Effects of brefeldin A on endocytosis, transcytosis and transport to the Golgi complex in polarized MDCK cells

We have studied the effects of brefeldin A (BFA) on endocytosis and intracellular traffic in polarized MDCK cells by using the galactose-binding protein toxin ricin as a membrane marker and HRP as a marker of fluid phase transport. We found that BFA treatment rapidly increased apical endocytosis of both ricin and HRP, whereas basolateral endocytosis was unaffected, as was endocytosis of HRP in the poorly polarized carcinoma cell lines HEp-2 and T47D. Tubular endosomes were induced by BFA both apically and basolaterally in some MDCK cells, comparable with those seen in HEp-2 and T47D cells. In addition, in MDCK cells, BFA induced formation of small (< 300 nm) vesicles, labeled both after apical and basolateral uptake of HRP, as well as some very large (> 700 nm) vacuoles, which were only labeled when HRP was present in the apical medium. In contrast, neither in MDCK nor in HEp-2 or T47D cells, did BFA have any effect on lysosomal morphology. Moreover, transcytosis in the basolateral-apical direction was stimulated both for HRP and ricin. Other vesicular transport routes were less affected or unaffected by BFA treatment. Two closely related structural analogues of BFA (B16 and B21), unable to produce the changes in Golgi and endosomal morphology seen after BFA treatment in a number of different cell lines, were also unable to mimic the effects of BFA on MDCK cells.     

Brefeldin A enhances receptor-mediated transcytosis of transferrin in filter-grown Madin-Darby canine kidney cells.

The effects of brefeldin A (BFA) on transferrin (Tf) transcellular transport, Tf receptor (TfR) distribution, and TfR-mediated endocytosis in filter-grown Madin-Darby canine kidney (MDCK) cells were studied. BFA (1.6 micrograms/ml) markedly enhanced the transcytosis of 125I-labeled Tf (125I-Tf) in both apical-to-basal and basal-to-apical directions; yet, BFA did not enhance the transcytosis of either native horseradish peroxidase (HRP) or membrane-bound HRP-poly(L-lysine) conjugates. Furthermore, this enhanced transcytosis of 125I-Tf was abolished either by competition with excess unlabeled Tf or by incubation at temperatures less than or equal to 25 degrees C. In addition, BFA treatment to MDCK cells: (a) increased 125I-Tf specific binding to the apical membrane and decreased 125I-Tf specific binding to the basal membrane; (b) decreased TfR recycling at the basolateral membrane; (c) altered the apical/basolateral distribution of TfRs in favor of the apical side; and (d) markedly increased 59Fe extraction, but not transcytosis, from apically endocytosed 59Fe-loaded Tf. These effects are consistent with a model in which BFA alters the traffic pattern of internalized Tf by decreasing basolateral TfR recycling, while diverting the nonrecycled fraction to the apical side of the cell. Our results indicate that, unlike the reported inhibition of polymeric IgA transcytosis (Hunziker, W., Whitney, J. A., and Mellman, I. (1991) Cell 67, 617-627), BFA can enhance the transcytosis of Tf in MDCK cells. Thus, by altering the intracellular traffic of ligand-receptor complexes, BFA can elicit either a decrease or an increase in transcytosis depending on the nature of the intracellular receptor processing.     

Cell-specific basolateral membrane sorting of the human liver Na(+)-dependent bile acid cotransporter

The human Na(+)-taurocholate cotransporting polypeptide (Ntcp) is located exclusively on the basolateral membrane of hepatocyte, but the mechanisms underlying its membrane sorting domain have not been fully elucidated. In the present study, a green fluorescent protein-fused human NTCP (NTCP-GFP) was constructed using the polymerase chain reaction and was stably transfected into Madin-Darby canine kidney (MDCK) and Caco-2 cells. Taurocholate uptake studies and confocal microscopy demonstrated that the polarity of basolateral surface expression of NTCP-GFP was maintained in MDCK cells but was lost in Caco-2 cells. Nocodazole (33 microM), an agent that causes microtubular depolymerization, partially disrupted the basolateral localization of NTCP-GFP by increasing apical surface expression to 33.5% compared with untreated cells (P < 0.05). Brefeldin A (BFA; 1-2 microM) disrupted the polarized basolateral localization of NTCP, but monensin (1.4 microM) had no affect on NTCP-GFP localization. In addition, low-temperature shift (20 degrees C) did not affect the polarized basolateral surface sorting of NTCP-GFP and repolarization of this protein after BFA interruption. In summary, these data suggest that the polarized basolateral localization of human NTCP is cell specific and is mediated by a novel sorting pathway that is BFA sensitive and monensin and low-temperature shift insensitive. The process may also involve microtubule motors.     

Epithelial cell polarity alters Rho-GTPase responses to Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that preferentially infects damaged epithelial tissues. Previous studies have failed to distinguish whether the increased susceptibility of injured epithelium results from the loss of cell polarity or increased access to the basolateral surface. We have used confluent monolayers of Madin-Darby canine kidney (MDCK) cells cultured on porous filter supports for 1-3 d as a model system to investigate whether the differentiation state of a polarized model epithelium affected the response of epithelial cells to this pathogen. Confluent incompletely polarized MDCK cell monolayers (day 1) efficiently internalized apically applied P. aeruginosa via a pathway that required actin polymerization and activation of Rho-family GTPases and was accompanied by an increase in the amount of activated RhoA. In contrast, P. aeruginosa entry into highly polarized MDCK monolayers (day 3) was 10- to 100-fold less efficient and was insensitive to inhibitors of actin polymerization or of Rho-family GTPase activation. There was no activation of RhoA; instead, Cdc42-GTP levels increased significantly. Basolateral infection of highly polarized MDCK monolayers was less efficient and insensitive to Clostridium difficile Toxin B, whereas basolateral infection of incompletely polarized MDCK monolayers was more efficient and required activation of Rho-family GTPases. Together, our findings suggest that as epithelial barrier differentiates and becomes highly polarized, it becomes resistant to P. aeruginosa infection. Nevertheless, polarized epithelial cells still sense the presence of apically infecting P. aeruginosa, but they may do so through a different group of surface proteins and/or downstream signaling pathways than do incompletely polarized cells.     

In Polarized MDCK Cells Basolateral Vesicles Arise from Clathrin-γ-adaptin–coated Domains on Endosomal Tubules

Human transferrin receptors (TR) and receptors for polymeric immunoglobulins (pIgR) expressed in polarized MDCK cells maintain steady-state, asymmetric distributions on the separate basolateral and apical surfaces even though they are trafficking continuously into and across these cells. The intracellular mechanisms required to maintain these asymmetric distributions have not been located. Here we show that TR and pIgR internalize from both surfaces to a common interconnected endosome compartment that includes tubules with buds coated with clathrin lattices. These buds generate vesicles that carry TR to the basolateral border. The lattices contain γ-adaptin and are dispersed by treatment with brefeldin A (BFA). Since BFA treatment abrogates the vectorial trafficking of TR in polarized MDCK cells, we propose that the clathrin-coated domains of the endosome tubules contain the polarized sorting mechanism responsible for their preferential basolateral distribution.     

Targeting of an apical endosomal protein to endosomes in Madin-Darby canine kidney cells requires two sorting motifs

The efficient sorting and targeting of endocytosed macromolecules is critical for epithelial function. However, the distribution of endosomal compartments in these cells remains controversial. In this study, we show that polarized Madin–Darby canine kidney (MDCK) cells target the apical endosomal protein endotubin into an apical early endosomal compartment that is distinct from the apical recycling endosomes. Furthermore, through a panel of site-directed mutations we show that signals required for apical endosomal targeting of endotubin are composed of two distinct motifs on the cytoplasmic domain, a hydrophobic motif and a consensus casein kinase II site. Endotubin-positive endosomes in MDCK cells do not label with basolaterally internalized transferrin or ricin, do not contain the small guanosine triphosphate-binding protein rab11, and do not tubulate in response to low concentrations of brefeldin-A (BFA). Nevertheless, high concentrations of BFA reversibly inhibits the sorting of endotubin from transferrin and cause colocalization in tubular endosomes. These results indicate that, in polarized cells, endotubin targets into a distinct subset of apical endosomes, and the targeting information required both for polarity and endosomal targeting is provided by the cytoplasmic portion of the molecule.     

Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras

The effects of viral Kirsten ras oncogene expression on the polarized phenotype of MDCK cells were investigated. Stable transformed MDCK cell lines expressing the v-K-ras oncogene were generated via infection with a helper-independent retroviral vector construct. When grown on plastic substrata, transformed cells formed continuous monolayers with epithelial-like morphology. However, on permeable filter supports where normal cells form highly polarized monolayers, transformed MDCK cells detached from the substratum and developed multilayers. Morphological analysis of the multilayers revealed that oncogene expression perturbed the polarized organization of MDCK cells such that the transformed cells lacked an apical--basal axis around which the cytoplasm is normally organized. Evidence for selective disruption of apical membrane polarity was provided by immunolocalization of membrane proteins; a normally apical 114-kD protein was randomly distributed on the cell surface in the transformed cell line, whereas normally basolateral proteins remained exclusively localized to areas of cell contact and did not appear on the free cell surface. The discrete distribution of the tight junction-associated ZO-1 protein as well as transepithelial resistance and flux measurements suggested that tight junctions were also assembled. These findings indicate that v-K-ras transformation alters cell-substratum and cell-cell interactions in MDCK cells. Furthermore, v-K-ras expression perturbs apical polarization but does not interfere with the development of a basolateral domain, suggesting that apical and basolateral polarity in epithelial cells may be regulated independently.     

Micron-scale supramolecular myosin arrays help mediate cytoskeletal assembly at mature adherens junctions

Epithelial cells assemble specialized actomyosin structures at E-Cadherin–based cell–cell junctions, and the force exerted drives cell shape change during morphogenesis. The mechanisms that build this supramolecular actomyosin structure remain unclear. We used ZO-knockdown MDCK cells, which assemble a robust, polarized, and highly organized actomyosin cytoskeleton at the zonula adherens, combining genetic and pharmacologic approaches with superresolution microscopy to define molecular machines required. To our surprise, inhibiting individual actin assembly pathways (Arp2/3, formins, or Ena/VASP) did not prevent or delay assembly of this polarized actomyosin structure. Instead, as junctions matured, micron-scale supramolecular myosin arrays assembled, with aligned stacks of myosin filaments adjacent to the apical membrane, overlying disorganized actin filaments. This suggested that myosin arrays might bundle actin at mature junctions. Consistent with this idea, inhibiting ROCK or myosin ATPase disrupted myosin localization/organization and prevented actin bundling and polarization. We obtained similar results in Caco-2 cells. These results suggest a novel role for myosin self-assembly, helping drive actin organization to facilitate cell shape change.     

pH-induced microtubule-dependent redistribution of late endosomes in neuronal and epithelial cells

The interaction between late endocytic structures and microtubules in polarized cells was studied using a procedure previously shown to cause microtubule-dependent redistribution of lysosomes in fibroblasts and macrophages (Heuser, J. 1989. J. Cell Biol. 108:855-864). In cultured rat hippocampal neurons, low cytoplasmic pH caused cation-independent mannose-6-phosphate receptor-enriched structures to move out of the cell body and into the processes. In filter grown MDCK cells lowering the cytosolic pH to approximately 6.5 caused late endosomes to move to the base of the cell and this process was shown to be microtubule dependent. Alkalinization caused a shift in distribution towards the apical pole of the cell. The results are consistent with low pH causing the redistribution of late endosomes towards the plus ends of the microtubules. In MDCK cells the microtubules orientated vertically in the cell may play a role in this process. The shape changes that accompanied the redistribution of the late endosomes in MDCK cells were examined by electron microscopy. On low pH treatment fragmentation of the late endosomes was observed whereas after microtubule depolymerization individual late endosomal structures appeared to fuse together. The late endosomes of the MDCK cell appear to be highly pleomorphic and dependent on microtubules for their form and distribution in the cell.     

Ricin transport in brefeldin A-treated cells: correlation between Golgi structure and toxic effect

Whereas brefeldin A (BFA) protected a number of cell lines against the protein toxin ricin, two of the cell lines tested were not protected but rather sensitized to ricin by BFA. EM studies revealed that upon addition of BFA the Golgi stacks in cells which were protected against the toxin rapidly transformed into a characteristic tubulo-vesicular reticulum connected to the endoplasmic reticulum, and subcellular fractionation experiments showed that galactosyl transferase disappeared from the Golgi fractions where it was normally located. EM and subcellular fractionation also indicated that in contrast to the Golgi stacks, the trans-Golgi network (TGN) remained intact and that internalized ricin was still localized in the TGN both when BFA was added before and after the toxin. Thus, BFA does not prevent fusion of ricin-containing vesicles with the TGN, and unlike resident proteins in Golgi stacks, ricin is not transported back to ER upon treatment of cells with BFA. Two kidney epithelial cell lines, MDCK and PtK2, were not protected against ricin by BFA, and EM studies of MDCK cells revealed that BFA did not alter the morphology of the Golgi complex in these cells. Also, subcellular fractionation revealed that, in contrast to the other cell types tested, the localization of galactosyl transferase in the gradients was not affected by BFA treatment. The data show that there is a correlation between BFA-induced disassembly of the Golgi stacks and protection against ricin, and they demonstrate that the structural organization of the Golgi apparatus is affected by BFA to different extents in various cell lines.     

Modulation of endocytic traffic in polarized Madin-Darby canine kidney cells by the small GTPase RhoA

Efficient postendocytic membrane traffic in polarized epithelial cells is thought to be regulated in part by the actin cytoskeleton. RhoA modulates assemblies of actin in the cell, and it has been shown to regulate pinocytosis and phagocytosis; however, its effects on postendocytic traffic are largely unexplored. To this end, we expressed wild-type RhoA (RhoAWT), dominant active RhoA (RhoAV14), and dominant inactive RhoA (RhoAN19) in Madin-Darby canine kidney (MDCK) cells expressing the polymeric immunoglobulin receptor. RhoAV14 expression stimulated the rate of apical and basolateral endocytosis, whereas RhoAN19 expression decreased the rate from both membrane domains. Polarized basolateral recycling of transferrin was disrupted in RhoAV14-expressing cells as a result of increased ligand release at the apical pole of the cell. Degradation of basolaterally internalized epidermal growth factor was slowed in RhoAV14-expressing cells. Although apical recycling of immunoglobulin A (IgA) was largely unaffected in cells expressing RhoAV14, transcytosis of basolaterally internalized IgA was severely impaired. Morphological and biochemical analyses demonstrated that a large proportion of IgA internalized from the basolateral pole of RhoAV14-expressing cells remained within basolateral early endosomes and was slow to exit these compartments. RhoAN19 and RhoAWT expression had little effect on these postendocytic pathways. These results indicate that in polarized MDCK cells activated RhoA may modulate endocytosis from both membrane domains and postendocytic traffic at the basolateral pole of the cell.     

Staphylococcal ADP-ribosyltransferase-sensitive small G protein is involved in brefeldin A action.

An early event in the action of brefeldin A (BFA) is the dissociation of beta-coat protein (beta-COP) from the Golgi membrane. We have recently shown that staphylococcal ADP-ribosyltransferase (epidermal cell differentiation inhibitor (EDIN)), which specifically modifies a small G protein, rho, mimics the action of BFA and disassembles the Golgi apparatus in Vero cells (Sugai, M., Chen, C-h., and Wu, H. C. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8903-8907). Three independent BFA-resistant cell lines (BER-40 from Vero cells, PtK1, and MDCK) showed cross-resistance to EDIN regarding the release of the beta-COP from the Golgi membrane by EDIN or BFA. BFA as well as EDIN induced disassembly of the actin microfilaments in Vero cells, and they both failed to induce the disassembly of actin microfilaments in BER-40, PtK1, and MDCK cells. BFA inhibited protein secretion in Vero cells but not in BFA-resistant cell lines, whereas EDIN did not inhibit protein secretion in either Vero or other cell lines. AlF-4 inhibited the effect of EDIN as well as that of BFA on the distribution of the beta-COP. These results suggest that an EDIN-sensitive rho protein together with trimeric and other small G protein(s) is involved in the regulation of the assembly of coated vesicles and vesicular transport in the Golgi apparatus.     

Fluid-Phase Markers in the Basolateral Endocytic Pathway Accumulate in Response to the Actin Assembly-promoting Drug Jasplakinolide

To investigate the role of filamentous actin in the endocytic pathway, we used the cell-permeant drug Jasplakinolide (JAS) to polymerize actin in intact polarized Madin–Darby canine kidney (MDCK) cells. The uptake and accumulation of the fluid-phase markers fluorescein isothiocyanate (FITC)-dextran and horseradish peroxidase (HRP) were followed in JAS-treated or untreated cells with confocal fluorescence microscopy, biochemical assays, and electron microscopy. Pretreatment with JAS increased the uptake and accumulation of fluid-phase markers in MDCK cells. JAS increased endocytosis in a polarized manner, with a marked effect on fluid-phase uptake from the basolateral surface but not from the apical surface of polarized MDCK cells. The early uptake of FITC-dextran and HRP was increased more than twofold in JAS-treated cells. At later times, FITC-dextran and HRP accumulated in clustered endosomes in the basal and middle regions of JAS-treated cells. The large accumulated endosomes were similar to late endosomes but they were not colabeled for other late endosome markers, such as rab7 or mannose-6-phosphate receptor. JAS altered transport in the endocytic pathway at a later stage than the microtubule-dependent step affected by nocodazole. JAS also had a notable effect on cell morphology, inducing membrane bunching at the apical pole of MDCK cells. Although other studies have implicated actin in endocytosis at the apical cell surface, our results provide novel evidence that filamentous actin is also involved in the endocytosis of fluid-phase markers from the basolateral membrane of polarized cells.     

Brefeldin A (BFA) disrupts the organization of the microtubule and the actin cytoskeletons

Previous inquiries into the effects of Brefeldin A (BFA) have largely concentrated on dynamics of ER-Golgi membrane traffic, predominantly after relatively short treatments with the drug. We have now analyzed the effects of long BFA treatment on overall cell morphology, behavior of resident and cycling Golgi proteins, and microtubular and actin cytoskeletons organization. Prolonged (15 h or 40 h) treatment of normal rat kidney (NRK) cells with BFA caused dramatic swelling of the Endoplasmic Reticulum (ER) and shifted its localization to the periphery of the cells. The Golgi complex was disassembled and Golgi proteins redistributed and persisted in partially distinct compartments. Prolonged BFA treatment resulted in marked disruption of the MT and actin cytoskeleton. Peripheral MT were absent and tubulin staining was concentrated in short astral MT emanating from the microtubule organizing center (MTOC). Actin stress fibers were largely absent and actin staining was concentrated within a perinuclear area. Within this region, actin localization overlapped that of the membrane transport factor p115. BFA effects on Golgi structure and on MT and actin organization showed the same threshold -- all could be partially reversed after 30 min and 15 h BFA treatment but were irreversible after 40h incubation with the drug. The observed effects were not induced by signaling pathways involved in apoptotic phenomena or in ER stress response pathways. These results suggest that BFA inhibits the activity of key molecules that regulate MT and actin cytoskeleton dynamics. The findings can be used as the basis for elucidating the molecular mechanism of BFA action on the cytoskeleton.     

Actin filament content and organization in unstimulated platelets

The extent of actin polymerization in unstimulated, discoid platelets was measured by DNase I inhibition assay in Triton X-100 lysates of platelets washed at 37 degrees C by gel filtration, or in Triton X-100 lysates of platelets washed at ambient temperatures by centrifugation in the presence of prostacyclin. About 40% of the actin in the discoid platelets obtained by either method existed as filaments. These filaments could be visualized by electron microscopy of thin sections. Similar results were obtained when the actin filament content of discoid platelets was measured by sedimentation of filaments from Triton X-100 lysates at high g forces (145,000 g for 45 min). However, few of these filaments sedimented at the lower g forces often used to isolate networks of actin filaments from cell extracts. These results indicate that actin filaments in discoid cells are not highly crosslinked. Platelets isolated by centrifugation in the absence of prostacyclin were not discoid, but were instead irregular with one or more pseudopodia. These platelets also contained approximately 40-50% of their actin in a filamentous form; many of these filaments sedimented at low g forces, however, indicating that they were organized into networks. The discoid shape of these centrifuged platelets could be restored by incubating them for 1-3 h at 37 degrees C, which resulted in the reversal of filament organization. High g forces were then required for the sedimentation of the actin. Approximately 80-90% of the actin in platelets washed at 4 degrees C was filamentous; this high actin filament content could be attributed to actin polymerization during the preparation of the platelets at low temperatures. These studies show that platelet activation involves mechanisms for the structural reorganization of existing filaments, in addition to those previously described for mediating actin polymerization.     

Apical localization of PMCA2w/b is enhanced in terminally polarized MDCK cells

The “w” splice forms of PMCA2 localize to distinct membrane compartments such as the apical membrane of the lactating mammary epithelium, the stereocilia of inner ear hair cells or the post-synaptic density of hippocampal neurons. Previous studies indicated that PMCA2w/b was not fully targeted to the apical domain of MDCK cells but distributed more evenly to the lateral and apical membrane compartments. Overexpression of the apical scaffold protein NHERF2, however, greatly increased the amount of the pump in the apical membrane of these epithelial cells. We generated a stable MDCK cell line expressing non-tagged, full-length PMCA2w/b to further study the localization and function of this protein. Here we demonstrate that PMCA2w/b is highly active and shows enhanced apical localization in terminally polarized MDCK cells grown on semi-permeable filters. Reversible surface biotinylation combined with confocal microscopy of fully polarized cells show that the pump is stabilized in the apical membrane via the apical membrane cytoskeleton with the help of endogenous NHERF2 and ezrin. Disruption of the actin cytoskeleton removed the pump from the apical actin patches without provoking its internalization. Our data suggest that full polarization is a prerequisite for proper positioning of the PMCA2w variants in the apical membrane domain of polarized cells.     

Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts

Rab8 is a small Ras-like GTPase that regulates polarized membrane transport to the basolateral membrane in epithelial cells and to the dendrites in neurons. It has recently been demonstrated that fibroblasts sort newly synthesized proteins into two different pathways for delivery to the cell surface that are equivalent to the apical and the basolateral post-Golgi routes in epithelial cells (Yoshimori, T., P. Keller, M.G. Roth, and K. Simons. 1996. J. Cell Biol. 133:247-256). To determine the role of Rab8 in fibroblasts, we used both transient expression systems and stable cell lines expressing mutant or wild-type (wt) Rab8. A dramatic change in cell morphology occurred in BHK cells expressing both the wt Rab8 and the activated form of the GTPase, the Rab8Q67L mutant. These cells formed processes as a result of a reorganization of both their actin filaments and microtubules. Newly synthesized vesicular stomatitis virus G glycoprotein, a basolateral marker protein in MDCK cells, was preferentially delivered into these cell outgrowths. Based on these findings, we propose that Rab8 provides a link between the machinery responsible for the formation of cell protrusions and polarized biosynthetic membrane traffic.     

Elevated expression of pp60c-src alters a selective morphogenetic property of epithelial cells in vitro without a mitogenic effect.

Madin-Darby canine kidney (MDCK) cells are highly differentiated and have retained the morphogenetic properties necessary to form polarized, multicellular epithelial structures (cysts) in vitro that resemble epithelial tissues in vivo. We introduced the c-src gene into MDCK cells to elevate the level of the plasma membrane-associated cellular tyrosine kinase, pp60c-src, to levels two- to ninefold higher than that expressed in parent MDCK cells. Our results revealed a highly discriminatory biological action of pp60c-src on the morphogenetic properties of MDCK cells. Elevated expression of pp60c-src conferred on MDCK cells the ability to undergo dramatic changes of cell shape that includes the formation of long cell processes (100 to 200 microns), never observed in control MDCK cells. The morphogenesis of multicellular epithelial cysts was altered by elevated levels of pp60c-src and led to predictable distortions of their three-dimensional architecture. However, these cells established morphologically normal cell polarity, formed adhesive epithelial cell-cell contacts indistinguishable from those of control MDCK cells, and exhibited neither focus-forming ability or anchorage-independent growth potential. Finally, we showed that MDCK cells expressing elevated levels of pp60c-src exhibit increased phosphorylation of a more limited number of phosphotyrosine-containing proteins than MDCK cells expressing pp60v-src. We suggest that a natural function of pp60c-src is to regulate the morphogenetic properties which determine the shape of differentiated cells and multicellular structures.     

Uncoupling of gate and fence functions of MDCK cells by the actin-depolymerizing reagent mycalolide B

The tight junction serves as a paracellular gate to seal the paracellular space of apposing cells and as a molecular fence to prevent diffusion of membrane proteins and lipids in epithelial cells. Although involvement of the actin cytoskeleton has been considered to be important in these two functions, it remains to be elucidated whether both functions are regulated in a coupled manner or differentially by actin. Treatment of highly polarized MDCK cells with mycalolide B (MB), a recently developed actin-depolymerizing reagent, induced a decrease of transepithelial resistance in a dose- and time-dependent manner with reversibility when the reagent was washed out. Changes in cytoskeletal actin, such as a reduction of cortical actin, irregularity of stress fibers, and punctated actin aggregates, were observed after MB treatment. However, the fence function, as studied by diffusion of apically labeled sphingomyelin/BSA complex, remained intact in the MB-treated MDCK cells. Localization of junctional molecules and apical marker proteins such as E-cadherin, ZO-1, and 114-kDa protein was shown to be unaffected. Furthermore, freeze-fracture study showed apparent tight junction strands. Collectively, MB treatment abolished the paracellular gate but not the fence function of MDCK cells, suggesting that cytoskeletal actin may play differential roles in the gate and fence functions of the tight junction.     

Effect of brefeldin A and the dynamics of the actin plate on cytokinesis of zygotes in the brown alga,Silvetia babingtonii (Fucales,Phaeophyceae)

In the cytokinesis of brown algae, actin filaments appear like a plate at the intersecting region of microtubules (MTs) that emerge from the centrosomes after mitosis. The function of the actin plate itself is still unknown. To elucidate the relationship between the actin plate, MTs and membrane fusion, without inducing cytoskeleton depolymerization, the effect of brefeldin A (BFA), which prevents the production of vesicles from Golgi bodies, was examined in zygotes of Silvetia babingtonii. The beginning of mitosis was slightly delayed in zygotes under BFA compared with the controls. Almost all zygotes were inhibited for the progression of cytokinesis by BFA treatment. Ultrastructural observations showed that Golgi cisternae became fragmented or curled following continuous treatment with BFA, and the inhibitory status of cytokinesis between zygotes. The next cell cycle started before cytokinesis was completed. Although the appearance of the actin plate was not disturbed by BFA treatment, the behaviour of the actin plate during the transition between the first and second cell cycles could be classified into two patterns: it was either invisible upon the initiation of the next cell cycle, or a portion of it remained even though the next cell cycle had begun. In the latter case, a part of the actin plate seemed to associate with the new partially formed cell partition membrane, and MTs from the centrosomes were bound to it. The actin plate completely disappeared in the next mitosis, then re-emerged in the middle area of the four daughter nuclei. The results of the present study indicated that, under BFA treatment, the actin plate persisted until just before the beginning of the next mitotic phase, when the new, incomplete cell partition membrane was present, and MTs sustained the actin plate until next mitosis.