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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.Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter.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. (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.

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.Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter.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. (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.

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