Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca²⁺) concentration are powerful tools for visualizing intracellular signaling ...activity. However, despite a decade of availability, the palette of single FP-based Ca²⁺ indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca²⁺ imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca²⁺ was imaged in three subcellular compartments, and, in conjunction with a cyan FP—yellow FP—based indicator, Ca²⁺ and adenosine 5′-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca²⁺ imaging.
Interneurons, innervated by multiple sensory neurons, need to integrate information from these sensory neurons and respond to sensory stimuli adequately. Mechanisms how sensory information is ...integrated to form responses of interneurons are not fully understood. In Caenorhabditis elegans, loss‐of‐function mutations of egl‐4, which encodes a cGMP‐dependent protein kinase (PKG), cause a defect in chemotaxis to odorants. Our genetic and imaging analyses revealed that the response property of AIY interneuron to an odorant is reversed in the egl‐4 mutant, while the responses of two upstream olfactory neurons, AWA and AWC, are largely unchanged. Cell‐ ablation experiments show that AIY in the egl‐4 mutant functions to suppress chemotaxis. Furthermore, the reversal of AIY response occurs only in the presence of sensory signals from both AWA and AWC. These results suggest that sensory signals are inadequately integrated in the egl‐4 mutant. We also show that egl‐4 expression in AWA and another sensory neuron prevents the reversed AIY response and restores chemotaxis in the egl‐4 mutants. We propose that EGL‐4/PKG, by suppressing aberrant integration of signals from olfactory neurons, converts the response property of an interneuron to olfactory stimuli and maintains the role of the interneuron in the circuit to execute chemotactic behavior.
EGL‐4/PKG in C. elegans mediates integration of sensory signals and regulates the role of an interneuron in a circuit to execute chemotactic behavior.
Many animal species change their behavior according to their stage of development. However, the mechanisms involved in translating their developmental stage into the modifications of the neuronal ...circuits that underlie these behavioral changes remain unknown. Here we show that Caenorhabditis elegans changes its olfactory preferences during development. Larvae exhibit a weak chemotactic response to the food-associated odor diacetyl, whereas adults exhibit a strong response. We show that germline loss, caused either by laser ablation of germline precursor cells or mutations, results in a diacetyl-specific chemotactic defect in adult animals. These results suggest that germline cells, which proliferate dramatically during the larval stages, enhance chemotaxis to diacetyl. Removal experiments of specific neurons suggested that AWA olfactory neurons and their downstream interneurons, AIA and AIB, are required for germline-dependent chemotactic enhancement. Calcium imaging in animals lacking germline cells indicates that the neural responses of AWA and AIB to diacetyl stimuli are decreased compared with animals with an intact germline. These changes in neural activities may at least partly explain the behavioral change of animals lacking germline cells. Furthermore, this germline-dependent chemotactic change depends on the transcription factor DAF-16/FOXO. We find that organismal behavior changes throughout development by integrating information about physiological status from internal tissues to modify a simple sensory circuit.
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•Odor preference changes during development in C. elegans•Adults lacking a germline show chemotactic responses similar to those of larvae•Activities of AWA and AIB neurons in an olfactory circuit are affected by a germline•DAF-16/FOXO is required for germline-dependent chemotactic regulation
Fujiwara et al. demonstrate that germline cells of C. elegans, which proliferate in larvae, affect the chemotactic response of adult animals specifically to a food-associated odorant, diacetyl. This is accomplished through the function of DAF-16/FOXO. Activities of AWA and AIB neurons in an olfactory circuit are changed by germline cells.
The growth and behavior of higher organisms depend on the accurate perception and integration of sensory stimuli by the nervous system. We show that defects in sensory perception in
C. elegans result ...in abnormalities in the growth of the animal and in the expression of alternative behavioral states. Our analysis suggests that sensory neurons modulate neural or neuroendocrine functions, regulating both bodily growth and behavioral state. We identify genes likely to be required for these functions downstream of sensory inputs. Here, we characterize one of these genes as
egl-4, which we show encodes a cGMP-dependent protein kinase. We demonstrate that this cGMP-dependent kinase functions in neurons of
C. elegans to regulate multiple developmental and behavioral processes including the orchestrated growth of the animal and the expression of particular behavioral states.
Glutamylation is a post-translational modification found on tubulin that can alter the interaction between microtubules (MTs) and associated proteins. The molecular mechanisms regulating tubulin ...glutamylation in response to the environment are not well understood. Here, we show that in the sensory cilia of Caenorhabditis elegans, tubulin glutamylation is upregulated in response to various signals such as temperature, osmolality, and dietary conditions. Similarly, tubulin glutamylation is modified in mammalian photoreceptor cells following light adaptation. A tubulin glutamate ligase gene ttll-4, which is essential for tubulin glutamylation of axonemal MTs in sensory cilia, is activated by p38 MAPK. Amino acid substitution of TTLL-4 has revealed that a Thr residue (a putative MAPK-phosphorylation site) is required for enhancement of tubulin glutamylation. Intraflagellar transport (IFT), a bidirectional trafficking system specifically observed along axonemal MTs, is required for the formation, maintenance, and function of sensory cilia. Measurement of the velocity of IFT particles revealed that starvation accelerates IFT, which was also dependent on the Thr residue of TTLL-4. Similarly, starvation-induced attenuation of avoidance behaviour from high osmolality conditions was also dependent on ttll-4. Our data suggest that a novel evolutionarily conserved regulatory system exists for tubulin glutamylation in sensory cilia in response to the environment.
The body size of Caenorhabditis elegans is thought to be controlled by sensory inputs because many mutants with sensory cilium structure defects exhibit small body size. The EGL-4 cGMP-dependent ...protein kinase acts in sensory neurons to reduce body size when animals fail to perceive sensory signals. In addition to body size control, EGL-4 regulates various other behavioral and developmental pathways, including those involved in the regulation of egg laying and chemotaxis behavior. Here we have identified gcy-12, which encodes a receptor-type guanylyl cyclase, as a gene involved in the sensory regulation of body size. Analyses with GFP fusion constructs showed that gcy-12 is expressed in several sensory neurons and localizes to sensory cilia. Genetic analyses indicated that GCY-12 acts upstream of EGL-4 in body size control but does not affect other EGL-4 functions. Our studies indicate that the function of the GCY-12 guanylyl cyclase is to provide cGMP to the EGL-4 cGMP-dependent kinase only for limited tasks including body size regulation. We also found that the PDE-2 cyclic nucleotide phosphodiesterase negatively regulates EGL-4 in controlling body size. Thus, the cGMP level is precisely controlled by GCY-12 and PDE-2 to determine body size through EGL-4, and the defects in the sensory cilium structure may disturb the balanced control of the cGMP level. The large number of guanylyl cyclases encoded in the C. elegans genome suggests that EGL-4 exerts pleiotropic effects by partnering with different guanylyl cyclases for different downstream functions.
The control of memory retention is important for proper responses to constantly changing environments, but the regulatory mechanisms underlying forgetting have not been fully elucidated. Our genetic ...analyses in C. elegans revealed that mutants of the TIR-1/JNK-1 pathway exhibited prolonged retention of olfactory adaptation and salt chemotaxis learning. In olfactory adaptation, conditioning induces attenuation of odor-evoked Ca2+ responses in olfactory neurons, and this attenuation is prolonged in the TIR-1/JNK-1-pathway mutant animals. We also found that a pair of neurons in which the pathway functions is required for the acceleration of forgetting, but not for sensation or adaptation, in wild-type animals. In addition, the neurosecretion from these cells is important for the acceleration of forgetting. Therefore, we propose that these neurons accelerate forgetting through the TIR-1/JNK-1 pathway by sending signals that directly or indirectly stimulate forgetting.
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► Food signals actively regulate forgetting of olfactory adaptation ► TIR-1/JNK-1 pathway accelerates forgetting of adaptation and of associative learning ► Response to diacetyl in AWA neurons is diminished after exposure to diacetyl ► Neurosecretion from AWC neurons is important for the acceleration of forgetting
Forgetting is one of the important steps in the regulation of the retention of memories. Ishihara and colleagues find that forgetting signals, which sensory neurons secrete depending on the environment, accelerate forgetting of the memories in other sensory neurons and that the secretion of forgetting signals is regulated by the TIR-1/JNK-1 pathway. These findings demonstrate that forgetting is actively regulated in neuronal circuits.
Small interfering RNAs (siRNAs) and microRNAs (miRNAs) mediate gene silencing through evolutionarily conserved pathways. In Caenorhabditis elegans, the siRNA/miRNA pathways are also known to affect ...transgene expression. To identify genes that regulate the efficiencies of the siRNA/miRNA pathways, we used the expression level of a transgene as an indicator of gene silencing and isolated a transgene-silencing mutant, adbp-1 (ADR-2 binding protein). The adbp-1 mutation caused transgene silencing in hypodermal and intestinal cells in a cell-autonomous manner, depending on the RNA interference (RNAi) machinery. The adbp-1 gene encodes a protein with no conserved domains that is localized in the nucleus. Yeast two-hybrid screening and co-immunoprecipitation analysis demonstrated that ADBP-1 physically interacts with ADR-2, an RNA-editing enzyme from the ADAR (adenosine deaminase acting on dsRNA) family. In the adbp-1 mutant, as previously shown in adr-2 mutants, A-to-I RNA editing was not detected, suggesting that ADBP-1 is required for the RNA-editing activity of ADR-2. We found that ADBP-1 facilitates the nuclear localization of ADR-2. ADBP-1 may regulate ADR-2 activity and the consequent RNA editing and thereby antagonize RNAi-mediated transgene silencing in C. elegans.
Cilia are important sensory organelles, which are thought to be essential regulators of numerous signaling pathways. In Caenorhabditis elegans, defects in sensory cilium formation result in a ...small-body phenotype, suggesting the role of sensory cilia in body size determination. Previous analyses suggest that lack of normal cilia causes the small-body phenotype through the activation of a signaling pathway which consists of the EGL-4 cGMP-dependent protein kinase and the GCY-12 receptor-type guanylyl cyclase. By genetic suppressor screening of the small-body phenotype of a cilium defective mutant, we identified a chb-3 gene. Genetic analyses placed chb-3 in the same pathway as egl-4 and gcy-12 and upstream of egl-4. chb-3 encodes a novel protein, with a zf-MYND motif and ankyrin repeats, that is highly conserved from worm to human. In chb-3 mutants, GCY-12 guanylyl cyclase visualized by tagged GFP (GCY-12::GFP) fails to localize to sensory cilia properly and accumulates in cell bodies. Our analyses suggest that decreased GCY-12 levels in the cilia of chb-3 mutants may cause the suppression of the small-body phenotype of a cilium defective mutant. By observing the transport of GCY-12::GFP particles along the dendrites to the cilia in sensory neurons, we found that the velocities and the frequencies of the particle movement are decreased in chb-3 mutant animals. How membrane proteins are trafficked to cilia has been the focus of extensive studies in vertebrates and invertebrates, although only a few of the relevant proteins have been identified. Our study defines a new regulator, CHB-3, in the trafficking process and also shows the importance of ciliary targeting of the signaling molecule, GCY-12, in sensory-dependent body size regulation in C. elegans. Given that CHB-3 is highly conserved in mammal, a similar system may be used in the trafficking of signaling proteins to the cilia of other species.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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