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

TEM micrographs of sectioned control- (A-B), and di-h-HALI treated LSECs (C-D).(A) Sectioning through the nuclear region (N) reveals a complex sponge-like appearance of fenestrae lying around the nucleus (arrow). Notice the presence of vacuoles (arrowheads). Scale bar, 1 μm. (B) Section through the peripheral cytoplasm reveals fenestrae grouped in sieve plates (large arrow). Note the difference between fenestrae (small arrow), endocytotic vesicles (large arrowhead), and vacuoles (small arrowhead). Scale bar, 1 μm. (C) High magnification image of a FFC (large arrow) after 60 minutes of di-h-HALI treatment, showing small fenestrae (small arrowheads), which form rows of fenestrae with increasing size (large arrowheads). Notice filamentous structures (asterisks). Scale bar, 200 nm. (D) FFC (large arrow) after 120 minutes of di-h-HALI treatment. Notice the granular pattern of the FFC and the absence of connected fenestrae rows at this stage. Fenestrae (small arrow); filaments (asterisks). Scale bar, 200 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC101387&req=5

Figure 9: TEM micrographs of sectioned control- (A-B), and di-h-HALI treated LSECs (C-D).(A) Sectioning through the nuclear region (N) reveals a complex sponge-like appearance of fenestrae lying around the nucleus (arrow). Notice the presence of vacuoles (arrowheads). Scale bar, 1 μm. (B) Section through the peripheral cytoplasm reveals fenestrae grouped in sieve plates (large arrow). Note the difference between fenestrae (small arrow), endocytotic vesicles (large arrowhead), and vacuoles (small arrowhead). Scale bar, 1 μm. (C) High magnification image of a FFC (large arrow) after 60 minutes of di-h-HALI treatment, showing small fenestrae (small arrowheads), which form rows of fenestrae with increasing size (large arrowheads). Notice filamentous structures (asterisks). Scale bar, 200 nm. (D) FFC (large arrow) after 120 minutes of di-h-HALI treatment. Notice the granular pattern of the FFC and the absence of connected fenestrae rows at this stage. Fenestrae (small arrow); filaments (asterisks). Scale bar, 200 nm.

Mentions: In addition to the whole-mount TEM method, ultrathin sectioning was applied to cultured LSECs after treatment with 100 nM di-h-HALI at different time points. Examination of tangentially cut control LSECs shows two organizations of fenestrae as described previously [1,29]; i.e., a somewhat complex sponge-like appearance around the nucleus (Fig. 9A), and grouped fenestrae in the peripheral cytoplasm (Fig. 9B). Within 30–60 minutes of di-h-HALI treatment, FFCs could be observed within the peripheral cytoplasm. Sectioned FFCs (Fig. 9C) are comparable to those seen with the whole-mount TEM method (Fig. 7C). Despite complete microfilament-disruption (Fig. 1C), the cytoplasm surrounding the FFCs show a few thin electron dense filaments fanning out in the surrounding cytoplasm (Fig. 9C). After 120 minutes of treatment, FFCs could be observed within the sieve plates, and they did not show connected rows of fenestrae at this stage. Moreover, on the edges of the sieve plates some short and thin filaments could be observed. At high magnification, the FFCs show a fine granular pattern of intermediate electron density (Fig. 9D).


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)

TEM micrographs of sectioned control- (A-B), and di-h-HALI treated LSECs (C-D).(A) Sectioning through the nuclear region (N) reveals a complex sponge-like appearance of fenestrae lying around the nucleus (arrow). Notice the presence of vacuoles (arrowheads). Scale bar, 1 μm. (B) Section through the peripheral cytoplasm reveals fenestrae grouped in sieve plates (large arrow). Note the difference between fenestrae (small arrow), endocytotic vesicles (large arrowhead), and vacuoles (small arrowhead). Scale bar, 1 μm. (C) High magnification image of a FFC (large arrow) after 60 minutes of di-h-HALI treatment, showing small fenestrae (small arrowheads), which form rows of fenestrae with increasing size (large arrowheads). Notice filamentous structures (asterisks). Scale bar, 200 nm. (D) FFC (large arrow) after 120 minutes of di-h-HALI treatment. Notice the granular pattern of the FFC and the absence of connected fenestrae rows at this stage. Fenestrae (small arrow); filaments (asterisks). Scale bar, 200 nm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: TEM micrographs of sectioned control- (A-B), and di-h-HALI treated LSECs (C-D).(A) Sectioning through the nuclear region (N) reveals a complex sponge-like appearance of fenestrae lying around the nucleus (arrow). Notice the presence of vacuoles (arrowheads). Scale bar, 1 μm. (B) Section through the peripheral cytoplasm reveals fenestrae grouped in sieve plates (large arrow). Note the difference between fenestrae (small arrow), endocytotic vesicles (large arrowhead), and vacuoles (small arrowhead). Scale bar, 1 μm. (C) High magnification image of a FFC (large arrow) after 60 minutes of di-h-HALI treatment, showing small fenestrae (small arrowheads), which form rows of fenestrae with increasing size (large arrowheads). Notice filamentous structures (asterisks). Scale bar, 200 nm. (D) FFC (large arrow) after 120 minutes of di-h-HALI treatment. Notice the granular pattern of the FFC and the absence of connected fenestrae rows at this stage. Fenestrae (small arrow); filaments (asterisks). Scale bar, 200 nm.
Mentions: In addition to the whole-mount TEM method, ultrathin sectioning was applied to cultured LSECs after treatment with 100 nM di-h-HALI at different time points. Examination of tangentially cut control LSECs shows two organizations of fenestrae as described previously [1,29]; i.e., a somewhat complex sponge-like appearance around the nucleus (Fig. 9A), and grouped fenestrae in the peripheral cytoplasm (Fig. 9B). Within 30–60 minutes of di-h-HALI treatment, FFCs could be observed within the peripheral cytoplasm. Sectioned FFCs (Fig. 9C) are comparable to those seen with the whole-mount TEM method (Fig. 7C). Despite complete microfilament-disruption (Fig. 1C), the cytoplasm surrounding the FFCs show a few thin electron dense filaments fanning out in the surrounding cytoplasm (Fig. 9C). After 120 minutes of treatment, FFCs could be observed within the sieve plates, and they did not show connected rows of fenestrae at this stage. Moreover, on the edges of the sieve plates some short and thin filaments could be observed. At high magnification, the FFCs show a fine granular pattern of intermediate electron density (Fig. 9D).

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