Regulating cell–cell junctions from A to Z

Epithelial sheets often present a “cobblestone” appearance, but the mechanisms underlying the dynamics of this arrangement are unclear. In this issue, Choi et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201506115) show that afadin and ZO-1 regulate tension and maintain zonula adherens architecture in response to changes in contractility.The textbook view of epithelial cells is that once such cells adopt a close, hexagonal packing, their “honeycomb” or “cobblestone” arrangement is static. This fixed appearance is misleading, as these cells are more like players in a rugby scrum, locked in a tussle in which the forces exerted by each of the players on the others maintains their seemingly static arrangement, but by a very dynamic force balance. How such balance is maintained in epithelia is a subject of substantial interest. A crucial role is played by F-actin and nonmuscle myosin II isoforms, which are deployed in contractile networks that transiently attach to cell–cell junctions to generate tensile forces along cell–cell boundaries (Lecuit and Yap, 2015). Contractile arrays of actomyosin are regulated by the monomeric G protein Rho, its upstream regulators, including Rho guanine nucleotide exchange factors (Quiros and Nusrat, 2014), and its effectors ROCK/Rho kinase and Shroom3 (Nishimura and Takeichi, 2008), but also by tension-mediated feedback between the myosin network and the junction (Lecuit and Yap, 2015). Cell–cell adhesion, including cadherin-dependent adhesion, also plays a crucial role in this process. As cells engage with one another via interactions of the extracellular domains of their cadherin complexes, they transduce forces to the actomyosin cytoskeleton through catenins. β-Catenin binds to the cytoplasmic domain of classical cadherins and recruits α-catenin, which binds F-actin.Given the dynamic nature of epithelia, the attachment of contractile actomyosin networks to junctions are also subject to regulation. One aspect of epithelial architecture that has received relatively little attention is that a typical epithelial monolayer (Fig. 1 A) displays two main types of cell–cell interfaces: bilateral junctions (BCJs), in which two cells establish a relatively long stretch of contact, and cellular vertices, which represent a confluence of three or more cell edges to form tricellular junctions (TCJs) or multicellular junctions. TCJs are not well understood, but are known to contain unique molecular components (Furuse et al., 2014; Flores-Benitez and Knust, 2015). In this issue, Choi et al. show that the multivalent scaffolding proteins afadin and ZO-1/2 regulate the spacing of and tension along lateral contacts in cultured cells, thereby shedding light on how contractile arrays containing bilateral and tri- or multicellular contact points are regulated in epithelia.Open in a separate windowFigure 1.ZO proteins and afadin regulate junctional tension and organization in cultured cells. (A) Untreated MDCK cells have sinuous cell boundaries, whereas ZO KD cells show extremely straight boundaries. When ZO proteins and afadin are knocked down, cells adopt contact zones of irregular length with other cells, sometimes clustering into foci (asterisks). Images courtesy of Mark Peifer (Choi et al., 2016). (B) A model for actomyosin organization at adherens junctions (adapted from Choi et al., 2016). Contractile actomyosin arrays run parallel to bicellular junctions and are anchored by side-on attachments (pink circles). At TCJs, end-on binding of actin, likely stabilized by afadin, anchors actomyosin filaments. In ZO KD cells, contractile elements and cadherin complexes collapse toward TCJs, and myosin minifilaments adopt a regularly spaced arrangement.Afadin and ZO-1/2 are far from new players at junctions. Afadin binds α-catenin, actin, and other cytoskeletal and junctional proteins and associates with the transmembrane protein nectin, which appears to form an alternative adhesion system at adherens junctions (Mandai et al., 2013). The zonula occludens proteins ZO-1 and ZO-2 are tight junction proteins that bind claudins and are required for tight junction formation (Itoh et al., 1999; Balda and Matter, 2008). In addition, ZO proteins also bind to α-catenin (Itoh et al., 1997), are involved in establishing the zonula adherens (ZA; Ikenouchi et al., 2007), and potentiate cadherin-dependent adhesion in Caenorhabditis elegans (Lockwood et al., 2008) and Drosophila melanogaster (Choi et al., 2011). Knockdown of ZO-1 and ZO-2 (ZO KD) in MDCK cells has previously been shown (Fanning et al., 2012) to lead to dramatic alterations of the ZA: F-actin and myosin IIs assemble into striking apical arrays at the ZA, spaced at regular intervals. In addition, the normally sinuous boundaries between cells give way to very straight borders (Fig. 1 A).Using superresolution microscopy, diffraction-limited junctional laser ablation, cell morphometry, kinetic analysis, and a whole-monolayer approach to contractility, Choi et al. (2016) now extend this story. To test whether contractility is increased after ZO KD, the authors first measured the recoil after laser ablation of ZO KD cells; an increase in recoil velocity indicated that the straight junctional boundaries between ZO-depleted cells are under tension. Imaging analysis of BCJs showed that the increase in contractility in ZO KD cells is associated with a strikingly dynamic behavior of the BCJs. Individual BCJs were found to undergo periods of shortening and elongation, whereas neighboring BCJs underwent compensatory, opposite changes in length. These changes in contractility have effects on the entire tissue sheet as well: whereas control cell sheets remained flat when detached from the substratum, ZO KD cells contracted into a cup-like shape. This constriction was blocked by the myosin inhibitor blebbistatin. Overall, these experiments indicated that ZO proteins regulate myosin assembly and contractility across the cellular sheet.To dissect the protein network mediating increased contractility in ZO KD cells, Choi et al. (2016) examined the role of ROCK and found that ROCK inhibitors abolished the straight BCJs, which became curvilinear. Additionally, Shroom3, which is known to recruit ROCK (Nishimura and Takeichi, 2008), was cytoplasmic in control cells but junctional in ZO KD cells. Transient Shroom3 overexpression led to ROCK recruitment to the ZA and drove formation of an actomyosin network similar to that in ZO KD cells. Conversely, Shroom3 knockdown resulted in loss of the actomyosin arrays in ZO KD cells. Collectively, these data indicated that Shroom3 is an effector of increased apical contractility in ZO KD cells.The researchers used ZO KD cells to test how tissue integrity is maintained despite elevated contractibility and how junctions are remodeled to maintain integrity when increased tension is present. Afadin is a good candidate: the Drosophila homologue of afadin, Canoe, plays roles in convergent extension and collective cell migration; in its absence, actomyosin networks at the apex of constricting epithelial cells in the embryo contract in a catastrophic, uncontrolled fashion (Sawyer et al., 2009), suggesting a potential role for afadin in the maintenance of tissue integrity during morphogenetic movements. Choi et al. (2016) therefore turned their attention to afadin. ZO KD cells have significantly more afadin at their adherens junctions and TCJs, a pattern reminiscent of the normal distribution of Canoe in Drosophila (Sawyer et al., 2009). Knocking down afadin by shRNA in ZO KD cells led to further defects in cell–cell boundary maintenance. In addition to the taut appearance of bicellular borders, cell boundary length became much more irregular, with occasional foci of highly contracted cells (Fig. 1 A). Velocimetry analysis and live-cell imaging indicated that loss of both ZO proteins and afadin led to large-scale cell movements within the monolayer not seen after ZO KD alone.New imaging techniques used by Choi et al. (2016) revealed further details about the changes in actomyosin arrays in ZO KD cells. Superresolution imaging of myosin light chain kinase staining via structured illumination showed that myosin II assembles into arrays of myosin minifilaments spaced ∼415 nm apart along bicellular contacts. Superresolution and transmission electron microscopy also revealed reorganization of F-actin and E-cadherin at TCJs in ZO KD cells. Lateral F-actin bundles appeared to terminate end-on at TCJs at sites where E-cadherin was present. ZO KD therefore induces assembly of a remarkably ordered actomyosin array along BCJs, and these arrays appear to be separate contractile units that anchor end-on at the ZA. Moreover, based on staining for vinculin and a specific epitope in αE-catenin that serve as markers for regions under high tension (Yonemura et al., 2010), the end-on attachments of actin cables to the ZA at TCJs experience significant tensile stress. Strikingly, although vinculin and αE-catenin accumulation at TCJs was relatively uniform after ZO KD, their distribution was more heterogeneous after ZO/afadin KD. Differences in staining paralleled differences in cell border length and correlated with the level of tension measured at BCJs after laser cutting, suggesting that afadin contributes to the ability of cells to distribute forces at TCJ/multicellular junctions throughout the monolayer. Lastly, the researchers investigated whether internal cues downstream of ZO KD are sufficient for myosin recruitment or whether such recruitment depends on mechanical cues exerted by neighboring cells. They designed an assay mixing small islands of wild-type cells surrounded by ZO KD cells (or vice versa) and found that the development of the contractile array at the ZA depends on the contractility of neighboring cells; however, afadin recruitment to the ZA was less dependent on the sustained contractility of neighboring cells. Taking these data together, Choi et al. (2016) propose that cells respond to elevated contractility by increasing junctional afadin; because combined ZO/afadin knockdown dramatically alters cell shape and barrier function in response to elevated contractility, afadin acts as a robust scaffold that maintains ZA architecture most crucially at TCJs.Although many aspects of the model proposed by Choi et al. (2016) remain to be tested, their data suggest new features regarding the detailed assembly of actomyosin contractile arrays in confluent cells (Fig. 1 B). In control cells, actomyosin arrays presumably extend parallel to individual BCJs. Choi et al. (2016) propose that these actomyosin bundles act as separate contractile units that terminate near TCJs, allowing the generation of tension along BCJs. In ZO KD cells, excessive assembly of actomyosin filaments, perhaps exacerbated by the tendency of F-actin/myosin minifilament arrays to self-assemble, somehow leads to regularly spaced actomyosin arrays, and perhaps collapse of cadherin complexes and other components toward TCJs. There is a precedent for such lateral collapse of cadherin-dependent attachments: it is a prominent feature of cadherin complexes at sites of high tension in the epidermis of the C. elegans embryo (Choi et al., 2015). If the new model of Choi et al. (2016) is correct, then the foci seen in ZO KD/afadin KD cells may be similar to what happens in a game of tug of war when one team stops pulling. If some end-on attachments (assisted by afadin) fail, filaments might be expected to collapse along BCJs as the other, still tethered end of a set of filaments contracts toward the remaining attachment at the opposite cell vertex.Several other interesting questions remain. First, what is the relationship of the striking, regularly spaced bipolar myosin II minifilaments that form in ZO KD cells to myosin arrays in normal cells? It is clear that untreated cells have junctional actomyosin networks, but not with this strict periodicity. One possibility is that this spacing is simply an epiphenomenon; when not appropriately anchored along junctions, actomyosin networks may self-organize as they are known to do in other systems, such as in the contractile ring and in migrating cells (Srivastava et al., 2015; Fenix et al., 2016). More optimistically, the spacing may represent an intensified version of processes that operate in normal cells at bicellular and multicellular contact sites. If so, components of the model of Choi et al. (2016) will require further investigation. For example, the organization of F-actin along BCJs remains unclear, as are the proteins that mediate the side-on binding envisioned in this model. It is also uncertain whether proteins assist bundling of filaments and what role dynamic growth and shrinkage of actin filaments plays in end-on binding. In some contexts, junctions are capable of seeding polymerization of F-actin (Brieher and Yap, 2013), and it may be that actin dynamics are important in the processes studied here.A second question has to do with the community events within monolayers that Choi et al. (2016) describe. The neighbor effects on ZA morphology that they document are intriguing, as are the long-range accelerated movements of cells lacking both ZO proteins and afadin. Collective properties of monolayers are only beginning to be explored; connecting these properties with subcellullar events is an exciting future challenge. Whatever the answers to these new questions, the work of Choi et al. (2016) refines our understanding of the roles of key scaffolding proteins in organizing and anchoring junctions in epithelia.     

Feeder cell density—A key parameter in human embryonic stem cell culture

Summary A key issue in human embryonic stem (ES) cell culture that has largely been ignored is the high degree of variability in the murine embryonic fibroblast (MEF) feeder cell density, which has been reported by different studies and protocols. Presumably, too low a feeder cell density would result in insufficient levels of secreted factors, extracellular matrix, and cellular contacts provided by the feeder cells for the maintenance of human ES cells in the undifferentiated state. Too high a feeder cell density, on the other hand, may result in a more rapid depletion of nutrients and oxygen within the in vitro culture milieu, as well as physically hinder the attachment and growth of ES colonies during serial passaging. Preliminary investigations by our group revealed that an elevated MEF cell density of 32,000 cells/cm², above the recommended value of 20,000 cells/cm², appeared to be highly detrimental to the attachment and growth of serially passaged ES colonies of the H9 line (WiCell Research Institute Inc., Wilmington, MA, USA). At the edge of ES colonies that have attached to the higher density feeder layer (32,000 cells/cm²), the ES cells appear to stack up to form a “bulge.” This was not observed under the recommended feeder cell density of 20,000 cells/cm². By contrast, other established ES cell lines are routinely propagated at much higher feeder densities of 60,000 to 70,000 cells/cm². This report briefly discusses the issue of MEF feeder cell density in relation to our preliminary observations, and the results of other studies.     

A cell–cell repulsion model on a hyperbolic Keller–Segel equation

Journal of Mathematical Biology - In this work, we discuss a cell–cell repulsion model based on a hyperbolic Keller–Segel equation with two populations, which aims at describing the...     

A second signal for autophagic cell death?

Dictyostelium cells in monolayers in vitro lend themselves well to a study of autophagic cell death (ACD). There is no apoptosis machinery in the protist Dictyostelium, no caspase nor Bcl-2 family members (except a paracaspase whose inactivation does not alter cell death), thus there is no apoptosis that could interfere with, or substitute for, non-apoptotic cell death. Also, Dictyostelium, a eukaryote, has a haploid genome, which facilitates random insertional mutagenesis.     

A role for Wnt/planar cell polarity signaling during lens fiber cell differentiation?

Wnt signaling through frizzled (Fz) receptors plays key roles in just about every developmental system that has been studied. Several Wnt-Fz signaling pathways have been identified including the Wnt/planar cell polarity (PCP) pathway. PCP signaling is crucial for many developmental processes that require major cytoskeletal rearrangements. Downstream of Fz, PCP signaling is thought to involve the GTPases, Rho, Rac and Cdc42 and regulation of the JNK cascade. Here we report on the localization of these GTPases and JNK in the lens and assess their involvement in the cytoskeletal reorganisation that is a key element of FGF-induced lens fiber cell differentiation.     

LewisX: A neural stem cell specific glycan?

LewisX (LeX) detecting antibodies are routinely used for cell sorting of neural stem- and progenitor cells (NSPCs). Applications include the enrichment of NSPCs after neural differentiation of human induced pluripotent- or embryonic stem cells, as well as their direct isolation from mouse neural tissue. Nevertheless, only little is known about the role of LeX in the central nervous system. Here we review the current knowledge on LeX-containing glycans expressed by neural stem cells and their progeny. New LeX-carrier proteins and ligands have recently been identified which reveal further insights into the potential function(s) of LeX-glycans. Moreover, evidence accumulates that individual LeX detecting antibody clones vary in their suitability as neural stem cell specific biomarker. Each antibody clone detects a unique LeX-containing glycan epitope. This allows a versatile utilization of anti-LeX antibodies that goes beyond neural stem cell sorting applications.     

KLN205—A murine lung carcinoma cell line

Summary KLN205 cells, a cloned cell line established from the Nettesheim lung carcinoma, grow in various synthetic media such as MEM, Fisher's or Roswell Park Memorial Institute Medium (RPMI) with the addition of 5 to 20% fetal bovine serum (FBS), calf serum (CS) or horse serum (HS). They grow optimally in minimum Eagle's medium plus nonessential amino acids (NEAA) plus 5 to 10% FBS or HS. The cells are transplantable to DBA/2, BDF₁, AKD₂F₁, and BALB/c, but not to C3H/He or ICR mice. The growth curves, plating efficiency, ultrastructural characteristics, modal number of chromosomes and transplantability to mice of various strains are almost the same for early and late passages of cells passaged in vitro. These parameters for 16th and 36th passages were: doubling time, 31 and 33 hr; plating efficiency, 12.4±1.2 and 14.6±2.6%; modal number of chromosomes, 73 and 76; lung colony formation in DBA/2, 50 and 45.9/mouse; and subcutaneous tumor diameter 24.5 and 27.4 mm, respectively. Only the numbers of lung colonies formed in BDF₁ mice were different: 24.4/mouse with 16th passage cells, and 10.2/mouse with 36th passage cells. The results suggest that KLN205 is a relatively stable cultured cell line through 36 passages. As was expected, immunosuppression by higher concentrations of triaminolone acetonide (TA) enhanced lung colony formation in BDF₁ mice. On the other hand, a low concentration of TA inhibited lung colony formation in DBA/2 mice, which was unexpected. These results suggest that KLN205 offers a model for investigations on metastases to lungs as well as chemotherapy for lung carcinoma. This work was supported by the National Cancer Institute of Canada.     

A germ cell origin of embryonic stem cells?

Because embryonic stem (ES) cells are generally derived by the culture of inner cell mass (ICM) cells, they are often assumed to be the equivalent of ICM cells. However, various evidence indicates that ICM cells transition to a different cell type during ES-cell derivation. Historically, ES cells have been believed to most closely resemble pluripotent primitive ectoderm cells derived directly from the ICM. However, differences between ES cells and primitive ectoderm cells have caused developmental biologists to question whether ES cells really have an in vivo equivalent, or whether their properties merely reflect their tissue culture environment. Here, we review recent evidence that the closest in vivo equivalent of an ES cell is an early germ cell.     

Planar cell polarity: A bridge too far?

The mechanisms of planar cell polarity are being revealed by genetic analysis. Recent studies have provided new insights into interactions between three proteins involved in planar cell polarity: Flamingo, Frizzled and Van Gogh.     

A genome-wide screen identifies conserved protein hubs required for 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.     

A Highlights from MBoC Selection: Spatial distribution of cell–cell and cell–ECM adhesions regulates force balance while main-taining E-cadherin molecular tension in cell pairs

Mechanical linkage between cell–cell and cell–extracellular matrix (ECM) adhesions regulates cell shape changes during embryonic development and tissue homoeostasis. We examined how the force balance between cell–cell and cell–ECM adhesions changes with cell spread area and aspect ratio in pairs of MDCK cells. We used ECM micropatterning to drive different cytoskeleton strain energy states and cell-generated traction forces and used a Förster resonance energy transfer tension biosensor to ask whether changes in forces across cell–cell junctions correlated with E-cadherin molecular tension. We found that continuous peripheral ECM adhesions resulted in increased cell–cell and cell–ECM forces with increasing spread area. In contrast, confining ECM adhesions to the distal ends of cell–cell pairs resulted in shorter junction lengths and constant cell–cell forces. Of interest, each cell within a cell pair generated higher strain energies than isolated single cells of the same spread area. Surprisingly, E-cadherin molecular tension remained constant regardless of changes in cell–cell forces and was evenly distributed along cell–cell junctions independent of cell spread area and total traction forces. Taken together, our results showed that cell pairs maintained constant E-cadherin molecular tension and regulated total forces relative to cell spread area and shape but independently of total focal adhesion area.     

Dihydroconiferyl alcohol — A cell division factor from Acer species

A compound that stimulated growth of soybean callus was isolated from spring sap of sycamore (Acer pseudoplatanus L.). Insufficient compound was isolated to permit it to be characterised. A compound with identical properties was isolated from commercial maple syrup, the concentrated spring sap of Acer saccharum L. The compound was identified as 3-(3-methoxy-4-hydroxyphenyl)-propan-1-ol (dihydroconiferyl alcohol, DCA). DCA was also active in the tobacco callus and radish leaf senescence assays, but was inactive in four other tests for cytokinin activity. DCA acted synergistically with kinetin to promote soybean callus growth. It is concluded that DCA has properties distinct from those of purine cytokinins.Abbreviation DCA dihydroconiferyl acohol - GC gas chromatography - IAA indole-3-acetic acid - iP isopentenyladenine - [9R]iP isopentenyladenosine - LC liquid chromatography - MS mass spectrometry - NAA 1-napthylacetic acid - TLC thinlayer chromatography - TMSi trimethylsilyl     

Engineering cell–cell signaling

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A risk-based approach for cell line development,manufacturing and characterization of genetically engineered,induced pluripotent stem cell–derived allogeneic cell therapies

Advances in cellular reprogramming and gene-editing approaches have opened up the potential for a new class of ex vivo cell therapies based on genetically engineered, induced pluripotent stem cell (iPSC)-derived allogeneic cells. While these new therapies share some similarities with their primary cell-derived autologous and allogeneic cell therapy predecessors, key differences exist in the processes used for generating genetically engineered, iPSC-derived allogeneic therapies. Specifically, in iPSC-derived allogeneic therapies, donor selection and gene-editing are performed once over the lifetime of the product as opposed to as part of the manufacturing of each product batch. The introduction of a well-characterized, fully modified, clonally derived master cell bank reduces risks that have been inherent to primary-cell derived autologous and allogeneic therapies. Current regulatory guidance, which was largely developed based on the learnings gained from earlier generation therapies, leaves open questions around considerations for donor eligibility, starting materials and critical components, cell banking and genetic stability. Here, a risk-based approach is proposed to address these considerations, while regulatory guidance continues to evolve.     

Role of AKAP 149–PKA–PDE4A complex in cell survival and cell differentiation processes

The cellular localization of A-kinase anchoring proteins (AKAPs), protein kinase A (PKAs) and phosphodiesterases (PDEs) is a key step to the spatiotemporal regulation of the second messenger adenosine 3′,5′-cyclic monophosphate (cAMP). In this paper the cellular distribution of the mitochondrial AKAP 149–PKA–PDE4A complex and its implications in the cell death induced by YTX treatment, a known PDE modulator, was studied. K-562 cell line was incubated with YTX for 24 or 48 h. Under these conditions AKAP 149, PKA and type-4A PDE (PDE4A) levels were measured in the cytosol, in the plasma membrane and in the nucleus. Apoptotic hallmarks were also measured after the same conditions. In addition, YTX effect on cell viability was checked after AKAP 149 and PDE4A silencing. The results obtained show a decrease in AKAP 149–PKA–PDE4A levels in cytosol after YTX exposure. 24 h after the toxin addition, the complex expression increased in the plasma membrane and after 48 h in the nucleus domain. Furthermore Bcl-2 levels were decreased and the expression of caspase 3 together with caspase 8 activity were increased after 24 h of toxin incubation but not after 48 h. These results suggest apoptotic cell death at 24 h and a non-apoptotic cell death after 48 h. When AKAP 149 and PDE4A were silenced YTX did not induce cellular death. In summary, AKAP 149–PKA–PDE4A complex localization is related with YTX effect in K-562 cell line. When this complex is mainly located in the plasma membrane apoptosis is activated while when the complex is in the nuclear domain non-apoptotic cellular death or cellular differentiation is activated. Therefore AKAP 149–PKA–PDE4A distribution and integrity have a key role in cellular survival.     

A “new” low incidence red cell antigen,NFLD

Summary A new low incidence red cell antigen, NFLD, is described. It was found in a Caucasian family and is inherited as an autosomal dominant. The antigen is not part of the AB0, MNSs, Duffy, Kidd, or Yt blood group systems and probably does not belong to the Rh or Kell blood group systems.     

Fucosyl-GM1 — A ganglioside associated with small cell lung carcinomas

Characterization of monosialogangliosides of a small cell lung carcinoma showed a unique composition. The tumour contained G_M2 and Fucosyl-G_M1 (Fuc-G_M1) with 2-hydroxy fatty acids as major ganglioside components. Three out of four other small cell carcinomas analysed contained also Fuc-G_M1 as a characteristic ganglioside. Fuc-G_M1 is suggested to be a small-cell lung carcinoma associated ganglioside antigen.Nomenclature: The gangliosides have been designated according to Svennerholm [25] G_M3 II³NeuAc-Lac-Cer - G_D3 II³(NeuAc)₂-LacCer - G_M2 II³NeuAc-GgOse₃Cer - G_M1 II³NeuAc-GgOse₄Cer - Fuc-G_M1 FuclV²Neu-AcII³-GgOse₄Cer - 3-L_M1 IV³NeuAc-nLcOse₄Cer - 6-L_M1 IV⁶NeuAc-nLcOse₄Cer