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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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Effects of the rabx-5 mutation are specific to early endosomal and synaptic compartments.A) Fluorescence intensity of Punc-25HGRS::mCherry, a tyrosine kinase substrate targeted specifically to early endosomes, is increased in synaptic and intersynaptic regions in rabx-5 mutants. B) Fluorescence intensity of Punc-25Syntaxin-13::mCherry, a SNARE protein that marks early and recycling endosomes as well as synaptic vesicles is increased in synaptic and intersynaptic regions in rabx-5 mutants. C) Fluorescence intensity of Punc-25CFP::RAB-11 a marker of recycling endosomes is decreased in soma and synaptic regions in rabx-5 mutants. D) Fluorescence intensity of Punc-25CFP::RAB-7, a marker of late endosomes is similar in wild type and in rabx-5 mutant animals. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively. Bar represents the mean. *p<0.05, ***p<0.001.
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pone-0037930-g004: Effects of the rabx-5 mutation are specific to early endosomal and synaptic compartments.A) Fluorescence intensity of Punc-25HGRS::mCherry, a tyrosine kinase substrate targeted specifically to early endosomes, is increased in synaptic and intersynaptic regions in rabx-5 mutants. B) Fluorescence intensity of Punc-25Syntaxin-13::mCherry, a SNARE protein that marks early and recycling endosomes as well as synaptic vesicles is increased in synaptic and intersynaptic regions in rabx-5 mutants. C) Fluorescence intensity of Punc-25CFP::RAB-11 a marker of recycling endosomes is decreased in soma and synaptic regions in rabx-5 mutants. D) Fluorescence intensity of Punc-25CFP::RAB-7, a marker of late endosomes is similar in wild type and in rabx-5 mutant animals. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively. Bar represents the mean. *p<0.05, ***p<0.001.

Mentions: In rabx-5 mutants, HGRS::mCherry intensity is similar to wildtype within the cell soma but exhibits a significant increase in synaptic (1.63±0.03 IU, 1.0±.04 IU in WT, p<0.0001) and intersynaptic regions (.99±.06 IU, 0.4±.04 IU in WT, p<0.0001) (Figure 4A). A similar pattern is observed in localization of SYN-13::mCherry with significant increases in both synaptic (2.05±.05 IU, 1.00±.03 IU in WT, p<0.0001) and intersynaptic intensity (1.04±.05 IU, 0.45±.03 IU in WT, p<0.0001) (Figure 4B). In contrast, CFP::RAB-11 exhibits decreased synaptic intensity in rabx-5 mutants (0.69±0.03 IU, 1.00±0.02 IU in WT, p<0.0001) and CFP::RAB-7 exhibits no significant change in intensity from WT (Figure 4C and 4D). We conclude that rabx-5 regulates the membranous compartments of early endosomes and not simply the cytosolic versus membranous localization of RAB-5. We also observe that rabx-5 acts primarily on RAB-5 with much less, if any, influence on RAB-11 or RAB-7.


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)

Effects of the rabx-5 mutation are specific to early endosomal and synaptic compartments.A) Fluorescence intensity of Punc-25HGRS::mCherry, a tyrosine kinase substrate targeted specifically to early endosomes, is increased in synaptic and intersynaptic regions in rabx-5 mutants. B) Fluorescence intensity of Punc-25Syntaxin-13::mCherry, a SNARE protein that marks early and recycling endosomes as well as synaptic vesicles is increased in synaptic and intersynaptic regions in rabx-5 mutants. C) Fluorescence intensity of Punc-25CFP::RAB-11 a marker of recycling endosomes is decreased in soma and synaptic regions in rabx-5 mutants. D) Fluorescence intensity of Punc-25CFP::RAB-7, a marker of late endosomes is similar in wild type and in rabx-5 mutant animals. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively. Bar represents the mean. *p<0.05, ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366993&req=5

pone-0037930-g004: Effects of the rabx-5 mutation are specific to early endosomal and synaptic compartments.A) Fluorescence intensity of Punc-25HGRS::mCherry, a tyrosine kinase substrate targeted specifically to early endosomes, is increased in synaptic and intersynaptic regions in rabx-5 mutants. B) Fluorescence intensity of Punc-25Syntaxin-13::mCherry, a SNARE protein that marks early and recycling endosomes as well as synaptic vesicles is increased in synaptic and intersynaptic regions in rabx-5 mutants. C) Fluorescence intensity of Punc-25CFP::RAB-11 a marker of recycling endosomes is decreased in soma and synaptic regions in rabx-5 mutants. D) Fluorescence intensity of Punc-25CFP::RAB-7, a marker of late endosomes is similar in wild type and in rabx-5 mutant animals. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively. Bar represents the mean. *p<0.05, ***p<0.001.
Mentions: In rabx-5 mutants, HGRS::mCherry intensity is similar to wildtype within the cell soma but exhibits a significant increase in synaptic (1.63±0.03 IU, 1.0±.04 IU in WT, p<0.0001) and intersynaptic regions (.99±.06 IU, 0.4±.04 IU in WT, p<0.0001) (Figure 4A). A similar pattern is observed in localization of SYN-13::mCherry with significant increases in both synaptic (2.05±.05 IU, 1.00±.03 IU in WT, p<0.0001) and intersynaptic intensity (1.04±.05 IU, 0.45±.03 IU in WT, p<0.0001) (Figure 4B). In contrast, CFP::RAB-11 exhibits decreased synaptic intensity in rabx-5 mutants (0.69±0.03 IU, 1.00±0.02 IU in WT, p<0.0001) and CFP::RAB-7 exhibits no significant change in intensity from WT (Figure 4C and 4D). We conclude that rabx-5 regulates the membranous compartments of early endosomes and not simply the cytosolic versus membranous localization of RAB-5. We also observe that rabx-5 acts primarily on RAB-5 with much less, if any, influence on RAB-11 or RAB-7.

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