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The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells.

Braet F, Spector I, Shochet N, Crews P, Higa T, Menu E, de Zanger R, Wisse E - BMC Cell Biol. (2002)

Bottom Line: In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics.Halichondramide and dihydrohalichondramide disrupt microfilaments within 10 minutes and double the number of fenestrae in 30 minutes.Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Cell Biology and Histology, Free University of Brussels (VUB), Laarbeeklaan 103, 1090 Brussels-Jette, Belgium. filipbra@cyto.vub.ac.be

ABSTRACT

Background: Liver sinusoidal endothelial cells (LSECs) react to different anti-actin agents by increasing their number of fenestrae. A new structure related to fenestrae formation could be observed when LSECs were treated with misakinolide. In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics. High-resolution microscopy, including immunocytochemistry and a combination of fluorescence- and scanning electron microscopy was applied.

Results: Halichondramide and dihydrohalichondramide disrupt microfilaments within 10 minutes and double the number of fenestrae in 30 minutes. Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter. Correlative microscopy showed the absence of actin filaments (F-actin) in sieve plates and fenestrae-forming centers. Comparable experiments on umbilical vein endothelial cells and bone marrow sinusoidal endothelial cells revealed cell contraction without the appearance of fenestrae or fenestrae-forming centers.

Conclusion: (I) A comparison of all anti-actin agents tested so far, revealed that the only activity that misakinolide and dihydrohalichondramide have in common is their barbed end capping activity; (II) this activity seems to slow down the process of fenestrae formation to such extent that it becomes possible to resolve fenestrae-forming centers; (III) fenestrae formation resulting from microfilament disruption is probably unique to LSECs.

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Fluorescence micrographs showing the effects of HALI and di-h-HALI on actin organization in LSECs, monitored with rhodamine-phalloidin (F-actin / red) and fluorescein-DNase I staining (G-actin / green). Blue color represents the nucleus stained with DAPI. (A) F-actin distribution in control LSECs shows the presence of cytoplasmic stress fibers and peripheral bands of actin bundles that line the cell margin. Note that G-actin is mainly localized in the perinuclear region. (B) LSECs treated with 100 nM HALI for 10 minutes, show a loss of cytoplasmic F-actin bundles and peripheral F-actin bands are less dense, whereas the cytoplasm is faintly stained and only few small F-actin dots and short fine filaments remaining. G-actin fluorescence increased markedly as compared to control LSECs and is distributed thorough the cytoplasm. (C) LSECs treated with 100 nM of di-h-HALI for 10 minutes show loss of F-actin bundles and appearance of brightly stained F-actin patches. Peripheral F-actin bands are still present, and furthermore G-actin is diffuse and faintly stained as compared to HALI-treated LSECs. Scale bars, 5 μm.
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Figure 1: Fluorescence micrographs showing the effects of HALI and di-h-HALI on actin organization in LSECs, monitored with rhodamine-phalloidin (F-actin / red) and fluorescein-DNase I staining (G-actin / green). Blue color represents the nucleus stained with DAPI. (A) F-actin distribution in control LSECs shows the presence of cytoplasmic stress fibers and peripheral bands of actin bundles that line the cell margin. Note that G-actin is mainly localized in the perinuclear region. (B) LSECs treated with 100 nM HALI for 10 minutes, show a loss of cytoplasmic F-actin bundles and peripheral F-actin bands are less dense, whereas the cytoplasm is faintly stained and only few small F-actin dots and short fine filaments remaining. G-actin fluorescence increased markedly as compared to control LSECs and is distributed thorough the cytoplasm. (C) LSECs treated with 100 nM of di-h-HALI for 10 minutes show loss of F-actin bundles and appearance of brightly stained F-actin patches. Peripheral F-actin bands are still present, and furthermore G-actin is diffuse and faintly stained as compared to HALI-treated LSECs. Scale bars, 5 μm.

Mentions: In untreated LSECs, rhodamine-phalloidin staining reveals intense circular bundles lining the cell periphery and straight bundles traversing the cytoplasm. Whereas G-actin staining was most intense at the perinuclear region, and diffusely distributed in the cytoplasm (Fig. 1A). Exposure of cells to 100 nM HALI or di-h-HALI resulted in a loss of actin bundles and in the appearance of a drug-specific F-, and G-actin pattern (Fig. 1B, 1C). Maximal effects of HALI and di-h-HALI were obtained after 10 minutes treatment, and further incubation did not result in additional alteration of actin organization. With HALI, F-actin staining disappeared almost completely and only a weak speckled fluorescence could be observed in the entire cytoplasm, with only a few short and fine fibers remaining mainly at the cell margin. The rapid and massive F-actin depolymerization was reflected in a significant increase in G-actin, which was diffusely distributed in the cytoplasm (Fig. 1B). Di-h-HALI treatment caused complete dissolution of cytoplasmic F-actin bundles and appearance of numerous bright F-actin patches in the cytoplasm, whereas the peripheral F-actin bundles showed interruptions and became less dense. Furthermore, G-actin staining was not increased in intensity and was mainly localized around the nuclear area as in untreated cells (Fig. 1C).


The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells.

Braet F, Spector I, Shochet N, Crews P, Higa T, Menu E, de Zanger R, Wisse E - BMC Cell Biol. (2002)

Fluorescence micrographs showing the effects of HALI and di-h-HALI on actin organization in LSECs, monitored with rhodamine-phalloidin (F-actin / red) and fluorescein-DNase I staining (G-actin / green). Blue color represents the nucleus stained with DAPI. (A) F-actin distribution in control LSECs shows the presence of cytoplasmic stress fibers and peripheral bands of actin bundles that line the cell margin. Note that G-actin is mainly localized in the perinuclear region. (B) LSECs treated with 100 nM HALI for 10 minutes, show a loss of cytoplasmic F-actin bundles and peripheral F-actin bands are less dense, whereas the cytoplasm is faintly stained and only few small F-actin dots and short fine filaments remaining. G-actin fluorescence increased markedly as compared to control LSECs and is distributed thorough the cytoplasm. (C) LSECs treated with 100 nM of di-h-HALI for 10 minutes show loss of F-actin bundles and appearance of brightly stained F-actin patches. Peripheral F-actin bands are still present, and furthermore G-actin is diffuse and faintly stained as compared to HALI-treated LSECs. Scale bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC101387&req=5

Figure 1: Fluorescence micrographs showing the effects of HALI and di-h-HALI on actin organization in LSECs, monitored with rhodamine-phalloidin (F-actin / red) and fluorescein-DNase I staining (G-actin / green). Blue color represents the nucleus stained with DAPI. (A) F-actin distribution in control LSECs shows the presence of cytoplasmic stress fibers and peripheral bands of actin bundles that line the cell margin. Note that G-actin is mainly localized in the perinuclear region. (B) LSECs treated with 100 nM HALI for 10 minutes, show a loss of cytoplasmic F-actin bundles and peripheral F-actin bands are less dense, whereas the cytoplasm is faintly stained and only few small F-actin dots and short fine filaments remaining. G-actin fluorescence increased markedly as compared to control LSECs and is distributed thorough the cytoplasm. (C) LSECs treated with 100 nM of di-h-HALI for 10 minutes show loss of F-actin bundles and appearance of brightly stained F-actin patches. Peripheral F-actin bands are still present, and furthermore G-actin is diffuse and faintly stained as compared to HALI-treated LSECs. Scale bars, 5 μm.
Mentions: In untreated LSECs, rhodamine-phalloidin staining reveals intense circular bundles lining the cell periphery and straight bundles traversing the cytoplasm. Whereas G-actin staining was most intense at the perinuclear region, and diffusely distributed in the cytoplasm (Fig. 1A). Exposure of cells to 100 nM HALI or di-h-HALI resulted in a loss of actin bundles and in the appearance of a drug-specific F-, and G-actin pattern (Fig. 1B, 1C). Maximal effects of HALI and di-h-HALI were obtained after 10 minutes treatment, and further incubation did not result in additional alteration of actin organization. With HALI, F-actin staining disappeared almost completely and only a weak speckled fluorescence could be observed in the entire cytoplasm, with only a few short and fine fibers remaining mainly at the cell margin. The rapid and massive F-actin depolymerization was reflected in a significant increase in G-actin, which was diffusely distributed in the cytoplasm (Fig. 1B). Di-h-HALI treatment caused complete dissolution of cytoplasmic F-actin bundles and appearance of numerous bright F-actin patches in the cytoplasm, whereas the peripheral F-actin bundles showed interruptions and became less dense. Furthermore, G-actin staining was not increased in intensity and was mainly localized around the nuclear area as in untreated cells (Fig. 1C).

Bottom Line: In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics.Halichondramide and dihydrohalichondramide disrupt microfilaments within 10 minutes and double the number of fenestrae in 30 minutes.Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Cell Biology and Histology, Free University of Brussels (VUB), Laarbeeklaan 103, 1090 Brussels-Jette, Belgium. filipbra@cyto.vub.ac.be

ABSTRACT

Background: Liver sinusoidal endothelial cells (LSECs) react to different anti-actin agents by increasing their number of fenestrae. A new structure related to fenestrae formation could be observed when LSECs were treated with misakinolide. In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics. High-resolution microscopy, including immunocytochemistry and a combination of fluorescence- and scanning electron microscopy was applied.

Results: Halichondramide and dihydrohalichondramide disrupt microfilaments within 10 minutes and double the number of fenestrae in 30 minutes. Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter. Correlative microscopy showed the absence of actin filaments (F-actin) in sieve plates and fenestrae-forming centers. Comparable experiments on umbilical vein endothelial cells and bone marrow sinusoidal endothelial cells revealed cell contraction without the appearance of fenestrae or fenestrae-forming centers.

Conclusion: (I) A comparison of all anti-actin agents tested so far, revealed that the only activity that misakinolide and dihydrohalichondramide have in common is their barbed end capping activity; (II) this activity seems to slow down the process of fenestrae formation to such extent that it becomes possible to resolve fenestrae-forming centers; (III) fenestrae formation resulting from microfilament disruption is probably unique to LSECs.

Show MeSH
Related in: MedlinePlus