Cell Polarity

The intracellular distribution of organelles is a crucial aspect of effective cell function. Chloroplasts change their intracellular positions to optimize photosynthetic activity in response to ambient light conditions. Through screening of mutants of Arabidopsis defective in chloroplast photorelocation movement, we isolated six mutant clones in which chloroplasts gathered at the bottom of the cells and did not distribute throughout cells. These mutants, termed chloroplast unusual positioning ( chup ), were shown to belong to a single genetic locus by complementation tests. Observation of the positioning of other organelles, such as mitochondria, peroxisomes, and nuclei, revealed that chloroplast positioning and movement are impaired specifically in this mutant, although peroxisomes are distributed along with chloroplasts. The CHUP1 gene encodes a novel protein containing multiple domains, including a coiled-coil domain, an actin binding domain, a Pro-rich region, and two Leu zipper domains. The N-terminal hydrophobic segment of CHUP1 was expressed transiently in leaf cells of Arabidopsis as a fusion protein with the green fluorescent protein. The fusion protein was targeted to envelope membranes of chloroplasts in mesophyll cells, suggesting that CHUP1 may localize in chloroplasts. A glutathione S -transferase fusion protein containing the actin binding domain of CHUP1 was found to bind F-actin in vitro. CHUP1 is a unique gene identified that encodes a protein required for organellar positioning and movement in plant cells.

Summary:  Cell polarization is a key feature of T‐cell function. The immunological synapse (IS) between T cells and antigen‐presenting cells is a beautiful example of how polarization of cells is used to guide cell function. Receptors, signal transducers, the cytoskeleton, and organelles are enriched at or depleted from the IS after its formation, and in many cases these re‐localizations have already been linked with certain T‐cell functions. One key step for T‐cell activation is a rise in the cytoplasmic calcium concentration. Whereas it is undisputed that the IS initiates and controls calcium signals in T cells, very little is known about the role of T‐cell polarization for calcium signals and calcium‐dependent signal transduction. We briefly summarize the basic commonly agreed principles of IS‐dependent calcium signal generation but then focus on the less well understood influence of polarization on calcium signals. The discussion of the role of polarization for calcium signals l...

Background~Aims: Diagnosis of small nodular lesions in the liver is often difficult because polarization of hepatocytes under pathological conditions is not as easily determined as for glandular or squamous epithelia. The aim of the present study was to investigate whether the bile canalicular enzyme dipeptidyi peptidase IV (DPP IV) would be useful to assess the pattern of hepatocellular surface polarity in liver sections. Methods: Expression of DPP IV activity was determined by enzymatic cytochemistry and image cytometry in 25 human hepatoceilular carcinomas and five cirrhotic livers removed at transplantation. Samples from the central and/or peripheral portion of neoplastic nodules and from surrounding tissue were analyzed in each case. Control specimens were obtained from normal liver of seven patients who underwent surgery for non-neoplastic conditions. Results: In normal liver, DPP IV activity was confined to the bile canalicular plasma membrane with a zone 3 predominance in the hepatic acinus. This was also the case in the majority of pathological non-neo-plastic livers, but the cell distribution pattern of DPP IV was altered in all hepatocellular carcinomas: 2/25 cases were completely devoid of DPP IV activity and in the remaining 23 DPP IV expressing bepatocellular carcinomas, three different patterns were observed that deviated distinctly from the typical canalicular pattern: (i) canaliculi were distorted and convoluted and contained an abnormally high DPP IV activity; (ii) canalicular activity was lost and enzymatic activity was restricted to isolated spots; (iii) pseudoacinar structures of hepatocytes with both basolaterai and apical DPP IV expression appeared. Conclusions: It is concluded that DPP IV is a useful bile canalicular enzyme to assess the functional polarization of hepatocytes and that aberrant DPP IV expression occurs in human hepatocellular carcinoma.

The function of acidification in protein sorting along the biosynthetic pathway has been difficult to elucidate, in part because reagents used to alter organelle pH affect all acidified compartments and are poorly reversible. We have used a novel approach to examine the role of acidification in protein sorting in polarized Madin-Darby canine kidney (MDCK) cells. We expressed the influenza virus M2 protein, an acid-activated ion channel that equilibrates lumenal and cytosolic pH, in polarized MDCK cells and examined the consequences on the targeting and delivery of apical and basolateral proteins. M2 activity affects the pH of only a subset of acidified organelles, and its activity can be rapidly reversed using ion channel blockers (Henkel, J.R., G. Apodaca, Y. Altschuler, S. Hardy, and O.A. Weisz. 1998. Mol. Biol. Cell. 8:2477–2490; Henkel, J.R., J.L. Popovich, G.A. Gibson, S.C. Watkins, and O.A. Weisz. 1999. J. Biol. Chem. 274:9854–9860). M2 expression significantly decreased the k...

In an attempt to develop effective vaccines against central nervous system (CNS) tumors, we evaluated the ability of vaccines with standard dendritic cells (DC) versus type 1 polarizing DCs (DC1) to induce glioma-specific type 1 CTLs with CNS tumor-relevant homing properties and the mechanism of their action. C57BL/6 mouse–derived bone marrow cells were cultured with mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) for 6 days, and CD11c+ cells were subsequently cultured with GM-CSF, rmIFN-γ, rmIFN-α, rmIL-4, and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose for 24 hours to generate DC1s. In analogy to their human counterparts, mouse DC1s exhibited surface marker profiles of mature DCs and produced high levels of IL-12 and CXCL10. Importantly for their application as cancer vaccines, such DC1s stably retained their type 1 phenotype even when exposed to type 2–promoting or regulatory T cell (Treg)–promoting environments. Consistently, m...

The B cell coreceptor CD22 plays an important role in regulating signal transduction via the B cell Ag receptor. Studies have shown that surface expression of CD22 can be modulated in response to binding of ligand (i.e., mAb). Thus, it is possible that alterations in the level of CD22 expression following binding of natural ligand(s) may affect its ability to modulate the Ag receptor signaling threshold at specific points during B cell development and differentiation. Therefore, it is important to delineate the physiologic mechanism by which CD22 expression is controlled. In the current study, yeast two-hybrid analysis was used to demonstrate that CD22 interacts with AP50, the medium chain subunit of the AP-2 complex, via tyrosine-based internalization motifs in its cytoplasmic domain. This interaction was further characterized using yeast two-hybrid analysis revealing that Tyr 843 and surrounding amino acids in the cytoplasmic tail of CD22 comprise the primary binding site for AP50. Subsequent studies using transfectant Jurkat cell lines expressing wild-type or mutant forms of CD22 demonstrated that either Tyr 843 or Tyr 863 is sufficient for mAb-mediated internalization of CD22 and that these motifs are involved in its interaction with the AP-2 complex, as determined by coprecipitation of ␣-adaptin. Finally, experiments were performed demonstrating that treatment of B cells with either intact anti-Ig Ab or F(ab) 2 blocks ligand-mediated internalization of CD22. In conclusion, these studies demonstrate that internalization of CD22 is dependent on its association with the AP-2 complex via tyrosine-based internalization motifs.

When cells in a primary tumor work together to invade into nearby tissue, this can lead to cell dissociationscancer cells breaking off from the invading front-leading to metastasis. What controls the dissociation of cells and whether they break off singly or in small groups? Can this be determined by cell-cell adhesion or chemotactic cues given to cells? We develop a physical model for this question, based on experiments that mimic aspects of cancer cell invasion using microfluidic devices with microchannels of different widths. Experimentally, most dissociation events ("ruptures") involve single cells breaking off, but we observe some ruptures of large groups (∼20 cells) in wider channels. The rupture probability is nearly independent of channel width. We recapitulate the experimental results with a phase-field cell motility model by introducing three different cell states (follower, guided, and high-motility "leader" cells) based on their spatial position. These leader cells may explain why single-cell rupture is the universal most probable outcome. Our simulation results show that cell-channel adhesion is necessary for cells in narrow channels to invade, and strong cell-cell adhesion leads to fewer but larger ruptures. Chemotaxis also influences the rupture behavior: Strong chemotaxis strength leads to larger and faster ruptures. Finally, we study the relationship between biological jamming transitions and cell dissociations. Our results suggest unjamming is necessary but not sufficient to create ruptures.

The actomyosin cytoskeleton is a primary force-generating mechanism in morphogenesis, thus a robust spatial control of cytoskeletal positioning is essential. In this report, we demonstrate that actomyosin contractility and planar cell polarity (PCP) interact in post-mitotic Ciona notochord cells to self-assemble and reposition actomyosin rings, which play an essential role for cell elongation. Intriguingly, rings always form at the cells' anterior edge before migrating towards the center as contractility increases, reflecting a novel dynamical property of the cortex. Our drug and genetic manipulations uncover a tug-of-war between contractility, which localizes cortical flows toward the equator and PCP, which tries to reposition them. We develop a simple model of the physical forces underlying this tug-of-war, which quantitatively reproduces our results. We thus propose a quantitative framework for dissecting the relative contribution of contractility and PCP to the self-assembly...

The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP pr...

Despite its importance in development and physiology the planar cell polarity (PCP) pathway remains one of the most enigmatic signaling mechanisms. The notochord of the ascidian Ciona provides a unique model for investigating the PCP pathway. Interestingly, the notochord appears to be the only embryonic structure in Ciona activating the PCP pathway. Moreover, the Ciona notochord as a single-file array of forty polarized cells is a uniquely tractable system for the study of polarization dynamics and the transmission of the PCP pathway. Here, we test models for propagation of a polarizing signal, interrogating temporal, spatial and signaling requirements. A simple cell-cell relay cascading through the entire length of the notochord is not supported; instead a more complex mechanism is revealed, with interactions influencing polarity between neighboring cells, but not distant ones. Mechanisms coordinating notochord-wide polarity remain elusive, but appear to entrain general (i.e., global) polarity even while local interactions remain important. However, this global polarizer does not appear to act as a localized, spatially-restricted determinant. Coordination of polarity along the long axis of the notochord requires the PCP pathway, a role we demonstrate is temporally distinct from this pathway's earlier role in convergent extension and intercalation. We also reveal polarity in the notochord to be dynamic: a cell's polarity state can be changed and then restored, underscoring the Ciona notochord's amenability for in vivo studies of PCP.

ability- in dissociated embryonic rat spinal cord cells. We studied the expression of responses to veratridine, a sodium channel activator; muscimol, a GABA, receptor agonist; and kainic acid, an agonist at a class of glutamate receptors. Responses were consistently detectable in a percentage of cells dissociated from the earliest age examined, embryonic day 13, and increased progressively in later ages.

Axon-dendrite formation is a crucial milestone in the life history of neurons. During this process, historically referred as “the establishment of polarity,” newborn neurons undergo biochemical, morphological and functional transformations to generate the axonal and dendritic domains, which are the basis of neuronal wiring and connectivity. Since the implementation of primary cultures of rat hippocampal neurons by Gary Banker and Max Cowan in 1977, the community of neurobiologists has made significant achievements in decoding signals that trigger axo-dendritic specification. External and internal cues able to switch on/off signaling pathways controlling gene expression, protein stability, the assembly of the polarity complex (i.e., PAR3-PAR6-aPKC), cytoskeleton remodeling and vesicle trafficking contribute to shape the morphology of neurons. Currently, the culture of hippocampal neurons coexists with alternative model systems to study neuronal polarization in several species, from s...

The establishment of polarity is crucial for the physiology and wiring of neurons. Therefore, monitoring the axo-dendritic specification allows the mechanisms and signals associated with development, growth, and disease to be explored. Here, we describe major and minor steps to study polarity acquisition, using primary cultures of hippocampal neurons isolated from embryonic rat hippocampi, for in vitro monitoring. Furthermore, we use in utero electroporated, GFP-expressing embryonic mouse brains for visualizing cortical neuron migration and polarization in situ. Some underreported after-protocol steps are also included.

The functions of the actin cytoskeleton in post-Golgi trafficking are still poorly understood. Here we report the role of LIM Kinase 1 (LIMK1) and its substrate cofilin in the trafficking of apical and basolateral proteins in MDCK cells. Our data indicate that LIMK1 and cofilin organize a specialized population of actin filaments at the Golgi complex that is selectively required for the emergence of an apical cargo route to the plasma membrane (PM). Quantitative pulse-chase live imaging experiments showed that overexpression of kinase-dead LIMK1 (LIMK1-KD), or of LIMK1 siRNA, or of an activated cofilin mutant (cofilin S3A), selectively slowed down the exit from the trans Golgi network (TGN) of the apical PM marker p75-GFP but did not interfere with the apical PM marker, GPI-GFP, or the basolateral PM marker, NCAM-GFP. High resolution live imaging experiments of carrier formation and release by the TGN and analysis of peri-Golgi actin dynamics using photoactivatable GFP suggest a scenario in which TGN-localized LIMK1-cofilin regulate a population of actin filaments required for dynaminsyndapin-cortactin dependent generation and/or fission of precursors to p75 transporters.

In this study we have examined the cellular functions of ERM proteins in developing neurons. The results obtained indicate that there is a high degree of spatial and temporal correlation between the expression and subcellular localization of radixin and moesin with the morphological development of neuritic growth cones. More importantly, we show that double suppression of radixin and moesin, but not of ezrin–radixin or ezrin–moesin, results in reduction of growth cone size, disappearance of radial striations, retraction of the growth cone lamellipodial veil, and disorganization of actin filaments that invade the central region of growth cones where they colocalize with microtubules. Neuritic tips from radixin–moesin suppressed neurons displayed high filopodial protrusive activity; however, its rate of advance is 8–10 times slower than the one of growth cones from control neurons. Radixin–moesin suppressed neurons have short neurites and failed to develop an axon-like neurite, a phen...

The region of a cell that lies beneath the plasma membrane and contains a network of actin filaments and associated proteins. The interaction between microtubules and components of the cell cortex that results in stabilization of the microtubules.

guanine nucleotide exchange factors of RhoGTPase 2 Department of Cell and Development Biology proteins have drastic effects on neurite extension (re-Weill Medical College of Cornell University viewed in Fukata et al., 2002c). It is generally thought 1300 York Avenue that Rho is a negative regulator and Rac is a positive New York, New York 10012 regulator of neurite growth. Nevertheless, little is known 3 INIMEC-CONICET about how the local activation (or inactivation) of these Avenue Friuli 2434 RhoGTPase family proteins is regulated. Very recent 5016 Cordoba data suggest that selective enrichment of mPar3/mPar6/ Argentina atypic protein kinase C complex and phosphatidylinositol 3-kinase activity at axonal neurites may play a crucial role in axonal specification through a mechanism Summary upstream of RhoGTPase-mediated cytoskeletal modulation (Shi et al., 2003). Coordinated microtubule and microfilament changes Microtubules are also involved in establishing axonal are essential for the morphological development of polarity. This idea has been demonstrated by the obneurons; however, little is know about the underlying servation that nascent axons had more stable microtumolecular machinery linking these two cytoskeletal bules (Ferreira et al., 1989) and that more microtubules systems. Similarly, the indispensable role of Rhowere selectively shipped into neurites fated to be axons GTPase family proteins has been demonstrated, but (Baas et al., 1993). More recently, CRMP-2 (Collapsin it is unknown how their activities are specifically regresponse mediator protein-2) was found to selectively ulated in different neurites. In this paper, we show accumulate in the distal region and the growth cones that the cytoplasmic dynein light chain Tctex-1 plays of nascent axons (Inagaki et al., 2001). CRMP-2's ability a key role in multiple steps of hippocampal neuron to bind free tubulin subunits may assist in microtubule development, including initial neurite sprouting, axon assembly, thereby enhancing axonal neurite extension specification, and later dendritic elaboration. The neu-(Fukata et al., 2002b). The microtubule plus-end bindritogenic effects elicited by Tctex-1 are independent ing protein APC (Adenomatous polyposis coli) has also from its cargo adaptor role for dynein motor transbeen shown to play roles in nerve growth factorport. Finally, our data suggest that the selective high induced axonal outgrowth through its ability to promote level of Tctex-1 at the growth cone of growing axons microtubule assembly (Zhou et al., 2004). drives fast neurite extension by modulating actin dy-Studies on the dynamics of growth cones in Aplysia namics and also Rac1 activity. neurons have lent support to the idea that microtubule redistribution into the leading lamellipodium is secondary to F-actin disassembly (Forscher and Smith, 1988).

In a biological sense, polarity refers to the extremity of the main axis of an organelle, cell, or organism. In neurons, morphological polarity begins with the appearance of the first neurite from the cell body. In multipolar neurons, a second phase of polarization occurs when a single neurite initiates a phase of rapid growth to become the neuron's axon, while the others later differentiate as dendrites. Finally, during a third phase, axons and dendrites develop an elaborate architecture, acquiring special morphological and molecular features that commit them to their final identities. Mechanistically, each phase must be preceded by spatial restriction of growth activity. We will review recent work on the mechanisms underlying the polarized growth of neurons.

In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short-(6 h) and long-(2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.

We applied suppression subtractive hybridization and mirror orientation selection to compare gene expression profiles of isolated Nicotiana tabacum cv SR1 zygotes and egg cells. Our results revealed that many differentially expressed genes in zygotes were transcribed de novo after fertilization. Some of these genes are critical to zygote polarity and pattern formation during early embryogenesis. This suggests that the transcriptome is restructed in zygote and that the maternal‐to‐zygotic transition happens before the first zygotic division, which is much earlier in higher plants than in animals. The expressed sequence tags used in this study provide a valuable resource for future research on fertilization and early embryogenesis.

The Rho GTPases pattern the cell cortex in a variety of fundamental cell-morphogenetic processes, including division, wound repair, and locomotion. It has recently become apparent that this patterning arises from the ability of the Rho GTPases to self-organize into static and migrating spots, contractile pulses, and propagating waves in cells from yeasts to mammals.1 These self-organizing Rho GTPase patterns have been explained by a variety of theoretical models that require multiple interacting positive and negative feedback loops. However, it is often difficult, if not impossible, to discriminate between different models simply because the available experimental data do not simultaneously capture the dynamics of multiple molecular concentrations and biomechanical variables at fine spatial and temporal resolution. Specifically, most studies typically provide either the total Rho GTPase signal or the Rho GTPase activity, as reported by various sensors, but not both. Therefore, it remains largely unknown how membrane accumulation of Rho GTPases (i.e., Rho membrane enrichment) is related to Rho activity. Here, we dissect the dynamics of RhoA by simultaneously imaging both total RhoA and active RhoA in propagating waves of Rho activity and F-actin polymerization.2,3,4,5 We find that within nascent waves, accumulation of active RhoA precedes that of total RhoA, and we exploit this finding to distinguish between two popular theoretical models previously used to explain propagating cortical Rho waves.

ABSTRACTUlcerative colitis and Crohn's disease are inflammatory bowel diseases thought to involve strains ofEscherichia coli. We report here that two wild-type Afa/Dr diffusely adheringE. coli(DAEC) strains, C1845 and IH11128, which harbor the fimbrial F1845 adhesin and the Dr hemagglutinin, respectively, and theE. colilaboratory strain HB101, transformed with the pSSS1 plasmid to produce Afa/Dr F1845 adhesin, all induced interleukin-8 (IL-8) production and transepithelial migration of polymorphonuclear leukocytes (PMNL) in polarized monolayers of the human intestinal cell line T84 grown on semipermeable filters. We observed that after PMNL migration, expression of decay-accelerating factor (DAF, or CD55), the brush border-associated receptor for Afa/Dr adhesins, was strongly enhanced, increasing the adhesion of Afa/Dr DAEC bacteria. When examining the mechanism by which DAF expression was enhanced, we observed that the PMNL transepithelial migration induced epithelial synthesis...

Gram-negative pathogens. Here we report a previously unsuspected role of human CEACAM1-4L (hCEACAM1-4L) in polarized epithelial cells. We find that in contrast with nontransfected cells, Madin Darby Canine Kidney strain II (MDCK) engineered for the apical expression of the long cytoplasmic chain protein hCEACAM1-4L showed a serum-independent increase in the phosphorylation of the extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinases (MAPKs) after treatment with lipopolysaccharide (LPS) of wild-type, diffusely adhering Afa/Dr Escherichia coli (Afa/Dr DAEC) strain IH11128. Aggregates of FITC-LPS bind the apical domain of MDCK-hCEACAM1-4L cells colocalizing with the apically expressed hCEACAM1-4L protein and do not bind MDCK-pCEP cells, and surface plasmon resonance analysis shows that LPS binds to the extracellular domain of the CEACAM1-4L protein. We showed that cell polarization and lipid rafts positively control the LPS-IH11128-induced phosphorylation of Erk1/2 in MDCK-hCEACAM1-4L cells. Structure-function analysis using mutated hCEACAM1-4L protein shows that the cytoplasmic domain of the protein is needed for LPS-induced MAPK signalling, and that phosphorylation of Tyr-residues is not increased in association with MAPK signalling. The hCEACAM1-4L-dependent Erk1/2 phosphorylation develops in the presence of lipid A and does not develop in the presence of penta-acylated LPS. Finally, small interfering RNA (siRNA) silencing of canine TLR4 abolishes the hCEACAM1-4L-dependent, LPS-induced phosphorylation of Erk1/2. Collectively, our results support the notion that the apically expressed, full-length hCEACAM1-4L protein functions as a novel LPS-conveying molecule at the mucosal surface of polarized epithelial cells for subsequent MD-2/TLR4 receptor-dependent MAPK Erk1/2 and p38 signalling.c mi_1575 764..785

Genetic and acquired loss-of-function defect of the cystic fibrosis transmembrane conductance regulator (cftR) compromise airway surface liquid homeostasis and mucociliary clearance (Mcc), culminating in recurrent lung inflammation/infection. While chronic cigarette smoke (CS), CS extract (cSe; water-soluble compounds) and cS condensate (cSc; particulate, organic fraction) exposure inhibit cftR activity at transcriptional, biochemical, and functional levels, the acute impact of cSc remains incompletely understood. We report that CSC transiently activates CFTR chloride secretion in airway epithelia. the comparable cftR phospho-occupancy after cSc-and forskolin-exposure, determined by affinity-enriched tandem mass spectrometry and pharmacology, suggest that localised cAMPdependent protein kinase (pKA) stimulation by cSc causes the channel opening. Due to the inhibition of the MRP4/ABCC4, a cAMP-exporter confined to the CFTR macromolecular signalling-complex, PKA activation is accomplished by the subcompartmentalised elevation of cytosolic cAMP. In line, MRP4 inhibition results in cftR activation and phospho-occupancy similar to that by forskolin. in contrast, acute cSc exposure reversibly inhibits the phosphorylated cftR both in vivo and in phospholipid bilayers, without altering its cell surface density and phospho-occupancy. We propose that components of cSc elicit both a transient protective cftR activation, as well as subsequent channel block in airway epithelia, contributing to the subacute Mcc defect in acquired cf lung diseases. The adverse long-term effects of inhaled, combusted tobacco or cigarette smoke (CS) on the lung's cellular and molecular physiology have been established and include DNA damage, goblet cell metaplasia, increased inflammation, autophagy and proteolysis 1 . These processes account for higher incidence of chronic obstructive pulmonary disease (COPD), the third leading cause of death in the US 2 , which is compounded by significant extrapulmonary pathologies 1,3 and lung cancer. The acquired loss of the cystic fibrosis transmembrane conductance regulator (CFTR) function, a chloride and bicarbonate-selective, 3′,5′-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) regulated anion channel 4 , is invoked in the pathogenesis of COPD partly as a consequence of CS exposure . COPD exhibits hallmarks of chronic bronchitis and emphysema, with overlapping clinical manifestation and molecular pathology to that of cystic fibrosis (CF) . Both acquired and inherited expression defects of CFTR result in the periciliary liquid layer depletion, acidification, mucus dehydration, increased bacterial adhesion, and decreased mucociliary clearance (MCC), causing recurrent infections and sustained inflammation with progressive deterioration of the lung tissue 6,9-12 . Compelling evidence indicates that subacute (2-24 h) and chronic (>24 h) cigarette smoke (CS) exposure compromises CFTR activity at the protein and mRNA level in a variety of cell types, including human nasal and bronchial epithelia . In contrast, the acute effect of CS on CFTR is incompletely understood. CFTR, a member of the adenosine triphosphate (ATP)-binding cassette transporter (ABC) superfamily, is predominantly localised at the apical plasma membrane (PM) of secretory and resorptive epithelia in various organs,

Plasmids for high-level expression of penicillin-binding protein 6 (PBP6) were constructed, giving rise to overproduction of PBP6 under the control of the lambdapR promoter in either the periplasmic or the cytoplasmic space. In contrast to penicillin-binding protein 5 (PBP5), the presence of high amounts of PBP6 in the periplasm as well as in the cytoplasm did not result in growth as spherical ceUls or in lysis. Deletion of the C-terminal membrane anchor of PBP6 resulted in a soluble form of the protein (PBP6'3-). Electron micrographs of thin sections of cells overexpressing both native membrane-bound and soluble PBP6 in the periplasm revealed a polar retraction of the cytoplasmic membrane. Cytoplasmic overexpression of native PBP6 gave rise to the formation of membrane vesicles, whereas the soluble PBP6 formed inclusion bodies in the cytoplasm. Both the membrane- bound and the soluble forms of PBP6 were purifie to homogeneity by using the immobilized dye Procion rubine MX-B. Purified preparations of PBP6 and PBP6S3se formed a 14[Cjpenicillin-protein complex at a 1:1 stoichiometry. The half-lives of the complexes were 8.5 and 6 min, respectively. In contrast to PBP5, no DD-carboxypeptidase activity could be detected for PBP6 by using bisacetyl-L-Lys-D-Ala-D-Ala and several other substrates. These findings led us to conclude that PBP6 has a biological function clearly distinct from that ofPBP5 and to suggest a role for PBP6 in the stabilization of the peptidogiycan during stationary phase.

Naturally photoswitchable proteins offer a means of directly manipulating the formation of protein complexes that drive a diversity of cellular processes. We have developed tunable light-inducible dimerization tags (TULIPs) based on a synthetic interaction between the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) and an engineered PDZ domain (ePDZ). TULIP tags can recruit proteins to diverse structures in living yeast and mammalian cells, either globally or with precise spatial control using a steerable laser. The equilibrium binding and kinetic parameters of the interaction are tunable by mutation, making TULIPs readily adaptable to signaling pathways with varying sensitivities and response times. We demonstrate the utility of TULIPs by conferring light sensitivity to functionally distinct components of the yeast mating pathway and by directing the site of cell polarization. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Background aims. Toll-like receptor (TLR)-3 synthetic agonist polyinosinic-polycytidylic acid (poly(I:C)) is a promising agent for dendritic cell (DC)-based anti-tumor vaccines because of its ability to induce a strong maturation of DCs, but such an effect is followed by stimulation of DC apoptosis. Tumor necrosis factor (TNF)-a may promote the survival of poly(I:C)stimulated DCs, but it is not known in detail how this combination affects the maturation and polarization capacity of monocyte-derived (Mo)DCs. Methods. Immature MoDCs, generated from human monocytes, were treated with different concentrations of poly(I:C) combined with TNF-a, and the effect on survival, phenotype, production of cytokines, allostimulatory and Th polarization capacity was assessed after 24 and 48 h. Results. We showed that TNF-a inhibited the dosedependent pro-apoptotic effect of poly(I:C). However, TNF-a also decreased poly(I:C)-induced production of interleukin (IL)-12 and IL-23 by MoDCs, which correlated with their diminished capacity to stimulate cellular proliferation, interferong and IL-17 production by allogeneic CD4 þ T cells in co-culture. Such an effect was more pronounced after 24 h and could not be restored by CD40 ligation. In the presence of CD40L, TNF-a even stimulated IL-10 production and immunoglobulin-like transcript 3 expression by poly(I:C)-matured DCs, which correlated with their increased capacity to induce IL-10 production by CD4 þ T cells. Conclusion. Even though TNF-a could promote the survival of poly(I:C)-matured MoDCs, it also suppresses key anti-tumor functions of these cells, which could have important implications when considering this, already suggested, protocol for the DC-based anti-tumor therapy.

Activated macrophages are classified into two different forms: classically activated (M1) or alternatively activated (M2) macrophages. The presence of M1/M2 phenotypic polarization has also been suggested for microglia. Here, we report that the secreted protein lipocalin 2 (LCN2) amplifies M1 polarization of activated microglia. LCN2 protein (EC 50 1 g/ml), but not glutathione S-transferase used as a control, increased the M1-related gene expression in cultured mouse microglial cells after 8 -24 h. LCN2 was secreted from M1-polarized, but not M2-polarized, microglia. LCN2 inhibited phosphorylation of STAT6 in IL-4-stimulated microglia, suggesting LCN2 suppression of the canonical M2 signaling. In the lipopolysaccharide (LPS)-induced mouse neuroinflammation model, the expression of LCN2 was notably increased in microglia. Primary microglial cultures derived from LCN2-deficient mice showed a suppressed M1 response and enhanced M2 response. Mice lacking LCN2 showed a markedly reduced M1-related gene expression in microglia after LPS injection, which was consistent with the results of histological analysis. Neuroinflammationassociated impairment in motor behavior and cognitive function was also attenuated in the LCN2-deficient mice, as determined by the rotarod performance test, fatigue test, open-field test, and object recognition task. These findings suggest that LCN2 is an M1-amplifier in brain microglial cells.-

By analysing the cellular and subcellular events that occur in the centre of the developing zebrafish neural rod, we have uncovered a novel mechanism of cell polarisation during lumen formation. Cells from each side of the neural rod interdigitate across the tissue midline. This is necessary for localisation of apical junctional proteins to the region where cells intersect the tissue midline. Cells assemble a mirror-symmetric microtubule cytoskeleton around the tissue midline, which is necessary for the trafficking of proteins required for normal lumen formation, such as partitioning defective 3 and Rab11a to this point. This occurs in advance and is independent of the midline cell division that has been shown to have a powerful role in lumen organisation. To our knowledge, this is the first example of the initiation of apical polarisation part way along the length of a cell, rather than at a cell extremity. Although the midline division is not necessary for apical polarisation, it confers a morphogenetic advantage by efficiently eliminating cellular processes that would otherwise bridge the developing lumen.

Given the central role of dendritic cells (DCs) in directing T cell phenotypes, the ability of biomaterial-treated DCs to dictate autologous T cell phenotype was investigated. Here, we demonstrate that differentially biomaterial-treated DCs differentially directed autologous T cell phenotype and polarization, depending on the biomaterial used to pre-treat the DCs. Immature DCs (iDCs) were derived from human peripheral blood monocytes, and treated with biomaterial films of alginate, agarose, chitosan, hyaluronic acid, or 75:25 poly(lactic-co-glycolic acid) (PLGA), followed by co-culture of these biomaterial-treated DCs and autologous T cells. When autologous T cells were co-cultured with DCs treated with biomaterial film/antigen (ovalbumin, OVA) combinations, different biomaterial films induced differential levels of T cell marker (CD4, CD8, CD25, CD69) expression, as well as differential cytokine profiles [interferon (IFN)-γ, interleukin (IL)-12p70, IL-10, IL-4] in the polarization of T helper types. Dendritic cells treated with agarose films/OVA induced CD4+CD25+FoxP3+ (T regulatory cells) expression, comparable to untreated iDCs, on autologous T cells in the DC-T co-culture system. Furthermore, in this co-culture, agarose treatment induced release of IL-12p70 and IL-10 at higher levels, as compared to DC treatment with other biomaterial films/OVA, suggesting Th1 and Th2 polarization, respectively. Dendritic cells treated with PLGA film/OVA treatment induced release of IFN-γ at higher levels compared to that observed for co-cultures with iDCs or DCs treated with all other biomaterial films. These results indicate that DC treatment with different biomaterial films has potential as a tool for immunomodulation by directing autologous T cell responses.

Altered expression of human Scribble is associated with invasive epithelial cancers, however, its role in tumour development remains unclear. Mutations in Drosophila Scribble result in loss of polarity, overproliferation and 3D-tumourous overgrowth of epithelial cells. Using complementation studies in Drosophila we recently demonstrated that expression of human Scribble can also regulate polarity and restrict tissue overgrowth. Here, we have undertaken a detailed study of human Scribble function in the polarized mammary cell line, MCF10A. We show that although Scribble does not seem to be required for apical-basal polarity or proliferation control in MCF10A cells, Scribble is essential for the control of polarity associated with directed epithelial cell migration. Scribble-depleted MCF10A cells show defective in vitro wound closure and chemotactic movement. The cells at the wound edge fail to polarize, show reduced lamellipodia formation and impaired recruitment of Cdc42 and Rac1 to the leading edge. Furthermore, we show that this function is relevant in vivo as Scribble mutant mice show defective epidermal wound healing. This data identifies an essential role for mammalian Scribble in the regulation of the polarity specifically involved in directed epithelial migration.

CD14 + dermal DCs (CD14 + DDCs) have a natural capacity to activate naı ¨ve B-cells. Targeting CD14 + DDCs is therefore a rational approach for vaccination strategies aimed at improving humoral responses towards poorly immunogenic antigens, for example, HIV-1 envelope glycoproteins (Env). Here, we show that two clinically relevant TLR ligand combinations, Hiltonol plus Resiquimod and Glucopyranosyl lipid A plus Resiquimod, potently activate CD14 + DDCs, as shown by enhanced expression of multiple cytokines (IL-6, IL-10, IL-12p40 and TNF-a). Furthermore, the responses of CD14 + DDCs to these TLR ligands were not compromised by the presence of HIV-1 gp120, which can drive immunosuppressive effects in vitro and in vivo. The above TLR ligand pairs were better than the individual agents at boosting the inherent capacity of CD14 + DDCs to induce naı ¨ve B-cells to proliferate and differentiate into CD27 + CD38 + B-cells that secrete high levels of immunoglobulins. CD14 + DDCs stimulated by these TLR ligand combinations also promoted the differentiation of Th1 (IFNc-secreting), but not Th17, CD4 + T-cells. These observations may help to identify adjuvant strategies aimed at inducing better antibody responses to vaccine antigens, including, but not limited to HIV-1 Env.

Recently, the LAP (for 'LRR and PDZ') family of adaptor proteins was described in vertebrates and invertebrates. The proteins are involved in cell polarity and receptor targeting, and they contain both LRR and PDZ domains in the same molecule, a combination that is specifically bilaterian. A search of available genome sequences reveals a limited number of lap genes, which we have classified by phylogenetic analysis. We propose a functional hypothesis for the origin of this protein family in bilaterians, and give a phylogenetic interpretation of their diversity.

The interplay between tension and adhesion is a fundamental aspect of multicellular organisms, dictating the mechanical behavior of cell aggregates during tissue morphogenesis, cancer metastasis, and wound healing. However, the processes driving this dynamic interaction are not well-characterized nor quantified. This dissertation investigated the essential elements impacting cortical tension and its interaction with cell adhesion across various scales. This dissertation focuses on two important aspects of this subject and provides a comprehensive analysis of both. First, the mechanical behavior and cortical tension of single cells are analyzed using electrodeformation-relaxation. Four distinct cell types, namely, MCF-10A, MCF-7, MDA-MB-231, and GBM, are studied, with pulse durations ranging from 0.01 to 10 seconds. In the long-pulse regime, the mechanical response exhibits a power-law behavior, which indicates soft glassy rheology (SGR) resulting from unbinding events within the cortex network. On the other hand, the short-pulse regime is characterized by a single timescale, providing insights into the ”naive” pre-stressed state of the cortex with minimal force-induced alterations. An analytical solution, supported by mathematical modeling is employed to extract the cortical tension. The tension values for all four cell types at the shortest pulse duration of 0.01 seconds are on the order of 10−2 N/m. Second, the crucial role of N-cadherin in activating signaling pathways that result in ”mechanical polarization” throughout the actin cortical network is elucidated. This polarization is characterized by increased cortical tension at the non-contacting boundary of cells and decreased interfacial tension at the cell-cell interface, both significantly contribute to adhesive strength between cells. These tension regulations are found to induce remodeling in the F-actin cortex and strongly rely on the functionality of the non-muscle myosin II motor. Rac1 activation is identified as a molecular switch that regulates contraction in the actin cortex, providing a basic and general mechanism for tension regulation and aggregation control in solid tumors and spheroids. In summary, this dissertation provides a quantitative and contextual understanding of the complex mechanical interaction within multicellular organisms, shedding light on the fundamental factors that regulate cortical tension and collective behavior in clusters, aggregates, and tissues.

To use nanoscopically defined, two-dimensional matrices assembled from aligned collagen type I fibrils as a sheet substratum for in vitro cultivation of human corneal endothelial cells (HCECs). To assess the effect of matrix architecture on HCEC morphology and to characterize integrin-mediated HCEC-matrix interaction. METHODS. Cell alignment and cell-matrix interactions of primary HCECs and three different immortalized HCEC populations on native and UV-cross-linked collagen type I matrices were examined by time-lapse microscopy. Specific integrin ␣ 2 ␤ 1 binding to the collagen matrix was demonstrated using a function-blocking ␣ 2 antibody. Integrin ␣ 2 subunit expression levels of the four HCEC populations were analyzed by Western blot analysis. All HCEC populations aligned along the oriented collagen fibrils. Primary HCECs and, to a lesser extent, the other tested HCEC populations exerted high traction forces, leading to progressive matrix destruction. Cross-linking of the collagen matrices considerably increased matrix stability. Integrin subunit ␣ 2 expression levels of the four cell types correlated with the degree of cell alignment and exertion of traction forces. In turn, blocking integrin subunit ␣ 2 reduced cell alignment and prevented matrix destruction. HCECs align directionally along parallel arrays of collagen type I fibrils. The interactions of HCECs with collagen type I are primarily mediated by integrin ␣ 2 ␤ 1 . Integrin subunit ␣ 2 levels correlate with matrix contraction and subsequent destruction. Sustained cultivation of HCECs on ultrathin collagen matrices thus requires matrix cross-linking and moderate integrin ␣ 2 ␤ 1 expression levels. (Invest

To test nanosize surface patterning for application as implant material, a suitable titanium composition has to be found first. Therefore we investigated the effect of surface chemistry on attachment and differentiation of osteoblast-like cells on pure titanium prepared by pulsed laser deposition (TiPLD) and different Ti alloys (Ti6Al4V, TiNb30 and TiNb13Zr13). Early attachment (30 min) and alkaline phosphatase (ALP) activity (day 5) was found to be fastest and highest, respectively, in cells grown on TiPLD and Ti6Al4V. Osteoblasts seeded on TiPLD produced most osteopontin (day 10), whereas expression of this extracellular matrix protein was an order of magnitude lower on the TiNb30 surface. In contrast, expression of the corresponding receptor, CD44, was not influenced by surface chemistry. Thus, TiPLD was used for further experiments to explore the influence of surface nanostructures on osteoblast adhesion, differentiation and orientation. By laser-induced oxidation, we produced p...

Cell and tissue polarity guides a large variety of developmental processes, including the choice between symmetric and asymmetric cell division. Asymmetric divisions create cell diversity and are needed for the maintenance of tissue-specific stem cells. Symmetric divisions, on the other hand, promote exponential cell proliferation. Polarized cells often divide symmetrically by cleaving along the axis of polarity. Alternatively, cell cleavage in a plane perpendicular to the polarity axis results in asymmetric division. To control this decision, developmental cues position the mitotic spindle, which instructs the plane of cell cleavage. In animal cells, the positioning of the spindle depends on evolutionarily conserved interactions between a heterotrimeric G-protein alpha subunit, TPR-GoLoco domain protein, and NuMA-related coiled-coil protein. This trimeric complex recruits the dynein microtubule motor and captures astral microtubules at the cortex. The interplay between dynein movement and depolymerizing microtubules generates cortical pulling forces that promote aster movement and spindle positioning. Through mechanisms that are poorly understood, cell polarity and other developmental signals control the microtubule-pulling forces to instruct the orientation and plane of cell division. In this chapter, we review the current understanding of the connection between cell polarity and spindle positioning, with a focus on studies of the early C. elegans embryo. The nematode C. elegans develops through a highly reproducible division pattern and has proven to be a powerful model for studying the regulation and execution of asymmetric cell division.

ABSTRACTAfa/Dr diffusely adheringEscherichia coli(Afa/Dr DAEC) strains cause symptomatic urinary tract and intestinal infections. The proinflammatory effects of Afa/Dr DAEC strains in vitro have been not investigated to date. In the present study, we used confluent polarized monolayers of intestinal cell line T84 to evaluate the consequences of epithelial infection by Afa/Dr DAEC strains in terms of proinflammatory response. Polymorphonuclear leukocyte (PMNL) migration across the epithelial barrier was induced after incubation of the T84 monolayers with the wild-type Afa/Dr DAEC strain C1845 harboring the fimbrial F1845 adhesin and strain IH11128 harboring the Dr hemagglutinin, and theE. colilaboratory strain HB101 was transformed with the pSSS1 plasmid, producing Afa/Dr F1845 adhesin. PMNL migrations were correlated with a basolateral secretion of interleukin-8 by T84 cells and were abolished after incubation of epithelial cells with an anti-decay accelerating factor (DAF) antibody...

ABSTRACTUlcerative colitis and Crohn's disease are inflammatory bowel diseases thought to involve strains ofEscherichia coli. We report here that two wild-type Afa/Dr diffusely adheringE. coli(DAEC) strains, C1845 and IH11128, which harbor the fimbrial F1845 adhesin and the Dr hemagglutinin, respectively, and theE. colilaboratory strain HB101, transformed with the pSSS1 plasmid to produce Afa/Dr F1845 adhesin, all induced interleukin-8 (IL-8) production and transepithelial migration of polymorphonuclear leukocytes (PMNL) in polarized monolayers of the human intestinal cell line T84 grown on semipermeable filters. We observed that after PMNL migration, expression of decay-accelerating factor (DAF, or CD55), the brush border-associated receptor for Afa/Dr adhesins, was strongly enhanced, increasing the adhesion of Afa/Dr DAEC bacteria. When examining the mechanism by which DAF expression was enhanced, we observed that the PMNL transepithelial migration induced epithelial synthesis...

ABSTRACTDiffusely adhering Escherichia coli (DAEC) strains expressing adhesins of the Afa/Dr family bind to epithelial cells in a diffuse adherence pattern by recognizing a common receptor, the decay-accelerating factor (CD55). Recently, a novel CD55-binding adhesin, named Dr-II, was identified from the pyelonephritogenic strain EC7372. In this report, we show that despite the low level of sequence identity between Dr-II and other members of the Afa/Dr family, EC7372 induces pathophysiological effects similar to those induced by other Afa/Dr DAEC strains on the polarized epithelial cell line Caco-2/TC7. Specifically, the Dr-II adhesin was sufficient to promote CD55 and CD66e clustering around adhering bacteria and apical cytoskeleton rearrangements. Unlike other Afa/Dr DAEC strains, EC7372 expresses a functional hemolysin that promotes a rapid cellular lysis. In addition, cell death by apoptosis or necrosis was observed in EC7372-infected Caco-2/TC7 cells, depending on infection tim...

Summary Schizosaccharomyces pombe Rho2 GTPase regulates α‐D‐glucan synthesis and acts upstream of Pck2 to activate the MAP kinase pathway for cell integrity. However, little is known about its regulation. Here we describe Rga2 as a Rho2 GTPase‐activating protein (GAP) that regulates cell morphology. rga2+ gene is not essential for growth but its deletion causes longer and thinner cells whereas rga2+ overexpression causes shorter and broader cells. rga2+ overexpression also causes abnormal accumulation of Calcofluor‐stained material and cell lysis, suggesting that it also participates in cell wall integrity. Rga2 localizes to growth tips and septum region. The N‐terminal region of the protein is required for its correct localization whereas the PH domain is necessary exclusively for Rga2 localization to the division area. Also, Rga2 localization depends on polarity markers and on actin polymerization. Rga2 interacts with Rho2 and possesses in vitro and in vivo GAP activity for this G...

Allergic contact dermatitis (ACD) represents a severe health problem with increasing worldwide prevalence. It is a T-cell-mediated inflammatory skin disease caused by chemicals present in the daily or professional environment. NiSO4 and 2,4-dinitrochlorobenzene (DNCB) are 2 chemicals involved in ACD. These contact sensitizers are known to induce an up-regulation of phenotypic markers and cytokine secretion in dendritic cells (DCs; professional APCs), leading to the generation of CD8(+) Tc1/Tc17 and CD4(+) Th1/Th17 effector T cells. In the present study, using a peptide array approach, we identified protein kinase CK2 as a novel kinase involved in the activation of human monocyte-derived DCs (MoDCs) in response to NiSO4 and DNCB. Inhibition of CK2 activity in MoDCs led to an altered mature phenotype with lower expression of CD54, PDL-1, CD86, and CD40 in response to NiSO4 or DNCB. CK2 activity also regulated proinflammatory cytokine production, such as TNF-α, IL-1β, and IL-23 in MoDC...

We recently showed that mucus from patients with ulcerative colitis, a chronic inflammatory disorder of the colon, is characterized by a low level of phosphatidylcholine (PC) while clinical studies reveal that therapeutic addition of PC using slow release preparations is beneficial. The positive role of PC in this disease is still elusive. Here we tested the hypothesis that exogenous application of PC has anti-inflammatory properties using three model systems. First, human Caco-2 cells were treated with TNF-a? to induce a proinflammatory response via activation of NF-?B. Second, latex bead phagosomes were analyzed for their ability to assemble actin in vitro, a process linked to pro-inflammatory signaling and correlating with the growth versus killing of mycobacteria in macrophages. The third system used was the rapid assembly of plasma membrane actin in macrophages in response to sphingosine 1-phosphate (S1P). TNF-a induced a pro-inflammatory response in Caco-2 cells including 1) assembly of plasma membrane actin; 2) activation of both MAP kinases ERK and p38; 3) transport of NF-?B subunits to the nucleus and 4) subsequent up-regulation of the synthesis of pro-inflammatory gene products. Exogenous addition of most PCs tested significantly inhibited these processes. Other phospholipids like sphingomyelin or phosphatidyl-ethanolamine showed no effects in these assays. PC also inhibited latex bead phagosome actin assembly, the killing of M. tuberculosis in macrophages and the S1P-induced actin assembly in macrophages. TNF-a induces the activation of signaling molecules and the reorganization of the actin cytoskeleton in human intestinal cells. Exogenous application of PC blocks pro-inflammatory signaling in Caco-2 cells, in phagosomes in vitro and facilitates intracellular survival of mycobacteria. We provide further evidence that actin assembly by membranes is part of the pro-inflammatory response. Collectively, these results provide a molecular foundation for the clinical studies showing a beneficial effect of PC therapy in ulcerative colitis.

To investigate whether the secretion of phosphatidylcholine (PC) in intestinal mucus occurs by apical secretion or via basolateral excretion and to determine its subsequent passage across the tight junctions to the apical mucus. We addressed this question using the polarized intestinally differentiated tumor cell line CaCo-2 grown on filters to confluence in Transwell culture chambers. The released PC and sphingomyelin (Sph) from apical and basolateral media were analyzed by mass spectrometry. The secreted PC species were identical in both compartments indicating the same intracellular origin of PC. However, PC secretion into the basolateral compartment was more effective, and the PC:Sph ratio in the basolateral compartment was significantly higher than that in the apical compartment (8.18 ± 1.84 vs 4.31 ± 1.22, P = 0.01). Both pathways were temperature sensitive and were unaltered in the presence of cyclosporine. The data demonstrate the PC secretion capacity of CaCo-2 cells and indicate two separated apical and basolateral release mechanisms.