Fluorescent phallotoxins as probes for filamentous actin

Faulstich, Heinz; Zobeley, Suse; Rinnerthaler, G.; Small, J. Victor

doi:10.1007/bf01774064

Cited by 161 publications

(74 citation statements)

References 118 publications

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“…Macrophages, stimulated with purified ␤ig-h3, were fixed and stained using phalloidin-rhodamine. 25 As shown in Figure 6A, untreated macrophages had few membrane ruffles. Stimulation of macrophages with CD14 did not induce significant membrane ruffles ( Figure 6B).…”

Section: ␤Ig-h3 Induces Membrane Rufflesmentioning

confidence: 81%

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A transforming growth factor–β–induced protein stimulates endocytosis and is up-regulated in immature dendritic cells

Cao

Lee²,

Zhang³

et al. 2006

Blood

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Dendritic cells (DCs) exhibit distinct functional properties at immature and mature states. To identify genes preferentially regulated in monocyte-derived immature DCs (imDCs), 13 000-element microarrays were hybridized with RNA isolated from imDCs, mature DCs (mDCs), monocytes, and macrophages and a TGF-␤-induced protein (␤ig-h3) was identified as being most prominently up-regulated in imDCs. By polymerase chain reaction (PCR), little ␤ig-h3 mRNA was detected in monocytes and macrophages, but it was abundant in imDCs. On DC activation with LPS, ␤ig-h3 mRNA became diminished, and in tissues, ␤ig-h3 mRNA was abundantly expressed in lymphoid-rich tissues such as the spleen, bone marrow, small intestines, and colon. ␤ig-h3 was expressed in 293T cells and purified as a 70-kDa protein and, by Western blotting, ␤ig-h3 was predominantly detected in the medium of imDCs. We demonstrate that ␤ig-h3 binds to macrophages and imDCs but not to mDCs and activates the Rac GTPase in macrophages, stimulating macrophage membrane ruffling and enhancing macrophage endocytosis. imDC endocytosis was also inhibited by purified anti-␤ig-h3 antibodies. Therefore, ␤ig-h3 appears to be selectively up-regulated in imDCs to regulate antigen uptake through endocytosis. (Blood. 2006;107:2777-2785)

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Section: ␤Ig-h3 Induces Membrane Rufflesmentioning

confidence: 81%

“…25 Macrophages were cultured for 24 hours on glass coverslips. After stimulation for 30 minutes with purified ␤ig-h3 or CD14 (10 g/mL) or left unstimulated, the cells were fixed for 15 minutes in 3.7% (wt/vol) formaldehyde in PBS and permeabilized for 5 minutes in 0.2% (vol/vol) Triton X-100.…”

Section: Detection Of Membrane Ruffles By Fluorescence Microscopymentioning

confidence: 99%

A transforming growth factor–β–induced protein stimulates endocytosis and is up-regulated in immature dendritic cells

Cao

Lee²,

Zhang³

et al. 2006

Blood

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“…5, group II, second pair). This GTP-y-S-induced redistribution of the filaments was completely prevented when the concentration of the prelabeling RP was increased to 2.0 pM, nearer to the saturating concentration of RP for F-actin (Faulstich et al, 1988) (Figs. 4 c and 5, group II, third pair).…”

Section: (A-d) (Ii1) Aif4-mentioning

confidence: 94%

Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins.

Norman

Price

Ridley

et al. 1994

The Journal of Cell Biology

135

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Abstract. Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-'y-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased.Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells.T HE activation of cell surface receptors leads to a multiplicity of responses, such as movement, secretion, mitosis, or phagocytosis. Most of these are accompanied by changes in the organization of the cytoskeleton. In secretory cells, such cytoskeletal reorganizations have been suggested to play a role in granule migration and in regulating the access of secretory vesicles to the plasma membrane, as well as endocytotic events after exocytosis (Burgoyne and Cheek, 1985;Linstedt and Kelly, 1987;Sontag et al., 1988).Cytoskeletal changes after cell activation have been attributed to changes in pH, Ca 2÷ or phosphorylation (Downey and Grinstein, 1989, Del Castillo et al., 1992, Stossel, 1989Downey et al., 1992) and also to the activation of GTPbinding proteins (Hall, 1992). The latter can work indirectly, via the production of second messengers (such as an increase in Ca2+i, cAME cGMP, or activation of protein kinase C) or via the increased turnover of phosphoinositides, which have been shown to bind to and regulate many actin-binding proteins (Lassing and Lindberg, 1988). However, evidence for direct control of microfilaments by GTPases is accumulating. In electropermeabilized neutrophils, GTP-3~-S was found to induce an increase in F-actin content, even when phospholipase C activity was inhibited (Bengtsson et al., 1990;Therrien and Naccache, 1989 cumulates at cell surface projections directed towards the chemoattractant, has sequence similarities with the/3 subunit of heterotrimeric G-proteins (de Hostos et al., 1991). These/3 subunits have also been found to be associated with the cytoskeleton of mouse lymphoma cells (Carlson et al., 1986). More recently, microinjection studies with cultured fibroblasts showed that two ms-related small GTPases, rho and rac, are essential for the ...

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“…Rhodamine-coupled phalloidin (a generous gift from Dr. H. Faulstich, M P Ifor Medicine, Heidelberg) and FITCcoupled phalloidin (Sigma, Deisenhofen, FRG) were used as specific probes for filamentous actin (Wulf et al 1979;Faulstich et al 1988). Each cryosection was incubated with 7 µl of one of the fluorescent phalloidins in phosphate-buffered saline, pH 7.2, (PBS) (0.01 mg/ml and 0.1 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

Distribution of F-actin in the compound eye of the blowfly, Calliphora erythrocephala (Diptera, Insecta)

Wolfrum

1991

Cell Tissue Res

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Summary.Filaments that probably consist of actin have been observed by electron microscopy in various cells and structures of the compound eye of insects and crustaceans. The overall distribution of F-actin in the compound eye of Calliphora erythrocephala was studied using fluorescent phalloidins as specific probes for F-actin. F-actin is localized (1) in the rhabdomeres, (2) in the Semper cells, (3) in the basement membrane, and (4) associated with desmosomes and membranes. It is concluded that the actin filaments present in the compound eye are involved in mechanical stabilization (e.g., within microvillar processes of the Semper cells and in association with intercellular contacts) and in motile phenomena (e.g., in the rhabdomeric microvilli). Key words: Compound eyesThe cytoskeleton in the cells of the ommatidia of the insect compound eye has only partially been examined and characterized (Blest 1988 ;Blest and Eddey 1984;Walz 1983). Most studies have focused on the axial filaments within the rhabdomeric microvilli of the photoreceptors (for example Blest et al. 1982a, b ; el-Gammalet al. 1987). In contrast to microtubules, microfilaments can be specifically characterized only by immunohisto-or cytochemical methods. Using these techniques, Arikawa and coworkers (1990) have shown in Drosophila melanogaster, that the axial filaments of the photoreceptive microvilli are composed of F-actin. However, a systematic study of the distribution of actin filaments within the insect compound eye is still lacking. In the present study it is shown that actin filaments in Calliphora erythrocephala occur within (1) the rhabdomeric microvilli, (2) the basement membrane, (3) the microvillar projections of the Semper cells, and are (4) associated with microtubules or with desmosomes or hemidesmosomes. Materials and methodsThe wild-type and the white-eyed "chalky" mutant of Calliphora erythrocephala (Diptera) were obtained from local cultures.Preparation. Specimens were fixed in a mixture of 0.2% glutaraldehyde and 3% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2, for 3 h at 4°C. They were subsequently washed in cold buffered 10% sucrose for 1 h. followed by infiltration with buffered 25% sucrose for 12 h at 4°C. The specimens were surrounded by boiled liver, embedded (medium: Reichert-Jung, Nussloch, FRG) and cryofixed in melting iso-pentane. They were sectioned at 6-12 µm thickness in a cryostate (Dittes, Heidelberg, FRG) between -15°C and -25°C, and placed on coverslips precoated with 0.05% aequous poly-L-lysine.Phalloidin staining. Rhodamine-coupled phalloidin (a generous gift from Dr. H. Faulstich, M P Ifor Medicine, Heidelberg) and FITCcoupled phalloidin (Sigma, Deisenhofen, FRG) were used as specific probes for filamentous actin (Wulf et al. 1979;Faulstich et al. 1988). Each cryosection was incubated with 7 µl of one of the fluorescent phalloidins in phosphate-buffered saline, pH 7.2, (PBS) (0.01 mg/ml and 0.1 mg/ml. respectively) for 1 h at room temperature and washed 3 times in PBS and one time in H2O to remove u...

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Fluorescent phallotoxins as probes for filamentous actin

Cited by 161 publications

References 118 publications

A transforming growth factor–β–induced protein stimulates endocytosis and is up-regulated in immature dendritic cells

A transforming growth factor–β–induced protein stimulates endocytosis and is up-regulated in immature dendritic cells

Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins.

Distribution of F-actin in the compound eye of the blowfly, Calliphora erythrocephala (Diptera, Insecta)

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