Vps4p (End13p) is an AAA-family ATPase that functions in membrane transport through endosomes, sorting of soluble vacuolar proteins to the vacuole, and multivesicular body (MVB) sorting of membrane proteins to the vacuole lumen. In a yeast two-hybrid screen with Vps4p as bait we isolated VPS20 (YMR077c) and the novel open reading frame YLR181c, for which the name VTA1 has recently been assigned (Saccharomyces Genome Database). Vps4p directly binds Vps20p and Vta1p in vitro and binding is not dependent on ATP - conversely, Vps4p binding to Vps20p is partially sensitive to ATP hydrolysis. Both ATP binding [Vps4p-(K179A)] and ATP hydrolysis [Vps4p-(E233Q)] mutant proteins exhibit enhanced binding to Vps20p and Vta1p in vitro. The Vps4p-Vps20p interaction involves the coiled-coil domain of each protein, whereas the Vps4p-Vta1p interaction involves the (non-coiled-coil) C-terminus of each protein. Deletion of either VPS20 (vps20Δ) or VTA1 (vta1Δ) leads to similar class E Vps- phenotypes resembling those of vps4Δ, including carboxypeptidase Y (CPY) secretion, a block in ubiquitin-dependent MVB sorting, and a delay in both post-internalisation endocytic transport and biosynthetic transport to the vacuole. The vacuole resident membrane protein Sna3p (whose MVB sorting is ubiquitin-independent) does not appear to exit the class E compartment or reach the vacuole in cells lacking Vps20p, Vta1p or Vps4p, in contrast to other proteins whose delivery to the vacuole is only delayed. We propose that Vps20p and Vta1p regulate Vps4p function in vivo.
Animals survive environmental challenges by adapting their physiology and behavior through homeostatic regulatory processes, mediated in part by specific neuropeptide release from the hypothalamus. Animals can also avoid environmental stressors within seconds, a fast behavioral adaptation for which hypothalamic involvement is not established. Using brain-wide neural activity imaging in behaving zebrafish, here we find that hypothalamic neurons are rapidly engaged during common avoidance responses elicited by various environmental stressors. By developing methods to register cellular-resolution neural dynamics to multiplexed in situ gene expression, we find that each category of stressor recruits similar combinations of multiple peptidergic cell types in the hypothalamus. Anatomical analysis and functional manipulations demonstrate that these diverse cell types play shared roles in behavior, are glutamatergic, and converge upon spinal-projecting brainstem neurons required for avoidance. These data demonstrate that hypothalamic neural populations, classically associated with slow and specific homeostatic adaptations, also together give rise to fast and generalized avoidance behavior.1 .
BackgroundLarval zebrafish, with a simple and transparent vertebrate brain composed of ~100 K neurons, is well suited for deciphering entire neural circuit activity underlying behavior. Moreover, their small body size (~4–5 mm in length) is compatible with 96-well plates, making larval zebrafish amenable to high content screening. Despite these attractive features, there is a scarcity of behavioral characterizations in larval zebrafish compared to other model organisms as well as adult zebrafish.ResultsIn this study, we have characterized the physiological and behavioral responses of larval zebrafish to several easily amenable stimuli, including heat, cold, UV, mechanical disturbance (MD), and social isolation (SI). These stimuli are selected based on their perceived aversive nature to larval zebrafish. Using a light/dark choice paradigm, in which larval zebrafish display an innate dark avoidance behavior (i.e. scotophobia), we find that heat, cold and UV stimuli significantly enhance their dark avoidance with heat having the most striking effect, whereas MD and SI have little influence on the behavior. Surprisingly, using the cortisol assay, a physiological measure of stress, we uncover that all stimuli but heat and SI significantly increase the whole body cortisol levels.ConclusionThese results identify a series of stressors that can be easily administered to larval zebrafish. Those stimuli that elicit differential responses at behavioral and physiological levels warrant further studies at circuit levels to understand the underlying mechanisms. The findings that various stressors enhance while anxiolytics attenuate dark avoidance further reinforce that the light/dark preference behavior in larval zebrafish is fear/anxiety-associated.
Ethanol, a widely abused substance, elicits evolutionarily conserved behavioral responses in a concentration-dependent manner in vivo. The molecular mechanisms underlying such behavioral sensitivity to ethanol are poorly understood. While locomotor-based behavioral genetic screening is successful in identifying genes in invertebrate models, such complex behavior-based screening has proven difficult for recovering genes in vertebrates. Here we report a novel and tractable ethanol response in zebrafish. Using this ethanol-modulated camouflage response as a screening assay, we have identified a zebrafish mutant named fantasma ( fan), which displays reduced behavioral sensitivity to ethanol. Positional cloning reveals that fan encodes type 5 adenylyl cyclase (AC5). fan/ac5 is required to maintain the phosphorylation of extracellular signal-regulated kinase (ERK) in the forebrain structures, including the telencephalon and hypothalamus. Partial inhibition of phosphorylation of ERK in wild-type zebrafish mimics the reduction in sensitivity to stimulatory effects of ethanol observed in the fan mutant, whereas, strikingly, strong inhibition of phosphorylation of ERK renders a stimulatory dose of ethanol sedating. Since previous studies in Drosophila and mice show a role of cAMP signaling in suppressing behavioral sensitivity to ethanol, our findings reveal a novel, isoform-specific role of AC signaling in promoting ethanol sensitivity, and suggest that the phosphorylation level of the downstream effector ERK is a critical "gatekeeper" of behavioral sensitivity to ethanol.
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