PubMed Health⌕ Search

Biomedical subjects

Enrique Rodriguez-Boulan

Publications and source records attributed to Enrique Rodriguez-Boulan.

At least 19 recordsLinked to original sources
PubMed2006

Regulatory dissociation of Tctex-1 light chain from dynein complex is essential for the apical delivery of rhodopsin.

Post-Golgi to apical surface delivery in polarized epithelial cells requires the cytoplasmic dynein motor complex. However, the nature of dynein-cargo interactions and their underlying regulation are largely unknown. Previous studies have shown that the apical surface targeting of rhodopsin requires the dynein light chain, Tctex-1, which binds directly to both dynein intermediate chain (IC) and rhodopsin. In this report, we show that the S82E mutant of Tctex-1, which mimics Tctex-1 phosphorylated at serine 82, has a reduced affinity for dynein IC but not for rhodopsin. Velocity sedimentation experiments further suggest that S82E is not incorporated into the dynein complex. The dominant-negative effect of S82E causes rhodopsin mislocalization in polarized Madin-Darby canine kidney (MDCK) cells. The S82A mutant, which mimics dephosphorylated Tctex-1, can be incorporated into dynein complex but is impaired in its release. Expression of S82A also causes disruption of the apical localization of rhodopsin in MDCK cells. Taken together, these results suggest that the dynein complex disassembles to release cargo due to the specific phosphorylation of Tctex-1 at the S82 residue and that this process is critical for the apical delivery of membrane cargoes.

Animals↗
PubMed2006

Open sesame! Coxsackieviruses conspire to trespass the tight junctional gate.

In the January 13 issue of Cell, Coyne and Bergelson describe an "Open sesame!" strategy developed by coxsackieviruses to invade the organism through the intestinal epithelium. The strategy involves coopting intrinsic signaling abilities of the apical GPI-anchored protein DAF to open the tight junction barrier, gain access to the primary receptor CAR, and activate virus internalization by a caveolin-dependent pathway.

Binding Sites↗
PubMed2005

Secramine inhibits Cdc42-dependent functions in cells and Cdc42 activation in vitro.

Inspired by the usefulness of small molecules to study membrane traffic, we used high-throughput synthesis and phenotypic screening to discover secramine, a molecule that inhibits membrane traffic out of the Golgi apparatus by an unknown mechanism. We report here that secramine inhibits activation of the Rho GTPase Cdc42, a protein involved in membrane traffic, by a mechanism dependent upon the guanine dissociation inhibitor RhoGDI. RhoGDI binds Cdc42 and antagonizes its membrane association, nucleotide exchange and effector binding. In vitro, secramine inhibits Cdc42 binding to membranes, GTP and effectors in a RhoGDI-dependent manner. In cells, secramine mimics the effects of dominant-negative Cdc42 expression on protein export from the Golgi and on Golgi polarization in migrating cells. RhoGDI-dependent Cdc42 inhibition by secramine illustrates a new way to inhibit Rho GTPases with small molecules and provides a new means to study Cdc42, RhoGDI and the cellular processes they mediate.

Actins↗
PubMed2005

Mechanisms regulating tissue-specific polarity of monocarboxylate transporters and their chaperone CD147 in kidney and retinal epithelia.

Proton-coupled monocarboxylate transporters (MCT) MCT1, MCT3, and MCT4 form heterodimeric complexes with the cell surface glycoprotein CD147 and exhibit tissue-specific polarized distributions that are essential for maintaining lactate and pH homeostasis. In the parenchymal epithelia of kidney, thyroid, and liver, MCT/CD147 heterocomplexes are localized in the basolateral membrane where they transport lactate out of or into the cell depending on metabolic conditions. A unique distribution of lactate transporters is found in the retinal pigment epithelium (RPE), which regulates lactate levels of the outer retina. In RPE, MCT1/CD147 is polarized to the apical membrane and MCT3/CD147 to the basolateral membrane. The mechanisms responsible for tissue-specific polarized distribution of MCTs are unknown. Here, we demonstrate that CD147 carries sorting information for polarized targeting of the MCT1/CD147 hetero-complexes in kidney and RPE cells. In contrast, MCT3 and MCT4 harbor dominant sorting information that cotargets CD147 to the basolateral membrane in both epithelia. RNA interference experiments show that MCT1 promotes CD147 maturation. Our results open a unique paradigm to study the molecular basis of tissue-specific polarity.

Adenosine Triphosphatases↗
PubMed2005

Bipolar assembly of caveolae in retinal pigment epithelium.

Caveolae and their associated structural proteins, the caveolins, are specialized plasmalemmal microdomains involved in endocytosis and compartmentalization of cell signaling. We examined the expression and distribution of caveolae and caveolins in retinal pigment epithelium (RPE), which plays key roles in retinal support, visual cycle, and acts as the main barrier between blood and retina. Electron microscopic observation of rat RPE, in situ primary cultures of rat and human RPE and a rat RPE cell line (RPE-J) demonstrated in all cases the presence of caveolae in both apical and basolateral domains of the plasma membrane. Caveolae were rare in RPE in situ but were frequent in primary RPE cultures and in RPE-J cells, which correlated with increased levels in the expression of caveolin-1 and -2. The bipolar distribution of caveolae in RPE is striking, as all other epithelial cells examined to date (liver, kidney, thyroid, and intestinal) assemble caveolae only at the basolateral side. This might be related to the nonpolar distribution of both caveolin-1 and 2 in RPE because caveolin-2 is basolateral and caveolin-1 nonpolar in other epithelial cells. The bipolar localization of plasmalemmal caveolae in RPE cells may reflect specialized roles in signaling and trafficking important for visual function.

Animals↗
PubMed2005

Protein sorting in the Golgi complex: shifting paradigms.

The paradigms for transport along the biosynthetic route have changed dramatically over the past 15 years. Unlike the situation 15 years ago, the current paradigm involves sorting signals practically at every step of the pathway. In particular, at the exit from the Golgi complex, apical, basolateral and lysosomal targeting signals result in the generation of a variety of routes. Furthermore, it is now quite clear that not all sorting in the biosynthetic route occurs in the Golgi complex or the Trans Golgi Network (TGN). Sorting may occur distally to the Golgi, in recycling endosomes or in budded tubulosaccular structures, or it may occur proximally to the Golgi complex, at the exit from the ER. Several adaptors are candidates to sort apical and basolateral proteins but only AP1B and AP4 are currently involved. Progress is fast and future work should elucidate many of the open questions.

Adaptor Proteins, Vesicular Transport↗
PubMed2005

Evolving endosomes: how many varieties and why?

The cell biologist's insight into endosomal diversity, in terms of both form and function, has increased dramatically in the past few years. This understanding has been promoted by the availability of powerful new techniques that allow imaging of both cargo and machinery in the endocytic process in real time, and by our ability to inhibit components of this machinery by RNA interference. The emerging picture from these studies is of a highly complex, dynamic and adaptable endosomal system that interacts at various points with the secretory system of the cell.

Animals↗
PubMed2005

Organization of vesicular trafficking in epithelia.

Experiments using mammalian epithelial cell lines have elucidated biosynthetic and recycling pathways for apical and basolateral plasma-membrane proteins, and have identified components that guide apical and basolateral proteins along these pathways. These components include apical and basolateral sorting signals, adaptors for basolateral signals, and docking and fusion proteins for vesicular trafficking. Recent live-cell-imaging studies provide a real-time view of sorting processes in epithelial cells, including key roles for actin, microtubules and motors in the organization of post-Golgi trafficking.

Adaptor Proteins, Vesicular Transport↗
PubMed2004

Par-1 promotes a hepatic mode of apical protein trafficking in MDCK cells.

Simple (i.e., nonstratified) epithelial cells use two different routes to target their newly synthesized luminal plasma membrane proteins to the cell surface: a direct route from the Golgi complex, as in the kidney-derived MDCK cell line, or an indirect route that involves a intermediate stop at the ab-luminal (basolateral) membrane, as in hepatocytes. The mechanisms or proteins responsible for these different protein targeting strategies are not known. Here, we show that increased expression of EMK1, a mammalian ortholog of Caenorhabditis elegans Par-1, in MDCK cells promotes a switch from a direct to a transcytotic mode of apical protein delivery and other trafficking changes typical of hepatocytes. These results, together with our recent demonstration that PAR-1 promotes morphological features of hepatocytes in MDCK cells, indicate that Par-1 modulates the developmental decision to build a columnar versus a hepatic epithelial cell. To our knowledge, Par-1 is the first gene assigned to this task in epithelial morphogenesis.

Animals↗
PubMed2004

The basolateral targeting signal of CD147 (EMMPRIN) consists of a single leucine and is not recognized by retinal pigment epithelium.

CD147, a type I integral membrane protein of the immunoglobulin superfamily, exhibits reversed polarity in retinal pigment epithelium (RPE). CD147 is apical in RPE in contrast to its basolateral localization in extraocular epithelia. This elicited our interest in understanding the basolateral sorting signals of CD147 in prototypic Madin-Darby canine kidney (MDCK) cells. The cytoplasmic domain of CD147 has basolateral sorting information but is devoid of well-characterized basolateral signals, such as tyrosine and di-leucine motifs. Hence, we carried out systematic site-directed mutagenesis to delineate basolateral targeting information in CD147. Our detailed analysis identified a single leucine (252) as the basolateral targeting motif in the cytoplasmic tail of CD147. Four amino acids (243-246) N-terminal to leucine 252 are also critical basolateral determinants of CD147, because deletion of these amino acids leads to mistargeting of CD147 to the apical membranes. We ruled out the involvement of adaptor complex 1B (AP1B) in the basolateral trafficking of CD147, because LLC-PK1 cells lacking AP1B, target CD147 basolaterally. At variance with MDCK cells, the human RPE cell line ARPE-19 does not distinguish between CD147 (WT) and CD147 with leucine 252 mutated to alanine and targets both proteins apically. Thus, our study identifies an atypical basolateral motif of CD147, which comprises a single leucine and is not recognized by RPE cells. This unusual basolateral sorting signal will be useful in unraveling the specialized sorting machinery of RPE cells.

Adaptor Protein Complex 1↗
PubMed2004

Mammalian PAR-1 determines epithelial lumen polarity by organizing the microtubule cytoskeleton.

Epithelial differentiation involves the generation of luminal surfaces and of a noncentrosomal microtubule (MT) network aligned along the polarity axis. Columnar epithelia (e.g., kidney, intestine, and Madin-Darby canine kidney [MDCK] cells) generate apical lumina and orient MT vertically, whereas liver epithelial cells (hepatocytes and WIFB9 cells) generate lumina at cell-cell contact sites (bile canaliculi) and orient MTs horizontally. We report that knockdown or inhibition of the mammalian orthologue of Caenorhabditis elegans Par-1 (EMK1 and MARK2) during polarization of cultured MDCK and WIFB9 cells prevented development of their characteristic lumen and nonradial MT networks. Conversely, EMK1 overexpression induced the appearance of intercellular lumina and horizontal MT arrays in MDCK cells, making EMK1 the first known candidate to regulate the developmental branching decision between hepatic and columnar epithelial cells. Our experiments suggest that EMK1 primarily promotes reorganization of the MT network, consistent with the MT-regulating role of this gene product in other systems, which in turn controls lumen formation and position.

Animals↗
PubMed2004

Epithelial trafficking: new routes to familiar places.

Research carried out in mammalian epithelial cell systems over the past 25 years has delineated pathways and sorting signals involved in polarized delivery of plasma membrane proteins. Recently some progress has been made in the identification of mechanisms underlying this polarized trafficking and in the visualization of trafficking routes in live cells. A promising area of research is the study of trafficking functions of novel polarity genes identified in Drosophila and Caenorhabditis elegans.

Animals↗
PubMed2003

Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells.

Targeted delivery of proteins to distinct plasma membrane domains is critical to the development and maintenance of polarity in epithelial cells. We used confocal and time-lapse total internal reflection fluorescence microscopy (TIR-FM) to study changes in localization and exocytic sites of post-Golgi transport intermediates (PGTIs) carrying GFP-tagged apical or basolateral membrane proteins during epithelial polarization. In non-polarized Madin Darby Canine Kidney (MDCK) cells, apical and basolateral PGTIs were present throughout the cytoplasm and were observed to fuse with the basal domain of the plasma membrane. During polarization, apical and basolateral PGTIs were restricted to different regions of the cytoplasm and their fusion with the basal membrane was completely abrogated. Quantitative analysis suggested that basolateral, but not apical, PGTIs fused with the lateral membrane in polarized cells, correlating with the restricted localization of Syntaxins 4 and 3 to lateral and apical membrane domains, respectively. Microtubule disruption induced Syntaxin 3 depolarization and fusion of apical PGTIs with the basal membrane, but affected neither the lateral localization of Syntaxin 4 or Sec6, nor promoted fusion of basolateral PGTIs with the basal membrane.

Animals↗
PubMed2002

Inhibitors of glycosphingolipid biosynthesis reduce transepithelial electrical resistance in MDCK I and FRT cells.

Madin-Darby canine kidney (MDCK) I and Fisher rat thyroid (FRT) cells exhibit transepithelial electrical resistance (TER) values in excess of 5,000 Omega. cm(2). When these cells were incubated in the presence of various inhibitors of sphingolipid biosynthesis, a >5-fold reduction of TER was observed without changes in the gate function for uncharged solutes or the fence function for apically applied fluorescent lipids. The localization of ZO-1 and occludin was not altered between control and inhibitor-treated cells, indicating that the tight junction was still intact. Furthermore, the complexity of tight junction strands, analyzed by freeze-fracture microscopy, was not reduced. Once the inhibitor was removed and the cells were allowed to synthesize sphingolipids, a gradual recovery of the TER was observed. Interestingly, these inhibitors did not attenuate the TER of MDCK II cells, a cell line that typically exhibits values below 800 omega x cm(2.) These results suggest that glycosphingolipids play a role in regulating the electrical properties of epithelial cells.

Animals↗
PubMed2002

The lipofuscin component A2E selectively inhibits phagolysosomal degradation of photoreceptor phospholipid by the retinal pigment epithelium.

Daily phagocytosis of spent photoreceptor outer segments is a critical maintenance function performed by the retinal pigment epithelium (RPE) to preserve vision. Aging RPE accumulates lipofuscin, which includes N-retinylidene-N-retinylethanolamine (A2E) as the major autofluorescent component. We studied the effect of physiological levels of A2E in RPE cultures on their ability to phagocytose outer segments. A2E localized to lysosomes in cultured RPE as well as in human RPE in situ. A2E-loaded RPE cells in culture bound and internalized identical numbers of outer segments as control RPE indicating that A2E does not alter early steps of phagocytosis. A2E-loaded RPE degraded outer segment proteins efficiently but, strikingly, failed to completely digest phospholipids within 24 h. Because of the circadian rhythm of RPE phagocytosis in the eye, a delay in lipid degradation would likely result in a build up of undigested material in RPE that could contribute to the development of age-related macular degeneration.

Cell Line↗
PubMed2002

Estrogen lowers Alzheimer beta-amyloid generation by stimulating trans-Golgi network vesicle biogenesis.

Estrogen reduces the risk of Alzheimer's disease in post-menopausal women, beta-amyloid (Abeta) burden in animal models of Alzheimer's disease, and secretion of Abeta from neuronal cultures. The biological basis for these effects remains unknown. Here, utilizing cell-free systems derived from both neuroblastoma cells and primary neurons, we demonstrate that 17beta-estradiol (17beta-E2) stimulates formation of vesicles containing the beta-amyloid precursor protein (betaAPP) from the trans-Golgi network (TGN). Accelerated betaAPP trafficking precludes maximal Abeta generation within the TGN. 17beta-E2 appears to modulate TGN phospholipid levels, particularly those of phosphatidylinositol, and to recruit soluble trafficking factors, such as Rab11, to the TGN. Together, these results suggest that estrogen may exert its anti-Abeta effects by regulating betaAPP trafficking within the late secretory pathway. These results suggest a novel mechanism through which 17beta-E2 may act in estrogen-responsive tissues and illustrate how altering the kinetics of the transport of a protein can influence its metabolic fate.

Alzheimer Disease↗
PubMed2002

The epithelial-specific adaptor AP1B mediates post-endocytic recycling to the basolateral membrane.

To perform vectorial secretory and transport functions that are critical for the survival of the organism, epithelial cells sort plasma membrane proteins into polarized apical and basolateral domains. Sorting occurs post-synthetically, in the trans Golgi network (TGN) or after internalization from the cell surface in recycling endosomes, and is mediated by apical and basolateral sorting signals embedded in the protein structure. Basolateral sorting signals include tyrosine motifs in the cytoplasmic domain that are structurally similar to signals involved in receptor internalization by clathrin-coated pits. Recently, an epithelial-specific adaptor protein complex, AP1B, was identified. AP-1B recognizes a subset of basolateral tyrosine motifs through its mu 1B subunit. Here, we characterized the post-synthetic and post-endocytic sorting of the fast recycling low density lipoprotein receptor (LDLR) and transferrin receptor (TfR) in LLC-PK1 cells, which lack mu 1B and mis-sort both receptors to the apical surface. Targeting and recycling assays in LLC-PK1 cells, before and after transfection with mu 1B, and in MDCK cells, which express mu 1B constitutively, suggest that AP1B sorts basolateral proteins post-endocytically.

Adaptor Proteins, Vesicular Transport↗