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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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Scanning electron microscopic (SEM) observations showing the surface topology of control-, and di-h-HALI-treated LSEC. (A) SEM micrograph of a control LSEC shows the presence of numerous fenestrae grouped in sieve plates (arrow). The bulging area contains the nucleus (N). Scale bar, 2 μm. (B) High-power SEM micrograph of the fenestrated cytoplasm obtained after 60 minutes exposure to 100 nM di-h-HALI. Note a typical cytoplasmic unfenestrated area (asterisk), surrounded by circular rows of very small fenestrae (arrow), suggesting nascent fenestrae fanning out into the surrounding fenestrated cytoplasm. Scale bar, 250 nm. (C) Shows a SEM micrograph of a LSEC treated with 100 nM di-h-HALI for 120 minutes, revealing a substantially increased number of fenestrae (large arrow). Thin nonfenestrated cytoplasmic arms (arrowheads) divide the cytoplasm into large sieve plates. In the fenestrated cytoplasm, small cytoplasmic unfenestrated areas devoid of connected fenestrae rows could be observed (small arrow), nucleus (N). Scale bar, 2 μm.
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Figure 2: Scanning electron microscopic (SEM) observations showing the surface topology of control-, and di-h-HALI-treated LSEC. (A) SEM micrograph of a control LSEC shows the presence of numerous fenestrae grouped in sieve plates (arrow). The bulging area contains the nucleus (N). Scale bar, 2 μm. (B) High-power SEM micrograph of the fenestrated cytoplasm obtained after 60 minutes exposure to 100 nM di-h-HALI. Note a typical cytoplasmic unfenestrated area (asterisk), surrounded by circular rows of very small fenestrae (arrow), suggesting nascent fenestrae fanning out into the surrounding fenestrated cytoplasm. Scale bar, 250 nm. (C) Shows a SEM micrograph of a LSEC treated with 100 nM di-h-HALI for 120 minutes, revealing a substantially increased number of fenestrae (large arrow). Thin nonfenestrated cytoplasmic arms (arrowheads) divide the cytoplasm into large sieve plates. In the fenestrated cytoplasm, small cytoplasmic unfenestrated areas devoid of connected fenestrae rows could be observed (small arrow), nucleus (N). Scale bar, 2 μm.

Mentions: To examine the effects of the two actin-binding agents on LSEC fenestration, purified cells were grown on collagen-coated cover slips and prepared for SEM. In these experiments, we treated cells with 100 nM of one of the compounds, because at this concentration the actin cytoskeleton was disassembled without further adverse effects on cell shape and viability. Untreated LSECs showed good preservation of their surface ultrastructural characteristics (Fig. 2A), revealing a central, bulging nucleus, surrounded by flat cytoplasmic extensions that contained fenestrae clustered in sieve plates. Within 30–60 minutes of di-h-HALI treatment, small cytoplasmic unfenestrated areas, surrounded by rows of very small fenestrae appeared, suggesting nascent fenestrae emerging from those areas (Fig. 2B). Curiously, HALI-treated LSECs did show such unfenestrated areas, and rows of very small fenestrae were absent even with different exposure times and concentrations. Because SEM gathers surface information with limited resolution, we prepared whole-mounts, and ultrathin sections for TEM to examine these peculiar areas in fine detail (vide infra).


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)

Scanning electron microscopic (SEM) observations showing the surface topology of control-, and di-h-HALI-treated LSEC. (A) SEM micrograph of a control LSEC shows the presence of numerous fenestrae grouped in sieve plates (arrow). The bulging area contains the nucleus (N). Scale bar, 2 μm. (B) High-power SEM micrograph of the fenestrated cytoplasm obtained after 60 minutes exposure to 100 nM di-h-HALI. Note a typical cytoplasmic unfenestrated area (asterisk), surrounded by circular rows of very small fenestrae (arrow), suggesting nascent fenestrae fanning out into the surrounding fenestrated cytoplasm. Scale bar, 250 nm. (C) Shows a SEM micrograph of a LSEC treated with 100 nM di-h-HALI for 120 minutes, revealing a substantially increased number of fenestrae (large arrow). Thin nonfenestrated cytoplasmic arms (arrowheads) divide the cytoplasm into large sieve plates. In the fenestrated cytoplasm, small cytoplasmic unfenestrated areas devoid of connected fenestrae rows could be observed (small arrow), nucleus (N). Scale bar, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Scanning electron microscopic (SEM) observations showing the surface topology of control-, and di-h-HALI-treated LSEC. (A) SEM micrograph of a control LSEC shows the presence of numerous fenestrae grouped in sieve plates (arrow). The bulging area contains the nucleus (N). Scale bar, 2 μm. (B) High-power SEM micrograph of the fenestrated cytoplasm obtained after 60 minutes exposure to 100 nM di-h-HALI. Note a typical cytoplasmic unfenestrated area (asterisk), surrounded by circular rows of very small fenestrae (arrow), suggesting nascent fenestrae fanning out into the surrounding fenestrated cytoplasm. Scale bar, 250 nm. (C) Shows a SEM micrograph of a LSEC treated with 100 nM di-h-HALI for 120 minutes, revealing a substantially increased number of fenestrae (large arrow). Thin nonfenestrated cytoplasmic arms (arrowheads) divide the cytoplasm into large sieve plates. In the fenestrated cytoplasm, small cytoplasmic unfenestrated areas devoid of connected fenestrae rows could be observed (small arrow), nucleus (N). Scale bar, 2 μm.
Mentions: To examine the effects of the two actin-binding agents on LSEC fenestration, purified cells were grown on collagen-coated cover slips and prepared for SEM. In these experiments, we treated cells with 100 nM of one of the compounds, because at this concentration the actin cytoskeleton was disassembled without further adverse effects on cell shape and viability. Untreated LSECs showed good preservation of their surface ultrastructural characteristics (Fig. 2A), revealing a central, bulging nucleus, surrounded by flat cytoplasmic extensions that contained fenestrae clustered in sieve plates. Within 30–60 minutes of di-h-HALI treatment, small cytoplasmic unfenestrated areas, surrounded by rows of very small fenestrae appeared, suggesting nascent fenestrae emerging from those areas (Fig. 2B). Curiously, HALI-treated LSECs did show such unfenestrated areas, and rows of very small fenestrae were absent even with different exposure times and concentrations. Because SEM gathers surface information with limited resolution, we prepared whole-mounts, and ultrathin sections for TEM to examine these peculiar areas in fine detail (vide infra).

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