XAIP1: a Xenopus homologue of yeast actin interacting protein 1 (AIP1), which induces disassembly of actin filaments cooperatively with ADF/cofilin family proteins

1999 ◽  
Vol 112 (10) ◽  
pp. 1553-1565 ◽  
Author(s):  
K. Okada ◽  
T. Obinata ◽  
H. Abe
Keyword(s):  
Actin Filaments ◽  
Electron Microscopic Observation ◽  
Affinity Column ◽  
Predicted Amino Acid Sequence ◽  
Cortical Actin ◽  
Electron Microscopic ◽  
Cell Stage ◽  
Interacting Protein ◽  
In Vivo Effects

We carried out affinity column chromatography using Xenopus ADF/cofilin (XAC), identified several polypeptides in oocytes specifically bound to this column with actin, and isolated a full-length cDNA clone for a 65 kDa protein in this fraction. The predicted amino acid sequence revealed that the 65 kDa protein has seven obvious WD repeats and exhibits striking homology with yeast actin interacting protein 1 (AIP1). Thus, we designated this protein Xenopus AIP1 (XAIP1). We purified XAIP1 from Xenopus oocytes, and its interaction with actin was characterized by a pelleting assay, photometrical analysis and electron microscopy. Although XAIP1 itself cosedimented with F-actin and increased unsedimented actin to some extent, it induced a rapid, drastic disassembly of actin filaments associated with XAC. Electron microscopic observation revealed that XAIP1 severs actin filaments in the presence of XAC. To elucidate the in vivo effects of XAIP1, the purified protein was injected into blastomeres at the two-cell stage. Although the localization of XAIP1 was similar to that of XAC, at the cortical cytoskeleton and diffusely in the cytoplasm, injection of a large amount of XAIP1 arrested development and abolished the strong cortical staining of both actin and XAC. From these results, we concluded that XAIP1 regulates the dynamics of the cortical actin cytoskeleton cooperatively with XAC in eggs.

scholarly journals Aip1p Interacts with Cofilin to Disassemble Actin Filaments

1999 ◽  
Vol 145 (6) ◽  
pp. 1251-1264 ◽  
Author(s):  
Avital A. Rodal ◽  
Jonathan W. Tetreault ◽  
Pekka Lappalainen ◽  
David G. Drubin ◽  
David C. Amberg
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Actin Filaments ◽  
Ternary Complex ◽  
Yeast Cells ◽  
Cortical Actin ◽  
Interacting Protein ◽  
Biochemical Analyses ◽  
Two Hybrid

Actin interacting protein 1 (Aip1) is a conserved component of the actin cytoskeleton first identified in a two-hybrid screen against yeast actin. Here, we report that Aip1p also interacts with the ubiquitous actin depolymerizing factor cofilin. A two-hybrid–based approach using cofilin and actin mutants identified residues necessary for the interaction of actin, cofilin, and Aip1p in an apparent ternary complex. Deletion of the AIP1 gene is lethal in combination with cofilin mutants or act1-159, an actin mutation that slows the rate of actin filament disassembly in vivo. Aip1p localizes to cortical actin patches in yeast cells, and this localization is disrupted by specific actin and cofilin mutations. Further, Aip1p is required to restrict cofilin localization to cortical patches. Finally, biochemical analyses show that Aip1p causes net depolymerization of actin filaments only in the presence of cofilin and that cofilin enhances binding of Aip1p to actin filaments. We conclude that Aip1p is a cofilin-associated protein that enhances the filament disassembly activity of cofilin and restricts cofilin localization to cortical actin patches.

scholarly journals Filopodia formation and endosome clustering induced by mutant plus-end–directed myosin VI

2017 ◽  
Vol 114 (7) ◽  
pp. 1595-1600 ◽  
Author(s):  
Thomas A. Masters ◽  
Folma Buss
Keyword(s):  
Actin Filaments ◽  
Leucine Zipper ◽  
Adaptor Protein ◽  
Cell Cortex ◽  
Myosin Vi ◽  
Actin Network ◽  
Ph Domain ◽  
Cortical Actin ◽  
Directed Movement ◽  
Interacting Protein

Myosin VI (MYO6) is the only myosin known to move toward the minus end of actin filaments. It has roles in numerous cellular processes, including maintenance of stereocilia structure, endocytosis, and autophagosome maturation. However, the functional necessity of minus-end–directed movement along actin is unclear as the underlying architecture of the local actin network is often unknown. To address this question, we engineered a mutant of MYO6, MYO6+, which undergoes plus-end–directed movement while retaining physiological cargo interactions in the tail. Expression of this mutant motor in HeLa cells led to a dramatic reorganization of cortical actin filaments and the formation of actin-rich filopodia. MYO6 is present on peripheral adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) signaling endosomes and MYO6+ expression causes a dramatic relocalization and clustering of this endocytic compartment in the cell cortex. MYO6+ and its adaptor GAIP interacting protein, C terminus (GIPC) accumulate at the tips of these filopodia, while APPL1 endosomes accumulate at the base. A combination of MYO6+ mutagenesis and siRNA-mediated depletion of MYO6 binding partners demonstrates that motor activity and binding to endosomal membranes mediated by GIPC and PI(4,5)P2 are crucial for filopodia formation. A similar reorganization of actin is induced by a constitutive dimer of MYO6+, indicating that multimerization of MYO6 on endosomes through binding to GIPC is required for this cellular activity and regulation of actin network structure. This unique engineered MYO6+ offers insights into both filopodia formation and MYO6 motor function at endosomes and at the plasma membrane.

scholarly journals Centriole Disassembly In Vivo and Its Effect on Centrosome Structure and Function in Vertebrate Cells

1998 ◽  
Vol 143 (6) ◽  
pp. 1575-1589 ◽  
Author(s):  
Y. Bobinnec ◽  
A. Khodjakov ◽  
L.M. Mir ◽  
C.L. Rieder ◽  
B. Eddé ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Electron Microscopic Observation ◽  
Cell Number ◽  
Animal Cells ◽  
Electron Microscopic ◽  
Long Term Stability ◽  
Pericentriolar Material ◽  
Immunofluorescence Labeling ◽  
And Function

Glutamylation is the major posttranslational modification of neuronal and axonemal tubulin and is restricted predominantly to centrioles in nonneuronal cells (Bobinnec, Y., M. Moudjou, J.P. Fouquet, E. Desbruyères, B. Eddé, and M. Bornens. 1998. Cell Motil. Cytoskel. 39:223–232). To investigate a possible relationship between the exceptional stability of centriole microtubules and the compartmentalization of glutamylated isoforms, we loaded HeLa cells with the monoclonal antibody GT335, which specifically reacts with polyglutamylated tubulin. The total disappearance of the centriole pair was observed after 12 h, as judged both by immunofluorescence labeling with specific antibodies and electron microscopic observation of cells after complete thick serial sectioning. Strikingly, we also observed a scattering of the pericentriolar material (PCM) within the cytoplasm and a parallel disappearance of the centrosome as a defined organelle. However, centriole disappearance was transient, as centrioles and discrete centrosomes ultimately reappeared in the cell population. During the acentriolar period, a large proportion of monopolar half-spindles or of bipolar spindles with abnormal distribution of PCM and NuMA were observed. However, as judged by a quasinormal increase in cell number, these cells likely were not blocked in mitosis. Our results suggest that a posttranslational modification of tubulin is critical for long-term stability of centriolar microtubules. They further demonstrate that in animal cells, centrioles are instrumental in organizing centrosomal components into a structurally stable organelle.

scholarly journals Intranuclear maturation pathways of rat liver ribosomal ribonucleic acids

1976 ◽  
Vol 160 (3) ◽  
pp. 495-503 ◽  
Author(s):  
M D Dabeva ◽  
K P Dudov ◽  
A A Hadjiolov ◽  
I Emanuilov ◽  
B N Todorov
Keyword(s):  
Secondary Structure ◽  
Rat Liver ◽  
Mammalian Cells ◽  
18S Rrna ◽  
Electron Microscopic Observation ◽  
28S Rrna ◽  
Electron Microscopic ◽  
Rrna Species ◽  
A Minor

The maturation of pre-rRNA (precursor to rRNA)in liver nuclei is studied by agar/ureagel electrophoresis, kinetics of labelling in vivo with [14C] orotate and electron-microscopic observation of secondary structure of RNA molecules. (1) Processing starts from primary pre-rRNA molecules with average mol. wt. 4.6×10(6)(45S) containing the segments of both 28S and 18S rRNA. These molecules form a heterogeneous peak on electrophoresis. The 28S rRNA segment is homogeneous in its secondary structure. However, the large transcribed spacer segment (presumably at the 5′-end) is heterogeneous in size and secondary structure. A minor early labelled RNA component with mol.wt. about 5.8×10(6) is reproducibly found, but its role as a pre-rRNA species remains to be determined. (2) The following intermediate pre-rRNA species are identified: 3.25×10(6) mol.wt.(41S), a precursor common to both mature rRNA species; 2.60×10(6)(36S) and 2.15×10(6)(32S) precursors to 28S rRNA; 1.05×10(6) (21S) precursor to 18S rRNA. The pre-rRNA molecules in rat liver are identical in size and secondary structure with those observed in other mammalian cells. These results suggest that the endonuclease-cleavage sites along the pre-rRNA chain are identical in all mammalian cells. (3) Labelling kinetics and the simultaneous existence of both 36S and 21S pre-rRNA reveal that processing of primary pre-rRNA in adult rat liver occurs simultaneously by at least two major pathways: (i) 45S → 41S → 32S+21S → 28S+18S rRNA and (ii) 45S → 41S → 36S+18S → 32S → 28S rRNA. The two pathways differ by the temporal sequence of endonuclease attack along the 41 S pre-rRNA chain. A minor fraction (mol.wt.2.9×10(6), 39S) is identified as most likely originating by a direct split of 28S rRNA from 45S pre-rRNA. These results show that in liver considerable flexibility exists in the order of cleavage of pre-rRNA molecules during processing.

scholarly journals High Rates of Actin Filament Turnover in Budding Yeast and Roles for Actin in Establishment and Maintenance of Cell Polarity Revealed Using the Actin Inhibitor Latrunculin-A

1997 ◽  
Vol 137 (2) ◽  
pp. 399-416 ◽  
Author(s):  
Kathryn R. Ayscough ◽  
Joel Stryker ◽  
Navin Pokala ◽  
Miranda Sanders ◽  
Phil Crews ◽  
...  
Keyword(s):  
Cell Surface ◽  
Actin Cytoskeleton ◽  
Cell Polarity ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Surface Growth ◽  
Interacting Protein ◽  
Capping Protein ◽  
Latrunculin A

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2–5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site. LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actindependent and the other actin-independent, underlie polarity development. The actin-dependent pathway localizes secretory vesicles and a putative vesicle docking complex to sites of cell surface growth, providing an explanation for the dependence of polarized cell surface growth on actin function. Unexpectedly, several proteins that function with actin during cell polarity development, including an unconventional myosin (Myo2p), calmodulin, and an actin-interacting protein (Bud6/Aip3p), achieved polarized localization by an actin-independent pathway, revealing interdependence among cell polarity pathways. Finally, transient actin depolymerization caused many cells to abandon one bud site or mating projection and to initiate growth at a second site. Thus, actin filaments are also required for maintenance of an axis of cell polarity.

Quantitative Electron-Microscopic Analysis of the in vivo Effects of Tetrahydrocannabinol on Rat Preovulatory Follicles

1987 ◽  
Vol 128 (3) ◽  
pp. 256-263 ◽  
Author(s):  
Lawrence C. Zoller ◽  
Joanne Quealy Buono ◽  
Kenneth Carr ◽  
Elizabeth Ninke ◽  
Kara Vegso
Keyword(s):  
Microscopic Analysis ◽  
Electron Microscopic Analysis ◽  
Electron Microscopic ◽  
Preovulatory Follicles ◽  
In Vivo Effects

The role of interstitial collagens in cleft formation of mouse embryonic submandibular gland during initial branching

Development ◽  
1988 ◽  
Vol 103 (2) ◽  
pp. 259-267 ◽  
Author(s):  
Y. Fukuda ◽  
Y. Masuda ◽  
J. Kishi ◽  
Y. Hashimoto ◽  
T. Hayakawa ◽  
...  
Keyword(s):  
Dental Pulp ◽  
Electron Microscopic Observation ◽  
Branching Morphogenesis ◽  
Collagen Fibrils ◽  
Electron Microscopic ◽  
Active Involvement ◽  
Interstitial Collagens ◽  
Cleft Formation

An interstitial collagenase was purified from the explant medium of bovine dental pulp and was shown to degrade collagens I and III but not IV and V. The enzyme halted cleft initiation in the epithelium of 12-day mouse embryonic submandibular glands in vitro, indicating the active involvement of interstitial collagens in the branching morphogenesis. Transmission electron microscopic observation of the intact 12-day gland without any clefts showed the scattered localization of a few collagen fibrils at the epithelial-mesenchymal interface of the bulb and also revealed the presence of numerous microfibrils around the stalk. Collagen bundles were regularly seen close to the wavy basal lamina at the bottom of clefts of the intact 13-day gland and 12-day gland cultured for 17 h under normal conditions. Mesenchymal cells were found in the clefts together with the frequent localization of peripheral nerve fibres and capillary endothelial cells. The collagen bundles were more often observed in the 12-day gland cultured in the presence of bovine dental pulp collagenase inhibitor, which had been shown to enhance cleft formation. In contrast, collagen fibrils were rarely found at the epithelial-mesenchymal interface of the 12-day gland cultured in the presence of Clostridial or bovine dental pulp collagenase. The findings indicated that the formation of interstitial collagen bundles is essential to form clefts in the epithelium both in vivo and in vitro.

scholarly journals Daip1, a Dictyostelium Homologue of the Yeast Actin-Interacting Protein 1, Is Involved in Endocytosis, Cytokinesis, and Motility

1999 ◽  
Vol 146 (2) ◽  
pp. 453-464 ◽  
Author(s):  
Angelika Konzok ◽  
Igor Weber ◽  
Evelyn Simmeth ◽  
Ulrike Hacker ◽  
Markus Maniak ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluid Phase ◽  
Gene Replacement ◽  
Cell Cortex ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Interacting Protein ◽  
Phagocytic Cups ◽  
Green Fluorescent

The 64-kD protein DAip1 from Dictyostelium contains nine WD40-repeats and is homologous to the actin-interacting protein 1, Aip1p, from Saccharomyces cerevisiae, and to related proteins from Caenorhabditis, Physarum, and higher eukaryotes. We show that DAip1 is localized to dynamic regions of the cell cortex that are enriched in filamentous actin: phagocytic cups, macropinosomes, lamellipodia, and other pseudopodia. In cells expressing green fluorescent protein (GFP)-tagged DAip1, the protein rapidly redistributes into newly formed cortical protrusions. Functions of DAip1 in vivo were assessed using null mutants generated by gene replacement, and by overexpressing DAip1. DAip1-null cells are impaired in growth and their rates of fluid-phase uptake, phagocytosis, and movement are reduced in comparison to wild-type rates. Cytokinesis is prolonged in DAip1-null cells and they tend to become multinucleate. On the basis of similar results obtained by DAip1 overexpression and effects of latrunculin-A treatment, we propose a function for DAip1 in the control of actin depolymerization in vivo, probably through interaction with cofilin. Our data suggest that DAip1 plays an important regulatory role in the rapid remodeling of the cortical actin meshwork.

Real-time measurement of the length of a single sarcomere in rat ventricular myocytes: a novel analysis with quantum dots

2011 ◽  
Vol 301 (5) ◽  
pp. C1116-C1127 ◽  
Author(s):  
Takahiro Serizawa ◽  
Takako Terui ◽  
Tatsuya Kagemoto ◽  
Akari Mizuno ◽  
Togo Shimozawa ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Real Time ◽  
Dynamic Properties ◽  
Electron Microscopic Observation ◽  
Left Ventricular ◽  
Time Measurement ◽  
Electron Microscopic ◽  
Real Time Measurement ◽  
T Tubules

As the dynamic properties of cardiac sarcomeres are markedly changed in response to a length change of even ∼0.1 μm, it is imperative to quantitatively measure sarcomere length (SL). Here we show a novel system using quantum dots (QDs) that enables a real-time measurement of the length of a single sarcomere in cardiomyocytes. First, QDs were conjugated with anti-α-actinin antibody and applied to the sarcomeric Z disks in isolated skinned cardiomyocytes of the rat. At partial activation, spontaneous sarcomeric oscillations (SPOC) occurred, and QDs provided a quantitative measurement of the length of a single sarcomere over the broad range (i.e., from ∼1.7 to ∼2.3 μm). It was found that the SPOC amplitude was inversely related to SL, but the period showed no correlation with SL. We then treated intact cardiomyocytes with the mixture of the antibody-QDs and FuGENE HD, and visualized the movement of the Z lines/T tubules. At a low frequency of 1 Hz, the cycle of the motion of a single sarcomere consisted of fast shortening followed by slow relengthening. However, an increase in stimulation frequency to 3–5 Hz caused a phase shift of shortening and relengthening due to acceleration of relengthening, and the waveform became similar to that observed during SPOC. Finally, the anti-α-actinin antibody-QDs were transfected from the surface of the beating heart in vivo. The striated patterns with ∼1.96-μm intervals were observed after perfusion under fluorescence microscopy, and an electron microscopic observation confirmed the presence of QDs in and around the T tubules and Z disks, but primarily in the T tubules, within the first layer of cardiomyocytes of the left ventricular wall. Therefore, QDs are a useful tool to quantitatively analyze the movement of single sarcomeres in cardiomyocytes, under various experimental settings.

scholarly journals Yeast actin-binding proteins: evidence for a role in morphogenesis.

1988 ◽  
Vol 107 (6) ◽  
pp. 2551-2561 ◽  
Author(s):  
D G Drubin ◽  
K G Miller ◽  
D Botstein
Keyword(s):  
Yeast Cell ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Binding Protein ◽  
Actin Binding ◽  
Cortical Actin ◽  
Actin Binding Protein

Three yeast actin-binding proteins were identified using yeast actin filaments as an affinity matrix. One protein appears to be a yeast myosin heavy chain; it is dissociated from actin filaments by ATP, it is similar in size (200 kD) to other myosins, and antibodies directed against Dictyostelium myosin heavy chain bind to it. Immunofluorescence experiments show that a second actin-binding protein (67 kD) colocalizes in vivo with both cytoplasmic actin cables and cortical actin patches, the only identifiable actin structures in yeast. The cortical actin patches are concentrated at growing surfaces of the yeast cell where they might play a role in membrane and cell wall insertion, and the third actin-binding protein (85 kD) is only detected in association with these structures. This 85-kD protein is therefore a candidate for a determinant of growth sites. The in vivo role of this protein was tested by overproduction; this overproduction causes a reorganization of the actin cytoskeleton which in turn dramatically affects the budding pattern and spatial growth organization of the yeast cell.

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