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 vitro entity only, but is a bona fide species in living cells.
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 NC 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 RA, 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 angiogenesis.
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 -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.Synaptic vesicle fusion and the subsequent release of neurotransmitter require the formation of heterotrimeric SNARE 2 complexes formed from plasma membrane proteins syntaxin1A and SNAP-25 (Q-SNAREs) with the synaptic vesicle membrane protein VAMP/synaptobrevin (R-SNARE) (1-3). Present on opposing membranes, these SNAREs combine and engage in thermodynamically stable coiled-coil interactions that bridge the two membranes and catalyze their fusion (4). The formation of SNARE complexes is spatially and temporally controlled by accessory components that lend additional specificity to SNARE pairing, arrest SNARE complex intermediates, and/or lower the energy required for fusion (4 -6). Ultimately, it is the functional activity of these regulators on SNARE complex assembly that determines the dynamics of the exocytotic event.Tomosyn is an important regulator of SNARE complex formation whose mechanism of action remains unclear. Initially identified in neurons (7-8), tomosyn, a soluble R-SNARE protein, was considered to be a negative effector of fusogenic SNARE complex assembly through interactions with syntaxin1A and SNAP-25 that preclude the binding of VAMP2,...
Although some of the principles of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function are well understood, remarkably little detail is known about sec1/munc18 (SM) protein function and its relationship to SNAREs. Popular models of SM protein function hold that these proteins promote or maintain an open and/or monomeric pool of syntaxin molecules available for SNARE complex formation. To address the functional relationship of the mammalian endoplasmic reticulum/Golgi SM protein rsly1 and its SNARE binding partner syntaxin 5, we produced a conformation-specific monoclonal antibody that binds only the available, but not the cis-SNARE-complexed nor intramolecularly closed form of syntaxin 5. Immunostaining experiments demonstrated that syntaxin 5 SNARE motif availability is nonuniformly distributed and focally regulated. In vitro endoplasmic reticulum-to-Golgi transport assays revealed that rsly1 was acutely required for transport, and that binding to syntaxin 5 was absolutely required for its function. Finally, manipulation of rsly1-syntaxin 5 interactions in vivo revealed that they had remarkably little impact on the pool of available syntaxin 5 SNARE motif. Our results argue that although rsly1 does not seem to regulate the availability of syntaxin 5, its function is intimately associated with syntaxin binding, perhaps promoting a later step in SNARE complex formation or function.
Vertebrate models, from zebrafish to mouse, and experimental tools, such as reporter transgenes, inhibitors of RA synthesis, and selective antagonists for RARs, have been successfully used to decipher retinoid functions during development at the cellular and molecular levels.Numerous examples from animal models and human diseases highlight the importance of RA in regulating neural crest contributions to craniofacial and ocular structures. The neural crest is a set of transient embryonic stem cells unique to vertebrates that originate at the border of the neural plate spanning the embryo from the diencephalon to the lumbosacral spinal cord. Neural crest cells undergo extensive and coordinated movements away from folds of the neural F I G U R E 1 Neural crest derivatives in the craniofacial region. (a) The neural crest is a transient population of embryonic stem cells that delaminate from the edge of the neural tube spanning from the diencephalon (Di) to the lumbosacral spinal cord (SpC). Neural crest cells that originate from the diencephalon and anterior mesencephalon (AM) migrate dorsal and ventral to the eye to populate the POM and frontonasal process (FNP). These cells migrate toward regions of high RA levels within the telencephalon (Te), POM, and frontonasal process. Neural crest cells from the posterior mesencephalon (PM) migrate into the first pharyngeal arch. Neural crest cells from the rhombencephalon, which are patterned by a RA gradient, migrate into the first through fourth pharyngeal arches in an anterior (AR) to posterior (PR) pattern. (b) The cranial neural crest is important in the development of the craniofacial skeleton. Neural crest cells in the frontonasal process give rise to the frontal bone (Fr), nasal bone (Na), and philtrum (Ph) in the midline of the face. The anterior portion of the first pharyngeal arch forms the maxillary (Mx) and zygomatic (Zy) bones while the posterior aspect gives rise to the mandible (Mn), The third and fourth pharyngeal arches both contribute to the hyoid (Ny) bone in the neck. (c) Neural crest cells, which are derived from the anterior mesenchyme and diencephalon, populate the periocular mesenchyme and migrate into the anterior segment of the eye. Ocular structures derived from the neural crest include the corneal stroma (CS), corneal endothelium (CEn), trabecular meshwork (TM), sclera (Scl), iris stroma (IS), uveal melanocytes (me), ciliary body muscle (CBM), and the tendons of the extraocular muscles (Tn). The corneal epithelium (CEp) and lens are derived from surface ectoderm while the ciliary body epithelium (CBE), retina (Ret), and retinal pigmented epithelium (RPE) arise from neuroepithelium. Schlemm's canal (SC) and extraocular muscles (EOM) originate from mesoderm. (d) Neural crest cells are also important in middle ear development. Vibrations are transmitted from the external ear, which consists of the pinna, auditory canal, and the tympanic membrane (Tym). The tympanic membrane is attached to the malleus in the middle ear. Both the malleus and the incus...
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