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Rabx-5 regulates RAB-5 early endosomal compartments and synaptic vesicles in C. elegans.

Sann SB, Crane MM, Lu H, Jin Y - PLoS ONE (2012)

Bottom Line: The rabx-5 mutation leads to decreased intensity of YFP::RAB-5 in the cell soma but increased intensity in the synaptic and intersynaptic regions of the axon.This effect is due to the bias of the cycling state of RAB-5, and results from a change in the organization of the early endosomal compartment as well as the membrane binding state of RAB-5.These results suggest that rabx-5 regulation of RAB-5 compartments is important for maintaining proper synaptic function throughout the lifetime.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology Section, Division of Biological Sciences, University of California San Diego, La Jolla, California, United States of America. ssann@ucsd.edu

ABSTRACT
Early endosomal membrane compartments are required for the formation and recycling of synaptic vesicles, but how these compartments are regulated is incompletely understood. We performed a forward genetic screen in C. elegans for mutations that affect RAB-5 labeled early endosomal compartments in GABAergic motoneurons. Here we report the isolation and characterization of one mutation, rabx-5. The rabx-5 mutation leads to decreased intensity of YFP::RAB-5 in the cell soma but increased intensity in the synaptic and intersynaptic regions of the axon. This effect is due to the bias of the cycling state of RAB-5, and results from a change in the organization of the early endosomal compartment as well as the membrane binding state of RAB-5. Synaptic vesicle accumulation is altered in rabx-5 mutants, and synaptic transmission from cholinergic neurons is decreased. Early endosomal membrane compartments show disorganization with ageing and rabx-5 mutant animals age faster. These results suggest that rabx-5 regulation of RAB-5 compartments is important for maintaining proper synaptic function throughout the lifetime.

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Rabx-5 mutations alter the RAB-5 organization in GABAergic motor neurons.A) A forward genetic screen was conducted by observing GABAergic motor neurons for changes in localization or intensity of Punc-25YFP::RAB-5. Initial screening isolated mutants in which YFP::RAB-5 was decreased in the cell soma. Further inspection determined if YFP::RAB-5 was increased at the synapse. B) Gene, transcript, and protein structure of rabx-5. The RABX-5 protein consists of a zinc finger motif (ZF) and a motif interacting with ubiquitin (U) that together regulate association with ubiquitinated endosomal cargo; a membrane binding motif and helical bundle required for association with the endosomal membrane; a Vps9 domain that along with the helical bundle promotes guanine exchange activity; and a coiled-coil region that binds rabaptin-5 and contains a motif for autoinhibition of guanine exchange activity. The rabx-5(qa7800) mutation leads to a truncation at the helical bundle. The rabx-5(tm1512) mutation deletes the start site and the exons encoding the zinc finger motif and the motif interacting with ubiquitn. C) Punc-25YFP::RAB-5 protein localization in the soma (left column) and dorsal cord (right column) of GABAergic motor neurons in wild type and mutant animals. D) Punc-25YFP::RAB-5 fluorescence intensity was decreased in the soma of mutant animals and increased in the synaptic and intersynaptic regions. This phenotype is recovered with expression of Punc-25CFP::RABX-5. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively. Bar represents the mean. **p<0.01, ***p<0.001. E) Punc-25CFP::RABX-5 expression recovers the rabx-5(qa7800) YFP::RAB-5 phenotype.
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pone-0037930-g001: Rabx-5 mutations alter the RAB-5 organization in GABAergic motor neurons.A) A forward genetic screen was conducted by observing GABAergic motor neurons for changes in localization or intensity of Punc-25YFP::RAB-5. Initial screening isolated mutants in which YFP::RAB-5 was decreased in the cell soma. Further inspection determined if YFP::RAB-5 was increased at the synapse. B) Gene, transcript, and protein structure of rabx-5. The RABX-5 protein consists of a zinc finger motif (ZF) and a motif interacting with ubiquitin (U) that together regulate association with ubiquitinated endosomal cargo; a membrane binding motif and helical bundle required for association with the endosomal membrane; a Vps9 domain that along with the helical bundle promotes guanine exchange activity; and a coiled-coil region that binds rabaptin-5 and contains a motif for autoinhibition of guanine exchange activity. The rabx-5(qa7800) mutation leads to a truncation at the helical bundle. The rabx-5(tm1512) mutation deletes the start site and the exons encoding the zinc finger motif and the motif interacting with ubiquitn. C) Punc-25YFP::RAB-5 protein localization in the soma (left column) and dorsal cord (right column) of GABAergic motor neurons in wild type and mutant animals. D) Punc-25YFP::RAB-5 fluorescence intensity was decreased in the soma of mutant animals and increased in the synaptic and intersynaptic regions. This phenotype is recovered with expression of Punc-25CFP::RABX-5. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively. Bar represents the mean. **p<0.01, ***p<0.001. E) Punc-25CFP::RABX-5 expression recovers the rabx-5(qa7800) YFP::RAB-5 phenotype.

Mentions: Several distinct endosomal compartments are present in the GABAergic motor neuron synapse of C. elegans[22]. The expression pattern of a fluorescently tagged RAB-5 reporter is specifically altered in mutant animals lacking the function of UNC-16, a kinesin adaptor protein [22], [24]. To identify additional genes involved in the endocytic pathway and axonal transport in neuronal development, we conducted a forward genetic screen using a YFP::RAB-5 reporter in GABAergic motor neurons. These neurons are pseudo-unipolar neurons, with their soma located in the ventral nerve cord, and nerve processes elongating along the ventral and dorsal nerve cord, connected by a circumferential commissure. Presynaptic terminals form en passant along the ventral (for VD neurons) and the dorsal (for DD neurons) processes. We have conducted the screen in two ways: a visual inspection and an automated screen. The visual screen was conducted in an unc-104 mutant background. UNC-104 is a KIF1A kinesin motor protein. Because of transport defects, unc-104 mutant worms have decreased numbers of synaptic vesicles marked with syntaptobrevin-GFP at the synapse as well as decreased localization of YFP::RAB-5 to the synapse [22]. Furthermore, unc-104 mutant worms are severely paralyzed, facilitating the examination of the YFP::RAB-5 protein localization phenotype. In this visual screen, 1,824 haploid genomes have been screened, resulting in the isolation of eight mutants with disrupted YFP::RAB-5 protein localization patterns. A parallel screen, based on a recent technological development using microfluidic automated screening [25] was carried out using YFP::RAB-5 in a wildtype background, screening 1500 haploid genomes and isolating nine mutations. Screening identified animals in which YFP::RAB-5 protein localization was decreased within D neuron cell bodies. Further inspection determined whether there were also synaptic changes (Figure 1a).


Rabx-5 regulates RAB-5 early endosomal compartments and synaptic vesicles in C. elegans.

Sann SB, Crane MM, Lu H, Jin Y - PLoS ONE (2012)

Rabx-5 mutations alter the RAB-5 organization in GABAergic motor neurons.A) A forward genetic screen was conducted by observing GABAergic motor neurons for changes in localization or intensity of Punc-25YFP::RAB-5. Initial screening isolated mutants in which YFP::RAB-5 was decreased in the cell soma. Further inspection determined if YFP::RAB-5 was increased at the synapse. B) Gene, transcript, and protein structure of rabx-5. The RABX-5 protein consists of a zinc finger motif (ZF) and a motif interacting with ubiquitin (U) that together regulate association with ubiquitinated endosomal cargo; a membrane binding motif and helical bundle required for association with the endosomal membrane; a Vps9 domain that along with the helical bundle promotes guanine exchange activity; and a coiled-coil region that binds rabaptin-5 and contains a motif for autoinhibition of guanine exchange activity. The rabx-5(qa7800) mutation leads to a truncation at the helical bundle. The rabx-5(tm1512) mutation deletes the start site and the exons encoding the zinc finger motif and the motif interacting with ubiquitn. C) Punc-25YFP::RAB-5 protein localization in the soma (left column) and dorsal cord (right column) of GABAergic motor neurons in wild type and mutant animals. D) Punc-25YFP::RAB-5 fluorescence intensity was decreased in the soma of mutant animals and increased in the synaptic and intersynaptic regions. This phenotype is recovered with expression of Punc-25CFP::RABX-5. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively. Bar represents the mean. **p<0.01, ***p<0.001. E) Punc-25CFP::RABX-5 expression recovers the rabx-5(qa7800) YFP::RAB-5 phenotype.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366993&req=5

pone-0037930-g001: Rabx-5 mutations alter the RAB-5 organization in GABAergic motor neurons.A) A forward genetic screen was conducted by observing GABAergic motor neurons for changes in localization or intensity of Punc-25YFP::RAB-5. Initial screening isolated mutants in which YFP::RAB-5 was decreased in the cell soma. Further inspection determined if YFP::RAB-5 was increased at the synapse. B) Gene, transcript, and protein structure of rabx-5. The RABX-5 protein consists of a zinc finger motif (ZF) and a motif interacting with ubiquitin (U) that together regulate association with ubiquitinated endosomal cargo; a membrane binding motif and helical bundle required for association with the endosomal membrane; a Vps9 domain that along with the helical bundle promotes guanine exchange activity; and a coiled-coil region that binds rabaptin-5 and contains a motif for autoinhibition of guanine exchange activity. The rabx-5(qa7800) mutation leads to a truncation at the helical bundle. The rabx-5(tm1512) mutation deletes the start site and the exons encoding the zinc finger motif and the motif interacting with ubiquitn. C) Punc-25YFP::RAB-5 protein localization in the soma (left column) and dorsal cord (right column) of GABAergic motor neurons in wild type and mutant animals. D) Punc-25YFP::RAB-5 fluorescence intensity was decreased in the soma of mutant animals and increased in the synaptic and intersynaptic regions. This phenotype is recovered with expression of Punc-25CFP::RABX-5. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively. Bar represents the mean. **p<0.01, ***p<0.001. E) Punc-25CFP::RABX-5 expression recovers the rabx-5(qa7800) YFP::RAB-5 phenotype.
Mentions: Several distinct endosomal compartments are present in the GABAergic motor neuron synapse of C. elegans[22]. The expression pattern of a fluorescently tagged RAB-5 reporter is specifically altered in mutant animals lacking the function of UNC-16, a kinesin adaptor protein [22], [24]. To identify additional genes involved in the endocytic pathway and axonal transport in neuronal development, we conducted a forward genetic screen using a YFP::RAB-5 reporter in GABAergic motor neurons. These neurons are pseudo-unipolar neurons, with their soma located in the ventral nerve cord, and nerve processes elongating along the ventral and dorsal nerve cord, connected by a circumferential commissure. Presynaptic terminals form en passant along the ventral (for VD neurons) and the dorsal (for DD neurons) processes. We have conducted the screen in two ways: a visual inspection and an automated screen. The visual screen was conducted in an unc-104 mutant background. UNC-104 is a KIF1A kinesin motor protein. Because of transport defects, unc-104 mutant worms have decreased numbers of synaptic vesicles marked with syntaptobrevin-GFP at the synapse as well as decreased localization of YFP::RAB-5 to the synapse [22]. Furthermore, unc-104 mutant worms are severely paralyzed, facilitating the examination of the YFP::RAB-5 protein localization phenotype. In this visual screen, 1,824 haploid genomes have been screened, resulting in the isolation of eight mutants with disrupted YFP::RAB-5 protein localization patterns. A parallel screen, based on a recent technological development using microfluidic automated screening [25] was carried out using YFP::RAB-5 in a wildtype background, screening 1500 haploid genomes and isolating nine mutations. Screening identified animals in which YFP::RAB-5 protein localization was decreased within D neuron cell bodies. Further inspection determined whether there were also synaptic changes (Figure 1a).

Bottom Line: The rabx-5 mutation leads to decreased intensity of YFP::RAB-5 in the cell soma but increased intensity in the synaptic and intersynaptic regions of the axon.This effect is due to the bias of the cycling state of RAB-5, and results from a change in the organization of the early endosomal compartment as well as the membrane binding state of RAB-5.These results suggest that rabx-5 regulation of RAB-5 compartments is important for maintaining proper synaptic function throughout the lifetime.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology Section, Division of Biological Sciences, University of California San Diego, La Jolla, California, United States of America. ssann@ucsd.edu

ABSTRACT
Early endosomal membrane compartments are required for the formation and recycling of synaptic vesicles, but how these compartments are regulated is incompletely understood. We performed a forward genetic screen in C. elegans for mutations that affect RAB-5 labeled early endosomal compartments in GABAergic motoneurons. Here we report the isolation and characterization of one mutation, rabx-5. The rabx-5 mutation leads to decreased intensity of YFP::RAB-5 in the cell soma but increased intensity in the synaptic and intersynaptic regions of the axon. This effect is due to the bias of the cycling state of RAB-5, and results from a change in the organization of the early endosomal compartment as well as the membrane binding state of RAB-5. Synaptic vesicle accumulation is altered in rabx-5 mutants, and synaptic transmission from cholinergic neurons is decreased. Early endosomal membrane compartments show disorganization with ageing and rabx-5 mutant animals age faster. These results suggest that rabx-5 regulation of RAB-5 compartments is important for maintaining proper synaptic function throughout the lifetime.

Show MeSH
Related in: MedlinePlus