The Golgi-localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus

Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells.     

Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation

Four isoforms of the Na+/H+ exchanger (NHE6-NHE9) are distributed to intracellular compartments in human cells. They are localized to Golgi and post-Golgi endocytic compartments as follows: mid- to trans-Golgi, NHE8; trans-Golgi network, NHE7; early recycling endosomes, NHE6; and late recycling endosomes, NHE9. No significant localization of these NHEs was observed in lysosomes. The distribution of these NHEs is not discrete in the cells, and there is partial overlap with other isoforms, suggesting that the intracellular localization of the NHEs is established by the balance of transport in and out of the post-Golgi compartments as the dynamic membrane trafficking. The overexpression of NHE isoforms increased the luminal pH of the compartments in which the protein resided from the mildly acidic pH to the cytosolic pH, suggesting that their in vivo function is to regulate the pH and monovalent cation concentration in these organelles. We propose that the specific NHE isoforms contribute to the maintenance of the unique acidic pH values of the Golgi and post-Golgi compartments in the cell.     

Immunocytochemical localization of alpha2,3(N)-sialyltransferase (ST3Gal III) in cell lines and rat kidney tissue sections: evidence for golgi and post-golgi localization

Sialylation is a biosynthetic process occurring in the trans compartments of the Golgi apparatus. Corresponding evidence is based on localization and biochemical studies of alpha2, 6(N)-sialyltransferase (ST6Gal I) as previously reported. Here we describe generation and characterization of polyclonal antibodies to recombinant rat alpha2,3(N)-sialyltransferase (ST3Gal III) expressed as a soluble enzyme in Sf9 cells or as a beta-galactosidase-human-ST3Gal III fusion- protein from E.coli , respectively. These antibodies were used to localize ST3Gal III by immunofluorescence in various cell lines and rat kidney tissue sections. In transiently transfected COS cells the antibodies directed to soluble sialyltransferase or the sialyltransferase portion of the fusion-protein only recognized the recombinant antigen retained in the endoplasmic reticulum. However, an antibody fraction crossreactive with beta-galactosidase recognized natively expressed ST3Gal III which was found to be colocalized with beta1, 4-galactosyltransferase in the Golgi apparatus of several cultured cell lines. Antibodies affinity purified on the beta- galactosidase-ST3Gal III fusion-protein column derived from both antisera have then been used to localize the enzyme in perfusion-fixed rat kidney sections. We found strong staining of the Golgi apparatus of tubular epithelia and a brush-border-associated staining which colocalized with cytochemical staining of the H+ATPase. This subcellular localization was not observed for ST6Gal I which localized to the Golgi apparatus. These data show colocalization in the Golgi apparatus and different post-Golgi distributions of the two sialyltransferases.      

Golvesin-GFP fusions as distinct markers for Golgi and post-Golgi vesicles in Dictyostelium cells

Golvesin is a new protein associated with membranes of the Golgi apparatus and post-Golgi vesicles in Dictyostelium cells. An internal hydrophobic sequence of 24 amino-acid residues is responsible for anchoring golvesin to the membranes of these organelles. In an attempt to visualize organelle dynamics in vivo, we have used specific antibody and other labels to localize golvesin-green fluorescent protein (GFP) constructs to different cellular compartments. With a GFP tag at its N-terminus, golvesin shows the same localization as the untagged protein. It is transferred to two post-Golgi compartments, the endosomal and contractile vacuole systems. Endosomes are decorated with GFP-golvesin within less than 10 min of their internalisation, and keep the label during the acidic phase of the pathway. Blockage of the C-terminus with GFP causes entrapment of the protein in the Golgi apparatus, indicating that a free C-terminus is required for transfer of golvesin to any of the post-Golgi compartments. The C-terminally tagged golvesin proved to be a reliable Golgi marker in Dictyostelium cells revealing protrusion of Golgi tubules at peak velocities of 3 to 4 microm x s(-1). The fusion protein is retained in Golgi vesicles during mitosis, visualizing Golgi disassembly and reorganization in line with cytokinesis.     

The small GTP-binding protein Rho1p is localized on the Golgi apparatus and post-Golgi vesicles in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the ras-related protein Rho1p is essentially the only target for ADP-ribosylation by exoenzyme C3 of Clostridium botulinum. Using C3 to detect Rho1p in subcellular fractions, Rho1p was found primarily in the 10,000 g pellet (P2) containing large organelles; small amounts also were detected in the 100,000 g pellet (P3), and cytosol. When P2 organelles were separated in sucrose density gradients Rho1p comigrated with the Kex-2 activity, a late Golgi marker. Rho1p distribution was shifted from P2 to P3 in several mutants that accumulate post-Golgi vesicles. Rho1p comigrated with post-Golgi transport vesicles during fractionation of P3 organelles from wild-type or sec6 cells. Vesicles containing Rho1p were of the same size but different density than those bearing Sec4p, a ras-related protein located both on post-Golgi vesicles and the plasma membrane. Immunofluorescence microscopy detected Rho1p as a punctate pattern, with signal concentrated towards the cell periphery and in the bud. Thus, in S. cerevisiae Rho1p resides primarily in the Golgi apparatus, and also in vesicles that are likely to be early post-Golgi vesicles.     

Cytochemical analysis of oligosaccharide processing in frog photoreceptors

Summary Lectin cytochemistry, together with exoglycosidase enzyme digestion, has been used to characterize partially glycoconjugates of several intracellular compartments in frog photoreceptors. In order to obtain uniform access of reagents to all intracellular compartments, the experiments were performed directly on semi-thin sections ofXenopus laevis retinal tissue embedded in a hydrophilic plastic resin. In the rod, the major photoreceptor intracellular binding sites for wheat germ agglutinin (WGA) are the outer segment, the Golgi complex, and other inner segment organelles which are probably involved in the transport of glycoconjugates from the Golgi complex to the outer segment. In addition, shed outer segment tips (phagosomes) are uniformly labelled with WGA. The WGA-binding sites of the outer segment and of the presumed transport organelles are resistant to neuraminidase digestion. This is consistent with the possibility that glycoconjugates (primarily opsin) are transported from the Golgi complex to the outer segment without further oligosaccharide processing. Specific staining of rod outer segments and of phagosomes is also obtained with theN-acetylglucosamine-specific lectin, succinyl-WGA (S-WGA). Outer segments and phagosomes stain the same with WGA, S-WGA and a variety of other lectins tested suggesting that no major post-Golgi oligosaccharide processing accompanies the shedding-phagocytosis event. Concanavalin A (Con A) staining of intracellular sites in rod inner segments reveals a striking difference compared to WGA staining in that the Con A binding sites are concentrated in the photoreceptor axon and presynaptic terminal. These results, and results from previous studies, indicate that the photoreceptor may utilize different mechanisms of oligosaccharide processing from the level of a single Golgi complex to the opposite ends of this cell. Furthermore, those glycoconjugates destined for the presynaptic terminal may undergo post-Golgi processing at or near their sites of insertion into the presynaptic plasma membrane.     

Light and electron microscopic localization of ACTH and pro-opiomelanocortin-derived peptides in human developmental and neoplastic cells

Oncofetal aspects of ACTH and pro-opiomelanocortin (POMC)-derived peptides were studied immunohistochemically at the light and electron microscopic level in human fetal pituitary glands, pituitary adenomas, and small-cell carcinoma of the lung. ACTH, beta-endorphin, and gamma-MSH were localized in the same cells of both fetal and adult pituitary, as well as in the above-mentioned neoplastic tissues. However, alpha-MSH was observed only in the early fetal pituitary, its concentration decreasing with advancing gestational age. The adult pituitary contained only a few alpha-MSH-positive cells. By immunoelectron microscopy, ACTH in the adult pituitary was localized exclusively in the secretory granules. In fetal pituitary at 9 weeks' gestation, ACTH was localized in the perinuclear spaces (PNS), cisternae of rough endoplasmic reticulum (RER), Golgi saccules, and secretory granules. The staining pattern of ACTH in these organelles varied from cell to cell. In fetal pituitaries of greater gestational ages, ACTH was localized in secretory granules. The pituitary adenomas mimicked the staining characteristics of the adult pituitary, i.e., negative or only very occasional alpha-MSH staining and localization of ACTH in the secretory granules. The ectopic ACTH-producing tumors showed a staining pattern similar to that of the early fetal pituitary, i.e., positive staining for alpha-MSH and the presence of ACTH in PNS and cisternae of RER.     

Tuberin is a component of lipid rafts and mediates caveolin-1 localization: role of TSC2 in post-Golgi transport

Mutations of the TSC2 gene lead to the development of hamartomas in tuberous sclerosis complex. Their pathology exhibits features indicative of defects in cell growth, proliferation, differentiation, and migration. We have previously shown that tuberin, the TSC2 protein, resides in multiple subcellular compartments and as such may serve multiple functions. To further characterize the microsomal pool of tuberin, we found that it cofractionated with caveolin-1 in a low-density, Triton X-100-resistant fraction (i.e., lipid rafts) and regulated its localization. In cells lacking tuberin, most of the endogenous caveolin-1 was displaced from the plasma membrane to a Brefeldin-A-sensitive, post-Golgi compartment distinct from the endosome and lysosome. Correspondingly, there was a paucity of caveolae at the plasma membrane of Tsc2-/- cells. Reintroduction of TSC2, but not a disease-causing mutant, reversed the caveolin-1 localization to the membrane. Exogenously expressed caveolin-1-GFP and vesicular stomatitis virus G protein, VSVG-GFP in the Tsc2-/- cells failed to be transported to the plasma membrane and were retained in distinct post-Golgi vesicles. Our data suggest a role of tuberin in regulating post-Golgi transport without apparent effects on protein sorting. The presence of mislocalized proteins in Tsc2-/- cells may contribute to the abnormal signaling and cellular phenotype of tuberous sclerosis.     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: I. Inhibition of glycoprotein migration in various rat cell types as shown by light microscope radioautography after injection of 3H-fucose

Previous studies have shown that colchicine and vinblastine inhibit secretion in many cell types by interrupting the normal intracellular migration of secretory products. In the present work, radioautography has been used to study the effects of these drugs on migration of membrane and secretory glycoproteins in a variety of cell types. Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for light microscope radioautography. Examination of secretory cell types such as ameloblasts and thyroid follicular cells in control animals revealed reactions of approximately equal intensity over the Golgi region and over extracellular secretion products, while in drug-treated rats most of the reaction was confined to the Golgi region. In a variety of other cell types, including endocrine cells (e.g., hepatocytes) and cells generally considered as nonsecretory (e.g., intestinal columnar cells), reaction in control animals occurred both over the Golgi region and over various portions of the cell surface. In drug-treated animals, a strong Golgi reaction was present, but reaction over the cell surface was weak or absent. These results indicate that in many cell types, colchicine and vinblastine inhibit migration out of the Golgi region not only of secretory glycoproteins, but also of membrane glycoproteins destined for the plasma membrane.     

Cytoplasmic Accumulation of Heterogeneous Nuclear Ribonucleoprotein K Strongly Promotes Tumor Invasion in Renal Cell Carcinoma Cells

Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a part of the ribonucleoprotein complex which regulates diverse biological events. While overexpression of hnRNP K has been shown to be related to tumorigenesis in several cancers, both the expression patterns and biological mechanisms of hnRNP K in renal cell carcinoma (RCC) cells remain unclear. In this study, we showed that hnRNP K protein was strongly expressed in selected RCC cell lines (ACHN, A498, Caki-1, 786–0), and knock-down of hnRNP K expression by siRNA induced cell growth inhibition and apoptosis. Based on immunohistochemical (IHC) analysis of hnRNP K expression in human clear cell RCC specimens, we demonstrated that there was a significant positive correlation between hnRNP K staining score and tumor aggressiveness (e.g., Fuhrman grade, metastasis). Particularly, the rate of cytoplasmic localization of hnRNP K in primary RCC with distant metastasis was significantly higher than that in RCC without metastasis. Additionally, our results indicated that the cytoplasmic distribution of hnRNP K induced by TGF-β stimulus mainly contributed to TGF-β-triggered tumor cell invasion in RCC cells. Dominant cytoplasmic expression of ectopic hnRNP K markedly suppressed the inhibition of invasion by knock-down of endogenous hnRNP K. The expression level of matrix metalloproteinase protein-2 was decreased by endogenous hnRNP K knock-down, and restored by ectopic hnRNP K. Therefore, hnRNP K may be a key molecule involved in cell motility in RCC cells, and molecular mechanism associated with the subcellular localization of hnRNP K may be a novel target in the treatment of metastatic RCC.     

ADPKD: a human disease altering Golgi function and basolateral exocytosis in renal epithelia

Epithelial cells explanted from autosomal dominant polycystic kidney disease (ADPKD) tissue exhibit impaired exocytosis, specifically between the Golgi and basolateral membrane (Charron A, Nakamura B, Bacallo R, Wandinger-Ness A. Compromised cytoarchitecture and polarized trafficking in autosomal dominant polycystic kidney disease cells. J Cell Biol 2000; 148: 111–124.). Here the defect is shown to result in the accumulation of the basolateral transport marker vesicular stomatitis virus (VSV) G protein in the Golgi complex. Golgi complex morphology is consequently altered in the disease cells, evident in the noticeable fenestration and dilation of the cisternae. Further detailed microscopic evaluation of normal kidney and ADPKD cells revealed that ineffective basolateral exocytosis correlated with modulations in the localization of select post-Golgi transport effectors. The cytosolic coat proteins p200/myosin II and caveolin exhibited enhanced association with the cytoskeleton or the Golgi of the disease cells, respectively. Most cytoskeletal components with known roles in vesicle translocation or formation were normally arrayed with the exception of Golgi β-spectrin, which was less prevalent on vesicles. The rab8 GTPase, important for basolateral vesicle targeting, was redistributed from the perinuclear Golgi region to disperse vesicles in ADPKD cells. At the basolateral membrane of ADPKD cells, there was a notable loss of the exocyst components sec6/sec8 and an unidentified syntaxin. It is postulated that dysregulated basolateral transport effector function precipitates the disruption of basolateral exocytosis and dilation of the ADPKD cell Golgi as basolateral cargo accumulates within the cisternae.     

Plant TGNs: dynamics and physiological functions

In all eukaryotic cells, a membrane trafficking system connects the post-Golgi organelles, including the trans-Golgi network (TGN), endosomes, and vacuoles. This complex network plays critical roles in several higher-order functions in multicellular organisms. The TGN, one of the important organelles for protein transport in the post-Golgi network, functions as a sorting station, where cargo proteins are directed to the appropriate post-Golgi compartments. The TGN has been considered to be a compartment belonging to the Golgi apparatus, located on the trans side of the Golgi apparatus. However, in plant cells, recent studies have suggested that the TGN is an independent, dynamic organelle that possesses features different than those of TGNs in animal and yeast cells. In this review, we summarize recent progress regarding the dynamics and physiological functions of the plant TGN.     

Stable Golgi-mitochondria complexes and formation of Golgi Ca(2+) gradients in pancreatic acinar cells

We have determined the localization of the Golgi with respect to other organelles in living pancreatic acinar cells and the importance of this localization to the establishment of Ca(2+) gradients over the Golgi. Using confocal microscopy and the Golgi-specific fluorescent probe 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine, we found Golgi structures localizing to the outer edge of the secretory granular region of individual acinar cells. We also assessed Golgi positioning in acinar cells located within intact pancreatic tissue using two-photon microscopy and found a similar localization. The mitochondria segregate the Golgi from lateral regions of the plasma membrane, the nucleus, and the basal part of the cytoplasm. The Golgi is therefore placed between the principal Ca(2+) release sites in the apical region of the cell and the important Ca(2+) sink formed by the peri-granular mitochondria. During acetylcholine-induced cytosolic Ca(2+) signals in the apical region, large Ca(2+) gradients form over the Golgi (decreasing from trans- to cis-Golgi). We further describe a novel, close interaction of the peri-granular mitochondria and the Golgi apparatus. The mitochondria and the Golgi structures form very close contacts, and these contacts remain stable over time. When the cell is forced to swell, the Golgi and mitochondria remain juxtaposed up to the point of cell lysis. The strategic position of the Golgi (closer to release sites than the bulk of the mitochondrial belt) makes this organelle receptive to local apical Ca(2+) transients. In addition the Golgi is ideally placed to be preferentially supplied by ATP from adjacent mitochondria.     

Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus

Huntingtin regulates post-Golgi trafficking of secreted proteins. Here, we studied the mechanism by which mutant huntingtin impairs this process. Colocalization studies and Western blot analysis of isolated Golgi membranes showed a reduction of huntingtin in the Golgi apparatus of cells expressing mutant huntingtin. These findings correlated with a decrease in the levels of optineurin and Rab8 in the Golgi apparatus that can be reverted by overexpression of full-length wild-type huntingtin. In addition, immunoprecipitation studies showed reduced interaction between mutant huntingtin and optineurin/Rab8. Cells expressing mutant huntingtin produced both an accumulation of clathrin adaptor complex 1 at the Golgi and an increase of clathrin-coated vesicles in the vicinity of Golgi cisternae as revealed by electron microscopy. Furthermore, inverse fluorescence recovery after photobleaching analysis for lysosomal-associated membrane protein-1 and mannose-6-phosphate receptor showed that the optineurin/Rab8-dependent post-Golgi trafficking to lysosomes was impaired in cells expressing mutant huntingtin or reducing huntingtin levels by small interfering RNA. Accordingly, these cells showed a lower content of cathepsin D in lysosomes, which led to an overall reduction of lysosomal activity. Together, our results indicate that mutant huntingtin perturbs post-Golgi trafficking to lysosomal compartments by delocalizing the optineurin/Rab8 complex, which, in turn, affects the lysosomal function.     

Alteration of terminal glycosylation sequences on N-linked oligosaccharides of Chinese hamster ovary cells by expression of beta-galactoside alpha 2,6-sialyltransferase

In this report we describe the alteration of the N-linked oligosaccharide terminal sequences of Chinese hamster ovary cell glycoproteins by expression of a beta-galactoside alpha 2,6-sialyltransferase cDNA. While wild type cells normally produce sugar chains terminating in the NeuAc alpha 2,3Gal linkage, the expressed enzyme competes with the endogenous sialyltransferase to attach an alternative terminal sequence, NeuAc alpha 2,6Gal. Subcellular localization of the NeuAc alpha 2,6Gal product by lectin-gold electron microscopy revealed localization throughout the Golgi apparatus cis to trans, post-Golgi membranes and vesicular structures. The results demonstrate the potential for purposefully altering terminal carbohydrate structures in vivo by "mis-expressing" terminal glycosyltransferases that compete with the endogenous enzyme normally produced by the cells.     

Two Separate Signals Act Independently to Localize a Yeast Late Golgi Membrane Protein through a Combination of Retrieval and Retention

The localization of proteins to late-Golgi membranes (TGN) of Saccharomyces cerevisiae is conferred by targeting motifs containing aromatic residues in the cytosolic domains of these proteins. These signals could act by directing retrieval from a post-Golgi compartment or by preventing exit from the TGN. To investigate the mechanism of localization of yeast TGN proteins, we used the heterologous protein A-ALP (consisting of the cytosolic domain of dipeptidyl aminopeptidase A [DPAP A] fused to the transmembrane and luminal domains of the vacuolar protein alkaline phosphatase [ALP]), which localizes to the yeast TGN. Insertion of the aromatic residue–based TGN localization motif (FXFXD) of DPAP A into the cytosolic domain of ALP results in a protein that resides in the TGN. We demonstrate that the FXFXD motif confers Golgi localization through retrieval from a post-Golgi compartment by detecting a post-Golgi processed form of this protein in the TGN. We present an assay that uncouples retrieval-mediated Golgi localization from static retention-based localization, allowing measurement of the rate at which proteins exit the yeast TGN. We also demonstrate that the cytosolic domain of DPAP A contains additional information, separate from the retrieval motif, that slows exit from the TGN. We propose a model for DPAP A localization that involves two distinct mechanisms: one in which the FXFXD motif directs retrieval from a post-Golgi compartment, and a second that slows the rate at which DPAP A exits the TGN.     

Concerted effort of centrosomal and Golgi-derived microtubules is required for proper Golgi complex assembly but not for maintenance

Assembly of an integral Golgi complex is driven by microtubule (MT)-dependent transport. Conversely, the Golgi itself functions as an unconventional MT-organizing center (MTOC). This raises the question of whether Golgi assembly requires centrosomal MTs or can be self-organized, relying on its own MTOC activity. The computational model presented here predicts that each MT population is capable of gathering Golgi stacks but not of establishing Golgi complex integrity or polarity. In contrast, the concerted effort of two MT populations would assemble an integral, polarized Golgi complex. Indeed, while laser ablation of the centrosome did not alter already-formed Golgi complexes, acentrosomal cells fail to reassemble an integral complex upon nocodazole washout. Moreover, polarity of post-Golgi trafficking was compromised under these conditions, leading to strong deficiency in polarized cell migration. Our data indicate that centrosomal MTs complement Golgi self-organization for proper Golgi assembly and motile-cell polarization.     

Correct targeting of plant ARF GTPases relies on distinct protein domains

Indispensable membrane trafficking events depend on the activity of conserved small guanosine triphosphatases (GTPases), anchored to individual organelle membranes. In plant cells, it is currently unknown how these proteins reach their correct target membranes and interact with their effectors. To address these important biological questions, we studied two members of the ADP ribosylation factor (ARF) GTPase family, ARF1 and ARFB, which are membrane anchored through the same N-terminal myristoyl group but to different target membranes. Specifically, we investigated how ARF1 is targeted to the Golgi and post-Golgi structures, whereas ARFB accumulates at the plasma membrane. While the subcellular localization of ARFB appears to depend on multiple domains including the C-terminal half of the GTPase, the correct targeting of ARF1 is dependent on two domains: an N-terminal ARF1 domain that is necessary for the targeting of the GTPase to membranes and a core domain carrying a conserved MxxE motif that influences the relative distribution of ARF1 between the Golgi and post-Golgi compartments. We also established that the N-terminal ARF1 domain alone was insufficient to maintain an interaction with membranes and that correct targeting is a protein-specific property that depends on the status of the GTP switch. Finally, an ARF1-ARFB chimera containing only the first 18 amino acids from ARF1 was shown to compete with ARF1 membrane binding loci. Although this chimera exhibited GTPase activity in vitro, it was unable to recruit coatomer, a known ARF1 effector, onto Golgi membranes. Our results suggest that the targeting of ARF GTPases to the correct membranes may not only depend on interactions with effectors but also relies on distinct protein domains and further binding partners on the Golgi surface.     

Establishment of the circumferential actin filament network is a prerequisite for localization of the cadherin-catenin complex in epithelial cells.

With the adhesion molecules, the actin cytoskeleton controls cell-cell and cell-substrate interactions and participates in transmembrane signaling. The relationships between actin and adhesion complexes at the sites of adhesion have been well documented. Here we investigate by a series of studies whether a relationship exists between actin organization and the localization and function of the components of the cadherin-catenin complex (CCC) that participates in the cell-cell adherens junction. Reversible actin depolymerization reversibly affects the peripheral distribution of CCCs. Mutations in adenovirus E1A and the small GTPase rac1, but not Ha-ras, disrupt the circumferential, cortical actin filament (CAF) network and the targeting of CCC components to the cell surface. Disruption of actin stress fibers or microtubules does not interfere with CCC localization and function. Constitutive loss of the apical cortical actin ring results in epithelial cells in which components of the CCCs are found only in intracellular vesicles and never at the surface. A kinetic analysis of the de novo appearance of the CAF network and the CCCs at the cell surface was also conducted. When F-actin was dissolved, surface CCC components were internalized. Reestablishment of CAFs required about 4 h, during which time E-cadherin and alpha-catenin were found first in a juxtanuclear location and then in intracellular vesicles or post-Golgi carriers, similar to what was observed in cells expressing mutant E1A or rac1. Thus, disruption of preexisting CCCs resulted in their internalization and recycling to the Golgi. It was only after the regeneration of the filamentous actin ring beneath the cell surface that peripheral localization of CCCs was observed. A similar result was observed with dominant negative rac1. These data suggest that the status of cortical actin is assessed and transduced and thereby regulates the transport and delivery of cadherin and catenins to the cell surface.