The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells - PubMed (original) (raw)
The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells
Filip Braet et al. BMC Cell Biol. 2002.
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.
Figures
Figure 1
Fluorescence micrographs showing the effects of HALI and di-h-HALI on actin organization in LSECs, monitored with rhodamine-phalloidin (F-actin / red) and fluorescein-DNase I staining (G-actin / green). Blue color represents the nucleus stained with DAPI. (A) F-actin distribution in control LSECs shows the presence of cytoplasmic stress fibers and peripheral bands of actin bundles that line the cell margin. Note that G-actin is mainly localized in the perinuclear region. (B) LSECs treated with 100 nM HALI for 10 minutes, show a loss of cytoplasmic F-actin bundles and peripheral F-actin bands are less dense, whereas the cytoplasm is faintly stained and only few small F-actin dots and short fine filaments remaining. G-actin fluorescence increased markedly as compared to control LSECs and is distributed thorough the cytoplasm. (C) LSECs treated with 100 nM of di-h-HALI for 10 minutes show loss of F-actin bundles and appearance of brightly stained F-actin patches. Peripheral F-actin bands are still present, and furthermore G-actin is diffuse and faintly stained as compared to HALI-treated LSECs. Scale bars, 5 μm.
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.
Figure 3
Effect of HALI and di-h-HALI on the number of fenestrae per micrometer squared in time. From this graph, we can conclude that both agents increase the number of fenestrae, although at a different rate and different maximum. Data are means plus or minus S.E.M. of triplicate determinations. Note the significant differences between control LSECs (0 minutes) and treated LSECs, as indicated by asterisks (p < 0.01) or by triangles (p < 0.05) (Mann-Whitney U test, two-sided). No significant difference in the number of fenestrae was observed at the 0.05 confidence level between 60 and 120 minutes of treatment with HALI or di-h-HALI.
Figure 4
Diameter distribution of fenestrae, showing values for control, HALI, and di-h-HALI – treated LSECs, extending the data of fenestrae diameter of Table1**.** From this graph, we can conclude that treatment of LSECs with HALI or di-h-HALI results in smaller fenestrae diameters.
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).
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.
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.
Figure 8
Frequency histogram of the number of fenestrae rows connected to one FFC after 10, 30, 60 and 120 min 100 nM di-h-HALI treatment, extending the static whole-mount TEM images of Figure7**.** From these histograms it becomes clear that the onset of fenestrae formation starts after 10 min treatment and that the number of fenestrae rows connected to FFCs reaches its maximum at 60 min di-h-HALI treatment. At 120 min of treatment, when the burst of fenestrae formation has subsided (see also, Fig. 7E), a significant shift towards a low number of connected fenestrae rows could be registered.
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.
References
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