Disassembly of filamentous actin in LSECs with the new microfilament-disrupting drug di-h-HALI enabled us to visualize FFCs (Figs. 2, 7, 9), most probably involved in the process of fenestrae formation, as demonstrated recently with the actin inhibitor misakinolide [20, 23]. Exposure of LSECs by other actin-perturbing agents including cytochalasin B , latrunculin A , swinholide A, jasplakinolide , and HALI (this paper) also produces a rapid increase in the number of fenestrae and the appearance of small unfenestrated areas which apparently represent inactive FFCs , indicating that actin disruption per se is sufficient to induce an increase in the number of fenestrae. However, the fact that only misakinolide and di-h-HALI resolved FFCs in the process of fenestrae formation indicate that specific alterations of the actin system are necessary to unmask active FFCs. Moreover, if our previous observations on FFCs as revealed by using misakinolide was an artefact of the drug, then it is most unlikely that di-h-HALI has the same side effect. The biochemical property that misakinolide and di-h-HALI have in common is their barbed end capping activity [20, 30], however misakinolide also forms actin dimers, whereas di-h-HALI possesses weak F-actin severing activity. In both cases an increased number of fenestrae (Fig. 3) and FFCs (Fig. 2, 7, 9)  could be observed, but misakinolide increases the number of fenestrae more rapidly than di-h-HALI (Fig. 3) , while di-h-HALI revealed approximately 40% more FFCs per squared micrometer (our unpublished data). While these differences may reflect distinct effects of these two agents on actin; it is possible that they also exert indirect effects on actin binding proteins as is the case with latrunculin . In contrast to the other anti-actin drugs that we tested, the specific alterations that misakinolide and di-h-HALI induce in the state of actin organization either by their barbed end capping activity or by indirect effects on the actin cytoskeleton appear to slow down the process of fenestrae formation to such an extent that it becomes possible to visualize active FFCs. In addition, beside these actin-related effects, possible membrane-associated interactions of di-h-HALI or misakinolide  cannot be excluded and may promote or inhibit the fusion/fission process of the cell membrane during fenestrae formation and as a consequence retard this process in such a way that FFCs appear with connected fenestrae rows. Intrinstingly, it has been recently reported that cytochalasin D facilitates apical membrane invagination and promotes exocytosis in pancreatic acinar cells ; whereas cytochalasin B inhibits membrane invagination recovery in neurons .
Endothelial cells of large vessels, which normally do not have fenestrae, have the ability to form fenestrae within minutes [34, 35]. This indicates that the process of fenestrae formation probably does not involve de novo synthesis of proteins, but rather a reorganization of preexisting cellular components. Indeed, specialized structures involved in the formation of diaphragmed fenestrae in the capillaries of the exocrine pancreas , adrenal cortex , kidney glomerulus  and tumor micro vascular endothelium  have been reported. It is presumed that peristomal rings of cholesterol, knob-like structures, vesiculo-vacuolar organelles and the endothelial pocket may represent important contributors to the formation of diaphragmed fenestrae. Therefore, it is conceivable that fenestrae increase, whether diaphragmed or not, is depending on pre-existing structures which promote fenestrae formation. Although these specialized structures may have an important role in the formation of diaphragmed fenestrae, their role in LSECs is less certain, primarily because LSEC fenestrae lack a diaphragm, are exceptionally abundant, and differ structurally from fenestrae in other blood vessels . The only fenestrae-related structure that LSECs and diaphragmed endothelial cells have in common in the complex process of fenestrae formation is the peristomal ring of sterols surrounding a fenestra . Nonetheless, our observations on the effect of di-h-HALI on large vessel endothelial cells and bone marrow sinusoidal endothelial cells (Fig. 5), demonstrated once more that the increase in the number of fenestrae and the appearance of FFCs by actin-disruption is probably a unique process for the hepatic sinusoidal endothelium. It has been reported that phorbol myristate acetate (PMA)  and vascular endothelial growth factor (VEGF) [25, 40] could induce diaphraghmed fenestrae in HUVECs. However, De Zanger et al.  showed that LSECs are insensible to PMA with regard to fenestrae induction. In addition, Krause and collaborators  recently noticed that the increase in the number of fenestrae with time in five days old LSECs cultures is independent of the presence of VEGF. Taking together, these observations clearly illustrate that LSECs respond in a different way to inducers of diaphragmed fenestrae, indicating once more that the machinery for the formation of diaphragmed versus non-diaphragmed fenestrae probably differs. Recent data are accumulating to show that VEGF-induced diaphragmed fenestrae are derived from fused caveolae . A recent study postulates that the same mechanism is used for the formation of LSEC-fenestrae [43, 44]. Evidence at the ultrastructural or molecular level about a possible relation between FFCs and interconnected caveolae is absent.
Fusion of two opposing cell membranes to form fenestrae probably requires the presence of unique compositional membrane microdomains and a cell membrane-associated cytoskeletal structure. Several theories have been used to model the possible mechanisms of membrane fusion for LSECs and other cell types. In general, the process leading to membrane fusion and fission is subdivided into different steps, i.e.: adhesion-dehydration; disappearance of the hydration barrier; contact between phospholipid bilayers, and; molecular rearrangement, resulting in pore formation [45, 46]. As for LSECs, the first step corresponds to the formation of intramembrane protein-free zones , while the appearance of peristomal rings of sterols probably corresponds with the final step . It seems reasonable to assume that these events take place in the rim of FFCs, and that these microdomains contain molecules to pull the bilayers of the cell membranes together at the edge of FFCs. Therefore, to define the FFC more precisely, we applied ultrathin sectioning and showed that filamentous structures of unknown nature are closely associated with these microdomains (Fig. 9C, 9D). In active FFCs, these filaments seem to serve as a guidance for the emerging nascent fenestrae. However, due to technical limitations it is impossible to get a nice plane overview picture of these filamentous structures on EM-sections. These limitations include the extremely small portion of the total cell volume that is included in a section, and the tendency of fine and thin structures cut obliquely or in cross section to disappear visually into the cytoplasmic ground substance. Together, these conditions make it almost impossible to correlate microscopical fluorescence data (Fig. 1B, 1C), with morphologically identified supramolecular structures in TEM (Fig. 9C, 9D). Therefore, in order to clarify the changes in actin organization that underlie the process of fenestrae formation correlative fluorescence- and SEM studies on the same cells was performed (vide infra). Surprisingly, examination of sectioned FFCs did not reveal additional structures regarding the architecture of this singular structure (Fig. 9C, 9D). Detailed investigation showed only a finely granular pattern of intermediate electron density. Although the molecular composition of FFCs remains unknown, F-actin was clearly found to be absent (Fig. 6I, 6L). Steffan et al.  postulated based on their in vitro and in situ studies with cytochalasin B that these pore-free microdomains may constitute an anchorage site for cytoskeletal elements. Our TEM sections (Fig. 9C, 9D) and correlative images (Fig. 6) support this statement but show that these cytoskeletal elements do not correspond with filamentous actin. In contrast, these F-actin patches clearly match with the fine globular topographic elevations present on the thin nonfenestrated cytoplasmic arms, and may represent anchor sites for linking F-actin with the plasma membrane .
Taking previous [20, 21, 23, 29, 47] and present findings together, we propose as an explanation for the events described that FFCs are anchored in the perinuclear area by the actin cytoskeleton where they cannot be resolved by electron microscopy due to their complex multi-fold organization. Disorganization of the filamentous actin cytoskeleton results in a centrifugally-like translocation of the FFCs towards the attenuated peripheral cytoplasm. Flattening of the FFCs occurs at the end of this movement and results in the appearance of FFCs with connected rows of fenestrae with increasing diameter. The spiraling rows may indicate that the translocated FFCs are rotating as they move into the peripheral region. The presence of a clear-cut FACR around every single fenestrae indicates that these centers already contain the necessary machinery and/or protein components for assembling the FACR around nascent fenestrae.
However, caution is required when interpreting this hypothesis, because electron microscopy provides only static information. Nevertheless, our data on the number of fenestrae rows connected to one FFC at different time points (Fig. 8), illustrates in one way a dynamic driven process at the level of the FFC. Recent attempts to confirm our hypothesis on the translocation of pre-existing FFC in real-time with atomic force microscopy failed [49, 50]. The unique low elastic modulus of living LSECs, together with the damaging tip-sample interactions constitute a problem to acquire sequential images of the process of fenestrae formation in real-time under the influence of cytoskeletal-disturbing agents.