Translation of the cell adhesion molecule ALCAM in axonal growth cones - regulation and functional importance.

ALCAM is a cell adhesion molecule that is present on extending axons and has been shown to be crucial for elongation and navigation of retinal ganglion cell (RGC) axons. In the present study, we show that ALCAM mRNA is present in axonal growth cones of RGCs in vivo and in vitro, and that translation of ALCAM occurs in RGC growth cones separated from their soma. This growth cone translation is regulated by the 3'-untranslated region (3'-UTR) of ALCAM and depends on the activity of the kinases ERK and TOR (target of rapamycin). We also investigated the impact of the growth cone translation of ALCAM on axonal functions. Growth cone translation of ALCAM is crucial for the enhanced elongation of axons extending in contact with ALCAM protein. The local translation of ALCAM in the growth cone is able to rapidly counterbalance experimentally induced ALCAM internalization, thereby contributing to the maintenance of constant ALCAM levels in the plasma membrane. Assays where RGC axons have the choice to grow on laminin or both ALCAM and laminin - as is the case in the developing retina - reveal that the axonal preference for ALCAM-containing lanes depends on translation of ALCAM in growth cones. Taken together, these results show for the first time that translation of a cell adhesion molecule in growth cones, as well as the impact of this local translation on the behavior of axon and growth cone.

Nanopatterns biofunctionalized with cell adhesion molecule DM-GRASP offered as cell substrate: spacing determines attachment and differentiation of neurons.

The density/spacing of plasma membrane proteins is thought to be crucial for their function; clear-cut experimental evidence, however, is still rare. We examined nanopatterns biofunctionalized with cell adhesion molecule DM-GRASP with respect to their impact on neuron attachment and neurite growth. Data analysis/modeling revealed that these cellular responses improve with increasing DM-GRASP density, with the exception of one spacing which does not allow for the anchorage of a cytoskeletal protein (spectrin) to three DM-GRASP molecules.

Adenomatous polyposis coli is differentially distributed in growth cones and modulates their steering.

Axonal steering reactions depend on the transformation of environmental information into internal, directed structures, which is achieved by differential modulation of the growth cone cytoskeleton; key elements are the microtubules, which are regulated in their dynamics by microtubule-associated proteins (MAPs). We investigated a potential role of the MAP adenomatous polyposis coli (APC) for growing axons, employing embryonic visual system as a model system. APC is concentrated in the distalmost (i.e., growing) region of retinal ganglion cell axons in vivo and in vitro. Within the growth cone, APC is enriched in the central domain; it only partially colocalizes with microtubules. When axons are induced to turn toward a cell or away from a substrate border, APC is present in the protruding and absent from the collapsing growth cone regions, thus indicating the future growth direction of the axon. To assess the functional role of the differential distribution of APC in navigating growth cones, the protein was inactivated via micro-scale chromophore-assisted laser inactivation in one half of the growth cone. If the N-terminal APC region (crucial for its oligomerization) is locally inactivated, the treated growth cone side collapses and the axon turns away. In contrast, if the 20 aa repeats in the middle region of APC (which can negatively regulate its microtubule association) are inactivated, protrusions are formed and the growth cone turns toward. Our data thus demonstrate a crucial role of APC for axon steering attributable to its multifunctional domain structure and differential distribution in the growth cone.

Cell adhesion molecule DM-GRASP presented as nanopatterns to neurons regulates attachment and neurite growth.

Adhesion and neurite formation of neurons and neuroblastoma cells critically depends on the lateral spacing of the cell adhesion molecule DM-GRASP offered as nanostructured substrate.

Accumulation of N-CAM 180 at Contact Sites Between Neuroblastoma Cells and Latex Beads Coated with Extracellular Matrix Molecules.

Neuronal cells expressing neural cell adhesion molecule (N-CAM) accumulate the largest N-CAM component (N-CAM 180) at cell - cell contact sites. To test whether this accumulation is induced by interactions at the surface membrane, latex beads coated with several purified adhesion molecules or extracellular matrix (ECM) components were co-cultured with neuroblastoma cells. Beads coated with L1, N-CAM, the L2/HNK-1 carbohydrate epitope-carrying molecules from adult mouse brain or laminin from Engelbreth-Holm-Swarm (EHS) sarcoma did not induce an accumulation of N-CAM 180 or L1 at sites of contact suggesting that these molecules are not directly involved in N-CAM 180 accumulation or that their mobility is required for this process. Beads coated with ECM components of the PF-HR9 cell line induced accumulation of N-CAM 180 at sites of contact with neuroblastoma cells. Accumulation was seen at cell bodies of undifferentiated and differentiated neuroblastoma cells, as well as on neurites and growth cones of differentiated neuroblastoma cells. Accumulation of the neural adhesion molecule L1 was also seen, but less prominently and reproducibly. These observations suggest that molecules of the ECM can directly or indirectly, e.g. via molecules linked to N-CAM 180 on the cell surface, induce accumulation of N-CAM 180.

Lateral Mobility of the Cell Adhesion Molecule L1 Within the Surface Membrane of Morphologically Undifferentiated and Differentiated Neuroblastoma Cells.

Lateral mobility and localization in the surface membrane of the adhesion molecule L1 was studied in morphologically undifferentiated and differentiated neuroblastoma cells to gain insight into its possible association with the different molecular forms of N-CAM. In undifferentiated cells, the fraction of mobile L1 molecules is high and similar to that of N-CAM 140. Upon long-term morphological differentiation, the fraction of mobile L1 molecules is reduced by a factor of three and is similar to that of N-CAM 180, the predominant molecular form of N-CAM in differentiated neuroblastoma cells. Comparable to N-CAM 180, L1 is also preferentially accumulated at contact sites between these cells as seen by indirect immunofluorescence. These observations raise the question of whether at least part of the L1 molecules may be directly or indirectly (e.g. via N-CAM 180) linked to the cytoskeleton, thus stabilizing cell contacts between differentiated cells.

Depending on its nano-spacing, ALCAM promotes cell attachment and axon growth.

ALCAM is a member of the cell adhesion molecule (CAM) family which plays an important role during nervous system formation. We here show that the two neuron populations of developing dorsal root ganglia (DRG) display ALCAM transiently on centrally and peripherally projecting axons during the two phases of axon outgrowth. To analyze the impact of ALCAM on cell adhesion and axon growth, DRG single cells were cultured on ALCAM-coated coverslips or on nanopatterns where ALCAM is presented in physiological amino-carboxyl terminal orientation at highly defined distances (29, 54, 70, 86, and 137 nm) and where the interspaces are passivated to prevent unspecific protein deposition. Some axonal features (branching, lateral deviation) showed density dependence whereas others (number of axons per neuron, various axon growth parameters) turned out to be an all-or-nothing reaction. Time-lapse analyses revealed that ALCAM density has an impact on axon velocity and advance efficiency. The behavior of the sensory axon tip, the growth cone, partially depended on ALCAM density in a dose-response fashion (shape, dynamics, detachment) while other features did not (size, complexity). Whereas axon growth was equally promoted whether ALCAM was presented at high (29 nm) or low densities (86 nm), the attachment of non-neuronal cells depended on high ALCAM densities. The attachment of non-neuronal cells to the rather unspecific standard proteins presented by conventional implants designed to enhance axonal regeneration is a severe problem. Our findings point to ALCAM, presented as 86 nm pattern, for a promising candidate for the improvement of such implants since this pattern drives axon growth to its full extent while at the same time non-neuronal cell attachment is clearly reduced.

Ubiquitination and endocytosis of cell adhesion molecule DM-GRASP regulate its cell surface presence and affect its role for axon navigation.

DM-GRASP, cell adhesion molecule of the immunoglobulin superfamily, has been shown to promote growth and navigation of axons. We here demonstrate that clustering of DM-GRASP in the plasma membrane induces its rapid internalization via dynamin- and clathrin-dependent endocytosis, which is controlled by phosphatidylinositol 3-kinase and mitogen-activated protein kinase ERK. The clustering of DM-GRASP activates ERK; the intensity and duration of ERK activation by DM-GRASP do not depend on rapid clathrin-mediated internalization of DM-GRASP. Moreover, the preference of retinal ganglion cell axons for DM-GRASP-coated micro-lanes requires clathrin-mediated endocytosis for the appropriate axonal turning reactions at substrate borders. Because the intracellular domain of DM-GRASP does not contain motifs for direct interactions with the endocytosis machinery, we performed a yeast two-hybrid screen to identify intracellular proteins mediating the uptake of DM-GRASP and isolated ubiquitin. Immunoprecipitation of DM-GRASP coexpressed with ubiquitin revealed that one or two ubiquitin(s) are attached to the intracellular domain of cell surface-resident DM-GRASP. Furthermore, elevated ubiquitination levels result in a decrease of cell surface-resident DM-GRASP as well as in the amount of total DM-GRASP. The endocytosis rate is not affected, but the delivery to multivesicular bodies is increased, indicating that DM-GRASP ubiquitination enhances its sorting into the degradation pathway. Together, our data show that ubiquitination and endocytosis of DM-GRASP in concert regulate its cell surface concentration, which is crucial for its function in axon navigation.

The cell adhesion molecule NrCAM is crucial for growth cone behaviour and pathfinding of retinal ganglion cell axons.

We investigated the role of the cell adhesion molecule NrCAM for axonal growth and pathfinding in the developing retina. Analysis of the distribution pattern of NrCAM in chick embryo retina sections and flat-mounts shows its presence during extension of retinal ganglion cell (RGC) axons; NrCAM is selectively present on RGC axons and is absent from the soma. Single cell cultures show an enrichment of NrCAM in the distal axon and growth cone. When offered as a substrate in addition to Laminin, NrCAM promotes RGC axon extension and the formation of growth cone protrusions. In substrate stripe assays, mimicking the NrCAM-displaying optic fibre layer and the Laminin-rich basal lamina, RGC axons preferentially grow on NrCAM lanes. The three-dimensional analysis of RGC growth cones in retina flat-mounts reveals that they are enlarged and form more protrusions extending away from the correct pathway under conditions of NrCAM-inhibition. Time-lapse analyses show that these growth cones pause longer to explore their environment, proceed for shorter time spans, and retract more often than under control conditions; in addition, they often deviate from the correct pathway towards the optic fissure. Inhibition of NrCAM in organ-cultured intact eyes causes RGC axons to misroute at the optic fissure; instead of diving into the optic nerve head, these axons cross onto the opposite side of the retina. Our results demonstrate a crucial role for NrCAM in the navigation of RGC axons in the developing retina towards the optic fissure, and also for pathfinding into the optic nerve.

Upstream AUGs in embryonic proinsulin mRNA control its low translation level.

Proinsulin is expressed prior to development of the pancreas and promotes cell survival. Here we study the mechanism affecting the translation efficiency of a specific embryonic proinsulin mRNA. This transcript shares the coding region with the pancreatic form, but presents a 32 nt extended leader region. Translation of proinsulin is markedly reduced by the presence of two upstream AUGs within the 5' extension of the embryonic mRNA. This attenuation is lost when the two upstream AUGs are mutated to AAG, leading to translational efficiency similar to that of the pancreatic mRNA. The upstream AUGs are recognized as initiator codons, because expression of upstream ORF is detectable from the embryonic transcript, but not from the mutated or the pancreatic mRNAs. Strict regulation of proinsulin biosynthesis appears to be necessary, since exogenous proinsulin added to embryos in ovo decreased apoptosis and generated abnormal developmental traits. A novel mechanism for low level proinsulin expression thus relies on upstream AUGs within a specific form of embryonic proinsulin mRNA, emphasizing its importance as a tightly regulated developmental signal.