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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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Correlative fluorescence-, and SEM micrographs of control (A-C) and microfilament-disrupted LSECs obtained after 10 min (D-F), 60 min (G-I) and 120 min (J-L) di-h-HALI treatment. Figure set shows simultaneous localization of fluorescent labeled F-actin (red) (left column) in combination with topographic SEM information (green) (middle column) and the merged image (right column) of the same cell. Control LSECs show features similar as those seen in Figs. 1A and 2A: i.e., a well developed filamentous actin cytoskeleton (A) and sieve plates (B). The merge image (C) reveals that the fenestrated areas are clearly interspersed between the actin filaments. Scale bar, 2 μm. After 10 min di-h-HALI treatment (D-F), the images show that the brightly stained F-actin dots matches with the fine globular topographic elevations present on the thin nonfenestrated cytoplasmic arms. Within one hour of di-h-HALI treatment (G-I), the merge image of the fenestrated area reveals that the FFC and the area around is devoid of F-actin. Scale bar, 200 nm. (J-L) Images obtained after 120 min of di-h-HALI treatment. Note that the F-actin dots are localized in the thin nonfenestrated cytoplasmic arms; while the highly fenestrated cytoplasm lacks F-actin. Scale bar, 2 μm.
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Figure 6: Correlative fluorescence-, and SEM micrographs of control (A-C) and microfilament-disrupted LSECs obtained after 10 min (D-F), 60 min (G-I) and 120 min (J-L) di-h-HALI treatment. Figure set shows simultaneous localization of fluorescent labeled F-actin (red) (left column) in combination with topographic SEM information (green) (middle column) and the merged image (right column) of the same cell. Control LSECs show features similar as those seen in Figs. 1A and 2A: i.e., a well developed filamentous actin cytoskeleton (A) and sieve plates (B). The merge image (C) reveals that the fenestrated areas are clearly interspersed between the actin filaments. Scale bar, 2 μm. After 10 min di-h-HALI treatment (D-F), the images show that the brightly stained F-actin dots matches with the fine globular topographic elevations present on the thin nonfenestrated cytoplasmic arms. Within one hour of di-h-HALI treatment (G-I), the merge image of the fenestrated area reveals that the FFC and the area around is devoid of F-actin. Scale bar, 200 nm. (J-L) Images obtained after 120 min of di-h-HALI treatment. Note that the F-actin dots are localized in the thin nonfenestrated cytoplasmic arms; while the highly fenestrated cytoplasm lacks F-actin. Scale bar, 2 μm.

Mentions: Fenestrae in vitro have a diameter in the order of 200 nm (Table 1), making it necessary to use electron microscopy for their study (Fig. 2). Therefore, to correlate fluorescently-labeled actin structures with SEM information, we combined one fluorescent and one SEM image of the same cell. This enables us to correlate actin and fenestrae-forming center topology. Examination of control LSECs stained for F-actin (Fig. 6A) and subsequently prepared for SEM (Fig. 6B), revealed features as shown in Fig. 1A and Fig. 2A, respectively. Projecting the green colored SEM micrograph on top of the corresponding red rhodamine-phalloidin stained LSEC, clearly illustrates a structural relation between F-actin and the surface topology of the cell (Fig. 6C). Dense peripheral actin filaments lined the cell margins, while straight actin fibers are mainly running in the peripheral cytoplasm. Close examination revealed negligible staining for F-actin in the nuclear area. Furthermore, F-actin was absent inside the fenestrated areas. However, actin filaments were running closely alongside the sieve plates (Fig. 6C). After 10 min of di-h-HALI treatment, the F-actin filaments are disrupted (Fig. 6D), sieve plates are clearly visible (Fig. 6E) and no sign of F-actin could be found inside these fenestrated areas (Fig. 6F). Moreover, the images show that the brightly stained F-actin patches as observed in Fig. 1C match with the fine globular topographic elevations present on the thin nonfenestrated arms which divide the cytoplasm in sieve plates (Fig. 6D, 6E, 6F). Thorough investigation of LSECs treated with di-h-HALI for 60 min, provided evidence that FFCs and the surrounding area are devoid of F-actin (Fig. 6G, 6H, 6I). Figure 6J illustrates that prolonged exposure to 100 nM di-h-HALI for 120 min did not result in additional alterations in actin organization (see also, Fig. 1C). At this time, the maximum effect of di-h-HALI on the fenestral number was reached (Fig. 6K), resulting in huge sieve plates which are lacking any sign of F-actin staining (Fig. 6L).


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)

Correlative fluorescence-, and SEM micrographs of control (A-C) and microfilament-disrupted LSECs obtained after 10 min (D-F), 60 min (G-I) and 120 min (J-L) di-h-HALI treatment. Figure set shows simultaneous localization of fluorescent labeled F-actin (red) (left column) in combination with topographic SEM information (green) (middle column) and the merged image (right column) of the same cell. Control LSECs show features similar as those seen in Figs. 1A and 2A: i.e., a well developed filamentous actin cytoskeleton (A) and sieve plates (B). The merge image (C) reveals that the fenestrated areas are clearly interspersed between the actin filaments. Scale bar, 2 μm. After 10 min di-h-HALI treatment (D-F), the images show that the brightly stained F-actin dots matches with the fine globular topographic elevations present on the thin nonfenestrated cytoplasmic arms. Within one hour of di-h-HALI treatment (G-I), the merge image of the fenestrated area reveals that the FFC and the area around is devoid of F-actin. Scale bar, 200 nm. (J-L) Images obtained after 120 min of di-h-HALI treatment. Note that the F-actin dots are localized in the thin nonfenestrated cytoplasmic arms; while the highly fenestrated cytoplasm lacks F-actin. Scale bar, 2 μm.
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Figure 6: Correlative fluorescence-, and SEM micrographs of control (A-C) and microfilament-disrupted LSECs obtained after 10 min (D-F), 60 min (G-I) and 120 min (J-L) di-h-HALI treatment. Figure set shows simultaneous localization of fluorescent labeled F-actin (red) (left column) in combination with topographic SEM information (green) (middle column) and the merged image (right column) of the same cell. Control LSECs show features similar as those seen in Figs. 1A and 2A: i.e., a well developed filamentous actin cytoskeleton (A) and sieve plates (B). The merge image (C) reveals that the fenestrated areas are clearly interspersed between the actin filaments. Scale bar, 2 μm. After 10 min di-h-HALI treatment (D-F), the images show that the brightly stained F-actin dots matches with the fine globular topographic elevations present on the thin nonfenestrated cytoplasmic arms. Within one hour of di-h-HALI treatment (G-I), the merge image of the fenestrated area reveals that the FFC and the area around is devoid of F-actin. Scale bar, 200 nm. (J-L) Images obtained after 120 min of di-h-HALI treatment. Note that the F-actin dots are localized in the thin nonfenestrated cytoplasmic arms; while the highly fenestrated cytoplasm lacks F-actin. Scale bar, 2 μm.
Mentions: Fenestrae in vitro have a diameter in the order of 200 nm (Table 1), making it necessary to use electron microscopy for their study (Fig. 2). Therefore, to correlate fluorescently-labeled actin structures with SEM information, we combined one fluorescent and one SEM image of the same cell. This enables us to correlate actin and fenestrae-forming center topology. Examination of control LSECs stained for F-actin (Fig. 6A) and subsequently prepared for SEM (Fig. 6B), revealed features as shown in Fig. 1A and Fig. 2A, respectively. Projecting the green colored SEM micrograph on top of the corresponding red rhodamine-phalloidin stained LSEC, clearly illustrates a structural relation between F-actin and the surface topology of the cell (Fig. 6C). Dense peripheral actin filaments lined the cell margins, while straight actin fibers are mainly running in the peripheral cytoplasm. Close examination revealed negligible staining for F-actin in the nuclear area. Furthermore, F-actin was absent inside the fenestrated areas. However, actin filaments were running closely alongside the sieve plates (Fig. 6C). After 10 min of di-h-HALI treatment, the F-actin filaments are disrupted (Fig. 6D), sieve plates are clearly visible (Fig. 6E) and no sign of F-actin could be found inside these fenestrated areas (Fig. 6F). Moreover, the images show that the brightly stained F-actin patches as observed in Fig. 1C match with the fine globular topographic elevations present on the thin nonfenestrated arms which divide the cytoplasm in sieve plates (Fig. 6D, 6E, 6F). Thorough investigation of LSECs treated with di-h-HALI for 60 min, provided evidence that FFCs and the surrounding area are devoid of F-actin (Fig. 6G, 6H, 6I). Figure 6J illustrates that prolonged exposure to 100 nM di-h-HALI for 120 min did not result in additional alterations in actin organization (see also, Fig. 1C). At this time, the maximum effect of di-h-HALI on the fenestral number was reached (Fig. 6K), resulting in huge sieve plates which are lacking any sign of F-actin staining (Fig. 6L).

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