Limits...
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.

Show MeSH

Related in: MedlinePlus

SEM observations of HUVECs (A-B) and BECs STR-4 (C-D) treated with 100 nM di-h-HALI for 120 min. (A) HUVECs exposed to di-h-HALI show significant signs of cell contraction; i.e. the cytoplasm is retracted and numerous fine cytoplasmic extensions appear (arrows). Scale bar, 5 μm. (B) High magnification image of the cytoplasm shows a smooth surface and the lack of fenestrated structures (compare with Fig. 2C for the difference with LSECs). Scale bar, 2.5 μm. (C-D) di-h-HALI treated BECs STR-4 show features similar to those seen in A and B (scale bars [C] 5 μm, [D] 2.5 μm).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC101387&req=5

Figure 5: SEM observations of HUVECs (A-B) and BECs STR-4 (C-D) treated with 100 nM di-h-HALI for 120 min. (A) HUVECs exposed to di-h-HALI show significant signs of cell contraction; i.e. the cytoplasm is retracted and numerous fine cytoplasmic extensions appear (arrows). Scale bar, 5 μm. (B) High magnification image of the cytoplasm shows a smooth surface and the lack of fenestrated structures (compare with Fig. 2C for the difference with LSECs). Scale bar, 2.5 μm. (C-D) di-h-HALI treated BECs STR-4 show features similar to those seen in A and B (scale bars [C] 5 μm, [D] 2.5 μm).

Mentions: To address the question whether actin-disruption could induce fenestrae and reveal FFCs in large vessel and other capillary endothelial cells as well, the effect of 25, 50, 100 and 200 nM HALI or di-h-HALI for 10, 20, 30, 60 and 120 minutes was examined on cultured HUVECs and BECs STR-4 (Fig. 5). At all concentrations and incubation times, both cell types changed their morphology from well-spread to a more retracted appearance, but they never rounded up completely (Fig. 5A, 5C). Rhodamine-phalloidin staining revealed a complete F-actin dissolution as early as 30 minutes after treatment for both anti-actin agents when concentrations higher than 50 nM were used (data not shown). Detailed investigation of the topology of Di-h-HALI-treated HUVECs (Fig. 5B) and BECs STR-4 (Fig. 5D) revealed no sign of fenestrae and connected fenestrae rows, instead smooth cytoplasmic processes surrounding the bulging nucleus could be observed.


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)

SEM observations of HUVECs (A-B) and BECs STR-4 (C-D) treated with 100 nM di-h-HALI for 120 min. (A) HUVECs exposed to di-h-HALI show significant signs of cell contraction; i.e. the cytoplasm is retracted and numerous fine cytoplasmic extensions appear (arrows). Scale bar, 5 μm. (B) High magnification image of the cytoplasm shows a smooth surface and the lack of fenestrated structures (compare with Fig. 2C for the difference with LSECs). Scale bar, 2.5 μm. (C-D) di-h-HALI treated BECs STR-4 show features similar to those seen in A and B (scale bars [C] 5 μm, [D] 2.5 μm).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC101387&req=5

Figure 5: SEM observations of HUVECs (A-B) and BECs STR-4 (C-D) treated with 100 nM di-h-HALI for 120 min. (A) HUVECs exposed to di-h-HALI show significant signs of cell contraction; i.e. the cytoplasm is retracted and numerous fine cytoplasmic extensions appear (arrows). Scale bar, 5 μm. (B) High magnification image of the cytoplasm shows a smooth surface and the lack of fenestrated structures (compare with Fig. 2C for the difference with LSECs). Scale bar, 2.5 μm. (C-D) di-h-HALI treated BECs STR-4 show features similar to those seen in A and B (scale bars [C] 5 μm, [D] 2.5 μm).
Mentions: To address the question whether actin-disruption could induce fenestrae and reveal FFCs in large vessel and other capillary endothelial cells as well, the effect of 25, 50, 100 and 200 nM HALI or di-h-HALI for 10, 20, 30, 60 and 120 minutes was examined on cultured HUVECs and BECs STR-4 (Fig. 5). At all concentrations and incubation times, both cell types changed their morphology from well-spread to a more retracted appearance, but they never rounded up completely (Fig. 5A, 5C). Rhodamine-phalloidin staining revealed a complete F-actin dissolution as early as 30 minutes after treatment for both anti-actin agents when concentrations higher than 50 nM were used (data not shown). Detailed investigation of the topology of Di-h-HALI-treated HUVECs (Fig. 5B) and BECs STR-4 (Fig. 5D) revealed no sign of fenestrae and connected fenestrae rows, instead smooth cytoplasmic processes surrounding the bulging nucleus could be observed.

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