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Schlemm's canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm's canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6 J SC, blood vessel, and lymphatic endothelial cells from limbal tissue (~4,500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6 J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize three molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics., Competing Interests: RB, KK, TL, NT, AB, GC, VB, RK, SZ, JP, MS, CM, WS, JQ, SJ No competing interests declared, (© 2024, Balasubramanian, Kizhatil et al.)

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

Chronically elevated intraocular pressure (IOP) is the major risk factor of primary open-angle glaucoma, a leading cause of blindness. Dysfunction of the trabecular meshwork (TM), which controls the outflow of aqueous humor (AqH) from the anterior chamber, is the major cause of elevated IOP. Here, we demonstrate that mice deficient in the Krüppel-like zinc finger transcriptional factor GLI-similar-1 (GLIS1) develop chronically elevated IOP. Magnetic resonance imaging and histopathological analysis reveal that deficiency in GLIS1 expression induces progressive degeneration of the TM, leading to inefficient AqH drainage from the anterior chamber and elevated IOP. Transcriptome and cistrome analyses identified several glaucoma- and extracellular matrix-associated genes as direct transcriptional targets of GLIS1. We also identified a significant association between GLIS1 variant rs941125 and glaucoma in humans (P = 4.73 × 10 -6 ), further supporting a role for GLIS1 into glaucoma etiology. Our study identifies GLIS1 as a critical regulator of TM function and maintenance, AqH dynamics, and IOP., (© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Variants in the LIM homeobox transcription factor 1-beta ( LMX1B ) gene predispose individuals to elevated intraocular pressure (IOP), a key risk factor for glaucoma. However, the effect of LMX1B mutations varies widely between individuals. To better understand the mechanisms underlying LMX1B-related phenotypes and individual differences, we backcrossed the Lmx1b V265D (also known as Lmx1b Icst ) allele onto the C57BL/6J (B6), 129/Sj (129), C3A/BLiA- Pde6b + /J (C3H) and DBA/2J- Gpnmb + (D2-G) mouse strain backgrounds. Strain background had a significant effect on the onset and severity of ocular phenotypes in Lmx1b V265D/+ mutant mice. Mice of the B6 background were the most susceptible to developing abnormal IOP distribution, severe anterior segment developmental anomalies (including malformed eccentric pupils, iridocorneal strands and corneal abnormalities) and glaucomatous nerve damage. By contrast, Lmx1b V265D mice of the 129 background were the most resistant to developing anterior segment abnormalities, had less severe IOP elevation than B6 mutants at young ages and showed no detectable nerve damage. To identify genetic modifiers of susceptibility to Lmx1b V265D -induced glaucoma-associated phenotypes, we performed a mapping cross between mice of the B6 (susceptible) and 129 (resistant) backgrounds. We identified a modifier locus on Chromosome 18, with the 129 allele(s) substantially lessening severity of ocular phenotypes, as confirmed by congenic analysis. By demonstrating a clear effect of genetic background in modulating Lmx1b -induced phenotypes, providing a panel of strains with different phenotypic severities and identifying a modifier locus, this study lays a foundation for better understanding the roles of LMX1B in glaucoma with the goal of developing new treatments., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Background: Glaucoma is characterized by the progressive dysfunction and loss of retinal ganglion cells. Recent work in animal models suggests that a critical neuroinflammatory event damages retinal ganglion cell axons in the optic nerve head during ocular hypertensive injury. We previously demonstrated that monocyte-like cells enter the optic nerve head in an ocular hypertensive mouse model of glaucoma (DBA/2 J), but their roles, if any, in mediating axon damage remain unclear., Methods: To understand the function of these infiltrating monocyte-like cells, we used RNA-sequencing to profile their transcriptomes. Based on their pro-inflammatory molecular signatures, we hypothesized and confirmed that monocyte-platelet interactions occur in glaucomatous tissue. Furthermore, to test monocyte function we used two approaches to inhibit their entry into the optic nerve head: (1) treatment with DS-SILY, a peptidoglycan that acts as a barrier to platelet adhesion to the vessel wall and to monocytes, and (2) genetic targeting of Itgam (CD11b, an immune cell receptor that enables immune cell extravasation)., Results: Monocyte specific RNA-sequencing identified novel neuroinflammatory pathways early in glaucoma pathogenesis. Targeting these processes pharmacologically (DS-SILY) or genetically (Itgam / CD11b knockout) reduced monocyte entry and provided neuroprotection in DBA/2 J eyes., Conclusions: These data demonstrate a key role of monocyte-like cell extravasation in glaucoma and demonstrate that modulating neuroinflammatory processes can significantly lessen optic nerve injury.

Primary congenital glaucoma (PCG) is a leading cause of blindness in children worldwide and is caused by developmental defects in 2 aqueous humor outflow structures, Schlemm's canal (SC) and the trabecular meshwork. We previously identified loss-of-function mutations in the angiopoietin (ANGPT) receptor TEK in families with PCG and showed that ANGPT/TEK signaling is essential for SC development. Here, we describe roles for the major ANGPT ligands in the development of the aqueous outflow pathway. We determined that ANGPT1 is essential for SC development, and that Angpt1-knockout mice form a severely hypomorphic canal with elevated intraocular pressure. By contrast, ANGPT2 was dispensable, although mice deficient in both Angpt1 and Angpt2 completely lacked SC, indicating that ANGPT2 compensates for the loss of ANGPT1. In addition, we identified 3 human subjects with rare ANGPT1 variants within an international cohort of 284 PCG patients. Loss of function in 2 of the 3 patient alleles was observed by functional analysis of ANGPT1 variants in a combined in silico, in vitro, and in vivo approach, supporting a causative role for ANGPT1 in disease. By linking ANGPT1 with PCG, these results highlight the importance of ANGPT/TEK signaling in glaucoma pathogenesis and identify a candidate target for therapeutic development.

Purpose: The molecular mechanisms controlling aqueous humor (AQH) outflow and IOP need much further definition. The mouse is a powerful system for characterizing the mechanistic basis of AQH outflow. To enhance outflow studies in mice, we developed a perfusion system that is based on human anterior chamber perfusion culture systems. Our mouse system permits previously impractical experiments., Methods: We engineered a computer-controlled, pump-based perfusion system with a platform for mounting whole dissected mouse eyes (minus lens and iris, ∼45% of drainage tissue is perfused). We tested the system's ability to monitor outflow and tested the effects of the outflow-elevating drug, Y27632, a rho-associated protein kinase (ROCK) inhibitor. Finally, we tested the system's ability to detect genetically determined decreases in outflow by determining if deficiency of the candidate genes Nos3 and Cav1 alter outflow., Results: Using our system, the outflow facility (C) of C57BL/6J mouse eyes was found to range between 7.7 and 10.4 nl/minutes/mm Hg (corrected for whole eye). Our system readily detected a 74.4% Y27632-induced increase in C. The NOS3 inhibitor L-NG-nitroarginine methyl ester (L-NAME) and a Nos3 null mutation reduced C by 28.3% and 35.8%, respectively. Similarly, in Cav1 null eyes C was reduced by 47.8%., Conclusions: We engineered a unique perfusion system that can accurately measure changes in C. We then used the system to show that NOS3 and CAV1 are key components of mechanism(s) controlling outflow.

Primary congenital glaucoma (PCG) is a devastating eye disease and an important cause of childhood blindness worldwide. In PCG, defects in the anterior chamber aqueous humor outflow structures of the eye result in elevated intraocular pressure (IOP); however, the genes and molecular mechanisms involved in the etiology of these defects have not been fully characterized. Previously, we observed PCG-like phenotypes in transgenic mice that lack functional angiopoietin-TEK signaling. Herein, we identified rare TEK variants in 10 of 189 unrelated PCG families and demonstrated that each mutation results in haploinsufficiency due to protein loss of function. Multiple cellular mechanisms were responsible for the loss of protein function resulting from individual TEK variants, including an absence of normal protein production, protein aggregate formation, enhanced proteasomal degradation, altered subcellular localization, and reduced responsiveness to ligand stimulation. Further, in mice, hemizygosity for Tek led to the formation of severely hypomorphic Schlemm's canal and trabecular meshwork, as well as elevated IOP, demonstrating that anterior chamber vascular development is sensitive to Tek gene dosage and the resulting decrease in angiopoietin-TEK signaling. Collectively, these results identify TEK mutations in patients with PCG that likely underlie disease and are transmitted in an autosomal dominant pattern with variable expressivity.

Rationale: The formation of the blood vasculature is achieved via 2 fundamentally different mechanisms, de novo formation of vessels from endothelial progenitors (vasculogenesis) and sprouting of vessels from pre-existing ones (angiogenesis). In contrast, mammalian lymphatic vasculature is thought to form exclusively by sprouting from embryonic veins (lymphangiogenesis). Alternative nonvenous sources of lymphatic endothelial cells have been suggested in chicken and Xenopus, but it is unclear whether they exist in mammals., Objective: We aimed to clarify the origin of the murine dermal lymphatic vasculature., Methods and Results: We performed lineage tracing experiments and analyzed mutants lacking the Prox1 transcription factor, a master regulator of lymphatic endothelial cell identity, in Tie2 lineage venous-derived lymphatic endothelial cells. We show that, contrary to current dogma, a significant part of the dermal lymphatic vasculature forms independently of sprouting from veins. Although lymphatic vessels of cervical and thoracic skin develop via sprouting from venous-derived lymph sacs, vessels of lumbar and dorsal midline skin form via assembly of non-Tie2-lineage cells into clusters and vessels through a process defined as lymphvasculogenesis., Conclusions: Our results demonstrate a significant contribution of nonvenous-derived cells to the dermal lymphatic vasculature. Demonstration of a previously unknown lymphatic endothelial cell progenitor population will now allow further characterization of their origin, identity, and functions during normal lymphatic development and in pathology, as well as their potential therapeutic use for lymphatic regeneration., (© 2015 American Heart Association, Inc.)

Schlemm's canal (SC) plays central roles in ocular physiology. These roles depend on the molecular phenotypes of SC endothelial cells (SECs). Both the specific phenotype of SECs and development of SC remain poorly defined. To allow a modern and extensive analysis of SC and its origins, we developed a new whole-mount procedure to visualize its development in the context of surrounding tissues. We then applied genetic lineage tracing, specific-fluorescent reporter genes, immunofluorescence, high-resolution confocal microscopy, and three-dimensional (3D) rendering to study SC. Using these techniques, we show that SECs have a unique phenotype that is a blend of both blood and lymphatic endothelial cell phenotypes. By analyzing whole mounts of postnatal mouse eyes progressively to adulthood, we show that SC develops from blood vessels through a newly discovered process that we name "canalogenesis." Functional inhibition of KDR (VEGFR2), a critical receptor in initiating angiogenesis, shows that this receptor is required during canalogenesis. Unlike angiogenesis and similar to stages of vasculogenesis, during canalogenesis tip cells divide and form branched chains prior to vessel formation. Differing from both angiogenesis and vasculogenesis, during canalogenesis SECs express Prox1, a master regulator of lymphangiogenesis and lymphatic phenotypes. Thus, SC development resembles a blend of vascular developmental programs. These advances define SC as a unique vessel with a combination of blood vascular and lymphatic phenotypes. They are important for dissecting its functions that are essential for ocular health and normal vision., Competing Interests: The authors have declared that no competing interests exist.

Costameres are cellular sites of mechanotransduction in heart and skeletal muscle where dystrophin and its membrane-spanning partner dystroglycan distribute intracellular contractile forces into the surrounding extracellular matrix. Resolution of a functional costamere interactome is still limited but likely to be critical for understanding forms of muscular dystrophy and cardiomyopathy. Dystrophin binds a set of membrane-associated proteins (the dystrophin-glycoprotein complex) as well as γ-actin and microtubules and also is required to align sarcolemmal microtubules with costameres. Ankyrin-B binds to dystrophin, dynactin-4, and microtubules and is required for sarcolemmal association of these proteins as well as dystroglycan. We report here that ankyrin-B interactions with β2 spectrin and dynactin-4 are required for localization of dystrophin, dystroglycan, and microtubules at costameres as well as protection of muscle from exercise-induced injury. Knockdown of dynactin-4 in adult mouse skeletal muscle phenocopied depletion of ankyrin-B and resulted in loss of sarcolemmal dystrophin, dystroglycan, and microtubules. Moreover, mutations of ankyrin-B and of dynactin-4 that selectively impaired binary interactions between these proteins resulted in loss of their costamere-localizing activity and increased muscle fiber fragility as a result of loss of costamere-associated dystrophin and dystroglycan. In addition, costamere-association of dynactin-4 did not require dystrophin but did depend on β2 spectrin and ankyrin-B, whereas costamere association of ankyrin-B required β2 spectrin. Together, these results are consistent with a functional hierarchy beginning with β2 spectrin recruitment of ankyrin-B to costameres. Ankyrin-B then interacts with dynactin-4 and dystrophin, whereas dynactin-4 collaborates with dystrophin in coordinating costamere-aligned microtubules.

Cyclic nucleotide-gated (CNG) channels localize exclusively to the plasma membrane of photosensitive outer segments of rod photoreceptors where they generate the electrical response to light. Here, we report the finding that targeting of CNG channels to the rod outer segment required their interaction with ankyrin-G. Ankyrin-G localized exclusively to rod outer segments, coimmunoprecipitated with the CNG channel, and bound to the C-terminal domain of the channel beta1 subunit. Ankyrin-G depletion in neonatal mouse retinas markedly reduced CNG channel expression. Transgenic expression of CNG channel beta-subunit mutants in Xenopus rods showed that ankyrin-G binding was necessary and sufficient for targeting of the beta1 subunit to outer segments. Thus, ankyrin-G is required for transport of CNG channels to the plasma membrane of rod outer segments.

Rod photoreceptors are highly polarized cells whose exquisite sensitivity to light depends on precise compartmentalization of ion channels/transporters within specialized membrane domains. Here, we report evidence for an ankyrin-B based mechanism for coordinated expression of the beta-2-spectrin-based membrane skeleton, and the Na/K-ATPase and Na/Ca exchanger in the inner segment of rod photoreceptors. We first discovered that ankyrin-B localizes to the inner segments but not outer segments of rod photoreceptors in vertebrates including humans, mice, and frogs. We found that haploinsufficiency of ankyrin-B in mice is accompanied by 50% reduction in inner segments of membrane proteins, including the Na/K-ATPase and the Na/Ca exchanger, as well as beta-2-spectrin, which is a component of the spectrin-actin membrane skeleton. These results are consistent with a mechanism where ankyrin-B is required to restrict the Na/K-ATPase and Na/Ca exchanger to the inner segment of rod photoreceptors by tethering these membrane proteins to beta-2-spectrin.

E-cadherin is a ubiquitous component of lateral membranes in epithelial tissues and is required to form the first lateral membrane domains in development. Here, we identify ankyrin-G as a molecular partner of E-cadherin and demonstrate that ankyrin-G and beta-2-spectrin are required for accumulation of E-cadherin at the lateral membrane in both epithelial cells and early embryos. Ankyrin-G binds to the cytoplasmic domain of E-cadherin at a conserved site distinct from that of beta-catenin. Ankyrin-G also recruits beta-2-spectrin to E-cadherin-beta-catenin complexes, thus providing a direct connection between E-cadherin and the spectrin/actin skeleton. In addition to restricting the membrane mobility of E-cadherin, ankyrin-G and beta-2-spectrin also are required for exit of E-cadherin from the trans-Golgi network in a microtubule-dependent pathway. Ankyrin-G and beta-2-spectrin co-localize with E-cadherin in preimplantation mouse embryos. Moreover, knockdown of either ankyrin-G or beta-2-spectrin in one cell of a two-cell embryo blocks accumulation of E-cadherin at sites of cell-cell contact. E-cadherin thus requires both ankyrin-G and beta-2-spectrin for its cellular localization in early embryos as well as cultured epithelial cells. We have recently reported that ankyrin-G and beta-2-spectrin collaborate in biogenesis of the lateral membrane ( Kizhatil, K., Yoon, W., Mohler, P. J., Davis, L. H., Hoffman, J. A., and Bennett, V. (2007) J. Biol. Chem. 282, 2029-2037 ). Together with the current findings, these data suggest a ankyrin/spectrin-based mechanism for coordinating membrane assembly with extracellular interactions of E-cadherin at sites of cell-cell contact.

Ankyrins are a family of adapter proteins required for localization of membrane proteins to diverse specialized membrane domains including axon initial segments, specialized sites at the transverse tubule/sarcoplasmic reticulum in cardiomyocytes, and lateral membrane domains of epithelial cells. Little is currently known regarding the molecular basis for specific roles of different ankyrin isoforms. In this study, we systematically generated alanine mutants of clusters of charged residues in the spectrin-binding domains of both ankyrin-B and -G. The corresponding mutants were evaluated for activity in either restoration of abnormal localization of the inositol trisphosphate receptor in the sarcoplasmic reticulum in mutant mouse cardiomyocytes deficient in ankyrin-B or in prevention of loss of lateral membrane in human bronchial epithelial cells depleted of ankyrin-G by small interfering RNA. Interestingly, ankyrin-B and -G share two homologous sites that result in loss of function in both systems, suggesting that common molecular interactions underlie diverse roles of these isoforms. Ankyrins G and B also exhibit differences; mutations affecting spectrin binding had no effect on ankyrin-B function but did abolish activity of ankyrin-G in restoring lateral membrane biogenesis. Depletion of beta(2)-spectrin by small interfering RNA phenocopied depletion of ankyrin-G and resulted in a failure to form new lateral membrane in interphase and mitotic cells. These results demonstrate that ankyrin-G and beta(2)-spectrin are functional partners in biogenesis of the lateral membrane of epithelial cells.

Ankyrin-G polypeptides are required for restriction of voltage-gated sodium channels, L1 cell adhesion molecules, and beta IV spectrin to axon initial segments and are believed to couple the Na/K-ATPase to the spectrin-actin network at the lateral membrane in epithelial cells. We report here that depletion of 190-kDa ankyrin-G in human bronchial epithelial cells by small interfering RNA results in nearly complete loss of lateral plasma membrane in interphase cells, and also blocks de novo lateral membrane biogenesis following mitosis. Loss of the lateral membrane domain is accompanied by an expansion of apical and basal plasma membranes and preservation of apical-basal polarity. Expression of rat 190-kDa ankyrin-G, which is resistant to human small interfering RNA, prevents loss of the lateral membrane following depletion of human 190-kDa ankyrin-G. Human 220-kDa ankyrin-B, a closely related ankyrin isoform, is incapable of preserving the lateral membrane following 190-kDa ankyrin-G depletion. Moreover, analysis of rat 190-kDa ankyrin G/ankyrin B chimeras shows that all three domains of 190-kDa ankyrin-G are required for preservation of the lateral membrane. These results demonstrate that 190-kDa ankyrin-G plays a pleiotropic role in assembly of lateral membranes of bronchial epithelial cells.

We report here that the system y+ cationic amino acid transporter ATRC1 localized to clusters within the basolateral membrane of polarized Madin-Darby canine kidney and human embryonic kidney (HEK) cells, suggesting that the transporters are restricted to discrete membrane microdomains in epithelial cells. Based on solubility in nonionic detergents, two populations of ATRC1 molecules existed: approximately half of the total ATRC1 in HEK cells associated with the actin membrane cytoskeleton, whereas another one-fourth resided in detergent-resistant membranes (DRM). In agreement with these findings, cytochalasin D reduced the amount of ATRC1 associated with the actin membrane cytoskeleton. Although some ATRC1 clusters in HEK cells colocalized with caveolin, the majority of ATRC1 did not colocalize with this marker protein for a type of DRM called caveolae. This distribution of ATRC1 is somewhat different from that reported for pulmonary artery endothelial cells in which transporters cluster predominantly in caveolae, suggesting that differences in the proportion of ATRC1 in specific membrane microdomains correlate with differences in the physiological role of the transporter in polarized kidney epithelial vs. vascular endothelial cells.

Doublecortin is a cytoplasmic protein mutated in the neuronal migration disorder X-linked lissencephaly. This study describes a novel activity of doublecortin in recognition of the FIGQY-phosphotyrosine motif present in the cytoplasmic domain of the L1 cell adhesion molecule neurofascin. Phospho-FIGQY-neurofascin (186 kDa) coimmunoprecipitated with doublecortin from detergent extracts of embryonic brain membranes, and this doublecortin-phospho-FIGQY neurofascin complex was disassociated by a synthetic phospho-FIGQY neurofascin peptide but not by a dephospho-FIGQY peptide. Doublecortin specifically recognized the phospho-FIGQY tyrosine in the context of a synthetic phospho-FIGQY neurofascin peptide and in phospho-FIGQY neurofascin isolated from cells treated with pervanadate. Mutations of doublecortin causing lissencephaly (R59H, D62N, and G253D) abolished binding to the phospho-FIGQY peptide and to phospho-FIGQY neurofascin. Finally, phospho-FIGQY neurofascin and doublecortin colocalize in developing axon tracts and in zones enriched in migrating neurons in the embryonic cerebral cortex. In the adult rostral migratory stream, doublecortin colocalizes in migrating neurons with a phospho-FIGQY bearing L1 CAM different from neurofascin. The finding that doublecortin associates with FIGQY-phosphorylated neurofascin provides the first connection of doublecortin with the plasma membrane and could be important for a function of doublecortin in directing neuronal migration.

Two second-site mutations in Moloney murine leukemia virus envelope surface protein (SU) were previously shown to rescue infection of two different SU mutants, a fusion-defective point mutant and a fusion-defective modified SU that exhibits weak subunit association. We report here that they also rescue infection of a third defective SU, one modified by insertion of the green fluorescent protein (GFP) between serine 6 and proline 7. GFP-SU assembled into virions and showed a strong association with the transmembrane protein (TM). However, these virions were noninfectious. GFP-SU expression was not maintained within cells, suggesting that the protein was toxic. Addition of the second-site mutations rendered the GFP-SU virus infectious and resulted in prolonged expression of the modified envelope protein. This virus showed a slight reduction in receptor binding but not in envelope protein processing, suggesting that addition of the GFP sequences results in subtle structural changes. Extrapolating these data, we see that the fundamental problem with the GFP-SU envelope protein appears to be a folding problem, suggesting that the second-site mutations rescue GFP-SU primarily by a mechanism that involves stabilizing the envelope protein structure.

Phosphorylation of neurofascin, a member of the L1 family of cell adhesion molecules (L1 CAMs), at the conserved FIGQY-tyrosine abolishes the ankyrin-neurofascin interaction. This study provides the first evidence, in Drosophila melanogaster and vertebrates, for the physiological occurrence of FIGQY phosphorylation in L1 family members. FIGQY tyrosine phosphorylation is localized at specialized cell junctions, including paranodes of sciatic nerve, neuromuscular junctions of adult rats and Drosophila embryos, epidermal muscle attachment sites of Drosophila, and adherens junctions of developing epithelial cells of rat and Drosophila. In addition, FIGQY-phosphorylated L1 CAMs are abundantly expressed in regions of neuronal migration and axon extension, including the embryonic cortex, the neonatal cerebellum and the rostral migratory stream, a region of continued neurogenesis and migration throughout adulthood in the rat. Based on our results, physiological FIGQY-tyrosine phosphorylation of the L1 family likely regulates adhesion molecule-ankyrin interactions establishing ankyrin-free and ankyrin-containing microdomains and participates in an ankyrin-independent intracellular signaling pathway at specialized sites of intercellular contact in epithelial and nervous tissue.

Infection by the neuropathogenic murine leukemia virus (MLV) TR1.3 results in hemorrhagic disease that correlates directly to in vivo syncytium formation of brain capillary endothelial cells (BCEC). This phenotype maps to amino acid 102 in the envelope (Env) protein of TR1.3. Substitution of glycine (G) for tryptophan (W) at this position (W102G Env) in the nonpathogenic MLV FB29 induces both syncytium formation and neurologic disease in vivo. Using an in vitro gene reporter cell fusion assay, we showed that fusion either with murine NIH 3T3 cells or with nonmurine target cells that expressed receptors at or below endogenous murine levels mirrored that seen in BCEC in vivo. In these instances only TR1.3 and W102G Env induced cell fusion. In contrast, when receptor levels on nonmurine cells were raised above endogenous murine levels, FB29 Env was as fusogenic as the neuropathogenic TR1.3 and W102G Env. These results indicate that TR1.3 Env and W102G Env are intrinsically more fusogenic than FB29 Env, that the induction of fusion requires a threshold number of receptors that is greater for FB29 Env than for TR1.3 or W102G Env, and that receptor density on murine NIH 3T3 cells and BCEC is below the threshold for FB29-dependent fusion. Surprisingly, receptor density on NIH 3T3 cells could not be increased by stable expression of exogenous receptors, and FB29-dependent fusion was not observed in NIH 3T3 cells that transiently expressed elevated receptor numbers. These results suggest that an additional undefined host cell factor(s) may limit both receptor expression and fusion potential in murine cells.