III Methods and materials
4.10 RAB-2 localizes to the Golgi, but not at the synapses
To understand how RAB-2 is involved in DCV biogenesis or maturation we studied its subcellular localization.
Figure 38. RAB-2 localizes to the Golgi apparatus
Fluorescently tagged RAB-2 protein was expressed in the nervous system along with different intracellular markers. Shown are confocal images of neuronal cell bodies in the ventral nerve cord. RAB-2 extensively colocalizes with Golgi markers Mannosidase II (MANS II) and COPI positive structure (ε-COP). RAB-2 shows no or only partial colocalization with ER (cytochrome b5, Cb-5) or early endosomes (FYVE-domain of EEA-1). (Scale bar 5μm).
mCherry-RAB-2
ER Golgi Endosome
GFP-Cb-5 MANS II-GFP 2xFYVE domain-GFP
mCherry-RAB-2 mCherry-RAB-2
Merge Merge Merge
RFP-εCOP
mYFP-RAB-2 mCherry-RAB-2
Merge
COPI vesicles
mCherry-RAB-2
ER Golgi Endosome
GFP-Cb-5 MANS II-GFP 2xFYVE domain-GFP
Merge Merge Merge
mCherry-RAB-2 mCherry-RAB-2
RFP-εCOP
Merge
COPI vesicles
mYFP-RAB-2 mCherry-RAB-2
When expressed as a fusion protein tagged N-terminally with either mYFPcitrine or mCherry, RAB-2 localizes to discrete cytoplasmic puncta in cell bodies of motorneurons (Fig. 38). Localization studies of RAB-2 showed its strong colocalization with Golgi marker Mannosidase II, while there was no colocalization with ER marker Cytochrome-b5 nor endosomal marker, FYVE domain of RAB-5 effector EEA-1.
mCherry-RAB-3
Merge
dorsal nerve cord
mYFP-RAB-2
Figure 39. RAB-2 is not present at the synapses
mYFP-RAB-2 (shown in red) does not accumulate at the synapses in the dorsal nerve cord like mCherry-RAB-3 (shown in green). The boxes represent the magnified synaptic punctae demonstrating no accumulation of RAB-2 at the synapses (Scale bar 10μm). Schematic drawing on the left represents the HSN neuron. The closed arrow heads represent the cell body of HSN neuron and the open arrowheads, the synapses formed by HSN neuron onto the vulval muscle and VC4 and VC5 neurons. HSN neuron axon enters the ventral nerve cord and stretches towards the head of the worm. In HSN neurons mCherry-RAB-2 is present only in cytoplasmic puncta in neuronal cell body, but not at the synapse with vulva muscle. HSN synapses are labeled by GFP-RAB-3. Scale bar is 5μm.
GFP-RAB-3HSN neuron mCherry-RAB-2 Merge mCherry-RAB-3
Merge
dorsal nerve cord
GFP-RAB-3HSN neuron
mCherry-RAB-2 Merge
mYFP-RAB-2
More importantly, RAB-2 does not localize to the synapses, as seen on light microscopy images. While for RAB-3 protein, which is a SV marker, strong accumulations were observed at the synapses of dorsal nerve cord, no accumulation was observed for RAB-2. Furthermore, looking at the single HSN neuron, strong accumulation of RAB-3 was observed at the synapse of the neuron with the vulva muscle, while RAB-2 is present only at the cell body of the neuron (Fig. 39).
To confirm the Golgi localization of the RAB-2, we performed immunogold labeling of endogenous RAB-2 on thin plastic sections immuno EM. For this purpose we generated monoclonal antibodies against C. elegans RAB-2.
RAB-2 broadly localizes to Golgi and vesicular structures around the Golgi with preference for cis side (Fig. 40). The staining was specific, because for rab-2 deletion mutant no gold labels were observed (Fig. 41). This suggests RAB-2 is active at the Golgi and not at the synapse, which is in agreement with possible function of RAB-2 in DCV biogenesis or maturation.
motor neuron cell body motor neuron cell body
Figure 40. RAB-2 is enriched at the cis side of the Golgi.
Immuno-gold labeling of endogenous RAB-2 in motorneuron cell bodies. 85 nm HPF EM thin plastic sections were probed with monoclonal RAB-2 antibodies. Gold labeling (white circles) is seen at the Golgi complex and vesicular structures around the Golgi. (Courtesy of Jan Hegermann).
N
G N G
wild type
wild type unc-unc-108 (nu415)108 (nu415)
N
G N
G N G
N G
wild type
wild type unc-unc-108 (nu415)108 (nu415) motorneuron cell bodies
N
G N G
wild type
wild type unc-unc-108 (nu415)108 (nu415)
N
G N
G N G
N G
wild type
wild type unc-unc-108 (nu415)108 (nu415) motorneuron cell bodies
Figure 41. Immunogoldlabeling of endogenous RAB-2 in wild type and null unc-108 (nu415) strain.
Immunogoldlabelling of endogenous RAB-2 in wild type and null unc-108 (nu415) strain, showing the specificity of the RAB-2 antibody. While in wild type most of the labels are at the Golgi or at the vesicles around the Golgi, in nu415 allele which has a nonsense mutation in rab-2 gene, only the background stains are observed. N, nucleus; G, Golgi stack. (Courtesy of Jan Hegermann).
Because the interaction of RAB-2 with Golgi matrix protein and its localization at the Golgi, one possibility would be that mutations in RAB-2 influence the morphology and therefore the function of the Golgi. Ultimately, this would influence the DCV formation at the trans Golgi network. However, the Golgi complexes appeared to be normal in unc-108 mutants (Fig. 43).
500 nm 500 nm
wild type
wild type uncunc--108 (n501)108 (n501) N
unc--108 (n777)108 (n777)
NN GG
uncunc--108 (nu415)108 (nu415) N
wild type uncunc--108 (n501)108 (n501) N
unc--108 (n777)108 (n777)
NN GG
uncunc--108 (nu415)108 (nu415) N
wild type uncunc--108 (n501)108 (n501) N
unc--108 (n777)108 (n777)
NN GG
uncunc--108 (nu415)108 (nu415) N
wild type uncunc--108 (n501)108 (n501) N
unc--108 (n777)108 (n777)
NN GG
uncunc--108 (nu415)108 (nu415) N
N GG
A A B B
C C D D
motorneuron cell bodies
Figure 43. Golgi morphology in neuronal cell bodies is not altered in unc-108 mutants.
Motorneuron cell bodies are shown for wild type (A) and unc-108 n501 (B), n777 (C) and nu415 (D) alleles. The morphology of the Golgi complexes in dominant or recessive unc-108 mutants appears normal when compared to wild type using HPF EM. (Courtesy of Jan Hegermann).