Improved visualization of F-actin in the green algaAcetabularia by microwave-accelerated fixation and simultaneous FITC-Phalloidin staining

1996 ◽  
Vol 28 (5) ◽  
pp. 353-360 ◽  
Author(s):  
Heiko Sawitzky ◽  
Julia Willingale-Theune ◽  
Diedrik Menzel
Keyword(s):  
Phalloidin Staining

scholarly journals F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments.

1996 ◽  
Vol 135 (5) ◽  
pp. 1291-1308 ◽  
Author(s):  
L G Tilney ◽  
P Connelly ◽  
S Smith ◽  
G M Guild
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Modular Design ◽  
Thin Sections ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Phalloidin Staining ◽  
Filament Bundles ◽  
End To End ◽  
As If

The actin bundles in Drosophila bristles run the length of the bristle cell and are accordingly 65 microns (microchaetes) or 400 microns (macrochaetes) in length, depending on the bristle type. Shortly after completion of bristle elongation in pupae, the actin bundles break down as the bristle surface becomes chitinized. The bundles break down in a bizarre way; it is as if each bundle is sawed transversely into pieces that average 3 microns in length. Disassembly of the actin filaments proceeds at the "sawed" surfaces. In all cases, the cuts in adjacent bundles appear in transverse register. From these images, we suspected that each actin bundle is made up of a series of shorter bundles or modules that are attached end-to-end. With fluorescent phalloidin staining and serial thin sections, we show that the modular design is present in nondegenerating bundles. Decoration of the actin filaments in adjacent bundles in the same bristle with subfragment 1 of myosin reveals that the actin filaments in every module have the same polarity. To study how modules form developmentally, we sectioned newly formed and elongating bristles. At the bristle tip are numerous tiny clusters of 6-10 filaments. These clusters become connected together more basally to form filament bundles that are poorly organized, initially, but with time become maximally cross-linked. Additional filaments are then added to the periphery of these organized bundle modules. All these observations make us aware of a new mechanism for the formation and elongation of actin filament bundles, one in which short bundles are assembled and attached end-to-end to other short bundles, as are the vertical girders between the floors of a skyscraper.

Rhodamine-Phalloidin Staining of F-Actin in Rhodophyta

1993 ◽  
Vol 68 (2) ◽  
pp. 91-98 ◽  
Author(s):  
A. Rosa McDonald ◽  
David J. Garbary ◽  
Jeffrey G. Duckett
Keyword(s):  
Rhodamine Phalloidin ◽  
Phalloidin Staining

A Crucial Role for Caspase 1 in Thrombo-Inflammatory Microparticle Release

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2806-2806
Author(s):  
Andrea S Rothmeier ◽  
Patrizia Marchese ◽  
Christian Furlan-Freguia ◽  
Brian G. Petrich ◽  
Mark H. Ginsberg ◽  
...  
Keyword(s):  
Cardiovascular Diseases ◽  
Cell Surface ◽  
P2x7 Receptor ◽  
Receptor Activation ◽  
Common Mechanism ◽  
Inflammasome Activation ◽  
Disulfide Exchange ◽  
Phalloidin Staining ◽  
Cytoskeletal Remodeling ◽  
Caspase 1

Abstract Macrophages are central orchestrators in the detrimental cycle of inflammation and coagulation in cardiovascular diseases. Cell injury signals trigger the macrophages P2X7 receptor and thereby induce the release both proinflammatory IL-1β and prothrombotic MP. Prothrombotic MP carry tissue factor (TF) and high content of phosphatidylserine (PS), and can induce thrombosis causing major clinical complications in patients. We previously identified the P2X7 receptor as a crucial component of thrombosis in mice, but the mechanistic details of macrophage MP release in this thrombo-inflammatory pathway remain incompletely understood. The the generation of these MP requires thiol-disulfide exchange-dependent activation of the inflammasome and is accompanied by the release of various soluble proteins into the extracellular space. We hypothesized that the released proteome presents regulators and structural components of the MP generation pathway and employed proteomics to unveil their identity. Amongst the most abundant proteins were γ-actin and actin cytoskeleton associated proteins, including PS-binding proteins annexin 1 and annexin 5. Cytoskeletal remodeling processes leading to formation of filopodia downstream of P2X7 receptor activation were crucial for the biogenesis of thrombo-inflammatory MP, since pharmacological inhibitors of inflammasome activation, cytoskeletal remodeling and the thioredoxin system attenuated both, filopodia formation and the release of highly procoagulant MP. Confocal microscopy demonstrated raft dependent translocation of TF onto filopodia that was prevented by the same inhibitory strategies. Surprisingly, phalloidin-staining of non-permeabilized macrophages revealed that F-actin is exposed to the cell surface decorating the base of filopodia. Positive phalloidin-staining of thrombo-inflammatory MP further demonstrated that F-actin remained associated with the MP surface. Strikingly, blocking surface actin by incubation with high concentration of phalloidin prevented the release of PS-rich MP, demonstrating that exposure of F-actin during filopodia formation is functionally linked to the biogenesis of thrombo-inflammatory MP. As the underlying common mechanism for thiol-disulfide exchange-dependent cell surface actin exposure and MP release, we showed that blockade of the cysteine protease caspase 1, which mediates processing and release of IL-1β downstream of inflammasome activation is also required for the release of thrombo-inflammatory MP. Although caspase 1-mediated activation of caplain was required for the release of filamin A localized TF to the cell cortex, calpain was not involved in the release of thrombo-inflammatory MP release. The N-terminus of γ-actin harbors a recognition and cleavage motif for caspase 1. Residual γ-actin released from caspase 1-blocked macrophages showed decreased electrophoretic mobility, indicating prior cleavage of actin that becomes exposed on the cell surface. We show here that the proteome released during thrombo-inflammatory activation of macrophages includes critical players in the biogenesis of MP and may provide diagnostic fingerprints in complex biological samples. Our data demonstrate an entirely unexpected role for caspase 1 and surface exposure of polymerized actin in the severing of prothrombotic MP from filopodia and thus position this protease upstream of both IL-1β processing and thrombo-inflammatory MP in cardiovascular diseases. Disclosures No relevant conflicts of interest to declare.

Involvement of anion channel(s) in the modulation of the transient outward K+ channel in rat ventricular myocytes

2004 ◽  
Vol 287 (1) ◽  
pp. C163-C170 ◽  
Author(s):  
Xiao-Gang Lai ◽  
Jun Yang ◽  
Shi-Sheng Zhou ◽  
Jun Zhu ◽  
Gui-Rong Li ◽  
...  
Keyword(s):  
Steady State ◽  
Ventricular Myocytes ◽  
Ionic Current ◽  
Outward Current ◽  
Anion Channel ◽  
K Channel ◽  
Transient Outward Current ◽  
Inactivation Curve ◽  
Phalloidin Staining ◽  
Steady State Inactivation

The cardiac Ca2+-independent transient outward K+ current ( Ito), a major repolarizing ionic current, is markedly affected by Cl− substitution and anion channel blockers. We reexplored the mechanism of the action of anions on Ito by using whole cell patch-clamp in single isolated rat cardiac ventricular myocytes. The transient outward current was sensitive to blockade by 4-aminopyridine (4-AP) and was abolished by Cs+ substitution for intracellular K+. Replacement of most of the extracellular Cl− with less permeant anions, aspartate (Asp−) and glutamate (Glu−), markedly suppressed the current. Removal of external Na+ or stabilization of F-actin with phalloidin did not significantly affect the inhibitory action of less permeant anions on Ito. In contrast, the permeant Cl− substitute Br− did not markedly affect the current, whereas F− substitution for Cl− induced a slight inhibition. The Ito elicited during Br− substitution for Cl− was also sensitive to blockade by 4-AP. The ability of Cl− substitutes to induce rightward shifts of the steady-state inactivation curve of Ito was in the following sequence: NO3− > Cl− ≈ Br− > gluconate− > Glu− > Asp−. Depolymerization of actin filaments with cytochalasin D (CytD) induced an effect on the steady-state inactivation of Ito similar to that of less permeant anions. Fluorescent phalloidin staining experiments revealed that CytD-pretreatment significantly decreased the intensity of FITC-phalloidin staining of F-actin, whereas Asp− substitution for Cl− was without significant effect on the intensity. These results suggest that the Ito channel is modulated by anion channel(s), in which the actin cytoskeleton may be implicated.

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