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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.

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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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TEM micrographs of whole mount, formaldehyde prefixed, cytoskeleton buffer-extracted control LSEC (A), and di-h-HALI treated LSECs (B-F). (A) Low magnification showing the area containing the nucleus (N) and extracted cytoplasm. Note that the sieve plates are well defined by a dark border (arrowheads). Inside the sieve plates, fenestrae can be observed (small arrows). Scale bar, 2 μm. (B) Treatment with di-h-HALI for 10 to 20 minutes resulted in the appearance of small cytoplasmic unfenestrated areas of intermediate density (small arrows) lying in the neighbourhood of the perinuclear area (pn). Scale bar, 1 μm. (C) Within 30–60 minutes of treatment, small cytoplasmic unfenestrated areas of intermediate density (arrows) could be observed within the peripheral cytoplasm. Scale bar, 2 μm. (D) Examination at high magnification of such cytoplasmic unfenestrated area or FFC (asterisks) show a peculiar structure, with centrally very small fenestrae (small arrow) which form rows of fenestrae with increasing size (large arrow), radiating into the surrounding cytoplasm as a whirlwind. Note the presence of microtubule bundles closely running along the sieve plates (arrowheads). Scale bar, 1 μm. (E) Low magnification showing the cell nucleus (N) and the highly fenestrated cytoplasm (small arrow) after 120 minutes of di-h-HALI treatment. Note the thin cytoplasmic arms (arrowheads) which run from the nucleus into the cytoplasm. Inactive FFCs (arrows). Scale bar, 5 μm. (F) Higher magnification of the fenestrated cytoplasm shows the presence of FACRs (arrow). From these rings, small interconnecting filaments (arrowheads) seem to cross-link the surrounding cytoskeleton. Scale bar, 250 nm.
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Figure 7: TEM micrographs of whole mount, formaldehyde prefixed, cytoskeleton buffer-extracted control LSEC (A), and di-h-HALI treated LSECs (B-F). (A) Low magnification showing the area containing the nucleus (N) and extracted cytoplasm. Note that the sieve plates are well defined by a dark border (arrowheads). Inside the sieve plates, fenestrae can be observed (small arrows). Scale bar, 2 μm. (B) Treatment with di-h-HALI for 10 to 20 minutes resulted in the appearance of small cytoplasmic unfenestrated areas of intermediate density (small arrows) lying in the neighbourhood of the perinuclear area (pn). Scale bar, 1 μm. (C) Within 30–60 minutes of treatment, small cytoplasmic unfenestrated areas of intermediate density (arrows) could be observed within the peripheral cytoplasm. Scale bar, 2 μm. (D) Examination at high magnification of such cytoplasmic unfenestrated area or FFC (asterisks) show a peculiar structure, with centrally very small fenestrae (small arrow) which form rows of fenestrae with increasing size (large arrow), radiating into the surrounding cytoplasm as a whirlwind. Note the presence of microtubule bundles closely running along the sieve plates (arrowheads). Scale bar, 1 μm. (E) Low magnification showing the cell nucleus (N) and the highly fenestrated cytoplasm (small arrow) after 120 minutes of di-h-HALI treatment. Note the thin cytoplasmic arms (arrowheads) which run from the nucleus into the cytoplasm. Inactive FFCs (arrows). Scale bar, 5 μm. (F) Higher magnification of the fenestrated cytoplasm shows the presence of FACRs (arrow). From these rings, small interconnecting filaments (arrowheads) seem to cross-link the surrounding cytoskeleton. Scale bar, 250 nm.

Mentions: One of the most straightforward ways to observe the complex architecture of the cytoskeleton is to examine whole mounts of cells by TEM [28]. This technique, allows the visualization of the cytoskeleton at the supramolecular level with a minimal disruption to the cells. Examination of control LSECs at low magnification showed the presence of an extensive network of cytoskeletal elements that fills the cytoplasm adjacent to the sieve plates (Fig. 7A). LSEC fenestration is characterized by the presence of a sieve plate-, and a fenestrae-associated cytoskeleton connected to a framework of microtubules and microfilaments [17]. Treatment with 100 nM HALI or di-h-HALI for 10 to 20 minutes resulted in the disappearance of microfilaments, and in the appearance of small cytoplasmic unfenestrated areas of intermediate electron density (gray centers), localized around the nucleus of all treated LSECs was apparent (Fig. 7B). Remarkably, between 30–60 minutes of di-h-HALI treatment, FFCs could be observed in all of these cells, consisting of rows of fenestrae, fanning out into the surrounding cytoplasm (Fig. 7C, 7D). Examination at higher magnification, reveals that these rows of fenestrae are clearly connected to the gray centers and that fenestrae with increasing diameter are emanating in the surrounding fenestrated cytoplasm (Fig. 7D). These structures are suggestive of de novo fenestrae formation and are similar to the recently described FFCs as revealed with the actin inhibitor misakinolide [23]. Even in static EM images these spiraling rows of fenestrae give the impression of a dynamic process, in which the newly formed fenestrae ebb away in the surrounding fenestrated cytoplasm as a tornado. This assumption was confirmed by quantifying the number of fenestrae rows connected to one FFC after different times of di-h-HALI treatment (Fig. 8), revealing that the onset of fenestrae formation starts after 10 min treatment and reaches its maximum after 60 min di-h-HALI treatment. At 120 min of treatment, when the burst of fenestrae formation has subsided (Fig. 7E), a significant shift towards a negligible low number of connected fenestrae rows could be registered (Fig. 8). At this time point, when the effect of di-h-HALI on the number of fenestrae reaches its maximum (Fig. 3), long cytoplasmic arms are extending from the nucleus into the cytoplasm and appear to divide the fenestrated cytoplasm into large sieve plates (Fig. 7E). At this stage, the burst of fenestrae formation has subsided and all fenestrae, including the newly formed ones, were delineated by the previously described FACR (Fig. 7F) with the same structure as in control LSECs [17]. Detailed and thorough investigation of HALI-treated LSECs at different times and concentrations only revealed the small unfenestrated areas (gray centers) that presumably represent inactive FFCs, but no sign of connected fenestrae rows.


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 whole mount, formaldehyde prefixed, cytoskeleton buffer-extracted control LSEC (A), and di-h-HALI treated LSECs (B-F). (A) Low magnification showing the area containing the nucleus (N) and extracted cytoplasm. Note that the sieve plates are well defined by a dark border (arrowheads). Inside the sieve plates, fenestrae can be observed (small arrows). Scale bar, 2 μm. (B) Treatment with di-h-HALI for 10 to 20 minutes resulted in the appearance of small cytoplasmic unfenestrated areas of intermediate density (small arrows) lying in the neighbourhood of the perinuclear area (pn). Scale bar, 1 μm. (C) Within 30–60 minutes of treatment, small cytoplasmic unfenestrated areas of intermediate density (arrows) could be observed within the peripheral cytoplasm. Scale bar, 2 μm. (D) Examination at high magnification of such cytoplasmic unfenestrated area or FFC (asterisks) show a peculiar structure, with centrally very small fenestrae (small arrow) which form rows of fenestrae with increasing size (large arrow), radiating into the surrounding cytoplasm as a whirlwind. Note the presence of microtubule bundles closely running along the sieve plates (arrowheads). Scale bar, 1 μm. (E) Low magnification showing the cell nucleus (N) and the highly fenestrated cytoplasm (small arrow) after 120 minutes of di-h-HALI treatment. Note the thin cytoplasmic arms (arrowheads) which run from the nucleus into the cytoplasm. Inactive FFCs (arrows). Scale bar, 5 μm. (F) Higher magnification of the fenestrated cytoplasm shows the presence of FACRs (arrow). From these rings, small interconnecting filaments (arrowheads) seem to cross-link the surrounding cytoskeleton. Scale bar, 250 nm.
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Figure 7: TEM micrographs of whole mount, formaldehyde prefixed, cytoskeleton buffer-extracted control LSEC (A), and di-h-HALI treated LSECs (B-F). (A) Low magnification showing the area containing the nucleus (N) and extracted cytoplasm. Note that the sieve plates are well defined by a dark border (arrowheads). Inside the sieve plates, fenestrae can be observed (small arrows). Scale bar, 2 μm. (B) Treatment with di-h-HALI for 10 to 20 minutes resulted in the appearance of small cytoplasmic unfenestrated areas of intermediate density (small arrows) lying in the neighbourhood of the perinuclear area (pn). Scale bar, 1 μm. (C) Within 30–60 minutes of treatment, small cytoplasmic unfenestrated areas of intermediate density (arrows) could be observed within the peripheral cytoplasm. Scale bar, 2 μm. (D) Examination at high magnification of such cytoplasmic unfenestrated area or FFC (asterisks) show a peculiar structure, with centrally very small fenestrae (small arrow) which form rows of fenestrae with increasing size (large arrow), radiating into the surrounding cytoplasm as a whirlwind. Note the presence of microtubule bundles closely running along the sieve plates (arrowheads). Scale bar, 1 μm. (E) Low magnification showing the cell nucleus (N) and the highly fenestrated cytoplasm (small arrow) after 120 minutes of di-h-HALI treatment. Note the thin cytoplasmic arms (arrowheads) which run from the nucleus into the cytoplasm. Inactive FFCs (arrows). Scale bar, 5 μm. (F) Higher magnification of the fenestrated cytoplasm shows the presence of FACRs (arrow). From these rings, small interconnecting filaments (arrowheads) seem to cross-link the surrounding cytoskeleton. Scale bar, 250 nm.
Mentions: One of the most straightforward ways to observe the complex architecture of the cytoskeleton is to examine whole mounts of cells by TEM [28]. This technique, allows the visualization of the cytoskeleton at the supramolecular level with a minimal disruption to the cells. Examination of control LSECs at low magnification showed the presence of an extensive network of cytoskeletal elements that fills the cytoplasm adjacent to the sieve plates (Fig. 7A). LSEC fenestration is characterized by the presence of a sieve plate-, and a fenestrae-associated cytoskeleton connected to a framework of microtubules and microfilaments [17]. Treatment with 100 nM HALI or di-h-HALI for 10 to 20 minutes resulted in the disappearance of microfilaments, and in the appearance of small cytoplasmic unfenestrated areas of intermediate electron density (gray centers), localized around the nucleus of all treated LSECs was apparent (Fig. 7B). Remarkably, between 30–60 minutes of di-h-HALI treatment, FFCs could be observed in all of these cells, consisting of rows of fenestrae, fanning out into the surrounding cytoplasm (Fig. 7C, 7D). Examination at higher magnification, reveals that these rows of fenestrae are clearly connected to the gray centers and that fenestrae with increasing diameter are emanating in the surrounding fenestrated cytoplasm (Fig. 7D). These structures are suggestive of de novo fenestrae formation and are similar to the recently described FFCs as revealed with the actin inhibitor misakinolide [23]. Even in static EM images these spiraling rows of fenestrae give the impression of a dynamic process, in which the newly formed fenestrae ebb away in the surrounding fenestrated cytoplasm as a tornado. This assumption was confirmed by quantifying the number of fenestrae rows connected to one FFC after different times of di-h-HALI treatment (Fig. 8), revealing that the onset of fenestrae formation starts after 10 min treatment and reaches its maximum after 60 min di-h-HALI treatment. At 120 min of treatment, when the burst of fenestrae formation has subsided (Fig. 7E), a significant shift towards a negligible low number of connected fenestrae rows could be registered (Fig. 8). At this time point, when the effect of di-h-HALI on the number of fenestrae reaches its maximum (Fig. 3), long cytoplasmic arms are extending from the nucleus into the cytoplasm and appear to divide the fenestrated cytoplasm into large sieve plates (Fig. 7E). At this stage, the burst of fenestrae formation has subsided and all fenestrae, including the newly formed ones, were delineated by the previously described FACR (Fig. 7F) with the same structure as in control LSECs [17]. Detailed and thorough investigation of HALI-treated LSECs at different times and concentrations only revealed the small unfenestrated areas (gray centers) that presumably represent inactive FFCs, but no sign of connected fenestrae rows.

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