関: phalloidine

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出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2014/12/27 17:14:50」(JST)

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Phalloidin is one of a group of toxins from the death cap mushroom (Amanita phalloides) known as phallotoxins. Its toxicity is due to its stabilizing of actin filaments within cells. Since the discovery that fluorescently labelled phalloidin still retained its actin binding properties,^[1] it has been widely used in biomedical research to visualize filamentous actin.

Background

Pioneering work on this toxin was done by the Nobel laureate Heinrich Wieland in the 1930s. Phalloidin was ultimately purified and crystallized in 1937 by Heinrich's student and son-in-law Feodor Lynen (who won a Nobel Prize in 1964 for his work on cholesterol metabolism), and Heinrich's nephew Ulrich Wieland.^[2]

Function

Phalloidin binds F-actin, preventing its depolymerization and poisoning the cell. Phalloidin binds specifically at the interface between F-actin subunits, locking adjacent subunits together. Phalloidin, a bicyclic heptapeptide, binds to actin filaments much more tightly than to actin monomers, leading to a decrease in the rate constant for the dissociation of actin subunits from filament ends, which essentially stabilizes actin filaments through the prevention of filament depolymerization.^[3] Moreover, phalloidin is found to inhibit the ATP hydrolysis activity of F-actin.^[4] Thus, phalloidin traps actin monomers in a conformation distinct from G-actin and it stabilizes the structure of F-actin by greatly reducing the rate constant for monomer dissociation, an event associated with the trapping of ADP.^[4] Overall, phalloidin is found to react stoichiometrically with actin, strongly promote actin polymerization, and stabilize actin polymers.^[5]

Phalloidin functions differently at various concentrations in cells. When introduced into the cytoplasm at low concentrations, phalloidin recruits the less polymerized forms of cytoplasmic actin as well as filamin into stable "islands" of aggregated actin polymers, yet it does not interfere with stress fibers, i.e. thick bundles of microfilaments.^[5] Wehland et al. also notes that at higher concentrations, phalloidin induces cellular contraction.

Use as an imaging tool

Fluorescent phalloidin (red) marking actin filaments in endothelial cells

The properties of phalloidin make it a useful tool for investigating the distribution of F-actin in cells by labeling phalloidin with fluorescent analogs and using them to stain actin filaments for light microscopy. Fluorescent derivatives of phalloidin have turned out to be enormously useful in localizing actin filaments in living or fixed cells as well as for visualizing individual actin filaments in vitro.^[3] A high-resolution technique was developed to detect F-actin at the light and electron microscopic levels by using phalloidin conjugated to the fluorophore eosin which acts as the fluorescent tag.^[6] In this method known as fluorescence photo-oxidation, fluorescent molecules can be utilized to drive the oxidation of diaminobenzidine (DAB) to create a reaction product that can be rendered electron dense and detectable by electron microscopy.^[6] The amount of fluorescence visualized can be used as a quantitative measure of the amount of filamentous actin there is in cells if saturating quantities of fluorescent phalloidin are used.^[3] Consequently, immunofluorescence microscopy along with microinjection of phalloidin can be used to evaluate the direct and indirect functions of cytoplasmic actin in its different stages of polymer formation.^[5] Therefore, fluorescent phalloidin can be used as an important tool in the study of actin networks at high resolution.

Limitations

Unmodified phalloidins do not permeate cell membranes, making them less effective in experiments with living cells. Derivatives of phalloidin with greatly increased cell permeability have been synthesized.^[7]

Cells treated with phalloidins exhibit a number of toxic effects and frequently die.^[3] Furthermore, it is important to note that phalloidin-treated cells will have greater levels of actin associated with their plasma membranes, and the microinjection of phalloidin into living cells will change actin distribution as well as cell motility.^[3]

References

^ Wulf E, Deboben A, Bautz FA, Faulatich H, Wieland T (September 1979). "Fluorescent phallotoxin, a tool for the visualization of cellular actin". PNAS 76 (9): 4498–502. doi:10.1073/pnas.76.9.4498. PMID 291981.
^ Feodor Lynen, Ulrich Wieland (1938). "Über die Giftstoffe des Knollenblätterpilzes". Justus Liebig's Annalen der Chemie 533 (1): 93–117. doi:10.1002/jlac.19385330105.
^ ^a ^b ^c ^d ^e Cooper JA (October 1987). "Effects of cytochalasin and phalloidin on actin". J. Cell Biol. 105 (4): 1473–8. doi:10.1083/jcb.105.4.1473. PMC 2114638. PMID 3312229.
^ ^a ^b Barden JA, Miki M, Hambly BD, Dos Remedios CG (February 1987). "Localization of the phalloidin and nucleotide-binding sites on actin". Eur. J. Biochem. 162 (3): 583–8. doi:10.1111/j.1432-1033.1987.tb10679.x. PMID 3830158.
^ ^a ^b ^c Wehland J, Osborn M, Weber K (December 1977). "Phalloidin-induced actin polymerization in the cytoplasm of cultured cells interferes with cell locomotion and growth". Proc. Natl. Acad. Sci. U.S.A. 74 (12): 5613–7. doi:10.1073/pnas.74.12.5613. PMC 431831. PMID 341163.
^ ^a ^b Capani F, Deerinck TJ, Ellisman MH, Bushong E, Bobik M, Martone ME (1 November 2001). "Phalloidin-eosin followed by photo-oxidation: a novel method for localizing F-actin at the light and electron microscopic levels". J. Histochem. Cytochem. 49 (11): 1351–61. doi:10.1177/002215540104901103. PMID 11668188.
^ emdmillipore.com

English Journal

Inhibition of prostate smooth muscle contraction and prostate stromal cell growth by the inhibitors of Rac, NSC23766 and EHT1864.

Wang Y1,2, Kunit T1,3, Ciotkowska A1, Rutz B1, Schreiber A1, Strittmatter F1, Waidelich R1, Liu C2, Stief CG1, Gratzke C1, Hennenberg M1.
British journal of pharmacology.Br J Pharmacol.2015 Jun;172(11):2905-17. doi: 10.1111/bph.13099. Epub 2015 May 5.
BACKGROUND AND PURPOSE: Medical therapy of lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH) targets smooth muscle contraction in the prostate, or prostate growth. However, current therapeutic options are insufficient. Here, we investigated the role of Rac in the c
PMID 25631101

EMMPRIN regulates β1 integrin-mediated adhesion through Kindlin-3 in human melanoma cells.

Delyon J1,2, Khayati F1, Djaafri I1,3, Podgorniak MP4, Sadoux A4, Setterblad N3, Boutalbi Z1, Maouche K5, Maskos U5, Menashi S6, Lebbé C1,2,3, Mourah S1,3,4.
Experimental dermatology.Exp Dermatol.2015 Jun;24(6):443-8. doi: 10.1111/exd.12693. Epub 2015 Apr 16.
EMMPRIN is known to promote tumor invasion through extracellular matrix (ECM) degradation. Here we report that EMMPRIN can regulate melanoma cell adhesion to the ECM through an interaction with β1 integrin involving kindlin-3. In this study, EMMPRIN knockdown in the human melanoma cell line M10 usi
PMID 25807898

Evaluation of an established pericardium patch for delivery of mesenchymal stem cells to cardiac tissue.

Vashi AV1, White JF, McLean KM, Neethling WM, Rhodes DI, Ramshaw JA, Werkmeister JA.
Journal of biomedical materials research. Part A.J Biomed Mater Res A.2015 Jun;103(6):1999-2005. doi: 10.1002/jbm.a.35335. Epub 2014 Sep 30.
The present study has evaluated a commercial pericardial material for its capacity to assist as a natural extracellular matrix (ECM) patch for the delivery and retention of mesenchymal stem cells for cardiac repair. The repair of cardiac tissue with cells delivered by an appropriate bioscaffold is e
PMID 25266083

Japanese Journal

Involvement of the phosphatidylinositol kinase pathway in augmentation of ATP-sensitive K+ channel currents by hypo-osmotic stress in rat ventricular myocytes

Mitsuyama Hirofumi,Yokoshiki Hisashi,Irie Yuki,Watanabe Masaya,Mizukami Kazuya,Tsutsui Hiroyuki
Canadian journal of physiology and pharmacology 91(9), 686-692, 2013-09
… Augmentation of I-K,I-ATP during hypo-osmotic stress was attenuated by wortmannin (50 mu mol/L), an inhibitor of phosphatidylinositol 3- and 4-kinases, but not by (i) phalloidin (30 mu mol/L), an actin filament stabilizer, (ii) the absence of Ca2+ from the internal and external solutions, and (iii) the presence of creatine phosphate (3 mmol/L), which affects creatine kinase regulation of the K-ATP channels. …
NAID 120005333605

Stem Cell Factor Combined with Matrix Proteins Regulates the Attachment and Migration of Melanocyte Precursors of Human Hair Follicles In Vitro

Wang Da-guang,Xu Xi-hui,Ma Hui-jun,Li Cheng-rang,Yue Xue-zhuang,Gao Jie,Zhu Wen-yuan
Biological and Pharmaceutical Bulletin, 2013
… F-actin was labeled by rhodamine-conjugated phalloidin, and then organization of the actin cytoskeleton was observed by confocal microscope. …
NAID 130003361507

Environmental chlamydiae alter the growth speed and motility of host acanthamoebae.

OKUDE MIHO,MATSUO JUNJI,NAKAMURA SHINJI [他],Kawaguchi Kouhei,Hayashi Yasuhiro,Sakai Haruna,Yoshida Mitsutaka,Takahashi Kaori,Yamaguchi Hiroyuki
Microbes and environments 27(4), 423-429, 2012-12-01
… Meanwhile, we observed a significant change in the phalloidin staining pattern and morphological changes in R18DOX (but not S13RFP) aposymbiotic amoebae, indicating a change in actin accumulation upon removal of the Protochlamydia. …
NAID 10031125940

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Phalloidin
Identifiers
CAS number	17466-45-4
PubChem	441542
Properties
Molecular formula	C35H48N8O11S
Molar mass	788.87 g mol−1
Appearance	Needles
Melting point	281 °C (538 °F; 554 K) (hyd)
	Except where noted otherwise, data are given for materials in their standard state (at 25 °C (77 °F), 100 kPa)
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	Infobox references

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英: phalloidin、phalloidine

[pmid291981-1] Wulf E, Deboben A, Bautz FA, Faulatich H, Wieland T (September 1979). "Fluorescent phallotoxin, a tool for the visualization of cellular actin". PNAS 76 (9): 4498–502. doi:10.1073/pnas.76.9.4498. PMID 291981.

[2] Feodor Lynen, Ulrich Wieland (1938). "Über die Giftstoffe des Knollenblätterpilzes". Justus Liebig's Annalen der Chemie 533 (1): 93–117. doi:10.1002/jlac.19385330105.

[Cooper87-3] Cooper JA (October 1987). "Effects of cytochalasin and phalloidin on actin". J. Cell Biol. 105 (4): 1473–8. doi:10.1083/jcb.105.4.1473. PMC 2114638. PMID 3312229.

[Barden87-4] Barden JA, Miki M, Hambly BD, Dos Remedios CG (February 1987). "Localization of the phalloidin and nucleotide-binding sites on actin". Eur. J. Biochem. 162 (3): 583–8. doi:10.1111/j.1432-1033.1987.tb10679.x. PMID 3830158.

[Wehland77-5] Wehland J, Osborn M, Weber K (December 1977). "Phalloidin-induced actin polymerization in the cytoplasm of cultured cells interferes with cell locomotion and growth". Proc. Natl. Acad. Sci. U.S.A. 74 (12): 5613–7. doi:10.1073/pnas.74.12.5613. PMC 431831. PMID 341163.

[Capani01-6] Capani F, Deerinck TJ, Ellisman MH, Bushong E, Bobik M, Martone ME (1 November 2001). "Phalloidin-eosin followed by photo-oxidation: a novel method for localizing F-actin at the light and electron microscopic levels". J. Histochem. Cytochem. 49 (11): 1351–61. doi:10.1177/002215540104901103. PMID 11668188.

[7] re.com

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phalloidin