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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 acts by biasing the cycling state of RAB-5.A) Soma and dorsal cord of mutant and wildtype GABAergic motor neurons expressing YFP::RAB-5(Q78L). The rabx-5 mutation does not exhibit as dramatic a phenotype as on YFP::RAB-5. B) Soma and dorsal cord of mutant and wildtype GABAergic motor neurons expressing YFP::RAB-5(S33N). The rabx-5 mutation does not exhibit as dramatic a phenotype as on YFP::RAB-5. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively. Bar represents the mean. *p<0.05, ***p<0.001 C) Fluorescent recovery after photobleaching (FRAP) of YFP::RAB-5 in the synaptic regions of WT, the synaptic and intersynaptic regions of rabx-5, and the synaptic region of unc-16 mutant worms in which RAB-5 is biased to the GTP bound state; and FRAP of free GFP. Arrow marks the region of bleaching. Fluorescence recovery after photobleaching of synaptic YFP::RAB-5 in rabx-5 mutants is significantly slower than WT but faster than unc-16 mutants (p<0.01 at time points from 5–15 sec; mean ± SEM). Intersynaptic YFP::RAB-5 in rabx-5 mutant animals exhibits similar dynamics to free GFP.
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pone-0037930-g003: Rabx-5 acts by biasing the cycling state of RAB-5.A) Soma and dorsal cord of mutant and wildtype GABAergic motor neurons expressing YFP::RAB-5(Q78L). The rabx-5 mutation does not exhibit as dramatic a phenotype as on YFP::RAB-5. B) Soma and dorsal cord of mutant and wildtype GABAergic motor neurons expressing YFP::RAB-5(S33N). The rabx-5 mutation does not exhibit as dramatic a phenotype as on YFP::RAB-5. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively. Bar represents the mean. *p<0.05, ***p<0.001 C) Fluorescent recovery after photobleaching (FRAP) of YFP::RAB-5 in the synaptic regions of WT, the synaptic and intersynaptic regions of rabx-5, and the synaptic region of unc-16 mutant worms in which RAB-5 is biased to the GTP bound state; and FRAP of free GFP. Arrow marks the region of bleaching. Fluorescence recovery after photobleaching of synaptic YFP::RAB-5 in rabx-5 mutants is significantly slower than WT but faster than unc-16 mutants (p<0.01 at time points from 5–15 sec; mean ± SEM). Intersynaptic YFP::RAB-5 in rabx-5 mutant animals exhibits similar dynamics to free GFP.

Mentions: Comparing YFP::RAB-5(Q78L) intensity between WT and rabx-5 mutant animals, we see no significant change in cell body intensity and a slight but significant increase in dorsal cord synaptic (WT 0.27±0.02, mutant 0.52±.03 IU) and intersynaptic (WT 0.082±.007, mutant 0.17±.02 IU) intensity (Figure 3A). This increase is not as dramatic as the approximately five-fold increase seen for YFP::RAB-5 intensity. YFP::RAB-5(S33N) exhibits an increase in cell body intensity in rabx-5 mutants (WT 1.4±0.3 IU, mutant 3.6±0.9 IU), the opposite of the effect on YFP::RAB-5 (Figure 3B). There is no significant change in intensity in the dorsal cord. These results suggest that the effect of the rabx-5 mutation on RAB-5 protein abundance and localization is at least partially due to a bias in RAB-5 guanine cycling state.


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 acts by biasing the cycling state of RAB-5.A) Soma and dorsal cord of mutant and wildtype GABAergic motor neurons expressing YFP::RAB-5(Q78L). The rabx-5 mutation does not exhibit as dramatic a phenotype as on YFP::RAB-5. B) Soma and dorsal cord of mutant and wildtype GABAergic motor neurons expressing YFP::RAB-5(S33N). The rabx-5 mutation does not exhibit as dramatic a phenotype as on YFP::RAB-5. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively. Bar represents the mean. *p<0.05, ***p<0.001 C) Fluorescent recovery after photobleaching (FRAP) of YFP::RAB-5 in the synaptic regions of WT, the synaptic and intersynaptic regions of rabx-5, and the synaptic region of unc-16 mutant worms in which RAB-5 is biased to the GTP bound state; and FRAP of free GFP. Arrow marks the region of bleaching. Fluorescence recovery after photobleaching of synaptic YFP::RAB-5 in rabx-5 mutants is significantly slower than WT but faster than unc-16 mutants (p<0.01 at time points from 5–15 sec; mean ± SEM). Intersynaptic YFP::RAB-5 in rabx-5 mutant animals exhibits similar dynamics to free GFP.
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Related In: Results  -  Collection

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pone-0037930-g003: Rabx-5 acts by biasing the cycling state of RAB-5.A) Soma and dorsal cord of mutant and wildtype GABAergic motor neurons expressing YFP::RAB-5(Q78L). The rabx-5 mutation does not exhibit as dramatic a phenotype as on YFP::RAB-5. B) Soma and dorsal cord of mutant and wildtype GABAergic motor neurons expressing YFP::RAB-5(S33N). The rabx-5 mutation does not exhibit as dramatic a phenotype as on YFP::RAB-5. Each point represents a single soma, synaptic puncta, or intersynaptic axonal region, respectively. Bar represents the mean. *p<0.05, ***p<0.001 C) Fluorescent recovery after photobleaching (FRAP) of YFP::RAB-5 in the synaptic regions of WT, the synaptic and intersynaptic regions of rabx-5, and the synaptic region of unc-16 mutant worms in which RAB-5 is biased to the GTP bound state; and FRAP of free GFP. Arrow marks the region of bleaching. Fluorescence recovery after photobleaching of synaptic YFP::RAB-5 in rabx-5 mutants is significantly slower than WT but faster than unc-16 mutants (p<0.01 at time points from 5–15 sec; mean ± SEM). Intersynaptic YFP::RAB-5 in rabx-5 mutant animals exhibits similar dynamics to free GFP.
Mentions: Comparing YFP::RAB-5(Q78L) intensity between WT and rabx-5 mutant animals, we see no significant change in cell body intensity and a slight but significant increase in dorsal cord synaptic (WT 0.27±0.02, mutant 0.52±.03 IU) and intersynaptic (WT 0.082±.007, mutant 0.17±.02 IU) intensity (Figure 3A). This increase is not as dramatic as the approximately five-fold increase seen for YFP::RAB-5 intensity. YFP::RAB-5(S33N) exhibits an increase in cell body intensity in rabx-5 mutants (WT 1.4±0.3 IU, mutant 3.6±0.9 IU), the opposite of the effect on YFP::RAB-5 (Figure 3B). There is no significant change in intensity in the dorsal cord. These results suggest that the effect of the rabx-5 mutation on RAB-5 protein abundance and localization is at least partially due to a bias in RAB-5 guanine cycling state.

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