III Methods and materials
4.14 Constitutively active RAB-5 blocks DCV secretion
We showed that overexpression of dominant active RAB-5 rescues the NLP-21 derived YFP levels at the unc-108 synapses. During the course of our experiment, we realized that the expression of dominant active RAB-5 although rescuing the axonal NLP-21 derived YFP levels, led to a block of DCV secretion (Fig. 64). In agreement with this, neuronal expression of dominant active RAB-5 decreased the DCV secretion by 80% (Fig. 65). Since dominant active RAB-5 is only expressed in neurons, it could not affect the endocytosis of coelomocytes.
nuIs183; unc-108 (n501); Ex[prab-3 ::rab-5 (DN)]nuIs183; unc-108 (n501); Ex[prab-3 ::rab-5 (DA)]
NLP-21-YFP DIC
10μm
nuIs183; unc-108 (n501); Ex[prab-3 ::rab-5 (DN)]nuIs183; unc-108 (n501); Ex[prab-3 ::rab-5 (DA)]
NLP-21-YFP DIC
10μm
Figure 64. Neuronal expression of dominant active RAB-5 blocks the DCV secretion from unc-108 neurons.
NLP-21-YFP was expressed in cholinergic motorneurons in the strain nuIs183. The expression of dominant active but not dominant negative RAB-5 blocks the NLP-21 derived YFP secretion in unc-108 mutant background (Scale bar is 10μm).
nuIs183; Ex[prab-3::
Figure 65. The overexpression of dominant active GTP bound RAB-5 blocks the secretion of DCVs.
mCherry tagged dominant active GTP bound form of RAB-5 was expressed panneuronally in nuIs183 strain which stably expresses neuropeptide NLP-21 tagged to YFP. Once secreted at
the synapse, NLP-21-YFP is taken up by coelomocytes. The overexpression of RAB-5 reduces the fluorescence in coelomocytes, thus reduces the secretion of the peptide at the synapses. (Scale bar is 5μm. Error bar is SEM, number of animals analyzed is indicated in the graph bars, ***p<0.005, Student’s T-test).
Accordingly, the overexpression of dominant active RAB-5 in neurons, leads to movement defects and aldicarb resistance (Fig. 66). This is in agreement with blockage of DCV signaling.
0 25 50 75 100
unc-108 (n501) rab-5 (DA)
Time (min.)
unc-108 (n501) rab-5 (DA)
Time (min.)
unc-108 (n501) rab-5 (DA)
Time (min.)
Figure 66. Overexpression of dominant active RAB-5 in neurons leads to movement defects and aldicarb resistance.
(A) Overexpression of dominant active RAB-5 in neurons decreases movement by 50%
compared to wild type (Number of animals analyzed is indicated in the graph bars, error bar is SEM, ***p<0.005, Student’s T-test)
(B) Overexpression of dominant active RAB-5 in neurons leads to aldicarb resistance similar to the one of unc-108 (n501) mutants (N=30 animals per strain, error bar is SEM).
Surprisingly, expression of dominant active GTP bound RAB-5 (Q78L) DA induces formation big vesicular structures in unc-108 synapses, but not in the wild type (Fig. 67). These could either present the early endosomes at the synapses or abnormal either synaptic or DCV fusions. We were not able to identify the identity of these vesicular structures, since the markers are not available: EEA-1 is not present at the synaptic early endosomes and RAB-3 and SNB-1 are present on both synaptic and DCVs.
motorneuron cell body synapse
unc
unc--108 (n501); 108 (n501);
prab
prab--3::mCherry3::mCherry--rabrab--5 (Q78L) DA5 (Q78L) DA uncunc--108 (n501); 108 (n501);
prab
prab--3::mCherry3::mCherry--rabrab--5 (Q78L) DA5 (Q78L) DA
motorneuron cell body synapse
unc
unc--108 (n501); 108 (n501);
prab
prab--3::mCherry3::mCherry--rabrab--5 (Q78L) DA5 (Q78L) DA uncunc--108 (n501); 108 (n501);
prab
prab--3::mCherry3::mCherry--rabrab--5 (Q78L) DA5 (Q78L) DA
Figure 67. Expression of dominant active GTP bound form of RAB-5 induces formation of big vesicular structures at unc-108 (n501) synapses.
HPF EM cross sections of motorneuron cell bodies and synapse of unc-108 (n501) expressing dominant active GTP bound RAB-5 (Q78L) DA.
To further investigate this, we looked at the SV marker RAB-3 fused to YFP in the wild type worms expressing dominant active GTP bound RAB-5 (Q78L) DA. In the wild type background, overexpression of dominant active form of
RAB-5 did not induce any changes in total number of fluorescence, nor synapse spacing or synaptic size (Fig. 68 and 69).
0.0 0.5 1.0 1.5
wild type
wild type; Ex[prab-3::mCherry-rab-5 (Q78L) DA
0.0 0.5 1.0
GFP-SNB-1 in dorsal nerve cord
normalized fluorescencenormalized fluorescence
wild type
number of punctaper mm
10 10
wild type; Ex[prab-3::mCherry-rab-5 (Q78L) DA
0.0 0.5 1.0
GFP-SNB-1 in dorsal nerve cord
normalized fluorescencenormalized fluorescence
wild type
number of punctaper mm
10 10
Figure 68. Overexpression of dominant active GTP bound form of RAB-5 does not induce synaptic morphology changes in wild type.
Dominant active GTP bound RAB-5 (Q78L) DA was expressed in strain expressing SNB-1-GFP in cholinergic motorneurons. The synapse morphology appears to be similar between wild type and wild type expressing dominant active RAB-5, as judged by SNB-1-GFP puncta number, size and average fluorescence. (Scale bar is 10μm, error bar is SEM, number of animals analyzed is indicated in the graph bars).
Figure 69. Expression of dominant active GTP bound form of RAB-5 does not change the morphology of wild type synapses.
wild type
wild type wild type; wild type; Ex[prabEx[prab--3::3::
mCherry
mCherry--rabrab--5 (Q78L) DA]5 (Q78L) DA]
wild type
wild type wild type; wild type; Ex[prabEx[prab--3::3::
mCherry
mCherry--rabrab--5 (Q78L) DA]5 (Q78L) DA]
HPF EM cross sections of synapses of wild type and wild type worms expressing dominant active GTP bound RAB-5 (Q78L) DA. (Courtesy of Jan Hegermann).
synaptic vesicle distribution
0 5 10 15 20 25
50 100 150 200 250 300 350 400 450 500
distance from synaptic density (nm)
percentage of SV / synapse
wild type rab-5 DA in WT
Furthermore, the distribution and the number of SVs does not change upon expression of dominant active RAB-5, as judged by the HPF EM data (Fig. 70 and Table 8). However, both SVs and DCVs appear to be smaller compared to wild type and DCV are more abundant. These data suggest that dominant active most likely affects DCV secretion specifically.
Figure 70. Expression of dominant active RAB-5 does not change the SV distribution in wild type animals.
The SV distribution relative to the presynaptic density at release sites was analyzed for wild type and wild type worms expressing dominant active GTP bound RAB-5 (Q78L) DA.
(Courtesy of Jan Hegermann and Christian Olendrowitz)
Table 9. SV and DCV statistics of dominant active RAB-5 expressing synapse.
Statistics on SV and DCV numbers and diameters as determined by HPF EM, the average synaptic area, number of synapses and independent animals analyzed are indicated.
(Courtesy of Jan Hegermann and Christian Olendrowitz)