Biomedical subjects

Ian G Macara

Publications and source records attributed to Ian G Macara.

At least 19 recordsLinked to original sources

PubMed2006

Depletion of E-cadherin disrupts establishment but not maintenance of cell junctions in Madin-Darby canine kidney epithelial cells.

E-cadherin forms calcium-dependent homophilic intercellular adhesions between epithelial cells. These contacts regulate multiple aspects of cell behavior, including the organization of intercellular tight junctions (TJs). To distinguish between the roles of E-cadherin in formation versus maintenance of junctions, Madin-Darby canine kidney (MDCK) cells were depleted of E-cadherin by RNA interference. Surprisingly, reducing E-cadherin expression had little effect on the protein levels or localization of adherens junction (AJ) or TJ markers. The cells underwent morphological changes, as the normally flat apical surface swelled into a dome. However, apical-basal polarity was not compromised, transmembrane resistance was normal, and zonula occludin protein 1 dynamics at the TJs were unchanged. Additionally, an E-cadherin/Cadherin-6 double knockdown also failed to disrupt established TJs, although beta-catenin was lost from the cell cortex. Nevertheless, cells depleted of E-cadherin failed to properly reestablish cell polarity after junction disassembly. Recovery of cell-cell adhesion, transepithelial resistance, and the localization of TJ and AJ markers were all delayed. In contrast, depletion of alpha-catenin caused long-term disruption of junctions. These results indicate that E-cadherin and Cadherin-6 function as a scaffold for the construction of polarized structures, and they become largely dispensable in mature junctions, whereas alpha-catenin is essential for the maintenance of functional junctions.

Adherens Junctions↗

PubMed2006

Structural analysis of septin 2, 6, and 7 complexes.

Mammalian septins comprise a family of 13 genes that encode GTP-binding proteins. Specific combinations of septins can hetero-oligomerize and form filaments in vivo and in vitro, by mechanisms that are not understood. Using fluorescence resonance energy transfer, size exclusion chromatography, and multi-angle light scattering techniques, we have characterized the conformation of a complex of filamentous human septins, Sept2, Sept6, and Sept7. We now show that Sept6 and Sept7 interact through a parallel coiled-coil, and that Sept2 interacts with Sept6 through their C-terminal domains. We have also been able to produce soluble, stable individual septins that behave as rod-like monomers and dimers. Taken together, these observations suggest that polymerized filaments could be comprised of laterally arranged septin core subunits.

Animals↗

PubMed2006

Closing the GAP between polarity and vesicle transport.

How are tight junctions maintained? In this issue of Cell, Wells et al. (2006) provide intriguing evidence for a new pathway that links polarity proteins and vesicle transport to the maintenance of tight junctions, through the control of Cdc42 by Rich1, a GTPase-activating protein.

Animals↗

PubMed2006

Par-3 mediates the inhibition of LIM kinase 2 to regulate cofilin phosphorylation and tight junction assembly.

The polarity protein Par-3 plays critical roles in axon specification and the establishment of epithelial apico-basal polarity. Par-3 associates with Par-6 and atypical protein kinase C and is required for the proper assembly of tight junctions, but the molecular basis for its functions is poorly understood. We now report that depletion of Par-3 elevates the phosphorylated pool of cofilin, a key regulator of actin dynamics. Expression of a nonphosphorylatable mutant of cofilin partially rescues tight junction assembly in cells lacking Par-3, as does the depletion of LIM kinase 2 (LIMK2), an upstream kinase for cofilin. Par-3 binds to LIMK2 but not to the related kinase LIMK1. Par-3 inhibits LIMK2 activity in vitro, and overexpressed Par-3 suppresses cofilin phosphorylation that is induced by lysophosphatidic acid. Our findings identify LIMK2 as a novel target of Par-3 and uncover a molecular mechanism by which Par-3 could regulate actin dynamics during cell polarization.

Actin Depolymerizing Factors↗

PubMed2006

The polarity protein PAR-3 and TIAM1 cooperate in dendritic spine morphogenesis.

PAR-3 (partitioning-defective gene 3) is essential for cell polarization in many contexts, including axon specification. However, polarity proteins have not been implicated in later steps of neuronal differentiation, such as dendritic spine morphogenesis. Here, we show that PAR-3 is necessary for normal spine development in primary hippocampal neurons. Depletion of PAR-3 causes the formation of multiple filopodia- and lamellipodia-like dendritic protrusions - a phenotype similar to neurons expressing activated Rac. PAR-3 regulates spine formation by binding the Rac guanine nucleotide-exchange factor (GEF) TIAM1, and spatially restricting it to dendritic spines. Thus, a balance of PAR-3 and TIAM1 is essential to modulate Rac-GTP levels and to allow spine morphogenesis.

Animals↗

PubMed2006

Zonula occludens-1 function in the assembly of tight junctions in Madin-Darby canine kidney epithelial cells.

Zonula occludens (ZO)-1 was the first tight junction protein to be cloned and has been implicated as an important scaffold protein. It contains multiple domains that bind a diverse set of junction proteins. However, the molecular functions of ZO-1 and related proteins such as ZO-2 and ZO-3 have remained unclear. We now show that gene silencing of ZO-1 causes a delay of approximately 3 h in tight junction formation in Madin-Darby canine kidney (MDCK) epithelial cells, but mature junctions seem functionally normal even in the continuing absence of ZO-1. Depletion of ZO-2, cingulin, or occludin, proteins that can interact with ZO-1, had no discernible effects on tight junctions. Rescue of junction assembly using murine ZO-1 mutants demonstrated that the ZO-1 C terminus is neither necessary nor sufficient for normal assembly. Moreover, mutation of the PDZ1 domain did not block rescue. However, point mutations in the Src homology 3 (SH3) domain almost completely prevented rescue. Surprisingly, the isolated SH3 domain of ZO-1 could also rescue junction assembly. These data reveal an unexpected function for the SH3 domain of ZO-1 in regulating tight junction assembly in epithelial cells and show that cingulin, occludin, or ZO-2 are not limiting for junction assembly in MDCK monolayers.

Animals↗

PubMed2006

RNA interference techniques to study epithelial cell adhesion and polarity.

Polarized epithelial cells are characterized by distinct plasma membrane domains and asymmetrical distribution of cell surface proteins and lipids. In vertebrates, tight junctions act as a fence between the apical and basolateral domains. Although many of the key components of the polarity machinery have been identified, their functions in cell polarization and junction formation remain to be determined. With the rapid improvement of the RNA interference (RNAi) technique, it is now possible to silence the expression of these polarity proteins in mammalian cells and to systematically analyze their distinct roles in orchestrating the polarization program. Here we describe approaches to achieve specific gene suppression in MDCK cells, a well-established cell culture model of canine kidney cells. We discuss the potential challenges and problems associated with the RNAi technique and describe basic protocols for suppressing gene expression using a vector-based short hairpin RNA (shRNA) expression system coupled with nucleofection.

Adaptor Proteins, Signal Transducing↗

PubMed2005

The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin.

Scribble (Scrib) is a conserved polarity protein required in Drosophila melanogaster for synaptic function, neuroblast differentiation, and epithelial polarization. It is also a tumor suppressor. In rodents, Scrib has been implicated in receptor recycling and planar polarity but not in apical/basal polarity. We now show that knockdown of Scrib disrupts adhesion between Madin-Darby canine kidney epithelial cells. As a consequence, the cells acquire a mesenchymal appearance, migrate more rapidly, and lose directionality. Although tight junction assembly is delayed, confluent monolayers remain polarized. These effects are independent of Rac activation or Scrib binding to betaPIX. Rather, Scrib depletion disrupts E-cadherin-mediated cell-cell adhesion. The changes in morphology and migration are phenocopied by E-cadherin knockdown. Adhesion is partially rescued by expression of an E-cadherin-alpha-catenin fusion protein but not by E-cadherin-green fluorescent protein. These results suggest that Scrib stabilizes the coupling between E-cadherin and the catenins and are consistent with the idea that mammalian Scrib could behave as a tumor suppressor by regulating epithelial cell adhesion and migration.

Animals↗

PubMed2005

mPins modulates PSD-95 and SAP102 trafficking and influences NMDA receptor surface expression.

Appropriate trafficking and targeting of glutamate receptors (GluRs) to the postsynaptic density is crucial for synaptic function. We show that mPins (mammalian homologue of Drosophila melanogaster partner of inscuteable) interacts with SAP102 and PSD-95 (two PDZ proteins present in neurons), and functions in the formation of the NMDAR-MAGUK (N-methyl-D-aspartate receptor-membrane-associated guanylate kinase) complex. mPins enhances trafficking of SAP102 and NMDARs to the plasma membrane in neurons. Expression of dominant-negative constructs and short-interfering RNA (siRNA)-mediated knockdown of mPins decreases SAP102 in dendrites and modifies surface expression of NMDARs. mPins changes the number and morphology of dendritic spines and these effects depend on its Galphai interaction domain, thus implicating G-protein signalling in the regulation of postsynaptic structure and trafficking of GluRs.

Animals↗

PubMed2005

Mammalian septins regulate microtubule stability through interaction with the microtubule-binding protein MAP4.

Mammalian septins constitute a family of at least 12 GTP-binding proteins that can form hetero-oligomers and that are sometimes found in association with actin or microtubule filaments. However, their functions are not understood. Using RNA interference, we found that suppression of septin expression in HeLa cells caused a pronounced increase in microtubule stability. Mass spectroscopic analysis of proteins coprecipitating with Sept6 identified the microtubule-associated protein MAP4 as a septin binding partner. A small, proline-rich region in the C-terminal half of MAP4 bound directly to a Sept 2:6:7 heterotrimer, and to the Sept2 monomer. The trimer blocked the ability of this MAP4 fragment to bind and bundle microtubules in vitro. In intact cells, MAP4 was required for the stabilization of microtubules induced by septin depletion. Moreover, septin depletion increased the number of cells with abnormal nuclei, and this effect was blocked by gene silencing of MAP4. These data identify a novel molecular function for septins in mammalian cells: the modulation of microtubule dynamics through interaction with MAP4.

Amino Acid Sequence↗

PubMed2005

A systems analysis of importin-{alpha}-{beta} mediated nuclear protein import.

Importin-beta (Impbeta) is a major transport receptor for Ran-dependent import of nuclear cargo. Impbeta can bind cargo directly or through an adaptor such as Importin-alpha (Impalpha). Factors involved in nuclear transport have been well studied, but systems analysis can offer further insight into regulatory mechanisms. We used computer simulation and real-time assays in intact cells to examine Impalpha-beta-mediated import. The model reflects experimentally determined rates for cargo import and correctly predicts that import is limited principally by Impalpha and Ran, but is also sensitive to NTF2. The model predicts that CAS is not limiting for the initial rate of cargo import and, surprisingly, that increased concentrations of Impbeta and the exchange factor, RCC1, actually inhibit rather than stimulate import. These unexpected predictions were all validated experimentally. The model revealed that inhibition by RCC1 is caused by sequestration of nuclear Ran. Inhibition by Impbeta results from depletion nuclear RanGTP, and, in support of this mechanism, expression of mRFP-Ran reversed the inhibition.

Active Transport, Cell Nucleus↗

PubMed2005

Par-3 controls tight junction assembly through the Rac exchange factor Tiam1.

The par (partitioning-defective) genes express a set of conserved proteins that function in polarization and asymmetric cell division. Par-3 has multiple protein-interaction domains, and associates with Par-6 and atypical protein kinase C (aPKC). In Drosophila, Par-3 is essential for epithelial cell polarization. However, its function in mammals is unclear. Here we show that depletion of Par-3 in mammalian epithelial cells profoundly disrupts tight junction assembly. Expression of a carboxy-terminal fragment plus the third PDZ domain of Par-3 partially rescues junction assembly, but neither Par-6 nor aPKC binding is required. Unexpectedly, Rac is constitutively activated in cells lacking Par-3, and the assembly of tight junctions is efficiently restored by a dominant-negative Rac mutant. The Rac exchange factor Tiam1 (ref. 7) binds directly to the carboxy-terminal region of Par-3, and knockdown of Tiam1 enhances tight junction formation in cells lacking Par-3. These results define a critical function for Par-3 in tight junction assembly, and reveal a novel mechanism through which Par-3 engages in the spatial regulation of Rac activity and establishment of epithelial polarity.

Actins↗

PubMed2005

Novel biosensors for the detection of estrogen receptor ligands.

There exists a significant need for the detection of novel estrogen receptor (ER) ligands for pharmaceutical uses, especially for treating complications associated with menopause. We have developed fluorescence resonance energy transfer (FRET)-based biosensors that permit the direct in vitro detection of ER ligands. These biosensors contain an ER ligand-binding domain (LBD) flanked by the FRET donor fluorophore, cyan fluorescent protein (CFP), and the acceptor fluorophore, yellow fluorescent protein (YFP). The ER-LBD has been modified so that Ala 430 has been changed to Asp, which increases the magnitude of the FRET signal in response to ligand-binding by more than four-fold compared to the wild-type LBD. The binding of agonists can be distinguished from that of antagonists on the basis of the distinct ligand-induced conformations in the ER-LBD. The approach to binding equilibrium occurs within 30min, and the FRET signal is stable over 24h. The biosensor demonstrates a high signal-to-noise, with a Z' value (a statistical determinant of assay quality) of 0.72. The affinity of the ER for different ligands can be determined using a modified version of the biosensor in which a truncated YFP and an enhanced CFP are used. Thus, we have developed platforms for high-throughput screens for the identification of novel estrogen receptor ligands. Moreover, we have demonstrated that this FRET technology can be applied to other nuclear receptors, such as the androgen receptor.

Androgens↗

PubMed2004

Overexpression of exportin 5 enhances RNA interference mediated by short hairpin RNAs and microRNAs.

Plasmids or viral vectors that express short hairpin RNAs (shRNAs) have emerged as important tools for the stable inhibition of specific genes by RNA interference. shRNAs are structural and functional homologs of pre-microRNAs, intermediates in the production of endogenously encoded microRNAs (miRNAs). Therefore, overexpressed shRNAs could inhibit miRNA function by competing for a limiting level of one or more factors involved in miRNA biogenesis or function. Here, we demonstrate that overexpressed shRNAs can saturate the activity of endogenous Exportin 5, a factor required for nuclear export of both shRNAs and pre-miRNAs. While shRNA overexpression can therefore inhibit miRNA function, simultaneous overexpression of Exportin 5 reverses this effect. Moreover, Exportin 5 overexpression can significantly enhance RNA interference mediated by shRNAs. These data have implications for the future clinical utilization of shRNAs and also provide a simple method to enhance RNA interference by shRNAs in culture.

Active Transport, Cell Nucleus↗

PubMed2004

Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import.

Ubiquitin is a small polypeptide that is conjugated to proteins and commonly serves as a degradation signal. The attachment of ubiquitin (Ub) to a substrate proceeds through a multi-enzyme cascade involving an activating enzyme (E1), a conjugating enzyme (E2), and a protein ligase (E3). We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11. We now show that the import mechanism for UbcM2 and two other human class III E2s (UbcH6 and UBE2E2) uniquely requires the covalent attachment of Ub to the active site cysteine of these enzymes. This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes. Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.

Active Transport, Cell Nucleus↗

PubMed2004

Mammalian Pins is a conformational switch that links NuMA to heterotrimeric G proteins.

During asymmetric cell divisions, mitotic spindles align along the axis of polarization. In invertebrates, spindle positioning requires Pins or related proteins and a G protein alpha subunit. A mammalian Pins, called LGN, binds Galphai and also interacts through an N-terminal domain with the microtubule binding protein NuMA. During mitosis, LGN recruits NuMA to the cell cortex, while cortical association of LGN itself requires the C-terminal Galpha binding domain. Using a FRET biosensor, we find that LGN behaves as a conformational switch: in its closed state, the N and C termini interact, but NuMA or Galphai can disrupt this association, allowing LGN to interact simultaneously with both proteins, resulting in their cortical localization. Overexpression of Galphai or YFP-LGN causes a pronounced oscillation of metaphase spindles, and NuMA binding to LGN is required for these spindle movements. We propose that a related switch mechanism might operate in asymmetric cell divisions in the fly and nematode.

Animals↗

PubMed2004

Isoforms of the polarity protein par6 have distinct functions.

PAR-6 is essential for asymmetric division of the Caenorhabditis elegans zygote. It is also critical for cell polarization in many other contexts throughout the Metazoa. The Par6 protein contains a PDZ domain and a partial CRIB (Cdc42/Rac interactive binding) domain, which mediate interactions with other polarity proteins such as Par3, Cdc42, Pals1, and Lgl. A family of mammalian Par6 isoforms (Par6A-D) has been described, but the significance of this diversification has been unclear. Here we demonstrate that Par6 family members localize differently when expressed in Madin-Darby canine kidney epithelial cells and have distinct effects on tight junction (TJ) assembly. Par6B localizes to the cytosol and inhibits TJ formation, but Par6A co-localizes predominantly with the TJ marker ZO-1 at cell-cell contacts and does not affect junctions. These functional differences correlate with differences in Pals1 binding; Par6B interacts strongly with Pals1, whereas Par6A binds weakly to Pals1 even in the presence of active Cdc42. Pals1 has a low affinity for the isolated CRIB-PDZ domain of Par6A, but analysis of chimeras showed that in addition Pals1 binding is blocked by an inhibitory property of the N terminus of Par6A. Unexpectedly, the localization of Par6A to cell-cell contacts is Cdc42-independent.

Amino Acid Sequence↗

PubMed2004

The brain-specific double-stranded RNA-binding protein Staufen2: nucleolar accumulation and isoform-specific exportin-5-dependent export.

The mammalian double-stranded RNA-binding proteins Staufen (Stau1 and Stau2) are involved in RNA localization in polarized neurons. In contrast to the more ubiquitously expressed Stau1, Stau2 is mainly expressed in the nervous system. In Drosophila, the third double-stranded RNA-binding domain (RBD3) of Staufen is essential for RNA interaction. When conserved amino acids within the RBD3 of Stau2 were mutated to render Stau2 defective for RNA binding, the mutant Stau2 proteins accumulate predominantly in the nucleolus. This is in contrast to wild type Stau2 that mostly localizes in the cytosol. The nuclear import is dependent on a nuclear localization signal in close proximity to the RBD3. The nuclear export of Stau2 is not dependent on CRM1 but rather on Exportin-5. We show that Exportin-5 interacts with the RBD3 of wild type Stau2 in an RNA-dependent manner in vitro but not with mutant Stau2. When Exportin-5 is down-regulated by RNA interference, only the largest isoform of Stau2 (Stau2(62)) preferentially accumulates in the nucleolus. It is tempting to speculate that Stau2(62) binds RNA in the nucleus and assembles into ribonucleoparticles, which are then exported via the Exportin-5 pathway to their final destination.

Animals↗