Antionette L. Williams

Subunit Structure of a Mammalian ER/Golgi SNARE Complex*

SNAP receptor (SNARE) complexes bridge opposing membranes to promote membrane fusion within the secretory and endosomal pathways. Because only the exocytic SNARE complexes have been characterized in detail, the structural features shared by SNARE complexes from different fusion steps are not known. We now describe the subunit structure, assembly, and regulation of a quaternary SNARE complex, which appears to mediate an early step in endoplasmic reticulum (ER) to Golgi transport. Purified recombinant syntaxin 5, membrin, and rbet1, three Q-SNAREs, assemble cooperatively to create a high affinity binding site for sec22b, an R-SNARE. The syntaxin 5 amino-terminal domain potently inhibits SNARE complex assembly. The ER/Golgi quaternary complex is remarkably similar to the synaptic complex, suggesting that a common pattern is followed at all transport steps, where three Q-helices assemble to form a high affinity binding site for a fourth R-helix on an opposing membrane. Interestingly, although sec22b binds to the combination of syntaxin 5, membrin, and rbet1, it can only bind if it is present while the others assemble; sec22b cannot bind to a pre-assembled ternary complex of syntaxin 5, membrin, and rbet1. Finally, we demonstrate that the quaternary complex containing sec22b is not an in vitroentity only, but is a bona fide species in living cells.

Structural and Functional Analysis of Tomosyn Identifies Domains Important in Exocytotic Regulation

Tomosyn is a 130-kDa cytosolic R-SNARE protein that associates with Q-SNAREs and reduces exocytotic activity. Two paralogous genes, tomosyn-1 and -2, occur in mammals and produce seven different isoforms via alternative splicing. Here, we map the structural differences between the yeast homologue of m-tomosyn-1, Sro7, and tomosyn genes/isoforms to identify domains critical to the regulation of exocytotic activity to tomosyn that are outside the soluble N-ethylmaleimide-sensitive attachment receptor motif. Homology modeling of m-tomosyn-1 based on the known structure of yeast Sro7 revealed a highly conserved functional conformation but with tomosyn containing three additional loop domains that emanate from a β-propeller core. Notably, deletion of loops 1 and 3 eliminates tomosyn inhibitory activity on secretion without altering its soluble N-ethylmaleimide-sensitive attachment receptor pairing with syntaxin1A. By comparison, deletion of loop 2, which contains the hypervariable splice region, did not reduce the ability of tomosyn to inhibit regulated secretion. However, exon variation within the hypervariable splice region resulted in significant differences in protein accumulation of tomosyn-2 isoforms. Functional analysis of s-tomosyn-1, m-tomosyn-1, m-tomosyn-2, and xb-tomosyn-2 demonstrated that they exert similar inhibitory effects on elevated K+-induced secretion in PC12 cells, although m-tomosyn-2 was novel in strongly augmenting basal secretion. Finally, we report that m-tomosyn-1 is a target substrate for SUMO 2/3 conjugation and that mutation of this small ubiquitin-related modifier target site (Lys-730) enhances m-tomosyn-1 inhibition of secretion without altering interaction with syntaxin1A. Together these results suggest that multiple domains outside the R-SNARE of tomosyn are critical to the efficacy of inhibition by tomosyn on exocytotic secretion.

Neural crest derivatives in ocular development: discerning the eye of the storm.

Neural crest cells (NCCs) are vertebrate-specific transient, multipotent, migratory stem cells that play a crucial role in many aspects of embryonic development. These cells emerge from the dorsal neural tube and subsequently migrate to different regions of the body, contributing to the formation of diverse cell lineages and structures, including much of the peripheral nervous system, craniofacial skeleton, smooth muscle, skin pigmentation, and multiple ocular and periocular structures. Indeed, abnormalities in neural crest development cause craniofacial defects and ocular anomalies, such as Axenfeld-Rieger syndrome and primary congenital glaucoma. Thus, understanding the molecular regulation of neural crest development is important to enhance our knowledge of the basis for congenital eye diseases, reflecting the contributions of these progenitors to multiple cell lineages. Particularly, understanding the underpinnings of neural crest formation will help to discern the complexities of eye development, as these NCCs are involved in every aspect of this process. In this review, we summarize the role of ocular NCCs in eye development, particularly focusing on congenital eye diseases associated with anterior segment defects and the interplay between three prominent molecules, PITX2, CYP1B1, and retinoic acid, which act in concert to specify a population of neural crest-derived mesenchymal progenitors for migration and differentiation, to give rise to distinct anterior segment tissues. We also describe recent findings implicating this stem cell population in ocular coloboma formation, and introduce recent evidence suggesting the involvement of NCCs in optic fissure closure and vascular development.

Tomosyn Expression Pattern in the Mouse Hippocampus Suggests Both Presynaptic and Postsynaptic Functions

The protein tomosyn decreases synaptic transmission and release probability of vesicles, and is essential for modulating synaptic transmission in neurons. In this study, we provide a detailed description of the expression and localization patterns of tomosyn1 and tomosyn2 in the subareas of the mouse hippocampus. Using confocal and two-photon high-resolution microscopy we demonstrate that tomosyn colocalizes with several pre- and postsynaptic markers and is found mainly in glutamatergic synapses. Specifically, we show that tomosyn1 is differentially distributed in the mouse hippocampus and concentrated mainly in the hilus and mossy fibers. Surprisingly, we found that tomosyn2 is expressed in the subiculum, CA1 and CA2 pyramidal cell bodies, dendrites and spines, and colocalizes with PSD95, suggesting a postsynaptic role. These results suggest that in addition to the well-characterized presynaptic function of tomosyn in neurotransmitter release, tomosyn2 might have a postsynaptic function, and place tomosyn as a more general regulator of synaptic transmission and plasticity.

Retinoic Acid and Pitx2 Regulate Early Neural Crest Survival and Migration in Craniofacial and Ocular Development.

Congenital eye and craniofacial anomalies are associated with the dysregulation of retinoic acid (RA) levels during embryogenesis. In the present study, we observed that RA and pitx2a cooperatively regulate early cranial neural crest migration from the rhombencephalon to the pharyngeal arches and from the mesencephalon and prosencephalon to the periocular mesenchyme and frontonasal processes. The cranial neural crest tracked toward areas of high RA activity (i.e., developing eye) and circumvented areas of low RA activity (i.e., mesencephalon). Although previous studies have shown that RA increased pitx2a expression at later stages of cranial neural crest development, in these studies we found that RA inhibited pitx2a expression in the early migrating ventral cranial neural crest. Increased RA or decreased Pitx2a expression decreased cell survival and inhibited ventral neural crest migration. Decreased RA or increased pitx2a expression markedly disrupted both dorsal and ventral neural crest migration. The tight control of RA and subsequent regulation of pitx2 were required for precise cranial neural crest survival and migration. These alterations in the neural crest in the periocular mesenchyme and frontonasal processes may reflect the craniofacial dysmorphism and microphthalmia observed in cases of increased (i.e., as resulting from isoretinoin exposure) or decreased (i.e., as may occur in fetal alcohol syndrome) RA signaling during pregnancy

Retinoic Acid and Pitx2 Regulate Early Neural Crest Survival and Migration in Craniofacial and Ocular Development

Congenital eye and craniofacial anomalies are associated with the dysregulation of retinoic acid (RA) levels during embryogenesis. In the present study, we observed that RA and pitx2a cooperatively regulate early cranial neural crest migration from the rhombencephalon to the pharyngeal arches and from the mesencephalon and prosencephalon to the periocular mesenchyme and frontonasal processes. The cranial neural crest tracked toward areas of high RA activity (i.e., developing eye) and circumvented areas of low RA activity (i.e., mesencephalon). Although previous studies have shown that RA increased pitx2a expression at later stages of cranial neural crest development, in these studies we found that RA inhibited pitx2a expression in the early migrating ventral cranial neural crest. Increased RA or decreased Pitx2a expression decreased cell survival and inhibited ventral neural crest migration. Decreased RA or increased pitx2a expression markedly disrupted both dorsal and ventral neural crest migration. The tight control of RA and subsequent regulation of pitx2 were required for precise cranial neural crest survival and migration. These alterations in the neural crest in the periocular mesenchyme and frontonasal processes may reflect the craniofacial dysmorphism and microphthalmia observed in cases of increased (i.e., as resulting from isoretinoin exposure) or decreased (i.e., as may occur in fetal alcohol syndrome) RA signaling during pregnancy

Cyp1b1 Regulates Ocular Fissure Closure Through a Retinoic Acid–Independent Pathway

Purpose Mutations in the CYP1B1 gene are the most commonly identified genetic causes of primary infantile-onset glaucoma. Despite this disease association, the role of CYP1B1 in eye development and its in vivo substrate remain unknown. In the present study, we used zebrafish to elucidate the mechanism by which cyp1b1 regulates eye development. Methods Zebrafish eye and neural crest development were analyzed using live imaging of transgenic zebrafish embryos, in situ hybridization, immunostaining, TUNEL assay, and methylacrylate sections. Cyp1b1 and retinoic acid (RA) levels were genetically (morpholino oligonucleotide antisense and mRNA) and pharmacologically manipulated to examine gene function. Results Using zebrafish, we observed that cyp1b1 was expressed in a specific spatiotemporal pattern in the ocular fissures of the developing zebrafish retina and regulated fissure patency. Decreased Cyp1b1 resulted in the premature breakdown of laminin in the ventral fissure and altered subsequent neural crest migration into the anterior segment. In contrast, cyp1b1 overexpression inhibited cell survival in the ventral ocular fissure and prevented fissure closure via an RA-independent pathway. Cyp1b1 overexpression also inhibited the ocular expression of vsx2, pax6a, and pax6b and increased the extraocular expression of shha. Importantly, embryos injected with human wild-type but not mutant CYP1B1 mRNA also showed colobomas, demonstrating the evolutionary and functional conservation of gene function between species. Conclusions Cyp1b1 regulation of ocular fissure closure indirectly affects neural crest migration and development through an RA-independent pathway. These studies provide insight into the role of Cyp1b1 in eye development and further elucidate the pathogenesis of primary infantile-onset glaucoma.

Differences in neural crest sensitivity to ethanol account for the infrequency of anterior segment defects in the eye compared with craniofacial anomalies in a zebrafish model of fetal alcohol syndrome

BACKGROUND Ethanol (ETOH) exposure during pregnancy is associated with craniofacial and neurologic abnormalities, but infrequently disrupts the anterior segment of the eye. In these studies, we used zebrafish to investigate differences in the teratogenic effect of ETOH on craniofacial, periocular, and ocular neural crest. METHODS Zebrafish eye and neural crest development was analyzed by means of live imaging, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, immunostaining, detection of reactive oxygen species, and in situ hybridization. RESULTS Our studies demonstrated that foxd3-positive neural crest cells in the periocular mesenchyme and developing eye were less sensitive to ETOH than sox10-positive craniofacial neural crest cells that form the pharyngeal arches and jaw. ETOH increased apoptosis in the retina, but did not affect survival of periocular and ocular neural crest cells. ETOH also did not increase reactive oxygen species within the eye. In contrast, ETOH increased ventral neural crest apoptosis and reactive oxygen species production in the facial mesenchyme. In the eye and craniofacial region, sod2 showed high levels of expression in the anterior segment and in the setting of Sod2 knockdown, low levels of ETOH decreased migration of foxd3-positive neural crest cells into the developing eye. However, ETOH had minimal effect on the periocular and ocular expression of transcription factors (pitx2 and foxc1) that regulate anterior segment development. CONCLUSION Neural crest cells contributing to the anterior segment of the eye exhibit increased ability to withstand ETOH-induced oxidative stress and apoptosis. These studies explain the rarity of anterior segment dysgenesis despite the frequent craniofacial abnormalities in fetal alcohol syndrome. Birth Defects Research 109:1212-1227, 2017.

Retinoic Acid Maintains Function of Neural Crest–Derived Ocular and Craniofacial Structures in Adult Zebrafish

Purpose Retinoic acid (RA) is required for embryonic formation of the anterior segment of the eye and craniofacial structures. The present study further investigated the role of RA in maintaining the function of these neural crest–derived structures in adult zebrafish. Methods Morphology and histology were analyzed by using live imaging, methylacrylate sections, and TUNEL assay. Functional analysis of vision and aqueous humor outflow were assayed with real-time imaging. Results Both decreased and increased RA signaling altered craniofacial and ocular structures in adult zebrafish. Exogenous treatment with all-trans RA for 5 days resulted in a prognathic jaw, while inhibition of endogenous RA synthesis through treatment with 4-diethylaminobenzaldehyde (DEAB) decreased head height. In adult eyes, RA activity was localized to the retinal pigment epithelium, photoreceptors, outer plexiform layer, inner plexiform layer, iris stroma, and ventral canalicular network. Exogenous RA increased apoptosis in the iris stroma and canalicular network in the ventral iridocorneal angle, resulting in the loss of these structures and decreased aqueous outflow. DEAB, which decreased RA activity throughout the eye, induced widespread apoptosis, resulting in corneal edema, cataracts, retinal atrophy, and loss of iridocorneal angle structures. DEAB-treated fish were blind with no optokinetic response and no aqueous outflow from the anterior chamber. Conclusions Tight control of RA levels is required for normal structure and function of the adult anterior segment. These studies demonstrated that RA plays an important role in maintaining ocular and craniofacial structures in adult zebrafish.

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.