Christine J. McNamee

Co-localisation, heterophilic interactions and regulated expression of IgLON family proteins in the chick nervous system.

The chick glycoprotein GP55 has been shown to inhibit the growth and adhesion of DRG and forebrain neurons. GP55 consists of several members of the IgLON family, a group of glycoproteins including LAMP, OBCAM, CEPU-1 (chick)/neurotrimin (rat) and neurotractin (chick)/kilon (rat) thought to play a role in the guidance of growing axons. IgLONs belong to the Ig superfamily and have three C2 domains and a glycosyl phosphatidylinositol anchor which tethers them to the neuronal plasma membrane. We have now completed the deduced amino acid sequence for two isoforms of chicken OBCAM and used recombinant LAMP, OBCAM and CEPU-1 to raise antisera specific to these three IgLONs. LAMP and CEPU-1 are co-expressed on DRG and sympathetic neurons, while both overlapping and distinct expression patterns for LAMP, OBCAM and CEPU-1 are observed in retina. Analysis of IgLON mRNA expression reveals that alternatively spliced forms of LAMP and CEPU-1 are developmentally regulated. In an attempt to understand how the IgLONs function, we have begun to characterise their molecular interactions. LAMP and CEPU-1 have already been shown to interact homophilically. We now confirm that OBCAM will bind homophilically and also that LAMP, OBCAM and CEPU-1 will interact heterophilically with each other. We propose that IgLON activity will depend on the complement of IgLONs expressed by each neuron.

Diglons are heterodimeric proteins composed of IgLON subunits, and Diglon-CO inhibits neurite outgrowth from cerebellar granule cells

IgLONs are a family of four cell adhesion molecules belonging to the Ig superfamily that are thought to play a role in cell-cell recognition and growth-cone migration. One member of the family, opioid-binding cell-adhesion molecule (OBCAM), might act as a tumour suppressor. Previous work has shown that limbic-system-associated protein (LAMP), CEPU-1/Neurotrimin and OBCAM interact homophilically and heterophilically within the family. Here, we show that, based on their relative affinities, CEPU-1 might be both a homo- and a heterophilic cell adhesion molecule, whereas LAMP and OBCAM act only as heterophilic cell adhesion molecules. A binding assay using recombinant IgLONs fused to human Fc showed that IgLONs are organized in the plane of the membrane as heterodimers, and we propose that IgLONs function predominantly as subunits of heterodimeric proteins (Diglons). Thus, the four IgLONs can form six Diglons. Furthermore, although singly transfected cell lines have little effect on neurite outgrowth, CHO cell lines expressing both CEPU-1 and OBCAM (Diglon-CO) inhibit neurite outgrowth from cerebellar granule cells.

Promotion of neuronal cell adhesion by members of the IgLON family occurs in the absence of either support or modification of neurite outgrowth

The IgLONs are a family of glycosyl phosphatidyl inositol‐linked cell adhesion molecules which are thought to modify neurite outgrowth and may play a role in cell–cell recognition. The family consists of LAMP, OBCAM, neurotrimin/CEPU‐1 and neurotractin/kilon. In this paper we report the effect of recombinant LAMP, CEPU‐1 and OBCAM, and transfected cell lines expressing these molecules, on the adhesion and outgrowth of dorsal root ganglion (DRG) and sympathetic neurones. CHO cells transfected with cDNA for CEPU‐1 adhered to a recombinant CEPU‐1‐Fc substrate. However, DRG or sympathetic neurones only adhered to CEPU‐1‐Fc when presented on protein A. Although DRG and sympathetic neurones express IgLONs on their surface, both types of neurones exhibited differential adhesion to CEPU‐1‐Fc, LAMP‐Fc and OBCAM‐Fc. Neither DRG nor sympathetic neurones extended neurites on a protein A/IgLON‐Fc substrate and overexpression of CEPU‐1‐GFP in DRG neurones also failed to stimulate neurite outgrowth on an IgLON‐Fc substrate. DRG neurones adhered to and extended neurites equally on transfected and non‐transfected cell lines and the recombinant proteins did not modulate the outgrowth of neurones on laminin. In contrast to previous reports we suggest that IgLONs may not have a primary role in axon guidance but may be more important for cell–cell adhesion and recognition.

Identification and Characterization of CEPU-Se—A Secreted Isoform of the IgLON Family Protein, CEPU-1

CEPU-1/Neurotrimin is a neuronal glycoprotein thought to play a role in axon guidance and cell-cell recognition. It is a member of the IgLON family, has three C2 domains, and is attached to the plasma membrane by a GPI-anchor. We report here the characterisation of an alternatively-spliced isoform of CEPU-1 that is secreted. This isoform, termed CEPU-Se, is coexpressed with CEPU-1 in retina, cerebellum, and DRG neurons. In the cerebellum CEPU-1/CEPU-Se is expressed predominantly on granule cells and in the molecular layer. Divalent but not monovalent CEPU-Se interacts with CEPU-1 and other IgLONs, suggesting that the ability of CEPU-Se to modify the activity of the IgLON family may require an additional cofactor. CEPU-Se does not support the outgrowth of DRG neurons or the extension of established growth cones; however, neurite outgrowth on laminin is unaffected by CEPU-Se. Our data suggest that CEPU-Se may act to modulate the ability of CEPU-1, LAMP, and OBCAM to influence neurite outgrowth.

Cell shape-dependent Control of Ca2+ influx and cell cycle progression in Swiss 3T3 fibroblasts.

The ability of adherent cells such as fibroblasts to enter the cell cycle and progress to S phase is strictly dependent on the extent to which individual cells can attach to and spread on a substratum. Here we have used microengineered adhesive islands of 22 and 45 μm diameter surrounded by a nonadhesive substratum of polyhydroxyl methacrylate to accurately control the extent to which individual Swiss 3T3 fibroblasts may spread. The effect of cell shape on mitogen-evoked Ca2+ signaling events that accompany entry into the cell cycle was investigated. In unrestricted cells, the mitogens bombesin and fetal calf serum evoked a typical biphasic change in the cytoplasmic free Ca2+ concentration. However, when the spreading of individual cells was restricted, such that progression to S phase was substantially reduced, both bombesin and fetal calf serum caused a rapid transient rise in the cytoplasmic free Ca2+ concentration but failed to elicit the normal sustained influx of Ca2+ that follows Ca2+ release. As expected, restricting cell spreading led to the loss of actin stress fibers and the formation of a ring of cortical actin. Restricting cell shape did not appear to influence mitogen-receptor interactions, nor did it influence the presence of focal adhesions. Because Ca2+ signaling is an essential component of mitogen responses, these findings implicate Ca2+ influx as a necessary component of cell shape-dependent control of the cell cycle.

DIgLONs inhibit initiation of neurite outgrowth from forebrain neurons via an IgLON-containing receptor complex.

IgLONs are a family of four GPI-anchored cell adhesion molecules that regulate neurite outgrowth, synaptogenesis and may act as tumour suppressor genes. IgLONs are thought to function as monomers or homodimers and we have proposed that IgLONs also act as heterodimeric complexes termed Dimeric IgLONs or DIgLONs. Here we show that the initiation of neurite outgrowth is inhibited from a subset of chick embryonic day (E) 7 or 8 forebrain neurons when they are cultured on CHO cell lines expressing DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP but not on CHO cells that express single IgLONs CEPU-1 or OBCAM. Surprisingly at the younger age of E6 forebrain neurons do not respond to DIgLONs. Since there is little difference in expression of IgLONs on the surface of chick forebrain neurons at these two ages we suggest IgLONs alone are not the receptor on the responding forebrain neurons. A DIgLON heterodimeric recombinant protein DIgLON:CEPU-1-OBCAM-Fc also blocked neurite outgrowth from E8 chick forebrain neurons. However, when IgLONs were removed from the surface of these E8 neurons they no longer responded to DIgLON:CEPU-1-OBCAM-Fc substrate, indicating that IgLONs form at least a component of the neuronal cell receptor complex involved in this inhibition of neurite outgrowth. Inhibitors pertussis toxin and Y27632 reversed the inhibition of neurite outgrowth on a DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP substrate. This suggests the involvement of a G-protein coupled receptor and activation of Rho A. In summary we provide evidence that DIgLON:CEPU-1-OBCAM and DIgLON:CEPU-1-LAMP complexes regulate initiation of neurite outgrowth on forebrain neurons via an IgLON-containing receptor complex.

Cell cycle‐dependent morphological changes in the actin cytoskeleton induced by agents which elevate cyclic AMP

Agents which increase the intracellular concentration of cyclic adenosine‐5′‐monophosphate, induce a highly arborised morphology in a proportion of sub‐confluent Swiss 3T3 fibroblasts. During this process the organisation of actin filaments progessively changes from a characteristic stress fibre pattern to leave a network of actin filaments within each and every arborisation. Despite this massive reorganisation of the actin cytoskeleton no changes are observed in the extent of polymerisation of actin during arborisation. The proportion of cells in asynchronous cultures undergoing arborisation at maximal concentrations of agents reaches a maximum of 30%; suggesting that the effect might be mediated only in cells during a restricted period of the cell cycle. More than 80% of serum‐starved cells responded to these agents between 1 and 8 hours after readdition of serum, but not at other times, suggesting that the arborisation response can occur only in the G1 phase of the cell cycle.

IgLONs form heterodimeric complexes on forebrain neurons

IgLONs are a family of four GPI‐anchored cell adhesion molecules that regulate neurite outgrowth and synaptogenesis and may act as tumour suppressor genes. Recently we have proposed that two members of the IgLON family act as a heterodimeric complex termed DIgLON. Neurons isolated from chick forebrain co‐express all six combinations of IgLONs and the intensity of fluorescence for each pair of IgLONs was highly correlated. Antibody‐patching experiments on forebrain neurons show complex formation for IgLON pairs but not between unrelated GPI‐anchored glycoproteins. Thus IgLONs are the first GPI‐anchored family of glycoproteins shown to form heterodimeric complexes in the plane of the membrane. Copyright

Defining drug response for stratified medicine

The premise for stratified medicine is that drug efficacy, drug safety, or both, vary between groups of patients, and biomarkers can be used to facilitate more targeted prescribing, with the aim of improving the benefit:risk ratio of treatment. However, many factors can contribute to the variability in response to drug treatment. Inadequate characterisation of the nature and degree of variability can lead to the identification of biomarkers that have limited utility in clinical settings. Here, we discuss the complexities associated with the investigation of variability in drug efficacy and drug safety, and how consideration of these issues a priori, together with standardisation of phenotypes, can increase both the efficiency of stratification procedures and identification of biomarkers with the potential for clinical impact.

No barrier to diffusion between cell soma and neurite membranes in sympathetic neurons for a GPI-anchored glycoprotein

As neurons extend their axons, it is thought that newly synthesised membrane components travel in vesicles along the axon, fuse with the growth cone membrane, and diffuse back along the axonal membrane. However, it is difficult to explain how axons continue to be populated with membrane proteins as they extend in length. To investigate this problem, we have used a CEPU-green fluorescent protein (GFP) chimeric protein to study the site of insertion of new glycosyl phosphatidyl inositol (GPI)-anchored glycoproteins and their subsequent behaviour in chick dorsal root ganglia (DRG) neurons. Infection of cultures grown for 24 h revealed rapid expression of CEPU-GFP over the whole surface of the neuron, more rapidly than could be accounted for by diffusion from the growth cone, and fluorescence intensity was uniform along the length of the neurite. Photobleaching experiments of neurite membrane revealed that recovery of fluorescence was due to diffusion from adjacent membranes and there was no evidence for membrane flow in either direction. Photobleaching of membrane adjacent to the cell body also showed rapid recovery, with chimera diffusing both from cell body membrane and the distal neurite membrane into the bleached area. These results suggest there is no barrier to diffusion between the cell body and neurite membrane in DRG and sympathetic neurons cultured for 1 or 2 days in vitro. We propose that the neurite is populated by newly synthesised chimera by diffusion from both regions. This situation may also occur in neurons in the early stages of extending axons in vivo prior to polarisation and the development of the dendritic field.