active Cdc42Hs to form typical adherens-type intercellular junctions enriched in cadherins (N-cadherin), beta-catenin, filamentous actin and myr 3. Recruitement of myr 3 to Cdc42-induced adherens junctions in HeLa cells was dependent on a short region of the tail domain and a functional myosin motor domain, but was independent of its myosin-I tail homology and SH3 regions. Overexpression of constitutively active Rac1 induced a distinct type of adherens junction in HeLa cells that was characterized by elaborate intercellular interdigitations enriched in N-cadherin, beta-catenin and F-actin. Myr 3 was often present, but not specifically enriched in the intercellular junctions induced by constitutively active Rac1.

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.

scholarly journals Small GTP-binding Protein TC10 Differentially Regulates Two Distinct Populations of Filamentous Actin in 3T3L1 Adipocytes

2002 ◽  
Vol 13 (7) ◽  
pp. 2334-2346 ◽  
Author(s):  
Makoto Kanzaki ◽  
Robert T. Watson ◽  
June Chunqiu Hou ◽  
Mark Stamnes ◽  
Alan R. Saltiel ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Actin Polymerization ◽  
Coat Proteins ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Amino Terminal ◽  
Gtp Binding ◽  
Subcellular Compartmentalization ◽  
Constitutively Active

TC10 is a member of the Rho family of small GTP-binding proteins that has previously been implicated in the regulation of insulin-stimulated GLUT4 translocation in adipocytes. In a manner similar to Cdc42-stimulated actin-based motility, we have observed that constitutively active TC10 (TC10/Q75L) can induce actin comet tails in Xenopus oocyte extracts in vitro and extensive actin polymerization in the perinuclear region when expressed in 3T3L1 adipocytes. In contrast, expression of TC10/Q75L completely disrupted adipocyte cortical actin, which was specific for TC10, because expression of constitutively active Cdc42 was without effect. The effect of TC10/Q75L to disrupt cortical actin was abrogated after deletion of the amino terminal extension (ΔN-TC10/Q75L), whereas this deletion retained the ability to induce perinuclear actin polymerization. In addition, alteration of perinuclear actin by expression of TC10/Q75L, a dominant-interfering TC10/T31N mutant or a mutant N-WASP protein (N-WASP/ΔVCA) reduced the rate of VSV G protein trafficking to the plasma membrane. Furthermore, TC10 directly bound to Golgi COPI coat proteins through a dilysine motif in the carboxyl terminal domain consistent with a role for TC10 regulating actin polymerization on membrane transport vesicles. Together, these data demonstrate that TC10 can differentially regulate two types of filamentous actin in adipocytes dependent on distinct functional domains and its subcellular compartmentalization.

scholarly journals Structure, function, and regulation of myosin 1C.

2005 ◽  
Vol 52 (2) ◽  
pp. 373-380 ◽  
Author(s):  
Barbara Barylko ◽  
Gwanghyun Jung ◽  
Joseph P Albanesi
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Subcellular Location ◽  
Lipid Binding ◽  
Tail Domain ◽  
Anionic Lipid ◽  
Myosin I ◽  
Docking Proteins ◽  
Neck Domain ◽  
Anionic Lipids

Myosin 1C, the first mammalian single-headed myosin to be purified, cloned, and sequenced, has been implicated in the translocation of plasma membrane channels and transporters. Like other forms of myosin I (of which eight exist in humans) myosin 1C consists of motor, neck, and tail domains. The neck domain binds calmodulins more tightly in the absence than in the presence of Ca(2+). Release of calmodulins exposes binding sites for anionic lipids, particularly phosphoinositides. The tail domain, which has an isoelectic point of 10.5, interacts with anionic lipid headgroups. When both neck and tail lipid binding sites are engaged, the myosin associates essentially irreversibly with membranes. Despite this tight membrane binding, it is widely believed that myosin 1C docking proteins are necessary for targeting the enzyme to specific subcellular location. The search for these putative myosin 1C receptors is an active area of research.

Distribution of the myosin I-like ninaC proteins in the Drosophila retina and ultrastructural analysis of mutant phenotypes

1992 ◽  
Vol 101 (1) ◽  
pp. 247-254 ◽  
Author(s):  
J.L. Hicks ◽  
D.S. Williams
Keyword(s):  
Compound Eye ◽  
Photoreceptor Cells ◽  
Amino Acid Sequences ◽  
Ultrastructural Analysis ◽  
Multivesicular Bodies ◽  
Filamentous Actin ◽  
Electron Microscopic ◽  
Myosin I ◽  
Specific Proteins ◽  
Mutant Phenotypes

The Drosophila ninaC gene encodes for two head-specific proteins of 132 kDa and 174 kDa. Their predicted amino acid sequences indicate that they may have myosin I and kinase properties. We have: (1) determined the cellular and subcellular distributions of the ninaC proteins in the Drosophila retina by electron microscopic immunocytochemistry with an antibody specific for epitopes shared by both proteins; (2) characterized the ultrastructure of the mutant phenotype. The proteins were detected only in the photoreceptor cells, but were detected in all classes of the compound eye photoreceptors. Within the photoreceptors, they were found in the rhabdomeral microvilli and the cytoplasm adjacent to the rhabdomeres. This distribution coincides with that shown previously for actin filaments. Immunolabelling of tissue from the ninaC P221 mutant, which lacks the 174 kDa protein, and two mutants whose rhabdomeres degenerate, suggests that the 132 kDa protein is present primarily in the cytoplasm adjacent to the rhabdomeres, and that the 174 kDa protein is concentrated in the rhabdomeres. Our ultrastructural analysis showed that the axial cytoskeleton of the rhabdomeral microvilli (which contains filamentous actin) was absent in both the null and P221 mutants. In the photoreceptor cell cytoplasm, the number of multivesicular bodies in the null mutant, but not the P221 mutant, was 3-fold greater in comparison with wild-type.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic changes of NuMA during the cell cycle and possible appearance of a truncated form of NuMA during apoptosis

1996 ◽  
Vol 109 (2) ◽  
pp. 277-288 ◽  
Author(s):  
H.L. Hsu ◽  
N.H. Yeh
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Structural Integrity ◽  
Chromatin Condensation ◽  
Tail Domain ◽  
Truncated Form ◽  
Cdc2 Kinase ◽  
Mitotic Apparatus ◽  
Interphase Cells ◽  
Late Stages

We have demonstrated that dynamic redistribution of nuclear-mitotic apparatus (NuMA) protein in the cell cycle is correlated temporally and spatially with its biochemical modifications. In interphase, NuMA behaves solely as a 220 kDa nuclear matrix-associated protein. After initiation of DNA condensation during mitosis, NuMA is phosphorylated by Cdc2 kinase into a 240 kDa form which is transported quickly to the centrosomal region. Once cells have passed the metaphase-anaphase transition, the 240 kDa form of NuMA either becomes a 180 kDa truncated form which is fated to be degraded completely before mitotic exit, or returns to the 220 kDa form that relocates to the daughter nuclei and remains throughout interphase. Apparently, a proteolytic enzyme is activated during the late stages of mitosis. After induction of a 180 kDa form of NuMA in interphase HeLa cells by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, nuclear apoptotic phenomena including chromatin condensation, DNA fragmentation, and micronucleation were observed. However, the same treatment did not induce apoptosis in mitotic phase-arrested HeLa cells. The 180 kDa form of NuMA was demonstrated to be a truncated product, at least lacking the tail domain. When HL60 cells were stimulated by diverse apoptosis inducers such as camptothecin, staurosporine, cycloheximide, and A23187, the extent of NuMA cleavage to produce a 180 kDa product was comparable with the degree of oligonucleosomal laddering. NuMA cleavage is likely to be a consequence of the onset of apoptosis. The intact 220 kDa NuMA functions in interphase cells to retain the nuclear structural integrity. Additionally, NuMA appears to act as a nuclear structural target for a death protease during apoptosis.

Cell confluence regulates tyrosine phosphorylation of adherens junction components in endothelial cells

1997 ◽  
Vol 110 (17) ◽  
pp. 2065-2077 ◽  
Author(s):  
M.G. Lampugnani ◽  
M. Corada ◽  
P. Andriopoulou ◽  
S. Esser ◽  
W. Risau ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Phosphorylation ◽  
Adherens Junction ◽  
Cell Junctions ◽  
Molecular Organization ◽  
Beta Catenin ◽  
Cell Contacts ◽  
Cell Type Specific ◽  
Dynamic Structures ◽  
Cell Cell

In src- and ras-transformed cells, tyrosine phosphorylation of adherens junction (AJ) components is related to impairment of cell-cell adhesion. In this paper we report that in human endothelial cells (EC), tyrosine phosphorylation of AJ can be a physiological process regulated by cell density. Immunofluorescence analysis revealed that a phosphotyrosine (P-tyr) antibody could stain cell-cell junctions only in sparse or loosely confluent EC, while the staining was markedly reduced in tightly confluent cultures. This process was reversible, since on artificial wounding of EC monolayers, the cells at the migrating front reacquired P-tyr labelling at cell contacts. In EC, the major cadherin at intercellular AJ is the cell-type-specific VE-cadherin. We therefore analyzed whether this molecule was at least in part responsible for the changes in P-tyr content at cell junctions. Tyrosine phosphorylation of VE-cadherin, beta-catenin and p120, occurred in looser AJ, i.e. in recently confluent cells, and was notably reduced in tightly confluent cultures. Changes in P-tyr content paralleled changes in the molecular organization of AJ. VE-cadherin was mostly associated with beta-catenin and p120 in loose EC monolayers, while in long-confluent cells, these two catenins were largely replaced by plakoglobin. Inhibition of P-tyr phosphatases (PTPases) by PV markedly augmented the P-tyr content of VE-cadherin, which bound p120 and beta-catenin more efficiently, but not plakoglobin. Transfection experiments in CHO cells showed that p120 could bind to a VE-cadherin cytoplasmic region different from that responsible for beta-catenin binding, and PV stabilized this association. Overall these data indicate that endothelial AJ are dynamic structures that can be affected by the state of confluence of the cells. Tyrosine phosphorylation of VE-cadherin and its association to p120 and beta-catenin characterizes early cell contacts, while the formation of mature and cytoskeleton-connected junctions is accompanied by dephosphorylation and plakoglobin association.

The involvement of adherens junction components in myofibrillogenesis in cultured cardiac myocytes

Development ◽  
1992 ◽  
Vol 114 (1) ◽  
pp. 173-183 ◽  
Author(s):  
E.J. Goncharova ◽  
Z. Kam ◽  
B. Geiger
Keyword(s):  
Alternative Pathway ◽  
Three Dimensional ◽  
Adherens Junction ◽  
Intercellular Junctions ◽  
Inhibitory Effect ◽  
Newborn Rats ◽  
Cell Surfaces ◽  
Cell Matrix ◽  
Adhesive Interactions ◽  
Lateral Cell

The distribution of adherens junction (AJ) components was investigated in cultured heart myocytes. These cells, derived from either newborn rats or chick embryos, develop elaborate arrays of myofibrils which become extensive and laterally aligned following several days in culture. The Z-disks in these cells, visualized by immunolabeling with antibodies to muscle-specific alpha-actinin, exhibit a characteristic periodicity of about 2 microns and are in register with those of neighboring myofibrils throughout the sarcoplasm. Vinculin, in these cells, associates with intercellular AJ and cell-matrix adhesions. In addition, this protein is detected in periodic bands located along the lateral cell membranes corresponding to “costamers” previously described by Pardo, J.V., Siliciano, J.D. and Craig, S.W. (Proc. Natn. Acad. Sci. USA, 80, 1008). Similarly, N-cadherin, which is predominantly associated with intercellular junctions, is also detected in periodic striations located mainly on the dorsal and lateral cell surfaces. Using computer-aided three-dimensional microscopy confirmed that these vinculin- and N-cadherin-containing structures are located in extrajunctional sites, apparently associated with Z-disks of peripheral myofibrils. Based on these findings an alternative pathway is proposed for the assembly of vinculin and N-cadherin, which is not triggered by adhesive interactions with extracellular surfaces but rather by interactions at the membrane-cytoplasm interphase with the periphery of the pre-assembled myofibrils. Moreover, we present evidence that antibodies to N-cadherin, which are capable of blocking AJ formation in culture, have an inhibitory effect also on the development and alignment of myofibrils. We discuss the functional significance of the “costameric” organization of vinculin and N-cadherin and consider its involvement both in the lateral alignment of neighboring muscle cells and in the stabilization of developing myofibrils.

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