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1.  β-catenin: A multi-functional protein’s role at the centrosome and implications for a broader role in cell division 
Beta-catenin is a multifunctional protein with critical roles in cell-cell adhesion, Wnt-signaling and the centrosome cycle. Whereas the roles of β-catenin in cell-cell adhesion and Wnt-signaling have been studied extensively, the mechanism(s) involving β-catenin in centrosome functions are poorly understood. β-Catenin localizes to centrosomes and promotes mitotic progression. NIMA-related protein kinase 2 (Nek2), which stimulates centrosome separation, binds to and phosphorylates β-catenin. β-Catenin interacting proteins involved in Wnt signaling such as APC, Axin and GSK3β, are also localized at centrosomes and play roles in promoting mitotic progression. Additionally, proteins associated with cell-cell adhesion sites, such as dynein, regulate mitotic spindle positioning. These roles of proteins at the cell cortex and Wnt signaling that involve β-catenin indicate a cross-talk between different sub-cellular sites in the cell at mitosis, and that different pools of β-catenin may co-ordinate centrosome functions and cell cycle progression.
PMCID: PMC3983869  PMID: 23804296
2.  The Evolutionary Origin of Epithelial Cell-Cell Adhesion Mechanisms 
Current topics in membranes  2013;72:267-311.
A simple epithelium forms a barrier between the outside and the inside of an organism, and is the first organized multicellular tissue found in evolution. We examine the relationship between the evolution of epithelia and specialized cell-cell adhesion proteins comprising the classical cadherin/β-catenin/α-catenin complex (CCC). A review of the divergent functional properties of the CCC in metazoans and non-metazoans, and an updated phylogenetic coverage of the CCC using recent genomic data reveal: 1) The core CCC likely originated before the last common ancestor of unikonts and their closest bikont sister taxa. 2) Formation of the CCC may have constrained sequence evolution of the classical cadherin cytoplasmic domain and β-catenin in metazoa. 3) The α-catenin binding domain in β-catenin appears to be the favored mutation site for disrupting β-catenin function in the CCC. 4) The ancestral function of the α/β-catenin heterodimer appears to be an actin-binding module. In some metazoan groups, more complex functions of α-catenin were gained by sequence divergence in the non-actin binding (N-, M-) domains. 5) Allosteric regulation of α-catenin, rather than loss of function mutations, may have evolved for more complex regulation of the actin cytoskeleton.
PMCID: PMC4118598  PMID: 24210433
Protein Evolution; Metazoan Evolution; Epithelia; Cell-cell Adhesion; Adherens Junction; α-catenin; β-catenin; Classical Cadherin; Actin Cytoskeleton; Vinculin
3.  A genome-wide screen identifies conserved protein hubs required for cadherin-mediated cell–cell adhesion 
The Journal of Cell Biology  2014;204(2):265-279.
A genome-wide screen identifies 17 regulatory hubs that modulate the levels of the core cadherin–catenin complex and coordinate cadherin-mediated cell–cell adhesion.
Cadherins and associated catenins provide an important structural interface between neighboring cells, the actin cytoskeleton, and intracellular signaling pathways in a variety of cell types throughout the Metazoa. However, the full inventory of the proteins and pathways required for cadherin-mediated adhesion has not been established. To this end, we completed a genome-wide (∼14,000 genes) ribonucleic acid interference (RNAi) screen that targeted Ca2+-dependent adhesion in DE-cadherin–expressing Drosophila melanogaster S2 cells in suspension culture. This novel screen eliminated Ca2+-independent cell–cell adhesion, integrin-based adhesion, cell spreading, and cell migration. We identified 17 interconnected regulatory hubs, based on protein functions and protein–protein interactions that regulate the levels of the core cadherin–catenin complex and coordinate cadherin-mediated cell–cell adhesion. Representative proteins from these hubs were analyzed further in Drosophila oogenesis, using targeted germline RNAi, and adhesion was analyzed in Madin–Darby canine kidney mammalian epithelial cell–cell adhesion. These experiments reveal roles for a diversity of cellular pathways that are required for cadherin function in Metazoa, including cytoskeleton organization, cell–substrate interactions, and nuclear and cytoplasmic signaling.
PMCID: PMC3897182  PMID: 24446484
4.  Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors 
eLife  2013;2:e00654.
The dynamic organization of signaling cascades inside primary cilia is key to signal propagation. Yet little is known about the dynamics of ciliary membrane proteins besides a possible role for motor-driven Intraflagellar Transport (IFT). To characterize these dynamics, we imaged single molecules of Somatostatin Receptor 3 (SSTR3, a GPCR) and Smoothened (Smo, a Hedgehog signal transducer) in the ciliary membrane. While IFT trains moved processively from one end of the cilium to the other, single SSTR3 and Smo underwent mostly diffusive behavior interspersed with short periods of directional movements. Statistical subtraction of instant velocities revealed that SSTR3 and Smo spent less than a third of their time undergoing active transport. Finally, SSTR3 and IFT movements could be uncoupled by perturbing either membrane protein diffusion or active transport. Thus ciliary membrane proteins move predominantly by diffusion, and attachment to IFT trains is transient and stochastic rather than processive or spatially determined.
eLife digest
Primary cilia are tiny protrusions from the cell surface, which have a central role in processing sensory stimuli, such as light or odorants. Cilia are also involved in mediating the response to developmental signaling molecules, including Sonic Hedgehog, and may help to convert mechanical signals into electrical or chemical ones. Primary cilia are made up of an axoneme—a core structure that consists of microtubules extending along the length of the cilium—ensheathed by a membrane that contains a number of receptor proteins.
These receptor proteins travel up and down the cilium, and it is generally assumed that an active process known as intraflagellar transport is responsible for their movement. This process is mediated by motor proteins called kinesins and dyneins, which carry cargo proteins along axonemal microtubules. However, it has been difficult to study the transport of individual receptor proteins directly because they are uniformly distributed over the membranes of the cilia.
Now, Ye et al. have shown that intraflagellar transport is not the most important mode of transport for membrane proteins within primary cilia. By labelling individual receptors with a fluorescent dye and then filming their movements under a microscope, Ye et al. found that the receptors generally did not show the directed, linear motion that would be expected from intraflagellar transport. Instead, much of their movement occurred through passive diffusion, with occasional short bursts of directed motion.
To investigate how rapidly receptor molecules could move through the cilium in this way, Ye et al. used a technique called fluorescence recovery after photobleaching (FRAP). This involves using light to bleach the fluorescent dye attached to receptor molecules in part of the cilium, and then measuring how long it takes for the fluorescence to return as a result of other labelled molecules moving into the bleached area: the shorter this time, the faster the movement of the molecules. It took less than a minute for fluorescence to be restored within a primary cilium, indicating that passive diffusion with occasional active transport can move proteins rapidly through the structure.
By using drugs to inhibit intraflagellar transport, Ye et al. confirmed that the majority of membrane protein transport within primary cilia occurs via diffusion. Further studies are now required to determine whether this is also the case for other molecules that travel along cilia, and whether intraflagellar transport may have a more important role in the assembly of these structures.
PMCID: PMC3736543  PMID: 23930224
cilia; IFT; diffusion; signaling; motors; Human; Mouse
5.  αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors 
Molecular Biology of the Cell  2013;24(23):3710-3720.
αE-catenin regulates transitions in actin organization between cell migration and cell–cell adhesion by controlling barbed-end polymerization of unbranched actin filaments and inhibiting Arp2/3 complex and cofilin regulation of actin filament branching and disassembly.
The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.
PMCID: PMC3842997  PMID: 24068324
6.  Mechano-Transduction: From Molecules to Tissues 
PLoS Biology  2014;12(11):e1001996.
Biological mechano-transduction and force-dependent changes scale from protein conformation (â„« to nm) to cell organization and multi-cell function (mm to cm) to affect cell organization, fate, and homeostasis.
External forces play complex roles in cell organization, fate, and homeostasis. Changes in these forces, or how cells respond to them, can result in abnormal embryonic development and diseases in adults. How cells sense and respond to these mechanical stimuli requires an understanding of the biophysical principles that underlie changes in protein conformation and result in alterations in the organization and function of cells and tissues. Here, we discuss mechano-transduction as it applies to protein conformation, cellular organization, and multi-cell (tissue) function.
PMCID: PMC4236045  PMID: 25405923
7.  α-Catenin and IQGAP Regulate Myosin Localization to Control Epithelial Tube Morphogenesis in Dictyostelium 
Developmental cell  2012;23(3):533-546.
Apical actomyosin activity in animal epithelial cells influences tissue morphology, and drives morphogenetic movements during development. The molecular mechanisms leading to myosin II accumulation at the apical membrane and its exclusion from other membranes are poorly understood. We show that in the non-metazoan Dictyostelium discoideum, myosin II localizes apically in tip epithelial cells that surround the stalk, and constriction of this epithelial tube is required for proper morphogenesis. IQGAP1 and its binding partner cortexillin I function downstream of α- and β-catenin to exclude myosin II from the basolateral cortex and promote apical accumulation of myosin II. Deletion of IQGAP1 or cortexillin compromises epithelial morphogenesis without affecting cell polarity. These results reveal that apical localization of myosin II is a conserved morphogenetic mechanism from non-metazoans to vertebrates, and identify a hierarchy of proteins that regulate the polarity and organization of an epithelial tube in a simple model organism.
PMCID: PMC3443284  PMID: 22902739
8.  Characterizing the initial encounter complex in cadherin adhesion 
Structure (London, England : 1993)  2009;17(8):1075-1081.
Cadherins are Ca2+-dependent cell-cell adhesion proteins with an extracellular region of five domains (EC1 to EC5). Adhesion is mediated by “strand-swapping” of a conserved tryptophan residue in position 2 between EC1 domains of opposing cadherins, but the formation of this structure is not well understood. Using single molecule Fluorescence Resonance Energy Transfer (FRET) and single molecule force measurements with the Atomic Force Microscope (AFM), we demonstrate that cadherins initially interact via EC1 domains without swapping tryptophan-2 to form a weak Ca2+ dependent initial encounter complex that has 25% of the bond strength of a strand-swapped dimer. We suggest that cadherin dimerization proceeds via an induced fit mechanism where the monomers first form a tryptophan-2 independent initial encounter complex and then undergo subsequent conformational changes to form the final strand-swapped dimer.
PMCID: PMC4113602  PMID: 19646884
9.  Nek2 phosphorylates and stabilizes β-catenin at mitotic centrosomes downstream of Plk1 
Molecular Biology of the Cell  2014;25(7):977-991.
Plk1 regulates Nek2 activity in stabilizing β-catenin at mitotic centrosomes and in promoting centrosome separation. Nek2 phosphorylates the same regulatory sites (S33/S37/T41) as GSK3β in β-catenin, as well as additional sites, and inhibits binding of the E3 ligase β-TrCP to β-catenin, thereby preventing β-catenin ubiquitination and degradation.
β-Catenin is a multifunctional protein with critical roles in cell–cell adhesion, Wnt signaling, and the centrosome cycle. Whereas the regulation of β-catenin in cell–cell adhesion and Wnt signaling are well understood, how β-catenin is regulated at the centrosome is not. NIMA-related protein kinase 2 (Nek2), which regulates centrosome disjunction/splitting, binds to and phosphorylates β-catenin. Using in vitro and cell-based assays, we show that Nek2 phosphorylates the same regulatory sites in the N-terminus of β-catenin as glycogen synthase kinase 3β (GSK3β), which are recognized by a specific phospho-S33/S37/T41 antibody, as well as additional sites. Nek2 binding to β-catenin appears to inhibit binding of the E3 ligase β-TrCP and prevents β-catenin ubiquitination and degradation. Thus β-catenin phosphorylated by Nek2 is stabilized and accumulates at centrosomes in mitosis. We further show that polo-like kinase 1 (Plk1) regulates Nek2 phosphorylation and stabilization of β-catenin. Taken together, these results identify a novel mechanism for regulating β-catenin stability that is independent of GSK3β and provide new insight into a pathway involving Plk1, Nek2, and β-catenin that regulates the centrosome cycle.
PMCID: PMC3967981  PMID: 24501426
10.  The ciliary diffusion barrier: the gatekeeper for the primary cilium compartment 
Cytoskeleton (Hoboken, N.J.)  2011;68(6):313-324.
The primary cilium is a cellular antenna that detects and transmits chemical and mechanical cues in the environment through receptors and downstream signal proteins enriched along the ciliary membrane. While it is known that ciliary membrane proteins enter the cilium by way of vesicular and intraflagellar transport, less is known about how ciliary membrane proteins are retained in, and how apical membrane proteins are excluded from the cilium. Here, we review evidence for a membrane diffusion barrier at the base of the primary cilium, and highlight the recent finding of a septin cytoskeleton diffusion barrier. We also discuss candidate ciliopathy genes that may be involved in formation of the barrier, and the role of a diffusion barrier as a common mechanism for compartmentalizing membranes and lipid domains.
PMCID: PMC3143192  PMID: 21634025
primary cilium; ciliary membrane proteins; ciliopathy; diffusion barrier; basal body; ciliary pocket; ciliary necklace; transition zone; septins; GTPases
11.  Tube morphogenesis: closure, but many openings remain 
Trends in Cell Biology  2003;13(12):615-621.
Epithelial and endothelial tubes form the basic structure of many organs and tissues in the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, zebrafish and mammals. Comparison of how tubes form during development defines several pathways that generate a single unbranched tube or dichotomously branching tubular networks. The formation of tubes can be induced directly by intrinsic signals within epithelial primordia or by inductive signaling between adjacent epithelia and the mesenchyme. Both processes are hierarchically controlled by master transcriptional regulators, growth factors and their receptors, directed cell migration and cellular reorganization, which is controlled by changes in the cytoskeleton and protein trafficking. This review provides a summary of these pathways based upon articles published in the Tube Morphogenesis Series in Trends in Cell Biology.
PMCID: PMC3368612  PMID: 14624839
12.  Protein Evolution in Cell and Tissue Development: Going Beyond Sequence and Transcriptional Analysis 
Developmental cell  2011;21(1):32-34.
Studies of animal evolution often focus on sequence and transcriptional analysis, based on an assumption that the evolution of development is driven by changes in gene expression. We argue that biochemical and cell biological approaches are also required, because sequence-conserved proteins can have different biochemical, cellular and developmental properties.
PMCID: PMC3145331  PMID: 21763606
13.  Microactuator device for integrated measurement of epithelium mechanics 
Biomedical microdevices  2013;15(1):117-123.
Mechanical forces are among important factors that drive cellular function and organization. We present a microfabricated device with on-chip actuation for mechanical testing of single cells. An integrated immersible electrostatic actuator system is demonstrated that applies calibrated forces to cells. We conduct stretching experiments by directly applying forces to epithelial cells adhered to device surfaces functionalized with collagen. We measure mechanical properties including stiffness, hysteresis and visco-elasticity of adherent cells.
PMCID: PMC3535526  PMID: 22927158
14.  A Polarized Epithelium Organized by β- and α-Catenin Predates Cadherin and Metazoan Origins 
Science (New York, N.Y.)  2011;331(6022):1336-1339.
A polarized epithelium in the non-metazoan Dictyostelium discoideum requires α-catenin and β-catenin but not classical cadherins, polarity proteins or Wnt signaling.
A fundamental characteristic of metazoans is the formation of a simple, polarized epithelium. In higher animals, the structural integrity and functional polarization of simple epithelia require a cell-cell adhesion complex containing a classical cadherin, the Wnt-signaling protein β-catenin and the actin-binding protein α-catenin. We show that the non-metazoan Dictyostelium discoideum forms a polarized epithelium that is essential for multicellular development. Although D. discoideum lacks a cadherin homolog, we identify an α-catenin ortholog that binds a β-catenin-related protein. Both proteins are essential for formation of the epithelium, polarized protein secretion and proper multicellular morphogenesis. Thus the organizational principles of metazoan multicellularity may be more ancient than previously recognized, and the role of the catenins in cell polarity predates the evolution of Wnt signaling and classical cadherins.
PMCID: PMC3152298  PMID: 21393547
15.  αE-catenin regulates actin dynamics independently of cadherin-mediated cell–cell adhesion 
The Journal of Cell Biology  2010;189(2):339-352.
αE-catenin has cell–cell contact–dependent and –independent functions in regulating actin and membrane dynamics.
αE-catenin binds the cell–cell adhesion complex of E-cadherin and β-catenin (β-cat) and regulates filamentous actin (F-actin) dynamics. In vitro, binding of αE-catenin to the E-cadherin–β-cat complex lowers αE-catenin affinity for F-actin, and αE-catenin alone can bind F-actin and inhibit Arp2/3 complex–mediated actin polymerization. In cells, to test whether αE-catenin regulates actin dynamics independently of the cadherin complex, the cytosolic αE-catenin pool was sequestered to mitochondria without affecting overall levels of αE-catenin or the cadherin–catenin complex. Sequestering cytosolic αE-catenin to mitochondria alters lamellipodia architecture and increases membrane dynamics and cell migration without affecting cell–cell adhesion. In contrast, sequestration of cytosolic αE-catenin to the plasma membrane reduces membrane dynamics. These results demonstrate that the cytosolic pool of αE-catenin regulates actin dynamics independently of cell–cell adhesion.
PMCID: PMC2856910  PMID: 20404114
16.  Adenomatous Polyposis Coli Regulates Endothelial Cell Migration Independent of Roles in β-Catenin Signaling and Cell–Cell Adhesion 
Molecular Biology of the Cell  2010;21(15):2611-2623.
Adenomatous polyposis coli is a cytoskeletal organizer and a scaffold for mediating degradation of the Wnt effector β-catenin. We uncouple these different APC functions and show that GSK3β/CKI phosphorylation regulates APC clusters and cell migration independently of cell–cell adhesion and β-catenin transcriptional activity.
Adenomatous polyposis coli (APC), a tumor suppressor commonly mutated in cancer, is a cytoskeletal organizer for cell migration and a scaffold for GSK3β/CKI-mediated phosphorylation and degradation of the Wnt effector β-catenin. It remains unclear whether these different APC functions are coupled, or independently regulated and localized. In primary endothelial cells, we show that GSK3β/CKI-phosphorylated APC localizes to microtubule-dependent clusters at the tips of membrane extensions. Loss of GSK3β/CKI-phosphorylated APC from these clusters correlates with a decrease in cell migration. GSK3β/CKI-phosphorylated APC and β-catenin at clusters is degraded rapidly by the proteasome, but inhibition of GSK3β/CKI does not increase β-catenin–mediated transcription. GSK3β/CKI-phosphorylated and -nonphosphorylated APC also localize along adherens junctions, which requires actin and cell–cell adhesion. Significantly, inhibition of cell–cell adhesion results in loss of lateral membrane APC and a concomitant increase in GSK3β/CKI-phosphorylated APC in clusters. These results uncouple different APC functions and show that GSK3β/CKI phosphorylation regulates APC clusters and cell migration independently of cell–cell adhesion and β-catenin transcriptional activity.
PMCID: PMC2912348  PMID: 20519433
17.  An Epithelial Tissue in Dictyostelium and the Origin of Metazoan Multicellularity 
Metazoans (multicellular animals) are defined by the presence of polarized epithelial tissues, and epithelial morphogenesis contributes to the diversity of animal body plans. The recent finding of a polarized epithelium in the non-metazoan social amoeba Dictyostelium discoideum demonstrates that an epithelial tissue is not a unique feature of metazoans and calls into question the traditional view that multicellularity evolved independently in social amoebae and metazoans. We propose the alternative hypothesis that animals, fungi and social amoebae evolved from an ancestor that spent a portion of its life cycle in a multicellular state and possessed molecular machinery necessary for forming an epithelial tissue. This hypothesis makes testable predictions regarding tissue organization in close relatives of metazoans and provides a novel conceptual framework for studies of early animal evolution.
PMCID: PMC3517009  PMID: 22930590
18.  Aquaporin-3 and Aquaporin-4 Are Sorted Differently and Separately in the Trans-Golgi Network 
PLoS ONE  2013;8(9):e73977.
Aquaporin-3 (AQP3) and aquaporin-4 (AQP4) are homologous proteins expressed in the basolateral plasma membrane of kidney collecting duct principal cells, where they mediate the exit pathway for apically reabsorbed water. Although both proteins are localized to the same plasma membrane domain, it is unknown if they are sorted together in the Golgi, or arrive in the same or different vesicles at the plasma membrane. We addressed these questions using high resolution deconvolution imaging, spinning disk and laser scanning confocal microscopy of cells expressing AQP3 and AQP4. AQP3 and AQP4 were observed mostly in separate post-Golgi carriers, and spinning disk microscopy showed that most of AQP3 and AQP4 were delivered to the plasma membrane in separate vesicles. In contrast, VSV-G and LDL-R, two well-charcterized basolateral proteins, co-localized to a high degree in the same post-Golgi carriers, indicating that the differential sorting of AQP3 and AQP4 is specific and regulated. Significantly, a chimeric AQP3 containing the AQP4 cytoplasmic tails co-localized with AQP4 in post-Golgi vesicles. These results indicate that AQP3 and AQP4 are separated into different post-Golgi carriers based on different cytoplasmic domain sorting signals, and are then delivered separately to the plasma membrane.
PMCID: PMC3776795  PMID: 24058510
19.  Bench to Bedside and Back Again: Molecular Mechanisms of α-Catenin Function and Roles in Tumorigenesis 
Seminars in cancer biology  2007;18(1):53-64.
The cadherin/catenin complex, comprised of E-cadherin, β-catenin and α-catenin, is essential for initiating cell-cell adhesion, establishing cellular polarity and maintaining tissue organization. Disruption or loss of the cadherin/catenin complex is common in cancer. As the primary cell-cell adhesion protein in epithelial cells, E-cadherin has long been studied in cancer progression. Similarly, additional roles for β-catenin in the Wnt signaling pathway has led to many studies of the role of β-catenin in cancer. Alpha-catenin, in contrast, has received less attention. However, recent data demonstrate novel functions for α-catenin in regulating the actin cytoskeleton and cell-cell adhesion, which when perturbed could contribute to cancer progression. In this review, we use cancer data to evaluate molecular models of α-catenin function, from the canonical role of α-catenin in cell-cell adhesion to non-canonical roles identified following conditional α-catenin deletion. This analysis identifies α-catenin as a prognostic factor in cancer progression.
PMCID: PMC2692220  PMID: 17945508
α-catenin; cancer; E-cadherin; cytoskeleton; adhesion
20.  Fabrication of a Dual Substrate Display to Test Roles of Cell Adhesion Proteins in Vesicle Targeting to Plasma Membrane Domains 
FEBS letters  2007;581(23):4539-4543.
While much is known of the molecular machinery involved in protein sorting during exocytosis, less is known about the spatial regulation of exocytosis at the plasma membrane (PM). This study outlines a novel method, Dual Substrate Display, used to formally test the hypothesis that E-cadherin-mediated adhesion directs basolateral vesicle exocytosis to specific sites at the PM. We show that vesicles containing the basolateral marker protein VSV-G preferentially target to sites of adhesion to E-cadherin rather than collagen VI or a control peptide. These results support the hypothesis that E-cadherin adhesion initiates signaling at the PM resulting in targeted sites for exocytosis.
PMCID: PMC2682434  PMID: 17803993
E-cadherin; exocytosis; protein micropatterning; dual substrate display; cell assay
21.  Epithelial polarity requires septin coupling of vesicle transport to polyglutamylated microtubules 
The Journal of Cell Biology  2008;180(2):295-303.
In epithelial cells, polarized growth and maintenance of apical and basolateral plasma membrane domains depend on protein sorting from the trans-Golgi network (TGN) and vesicle delivery to the plasma membrane. Septins are filamentous GTPases required for polarized membrane growth in budding yeast, but whether they function in epithelial polarity is unknown. Here, we show that in epithelial cells septin 2 (SEPT2) fibers colocalize with a subset of microtubule tracks composed of polyglutamylated (polyGlu) tubulin, and that vesicles containing apical or basolateral proteins exit the TGN along these SEPT2/polyGlu microtubule tracks. Tubulin-associated SEPT2 facilitates vesicle transport by maintaining polyGlu microtubule tracks and impeding tubulin binding of microtubule-associated protein 4 (MAP4). Significantly, this regulatory step is required for polarized, columnar-shaped epithelia biogenesis; upon SEPT2 depletion, cells become short and fibroblast-shaped due to intracellular accumulation of apical and basolateral membrane proteins, and loss of vertically oriented polyGlu microtubules. We suggest that septin coupling of the microtubule cytoskeleton to post-Golgi vesicle transport is required for the morphogenesis of polarized epithelia.
PMCID: PMC2213583  PMID: 18209106
22.  Localized zones of Rho and Rac activities drive initiation and expansion of epithelial cell–cell adhesion 
The Journal of Cell Biology  2007;178(3):517-527.
Spatiotemporal coordination of cell–cell adhesion involving lamellipodial interactions, cadherin engagement, and the lateral expansion of the contact is poorly understood. Using high-resolution live-cell imaging, biosensors, and small molecule inhibitors, we investigate how Rac1 and RhoA regulate actin dynamics during de novo contact formation between pairs of epithelial cells. Active Rac1, the Arp2/3 complex, and lamellipodia are initially localized to de novo contacts but rapidly diminish as E-cadherin accumulates; further rounds of activation and down-regulation of Rac1 and Arp2/3 occur at the contacting membrane periphery, and this cycle repeats as a restricted membrane zone that moves outward with the expanding contact. The cortical bundle of actin filaments dissolves beneath the expanding contacts, leaving actin bundles at the contact edges. RhoA and actomyosin contractility are activated at the contact edges and are required to drive expansion and completion of cell–cell adhesion. We show that zones of Rac1 and lamellipodia activity and of RhoA and actomyosin contractility are restricted to the periphery of contacting membranes and together drive initiation, expansion, and completion of cell–cell adhesion.
PMCID: PMC2064836  PMID: 17646397
23.  A molecular mechanism directly linking E-cadherin adhesion to initiation of epithelial cell surface polarity 
The Journal of Cell Biology  2007;178(2):323-335.
Mechanisms involved in maintaining plasma membrane domains in fully polarized epithelial cells are known, but when and how directed protein sorting and trafficking occur to initiate cell surface polarity are not. We tested whether establishment of the basolateral membrane domain and E-cadherin–mediated epithelial cell–cell adhesion are mechanistically linked. We show that the basolateral membrane aquaporin (AQP)-3, but not the equivalent apical membrane AQP5, is delivered in post-Golgi structures directly to forming cell–cell contacts where it co-accumulates precisely with E-cadherin. Functional disruption of individual components of a putative lateral targeting patch (e.g., microtubules, the exocyst, and soluble N-ethylmaleimide–sensitive factor attachment protein receptors) did not inhibit cell–cell adhesion or colocalization of the other components with E-cadherin, but each blocked AQP3 delivery to forming cell–cell contacts. Thus, components of the lateral targeting patch localize independently of each other to cell–cell contacts but collectively function as a holocomplex to specify basolateral vesicle delivery to nascent cell–cell contacts and immediately initiate cell surface polarity.
PMCID: PMC2064450  PMID: 17635938
24.  Transcriptional Modulation of Genes Encoding Structural Characteristics of Differentiating Enterocytes During Development of a Polarized Epithelium In Vitro 
Molecular Biology of the Cell  2007;18(11):4261-4278.
Although there is considerable evidence implicating posttranslational mechanisms in the development of epithelial cell polarity, little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized the temporal program of gene expression during cell–cell adhesion–initiated polarization of human Caco-2 cells in tissue culture, which develop structural and functional polarity similar to that of enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell–cell contacts between neighboring cells. Expression of genes involved in cell proliferation was down-regulated concomitant with induction of genes necessary for functional specialization of polarized epithelial cells. Transcriptional up-regulation of these latter genes correlated with formation of important structural and functional features in enterocyte differentiation and establishment of structural and functional cell polarity; components of the apical microvilli were induced as the brush border formed during polarization; as barrier function was established, expression of tight junction transmembrane proteins peaked; transcripts encoding components of the apical, but not the basal-lateral trafficking machinery were increased during polarization. Coordinated expression of genes encoding components of functional cell structures were often observed indicating temporal control of expression and assembly of multiprotein complexes.
PMCID: PMC2043570  PMID: 17699590
25.  Parallels between Global Transcriptional Programs of Polarizing Caco-2 Intestinal Epithelial Cells In Vitro and Gene Expression Programs in Normal Colon and Colon Cancer 
Molecular Biology of the Cell  2007;18(11):4245-4260.
Posttranslational mechanisms are implicated in the development of epithelial cell polarity, but little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized temporal patterns of gene expression during cell–cell adhesion-initiated polarization of cultured human Caco-2 cells, which develop structural and functional polarity resembling enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell–cell contacts. Comparison to gene expression patterns in normal human colon and colon tumors revealed that the pattern in proliferating, nonpolarized Caco-2 cells paralleled patterns seen in human colon cancer in vivo, including expression of genes involved in cell proliferation. The pattern switched in polarized Caco-2 cells to one more closely resembling that in normal colon tissue, indicating that regulation of transcription underlying Caco-2 cell polarization is similar to that during enterocyte differentiation in vivo. Surprisingly, the temporal program of gene expression in polarizing Caco-2 cells involved changes in signaling pathways (e.g., Wnt, Hh, BMP, FGF) in patterns similar to those during migration and differentiation of intestinal epithelial cells in vivo, despite the absence of morphogen gradients and interactions with stromal cells characteristic of enterocyte differentiation in situ. The full data set is available at
PMCID: PMC2043540  PMID: 17699589

Results 1-25 (90)