The Caenorhabditis elegans Kinesin-3 Motor UNC-104/KIF1A Is Degraded upon Loss of Specific Binding to Cargo

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DOI: 10.1371/journal.pgen.1001200 · Source: PubMed
Abstract
UNC-104/KIF1A is a Kinesin-3 motor that transports synaptic vesicles from the cell body towards the synapse by binding to PI(4,5)P(2) through its PH domain. The fate of the motor upon reaching the synapse is not known. We found that wild-type UNC-104 is degraded at synaptic regions through the ubiquitin pathway and is not retrogradely transported back to the cell body. As a possible means to regulate the motor, we tested the effect of cargo binding on UNC-104 levels. The unc-104(e1265) allele carries a point mutation (D1497N) in the PI(4,5)P(2) binding pocket of the PH domain, resulting in greatly reduced preferential binding to PI(4,5)P(2)in vitro and presence of very few motors on pre-synaptic vesicles in vivo. unc-104(e1265) animals have poor locomotion irrespective of in vivo PI(4,5)P(2) levels due to reduced anterograde transport. Moreover, they show highly reduced levels of UNC-104 in vivo. To confirm that loss of cargo binding specificity reduces motor levels, we isolated two intragenic suppressors with compensatory mutations within the PH domain. These show partial restoration of in vitro preferential PI(4,5)P(2) binding and presence of more motors on pre-synaptic vesicles in vivo. These animals show improved locomotion dependent on in vivo PI(4,5)P(2) levels, increased anterograde transport, and partial restoration of UNC-104 protein levels in vivo. For further proof, we mutated a conserved residue in one suppressor background. The PH domain in this triple mutant lacked in vitro PI(4,5)P(2) binding specificity, and the animals again showed locomotory defects and reduced motor levels. All allelic variants show increased UNC-104 levels upon blocking the ubiquitin pathway. These data show that inability to bind cargo can target motors for degradation. In view of the observed degradation of the motor in synaptic regions, this further suggests that UNC-104 may get degraded at synapses upon release of cargo.
The
Caenorhabditis elegans
Kinesin-3 Motor UNC-104/
KIF1A Is Degraded upon Loss of Specific Binding to
Cargo
Jitendra Kumar
1.
, Bikash C. Choudhary
1.
, Raghu Metpally
, Qun Zheng
2
, Michael L. Nonet
2
,
Sowdhamini Ramanathan
1
, Dieter R. Klopfenstein
3,4
, Sandhya P. Koushika
1
*
1 National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India, 2 Department of Anatomy and Neurobiology, Washington University
School of Medicine, St. Louis, Missouri, United States of America, 3 Center for Molecular Physiology of the Brain, Georg August Universita
¨
tGo
¨
ttingen, Go
¨
ttingen, Germany,
4 Drittes Physikalisches Institut, Go
¨
ttingen, Germany
Abstract
UNC-104/KIF1A is a Kinesin-3 motor that transports synaptic vesicles from the cell body towards the synapse by binding to
PI(4,5)P
2
through its PH domain. The fate of the motor upon reaching the synapse is not known. We found that wild-type
UNC-104 is degraded at synaptic regions through the ubiquitin pathway and is not retrogradely transported back to the cell
body. As a possible means to regulate the motor, we tested the effect of cargo binding on UNC-104 levels. The unc-
104(e1265) allele carries a point mutation (D1497N) in the PI(4,5)P
2
binding pocket of the PH domain, resulting in greatly
reduced preferential binding to PI(4,5)P
2
in vitro and presence of very few motors on pre-synaptic vesicles in vivo. unc-
104(e1265) animals have poor locomotion irrespective of in vivo PI(4,5)P
2
levels due to reduced anterograde transport.
Moreover, they show highly reduced levels of UNC-104 in vivo. To confirm that loss of cargo binding specificity reduces
motor levels, we isolated two intragenic suppressors with compensatory mutations within the PH domain. These show
partial restoration of in vitro preferential PI(4,5)P
2
binding and presence of more motors on pre-synaptic vesicles in vivo.
These animals show improved locomotion dependent on in vivo PI(4,5)P
2
levels, increased anterograde transport, and
partial restoration of UNC-104 protein levels in vivo. For further proof, we mutated a conserved residue in one suppressor
background. The PH domain in this triple mutant lacked in vitro PI(4,5)P
2
binding specificity, and the animals again showed
locomotory defects and reduced motor levels. All allelic variants show increased UNC-104 levels upon blocking the ubiquitin
pathway. These data show that inability to bind cargo can target motors for degradation. In view of the observed
degradation of the motor in synaptic regions, this further suggests that UNC-104 may get degraded at synapses upon
release of cargo.
Citation: Kumar J, Choudhary BC, Metpally R, Zheng Q, Nonet ML, et al. (2010) The Caenorhabditis elegans Kinesin-3 Motor UNC-104/KIF1A Is Degraded upon Loss
of Specific Binding to Cargo. PLoS Genet 6(11): e1001200. doi:10.1371/journal.pgen.1001200
Editor: Miriam B. Goodman, Stanford University School of Medicine, United States of America
Received February 17, 2010; Accepted October 7, 2010; Published November 4, 2010
Copyright: ß 2010 Kumar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by the funding agency Department of Science and Technology, http://dst.gov.in/, grant number SR/S0/AS-67/2006. BCC is
supported by a Ph.D. fellowship from CSIR, Government of India. CIFF, NCBS was supported by Department of Scien ce and Technology - Centre for
Nanotechnology (No. SR/55/NM-36-2005). The funders had no role in study design, data collection and analysis, deci sion to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: koushika@ncbs.res.in
. These authors contributed equally to this work.
¤ Current address: University of Iowa, Institute for Clinical and Translational Science (ICTS) Administration, Iowa City, Iowa, United States of America
Introduction
Transport of pre-synaptic vesicles from the neuronal cell body
to the synapse is an essential process to ensure that the nerve
terminals can effectively participate in synaptic transmission [1,2].
This transport is a regulated process that occurs primarily using
the Kinesin-3 family motor UNC-104, Imac, KIF1A and KIF1Bb,
respectively, in the model systems C. elegans, Drosophila, mouse and
humans [3-9]. In C. elegans, mutants in unc-104 have locomotory
defects that arise from the absence of transport of synaptic vesicles,
leading to reduced synaptic transmission at neuromuscular
junction synapses [3,10].
Molecular motors in neurons such as UNC-104 are thought to
bind to their cargoes in the cell body of the neuron, get transported
along microtubule tracks to synapses and release their cargo upon
reaching the synapse [2]. It has been proposed that upon release of
cargo the motor gets either inactivated or degraded [11], thus
suggesting cargo binding and cargo release as possible means to
regulate motor levels. UNC-104 recognizes its cargo by binding
PI(4,5)P
2
present on the carrier vesicle via its PH domain [12] and
its mammalian orthologue in addition uses other proteins to
recognize cargo [13].
Several effects of cargo binding on the Kinesin-3 family motors
have been shown. Cargo binding by a chimeric Kinesin-3 leads to
aggregation of the motor on the cargo surface and improved
processivity of the chimera [14,15]. Mutations in the cargo-
binding PH domain of UNC-104 that do not bind PI(4,5)P
2
efficiently have also been suggested to affect processivity of the
motor [12,14]. Further, it has been proposed that UNC-104
dimerizes upon cargo binding [14]. The mammalian KIF1A has
PLoS Genetics | www.plosgenetics.org 1 November 2010 | Volume 6 | Issue 11 | e1001200
recently been reported to exist in a dimeric autoinhibited state
from which it is released upon cargo binding [16,17], showing that
while the orthologues behave differently, they are both regulated
by cargo binding. Similarly another motor, Kinesin-1, is
maintained in an inactive folded state [18] and is activated by
binding to regulatory molecules/cargo adaptors. Simultaneous
binding by both JIP1 and Fez1 activates Kinesin-1 and allows the
motor to bind microtubules [19].
Cargo release has also been postulated to play important roles in
motor regulation [20]. Motors involved in anterograde axonal
transport such as Kinesin-1, Kinesin-3/KIF1A and heterotrimeric
Kinesin are all thought be regulated after releasing cargo at the
synapse. All three motors, although transported robustly in the
anterograde direction to the synapse, are not efficiently retro-
gradely transported [6,21-23]. These observations have led to the
hypothesis that once these motors release cargo at synapses, they
are largely degraded, thus maintaining directionality of axonal
transport [11].
We sought to test this hypothesis for the C. elegans UNC-104
motor protein. To do so, it is necessary to address the following
two questions. 1) Does the motor get degraded at the synapse? 2)
Does the motor get degraded once there is no binding to the
cargo? To answer the first question, we established that the wild
type motor is degraded in synaptic regions and that it does not
return to the cell body from the synapse. We further showed that
the degradation near the synapse takes place through the ubiquitin
pathway. To address the second question, we studied the effect of
lack of cargo binding on the C. elegans UNC -104 motor protein.
For this we used a mutant UNC-104 motor and showed that it has
greatly reduced ability to preferentially bind PI(4,5)P
2
in vitro as
well as greatly reduced presence on pre-synaptic vesicles in vivo.We
found that this leads to almost total loss of the motor in vivo, even
though the motor still retains the ability to bind other lipids. The
relationship between ability to bind cargo and motor levels was
verified by analyzing two intragenic suppressors of the original
mutation in the PH domain. The suppressors only moderately
reduce the ability to preferentially bind PI(4,5)P
2
and we see that
UNC-104 levels are partially restored. All three PH domain
variants of the motor are degraded via the ubiquitin pathway in
synapse rich regions of the animal. A triple mutant reversing the
effect of one of the suppressor mutations again does not
preferentially bind PI(4,5)P
2
in vitro, does not provide behavioural
rescue and does not show expression of UNC-104 in vivo. These
findings, together with the observed degradation of wild type
UNC-104 in synaptic regions, suggest that the synaptic vesicle
motor UNC-104 is degraded upon release from pre-synaptic
vesicles near the synapse.
Results
Wild-type UNC-104 is degraded at synapses
To determine whether the UNC-104/KIF1A motor is degraded
at synapses we used a transgenic line over-expressing UNC-
104::GFP in the six mechanosensory neurons of C. elegans.We
examined the posterior neurons (PLM) whose morphology and
synaptic locations are very well defined [24]. Further, a C-terminal
UNC-104::GFP fusion provides functional rescue and its localiza-
tion is similar to that of endogenous UNC-104 [25] suggesting that
the addition of GFP does not impair the motor’s in vivo function or
localization. In a wild type background the UNC-104::GFP is
present in the cell body, neuronal process and at synaptic regions
(Figure 1A: b1-b3).
To determine whether UNC-104 is degraded we crossed the
transgenic strain expressing UNC-104::GFP into a temperature
sensitive uba-1(it129ts) mutant. uba-1 encodes the only C. elegans E1
ubiquitin activating enzyme [26]. This activation is an early and
essential step in the ubiquitin-degradation pathway. Consequently
in uba-1 animals ubiquitin-mediated degradation is reduced. At the
lower growth temperature of 16uC the expression of UNC-
104::GFP in uba-1 animals is not significantly different from wild
type in the cell body, neuronal process or at synaptic regions
(Figure 1A: b1-b6, 1B). However, at the restrictive temperature of
22uC the expression of UNC-104::GFP significantly increases in
synaptic regions (Figure 1A: e1,e4, 1B). The expression remains
largely unchanged in the axon and in the cell body, although our
method may not be sensitive to small changes in protein levels,
especially in the narrow geometry of the neuronal process
(Figure 1A: e2,e5, 1C, Figure S4H).
To confirm that the morphology of the mechanosensory neuron
(including its synapses) is relatively unaffected in uba-1 animals, we
examined the localization and levels of soluble GFP and of the
synaptic vesicle marker GFP::RAB-3 [27-29]. No alteration in
expression levels of soluble GFP or GFP::RAB-3 was observed in
the synapses, cell body or axon in uba-1 animals (Figure 1A: d1-d6,
f1-f6, 1C, 1D, Figure S4F, S4G, S4H). Compared to wild type, no
changes were observed in the area and intensity of GFP in synaptic
regions marked either by soluble GFP or by GFP::RAB-3 in uba-1
animals, with the exception of a modest decrease observed in
synaptic area marked by GFP::RAB-3 at the restrictive temper-
ature (Figure 1A: d1-d6,f1-f6, 1B, 1C, 1G). This exception is
consistent with the known importance of degradation for synapse
formation in mechanosensory neurons [30]. Taken together these
data suggest that development of the mechanosensory neurons and
their synapses are not greatly altered in uba-1(it129ts) while there
are significant effects on the levels of expression of the synaptic
vesicle motor UNC-104 at synaptic regions.
The above observations also suggest that UNC-104 may get
degraded directly through attachment of ubiquitin (8 kDa)
molecules to the motor. To test if UNC-104 is ubiquitinated we
immunoprecipitated the endogenous UNC-104 motor (approxi-
mately 200 kDa) from a mixed-stage C. elegans extract. Western
blot analysis of immunoprecipitated UNC-104 motor showed that
the same band of about 200 kDa was recognized by both the anti-
UNC-104 and the anti-ubiquitin antibodies (Figure 2D). Further,
western analysis of the immunoprecipitate obtained using anti-
ubiquitin and probed with anti-UNC-104 showed an approxi-
mately 200 kDa band, which migrates identically to the
endogenous UNC-104 motor (Figure 2D). However, unlike the
Author Summary
The cell body and the synapse in a neuron are often
separated by significant distance, which is spanned by the
axon connecting the two. Transport of various cargoes
along the axonal highway is very important for neuronal
function. The regulation of this complex process is not well
understood. Using the Caenorhabditis elegans model
system, we have demonstrated for the first time the fate
of a motor after it carries its cargo to the synapse from the
cell body. We show that the UNC-104 motor, which carries
pre-synaptic vesicles to the synapse, is degraded once it
gets there. Moreover, our genetic studies show evidence
that loss of cargo binding targets the motor for
degradation, suggesting an attractive mechanism for the
regulation of motors at the synapse. Our study opens up
several further questions, such as the mechanism of motor
degradation, and has significant implications for regulation
of cargo transport.
UNC-104 Degrades upon Loss of Cargo Binding
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Figure 1. Degradation of UNC-104::GFP in mechanosensory neurons. (A) Effect of reduced ubiquitination (using the mutant uba-1) on sub-
cellular UNC-104 levels. Expression levels of different transgenes in uba-1(it129ts) background in mechanosensory neurons. Left: 16uC (permissive)
UNC-104 Degrades upon Loss of Cargo Binding
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immunoprecipitation with the anti-UNC-104, immunoprecipita-
tion using anti-ubiquitin showed the presence of UNC-104 in the
supernatant. This signal may rise from UNC-104 molecules that
are not ubiquitinated. Our observations suggest that UNC-104
can be ubiquitinated in vivo. Further, the data imply that UNC-104
transports synaptic vesicles to the synapse and upon reaching that
location UNC-104 is degraded through the ubiquitin pathway,
possibly through direct ubiquitination of the endogenous motor.
UNC-104 is not retrogradely transported from synapses
back to the cell body
Our observation that the motor reaching the synapse gets
degraded predicts that there would be little retrograde transport of
UNC-104 from the synapse back to the cell body. To test this
hypothesis we carried out a transport assay by laser microsurgery
of the mechanosensory neuron. We had observed that 1 hour after
axotomy, cargoes such as GFP::RAB-3 and SNB-1::GFP accu-
mulate on both sides of the cut site [31]. By contrast, wild type
UNC-104::GFP accumulates only in the proximal region, i.e., at
the end of the cut that is attached to the cell body (Figure 2A: b,d
arrowhead). The distal end shows no accumulation of UNC-
104::GFP, corroborating the hypothesis. Further the distal axon
shows much lower level of UNC-104::GFP than the same region
of the uncut axon (Figure 2A: b,d arrow). This reduction could
result from UNC-104 in the distal axon being degraded after
reaching the synapse. UNC-104::GFP has been shown in the
seconds time-scale to undergo microscopic motion in both
anterograde and retrograde directions but with a significant
anterograde bias [25], which may result in overall bulk flow of the
motor also being biased towards synaptic regions. Our results
show that additionally, degradation of the motor in the synaptic
region confers a macroscopic directionality to the movement of the
motor. To further test this explanation, we carried out laser
axotomy in uba-1 animals and also did bleach recovery
experiments to assess UNC-104 motor flow.
uba-1 animals, one hour after laser axotomy of mechanosensory
neurons expressing UNC-104::GFP, showed robust levels of
UNC-104::GFP at the proximal regions (Figure 2A: f,i arrow)
and significant levels in the distal regions as well (Figure 2A: e-i
arrowhead). Thus upon blocking ubiquitination, UNC-104 is
capable of macroscopic retrograde movement. To further confirm
predicted trends in macroscopic motor movement in an uninjured
neuron, we carried out a bleach recovery experiment of UNC-
104::GFP in both wild type and uba-1 animals. In either genotype,
the UNC-104 motor recovers in both anterograde and retrograde
directions in the time frame of seconds (Figure 2B). In wild type,
the anterograde recovery is faster than the retrograde recovery,
consistent with prior observations of anterograde bias of the
microscopic movements of UNC-104::GFP (Figure 2C) [25].
Further, supporting our hypothesis, we observed that the
retrograde recovery front moved faster in uba-1 animals compared
to wild type, while the anterograde recovery in the two genotypes
did not differ significantly (Figure 2B, 2C).
These observations show that in wild type, very little of the
anterograde motor UNC-104 is transported back to the cell body
from the synapse. By contrast, when ubiquitin-mediated degrada-
tion is blocked, there is significant retrograde transport of the
motor from the synapse towards the cell body, likely due to
increased UNC-104 levels at synapses.
The unc-104(e1265) allele encodes a D1497N change in
its PH domain, leading to reduced ability to bind synaptic
vesicle cargo
After establishing that the UNC-104 motor is degraded at
synapses, we wished to study a possible mechanism for this process.
One hypothesis is that once the motor gets to the synapse, it releases
cargo and is then targeted for degradation [11], suggesting that
degradation of the motor is linked to its being unbound to cargo. We
decided to test this by studying the fate of the UNC-104 motors in a
series of alleles that either strongly or moderately alter the ability of
the motor to bind cargo through its PH domain.
We first attempted to identify a pre-existing allele affecting
cargo binding by sequencing several unc-104 alleles (Figure S1A).
Of these, unc-104(e1265), a canonical allele, showed a single amino
acid change D1497 to N in the PH domain (Figure S1A, S1B). To
test participation of the highly conserved residue D1497 in binding
PI(4,5)P
2
, we built a homology model of the UNC-104 PH domain
using the crystal structure of the closest orthologue in the database,
the protein DAPP1/PHISH (Figure 3A, Figure S1C) [32,33]. The
residues (KK1463/4 and R1496), known to be important for lipid
binding [12], are respectively, 12 A
˚
/16 A
˚
and 3.8 A
˚
from
D1497N (Figure 3A). Thus the residue D1497 is on the surface
of the PH domain in a region known to be important in binding
PI(4,5)P
2
. Further, on docking the ligand PI(4,5)P
2
on to the
homology model using GRAMM [34] we observed that in 40% of
the models it preferentially binds to the region juxtaposed to the
D1497, R1496 and KK1463/4 residues (Figure 3A). The next two
most common models (25%, 20%) identified for ligand docking do
not show proximity to residues known to be important in PH
domain-PI(4,5)P
2
interactions.
To directly test the role of the D1497N mutation encoded by
the unc-104(e1265) allele (which encodes the protein UNC-
104(D1497N)) in binding to PI(4,5)P
2
, we carried out an in vitro
liposome binding assay. The wild type UNC-104 PH domain
binds preferentially to PI(4,5)P
2
, PI(4)P and brain lipids (Figure 3B)
[12]. By contrast, the UNC-104 PH domain with the D1497N
residue greatly reduces the preferential affinity for PI(4,5)P
2
, PI(4)P
and brain lipids (Figure 3B, 3C). However the binding to both PC
and PI increases compared to the wild type PH domain
(Figure 3B). This suggests that the D1497N PH domain variant
likely retains the ability to bind lipids even though the preferential
binding to PI(4,5)P
2
is highly decreased.
and right: 22uC (restrictive). Upper panel of each set is the transgene in a wild type background and the lower panel is the transgene in a uba-
1(it129ts) mutant background. a1-a6, d1-d6: Ventral synapses, axon and cell body of animals expressing soluble GFP (zdIs5). b1-b6, e1-e6: Ventral
synapses, axon and cell body of animals expressing UNC-104::GFP (jsIs1111). c1-c6, f1-f6: Ventral synapses, axon and cell body of animals expressing
GFP::RAB-3 (jsIs821). Only animals expressing UNC-104::GFP show a prominent increase in fluorescent signal in synaptic regions of animals in which
ubiquitination is reduced at 22uC. (B) Mean fluorescent intensity per pixel in arbitrary units (A.U.) of UNC-104::GFP and soluble GFP in wild type and
uba-1(it129ts) background grown at 16uC and 22 uC. UNC-104::GFP intensity per pixel nearly double in the uba-1(it129ts) background at 22uC
compared to the wild type background in synaptic regions (n = 25-35, *p = 10
-5
). (C, D) Mean fluorescent intensity per pixel in arbitrary units of UNC-
104::GFP and soluble GFP in wild type and uba-1(it129ts) background grown at 16uC and 22uC in the axon (C) and cell body (D). (E, F) Mean area of the
mechanosensory neuron cell body (E) and synapses (F) measured using UNC-104::GFP. (G) Mean area of the posterior mechanosensory neuron
synapses measured using soluble GFP and GFP::RAB-3 in wild type and uba-1(it129ts) background grown at 16uC and 22uC. A small change in synapse
area as measured by GFP::RAB-3 was seen in uba-1(it129ts) at both 16uC and 22uC (n = 30, p = 0.03). No such change was seen when using soluble GFP
zdIs5 as the marker. All data represented as mean 6 SEM and n = 25-35 in all cases. Scale bar: 10
mm.
doi:10.1371/journal.pgen.1001200.g001
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For further confirmation, we tested whether increasing PI(4,5)P
2
in vivo provided functional rescue. In the transgenic line gqIs25,
which over-expresses the PI(4,5)P
2
biosynthetic enzyme ppk-1 in
neurons, PI(4,5)P
2
levels are increased by 40% in vivo [35]. We
tested functional rescue of transport using a locomotory behav-
ioural assay and an aldicarb resistance assay. These assays depend
on the release of neurotransmitter filled vesicles at synapses [36]
that have been transported by the UNC-104 motor. (See materials
and methods for the inverse relationship between synaptic
transmission and paralysis induced by the acetylcholine esterase
Figure 2. UNC-104 transport in mechanosensory neurons and co-sedimentation with pre-synaptic vesicles. (A) Laser microsurgery of
UNC-104::GFP expressed in mechanosensory neurons (jsIs1111) upon block in ubiquitination using uba-1(it129ts). (a,c,e,g) Arrow points to
representative levels of UNC-104::GFP in the axon in wild type (a,c) and uba-1 (e,g) animals. (b,d,f,i) The main neuronal process was cut 20
mm away
from the cell body or 20
mm before the synaptic branch that ends in synapses. The UNC-104::GFP motor accumulated only in the proximal cut end
after 1 hour (b, d arrowhead) and is greatly reduced in the distal neuronal process (b,d arrow). Upon axotomy in uba-1 animals, UNC-104::GFP motor
increased significantly in distal neuronal process (f,i arrowhead and arrow). Scale bar: 10
mm. (n = 15-20 animals) (B,C) Fluorescence recovery after
photo bleaching of UNC-104::GFP in mechanosensory neurons upon block in ubiquitination using uba-1(it129ts). Bleached area, anterograde recovery
front and retrograde recovery front are marked by star, arrow and arrowhead respectively. The retrograde recovery of UNC-104::GFP is faster in uba-
1(it129ts) than in wild type. Rate of recovery of UNC-104::GFP is represented as velocity of the UNC-104::GFP front in wild type and uba-1(it129ts). Rate
of retrograde recovery of UNC-104::GFP is significantly increased in uba-1 animals. Data represented as mean 6 SEM (n = 30, *p,10
-4
). (D)
Immunoprecipitation using anti-UNC-104 antibody (upper two panels) and anti-ubiquitin antibody (lower panel). Immunoprecipitation using anti-
UNC-104 and subsequent western blot is probed with anti-UNC-104 and anti-ubiquitin. Immunoprecipitation using anti-ubiquitin and subsequent
western blot is probed with anti-UNC-104. Input: 2% of lysate used for immunoprecipitation, S: supernatant after immunoprecipitation, P:
immunoprecipitate. (E) Co-sedimentation of UNC-104 and pre-synaptic vesicles. Presence of pre-synaptic vesicles assayed using anti-synaptobrevin
antibodies in the genotypes wild type, unc-104(e1265) and unc-104(e1265tb120). The alleles unc-104(e1265), unc-104(e1265tb107) and unc-
104(e1265tb120) are labeled in the figure by the respective protein changes they encode, namely D1497N, D1497N R1501Q and D1497N M1540I.
doi:10.1371/journal.pgen.1001200.g002
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inhibitor aldicarb.) In unc-104 mutants, vesicles at synaptic regions
are greatly reduced [3], resulting in animals that are nearly
immobile due to reduced synaptic transmission and are greatly
resistant to paralysis induced by aldicarb (Figure 3D, 3E) [10].
Wild type and PI(4,5)P
2
over-expressing animals have robust
locomotion and are highly sensitive to aldicarb (Figure 3D, 3E). In
unc-104(e1265) animals over-expressing PI(4,5)P
2
, there is no
improvement in locomotory behaviour or sensitivity to aldicarb
when compared to unc-104(e1265) animals (Figure 3D, 3E). Thus
the protein encoded by the unc-104(e1265) allele with the D1497N
lesion is insensitive to PI(4,5)P
2
levels in vivo.
We wished to confirm that the reduced ability of the D1497N
PH domain to bind PI(4,5)P
2
in vitro results in correspondingly
reduced ability of the UNC-104(D1497N) motor to bind to its
synaptic vesicle cargo in vivo. For this we prepared pre-synaptic
vesicles from unc-104(e1265) and wild type animals. Both
genotypes have nearly identical levels of synaptic vesicles as
assayed by the vesicle marker synaptobrevin (Figure 2E). At the
same time, the vesicles prepared from unc-104(e1265) animals have
very low amounts of UNC-104 present on them when compared
to vesicles prepared from wild type animals (Figure 2E).
Thus UNC-104(D1497N), encoded by unc-104(e1265), loses its
ability to bind to PI(4,5)P
2
in vitro, has greatly reduced presence of
the mutant motor on its vesicular cargo in vivo and shows loss of
synaptic vesicle transport irrespective of PI(4,5)P
2
levels in vivo.
This suggests that the motor encoded by the unc-104(e1265) allele
is unable to transport synaptic vesicles through its inability to bind
to its cargo.
Intragenic suppressors of unc-104(e1265) improve
synaptic protein localization and behaviour
To ameliorate the effects of unc-104(e1265) and to improve in vivo
cargo binding, we screened ,40,000 genomes in a behavioural
suppressor screen using EMS mutagenesis. We identified four
independent intragenic suppressors within the PH domain that
improved the locomotory behaviour of unc-104(e1265) (Figure 3D).
Of these, three alter the same residue, M1540I, while the other
suppressor was an alteration at the highly conserved residue R1501
to Q1501 (Figure S1A, S1B). The three hits that result in the
M1540I change were independently isolated at different times and
did not always have the same nucleotide change (Figure S1A).
We wished to determine if the suppressors, unc-104(e1265tb107)
and unc-104(e1265tb120) (respectively encoding the proteins UNC-
104(D1497N R1501Q) and UNC-104(D1497N M1540I)), im-
proved behaviour by altering synaptic vesicle distribution. We
carried out an aldicarb resistance assay and also directly observed
the distribution of a synaptic vesicle protein in motor neurons. The
intragenic suppressors are less resistant to aldicarb compared to
unc-104(e1265) (Figure 3E, Figure S2C). This indicates that both
intragenic suppressors have greater release of acetylcholine at
synapses. Consistent with these observations, we found that
synaptobrevin-1::GFP (SNB-1::GFP), a synaptic vesicle protein
marker transgenically expressed in motor neurons, accumulates
largely in cell bodies rather than at synapses of unc-104(e1265)
animals (Figure 4A: b) [24,37] and the number of muscle arms
connecting with synapses is greatly reduced (Figure S2A, S2B)
[38]. In both intragenic suppressors the accumulation of SNB-
1::GFP in cell bodies is greatly reduced and correspondingly more
SNB-1::GFP is present at synapses and the number of muscle arms
increases significantly (Figure 4A: c,d, Figure S2A, S2B).
Another synaptic vesicle marker GFP::RAB-3 [27,28] behaves
identically to SNB-1::GFP in mechanosensory neurons. In unc-
104(e1265) the marker GFP::RAB-3 accumulates in the cell body
with nearly no protein present at synapses. In both intragenic
suppressors more GFP::RAB-3 is present in synaptic regions with lower
accumulations in the cell body (Figure S2D). Consistent with this
increase of GFP::RAB-3 in synaptic regions, the anterograde flux of
GFP::RAB-3 in mechanosensory neurons is higher in the intragenic
suppressors than in unc-104(e1265) (Videos S2, S3), but still significantly
less than in wild type (Figure 4D, 4E, Video S1). There is a reduction in
the anterograde velocity of GFP::RAB-3 only in unc-104(e1265)
animals (Figure 4F), suggesting that in the suppressors, the partially
functional UNC-104 motors that succeed in binding cargo are able to
transport it efficiently. The retrograde flux of GFP::RAB-3 is also
reduced in all three mutants, greatly so in unc-104(e1265) but only
moderately in the suppressors (Figure 4D). The retrograde velocity is
unaffected in all mutants (Figure 4F), suggesting that the reduced
retrograde flux is likely due to fewer cargo vesicles being available for
retrograde transport as a result of reduced anterograde transport.
Thus both intragenic suppressors that map to the PH domain
improve behaviour and cholinergic synaptic transmission by
increasing the transport of cargo in the axon and the number of
synaptic vesicles at the synapse.
Both intragenic suppressors partially restore preferential
PI(4,5)P
2
and cargo binding
To determine if the intragenic suppressors improve synaptic
vesicle transport through improved cargo binding, we carried out
Figure 3. Binding of UNC-104 variants
in vitro
to PI(4,5)P
2
and their
in vivo
sensitivity to PI(4,5)P
2
. (A) Homology model of the UNC-104
Pleckstrin homology (PH) domain docked with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P
2
). Left: The original lesion in unc-104(e1265), D1497N,
is marked along with the three residues (KK1463/4, R1496) known to be important for binding lipids. Note that the D1497N is a surface residue that
lies in the same PI(4,5)P
2
binding pocket as the other three residues. Right: Positions of the two residues that are altered in the intragenic suppressors
unc-104(e1265tb107) (having the R1501Q lesion) and unc-104(e1265tb120) (having the M1540I lesion) along with the original lesion D1497N. Blue
indicates basic residues; red indicates acidic residues and pink indicates mildly acidic residues. (B) Percentage binding of the UNC-104 PH domain
variants to various lipids in vitro. All data represented as mean 6 SD and obtained from four independent experiments assayed in triplicates. (C)
Normalized binding of PI(4,5)P
2
and brain lipids. Both D1497N and D1497N M1540I W1549A have very little specific binding to PI(4,5)P
2
or brain lipids
when normalized to PC binding. However the D1497N R1501Q and D1497N M1540I variants bind significantly better than D1497N to PI(4,5)P
2
. Data
represented as mean 6 SD (*p,0.005). PC Phosphatidylcholine, PI(4,5)P
2
Phosphatidyl inositol-4,5-bisphosphate. (D, E) Responsiveness of unc-
104(e1265), unc-104(e1265tb107) and unc-104(e1265tb120) to increase in PI(4,5)P
2
levels in vivo in all neurons. gqIs25 over-expresses Type I PIP kinase
(ppk-1) phosphatidylinositol-4-phosphate 59 kinase in neurons and increases PI(4,5)P
2
levels by 40% in vivo. (D) Locomotion of the various C. elegans
strains. Wild type and gqIs25 animals move well. Worm locomotion is unchanged in unc-104(e1265) upon increase in PI(4,5)P
2
. Locomotion is
significantly improved in unc-104(e1265tb107) and unc-104(e1265tb120) when PI(4,5)P
2
levels are increased (*p,10
-5
). n = 30 animals in all
experiments. All data represented as mean 6 SEM. (E) Aldicarb resistance of the various C. elegans strains as a measure of cholinergic transmission.
Aldicarb inhibits acetylcholine esterase in C. elegans and causes hyperstimulation of the muscle and thus paralysis. Aldicarb resistance is unchanged
in unc-104(e1265) upon increase in PI(4,5)P
2
. Time for paralysis is reduced in unc-104(e1265tb107) and unc-104(e1265tb120) when PI(4,5)P
2
levels are
increased (*p,0.001). n = 30 animals, done three times independently. Data represented as the average time (mean 6 SEM) taken for 50% of the
animals to be completely paralyzed. The alleles unc-104(e1265), unc-104(e1265tb107) and unc-104(e1265tb120) are labeled in the figure by the
respective protein changes they encode, namely D1497N, D1497N R1501Q and D1497N M1540I.
doi:10.1371/journal.pgen.1001200.g003
UNC-104 Degrades upon Loss of Cargo Binding
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Figure 4. Phenotypic characterization of the
unc-104
allelic series. (A) Cargo molecules marked by synaptobrevin-1::GFP in motor neurons
(juIs1) in wild type (a), unc-104(e1265) (b), unc-104(e1265tb107) (c) and unc-104(e1265tb120) (d). In all the images, the arrowhead points to the cell
body and the arrow to the puncta at the neuromuscular junctions. Intensity and numbers of puncta along the process reflect the numbers of synaptic
UNC-104 Degrades upon Loss of Cargo Binding
PLoS Genetics | www.plosgenetics.org 8 November 2010 | Volume 6 | Issue 11 | e1001200
a parallel analysis of the suppressors in a manner similar to unc-
104(e1265). The R1501Q mutation in unc-104(e1265tb107) is also
on the surface of the PH domain (Figure 3A). It lies ,7A
˚
away
from D1497N within the PI(4,5)P
2
binding pocket. The R1501Q
may reverse the loss of charge in the PH domain variant encoded
by unc-104(e1265) through compensatory local short range
interactions. The compensatory change M1540I in the unc-
104(e1265tb120) suppressor lies ,20 A
˚
from D1497N and is
likely to mediate any possible effect on PI(4,5)P
2
through long-
range interactions (Figure 3A).
In vitro lipid binding using the D1497 R1501Q and D1497N
M1540I PH domains showed that they partially restore preferen-
tial PI(4,5)P
2
binding (Figure 3B, 3C). D1497N R1501Q shows a
small increase in PI(4)P binding but D1497N M1540I does not
show a similar increase. These suppressor variants of the UNC-
104 PH domain, like the D1497N variant, continue to bind PC
and PI at higher levels than the PH domain encoded by wild type
(Figure 3B). These data suggest that along with partial restoration
of preferential binding to PI(4,5)P
2
, some non-specific binding to
lipids is still retained by the PH domains encoded by the intragenic
suppressors.
As a confirmation that the intragenic suppressors are able to
recognize PI(4,5)P
2
in vivo, we observed that increased PI(4,5)P
2
resulting from neuronal over-expression of ppk-1 [35] leads in each
suppressor to improved locomotion and reduced resistance to
aldicarb induced paralysis (Figure 3D, 3E). This indicates
increased transport to synapses resulting in increased vesicle
release in the intragenic suppressors over-expressing PI(4,5)P
2
.
The previously described [12] engineered mutants KK1463/4AA
and R1496A are also similarly sensitive to PI(4,5)P
2
levels in vivo
(Figure S3E), suggesting that they do not reduce PI(4,5)P
2
binding
as severely as the D1497N variant.
Consistent with the in vitro and in vivo data, we observe that pre-
synaptic vesicles prepared from unc-104(e1265tb120) animals have
larger amounts of UNC-104 present on them than those prepared
from unc-104(e1265) animals (Figure 2E). Both genotypes have
nearly identical levels of synaptic vesicles as assessed by levels of
the synaptic vesicle marker synaptobrevin (Figure 2E). Thus,
compared to unc-104(e1265), the proteins encoded by the
intragenic suppressors (1) partially restore preferential PI(4,5)P
2
binding in vitro, (2) are sensitive to PI(4,5)P
2
levels in vivo (3) have
more UNC-104 molecules on pre-synaptic vesicles and (4)
facilitate transport of synaptic vesicles to synapses through an
improved ability to bind cargo vesicles, leading to improved
behaviour.
Lack of binding to cargo results in loss of the UNC-104
motor, which is partially restored by both intragenic
suppressors
To investigate consequences of cargo binding ability on the
motor we examined the levels of the pan-neurally expressed UNC-
104 motor in several alleles (Figure S4D). Greatest levels of
endogenous UNC-104 are found in the synapse rich regions of the
nerve ring and of the ventral cord (Figure S4A:a, Figure 4B).
Lower levels of UNC-104 are present in the dorsal cord, sub-
lateral cords and in neuronal commissural processes (Figure S4B).
UNC-104 levels in unc-104(e1265) are greatly reduced compared
to wild type animals and residual protein is still localized in the
synapse rich regions of the nerve ring and ventral cord (Figure 4B,
4C:b, g, Figure S4A: c). As a comparison no change was observed
in the levels or localization of the neuronal plasma membrane t-
snare syntaxin (Figure 4C: k, l). In another pre-existing allele unc-
104(rh43), which encodes the motor UNC-104(G96E G314E) with
a mutation in the ATP binding pocket of the motor domain
(Figure S1A), the UNC-104 levels appear similar to wild type,
although altered in distribution with significant increases in
neuronal cell bodies (Figure 4C: c, h, Figure S4A: b). The altered
distribution may arise from a motor that is unable to hydrolyze
ATP and thus cannot walk efficiently along microtubules.
To see how partial restoration of the pattern that favours
PI(4,5)P
2
binding affects UNC-104 levels, we carried out
immunohistochemistry and Western blots on both intragenic
suppressors. We observed that the UNC-104 protein levels are also
partially restored in the intragenic suppressors (Figure 4B, 4C:
d,e,i,j). Moreover, this increase occurs where the endogenous levels
of UNC-104 were highest, namely in the synapse rich regions of
the nerve ring and of the ventral cord (Figure 4C: d,e,i,j). These
regions also contain axons, so some of the increase could be taking
place in axons. Upon increasing the in vivo levels of PI(4,5)P
2
in
intragenic suppressors, along with improved behaviour (Figure 3D,
3E), we see a further increase in UNC-104 levels (Figure S3F).
Again this additional increase in UNC-104 levels is detected only
in the synapse rich regions of the ventral cord (Figure S4I). This is
likely due to an increased number of partially functional motors
being recruited to cargo vesicles. (The above data are summarized
in Table 1)
Taken together, our observations show that the in vivo levels of
the UNC-104 motor are directly related to its ability to bind pre-
synaptic vesicles through PI(4,5)P
2
, suggesting a link between
specific binding of a motor to its cargo and levels of the motor in
neurons.
vesicles at motor neuron synapses. Scale bar: 10 mm. (B) Western blot analysis. Monoclonal anti-UNC-104 was used against wild type, unc-104(e1265)
and its intragenic suppressors. As a control the same blot was probed with anti-tubulin. (C) anti-UNC-104 immunoreactivity using MAb 25H11. All
experiments were done simultaneously and unsaturated images taken at identical exposures. UNC-104 is present in high levels in the nerve ring
(arrow) and in the ventral cord (arrowhead) in wild type animals (a,f) but is greatly reduced in unc-104(e1265) (b,g) and partially restored in unc-
104(e1265tb107) (d,i) and unc-104(e1265tb120) (e,j). Although UNC-104 is mis-localized no reduction in levels is seen in unc-104(rh43), another allele
with a lesion in the motor domain (c,h). Arrowhead in c,h points to a large number of cell bodies in the nerve ring and ventral cord region.
Immunoreactivity to syntaxin that marks all neurons is unchanged in all unc-104 alleles that encode PH domain variants (k,l,m,n,o). Scale bar: 10
mm.
(D) Anterograde and Retrograde flux of GFP::RAB-3 in mechanosensory neurons. These data are obtained using movies, examples of which are
provided in Videos S1, S2, S3. The number of particles moving to the synapse is highest in wild type animals and is significantly reduced in unc-
104(e1265) (*p = 10
-19
). Anterograde flux is partially restored in the intragenic suppressors unc-104(e1265tb107) and unc-104(e1265tb120) compared to
unc-104(e1265) (*p = 10
-15
,10
-14
respectively). Anterograde flux in unc-104(e1265tb107) and unc-104(e1265tb120) continues to be significantly lower
than in wild type animals (*p = 10
-12
,10
-11
respectively). Retrograde flux parallels the anterograde trends in all genotypes. (E) Representative
kymographs of various genotypes. A kymograph shows distance moved by GFP::RAB-3 particles (X-axis) over time (Y-axis). Anterogradely moving
GFP::RAB-3 containing vesicles are indicated by arrows. Arrowheads point to stationary particles. (F) Anterograde and retrograde velocity of GFP::RAB-
3 in wild type, unc-104(e1265) and intragenic suppressors [unc-104(e1265tb107), unc-104(e1265tb120)]. Only the anterograde velocity in unc-104(e1265)
shows any reduction (*p = 10
-29
) while all other measurements do not differ significantly from wild type. (G) Movement of UNC-104::GFP and UNC-
104(D1497N)::GFP motors. Both wild type and mutant motors show a similar anterograde bias in movement. All data represented as mean 6 SEM
and collected from 13-15 animals. The alleles unc-104(e1265), unc-104(e1265tb107), unc-104(e1265tb120) and unc-104(rh43) are labeled in the figure by
the respective protein changes they encode, namely D1497N, D1497N R1501Q, D1497N M1540I and G96E G314E.
doi:10.1371/journal.pgen.1001200.g004
UNC-104 Degrades upon Loss of Cargo Binding
PLoS Genetics | www.plosgenetics.org 9 November 2010 | Volume 6 | Issue 11 | e1001200
UNC-104 motors with reduced cargo binding ability are
degraded, at least partly through the ubiquitin pathway
We wished to test if the reduced UNC-104 levels in the unc-
104 variants are due to its degradation. To rule out reduction in
transcripts, we measured RNA levels of UNC-104 using real-
timePCR.WesawnochangeinUNC-104RNAlevelsbetween
wild type and unc -104(e1265) animals (Figure S3D). To study
other possible effects of the D1497N mutation on the UNC-104
motor such as altered localization or motility, we compared
transgenic animals expressing high levels of UNC-104::GFP and
UNC-104(D1497N)::GFP. High levels were used since at low
levels, there is almost no expression of the mutant motor in vivo.
Both variants show similar localization and nearly identical
microscopic movements (Figure 4G, see below). Nearly 85% of
UNC-104::GFP and 75% of UNC-104(D1497N)::GFP mole-
cules that move do so in the anterograde direction while
approximately 15-25% move in the retrograde direction
(Figure 4G). Thus mis-localization or immobility of the UNC-
104 motor are unlikely to underlie the observed phenotypes of
unc-104(e1265) animals.
To test if UNC-104 is degraded in the unc-104 allelic variants we
built double mutants between these variants and the temperature
sensitive allele of the E1 Ubiquitin ligase uba-1(it129ts) [26]. We
observed a small but consistent increase in UNC-104 levels on
Western blots in unc-104(e1265); uba-1 animals grown at 22uC
compared to unc-104(e1265) animals grown at the same temper-
ature (Figure 5A1). This increase, observed primarily in the nerve
ring (Figure 5B: b, f, j, n), did not result in any improvement in
resistance to aldicarb (Figure 5E), probably because the mutant
motors are still unable to bind cargo efficiently for transport.
Similar results were obtained for the two intragenic suppressors.
Western blots showed increased UNC-104 levels in each
suppressor in the uba-1 background (Figure 5A2). Again this
increase occurs in the synapse rich regions of the nerve ring and of
the ventral cord (Figure 5B: c,g,k,o,d,h,l,p). These regions also
contain axons, so the increase in motors may occur to some degree
in axons in addition to synapses.
Concomitant with the increase in motor levels, we observed a
significant increase in synaptic vesicles at neuromuscular junction
synapses in unc-104(e1265tb107) and unc-104(e1265tb120) in the
uba-1 background (Figure 5C, 5D). This was reflected in better
behaviour, namely we saw greater sensitivity to aldicarb at 22uCin
both suppressors in the uba-1 background (Figure 5E). These data
indicate that blocking the ubiquitin-mediated degradation path-
way in the suppressors increases the numbers of partially
functional motors, which likely improves the transport of pre-
synaptic vesicles, resulting in improved synaptic transmission.
Taken together, our observations suggest that loss of ability to
bind cargo can lead to motor degradation in neurons. Further, the
UNC-104 motors that have reduced binding ability to PI(4,5)P
2
Table 1. Summary of all UNC-104 alleles and transgenes and their behaviour in multiple assays.
Allele Transgene
In vitro
PIP
2
binding
In vivo
PIP
2
sensitivity
In vivo
motor
on synaptic
vesicles
Rescue of
unc-104
function
UNC-104 Protein
levels and
localization
Wild type High Insensitive
(saturated?)
High ---- High, largely in
synapse-rich regions
unc-104(e1265) encodes
UNC-104(D1497N)
Poor insensitive Very low ---- Very low, residual in
synapse-rich regions
unc-104(e1265tb107)
encodes UNC-104
(D1497N R1501Q)
Medium sensitive More than in
unc-104(e1265)
---- Medium, residual in
synapse-rich
unc-104(e1265tb120)
encodes UNC-104
(D1497N M1540I)
Medium sensitive More than in
unc-104(e1265)
---- Medium, residual in
synapse-rich
UNC-104::GFP High ------ ------ Full Similar to wild type
UNC-104(D1497N)::GFP Poor ------ ------ No No GFP tagged
protein observed
UNC-104(D1497N)::
GFP very high copy number
------ ------ ------ No Similar to wild type
UNC-104(M1540I)::GFP ------ ------ ------ Full Similar to wild type
UNC-104(D1497N M1540I)::
GFP
Medium ------ ------ Nearly full Similar to wild type
UNC-104(D1497N R1501Q)::
GFP
Medium ------ ------ Nearly full Similar to wild type
UNC-104(R1501Q)::GFP ------ ------ ------ Full Similar to wild type
UNC-104(W1549A)::GFP ------ ------ ------ Full Similar to wild type
UNC-104(D1497N M1540I
W1549A)::GFP
Poor ------ ------ Poor No GFP tagged
protein observed
UNC-104(KK1463/4AA)::
GFP high expression
Poor [12] sensitive ------- Poorer than UNC-
104(D1497N
M1540I)::GFP
Similar to wild type
UNC-104(R1496A)::
GFP high expression
Poor [12] sensitive ------- Poorer than UNC-
104(D1497N
M1540I)::GFP
Similar to wild type
doi:10.1371/journal.pgen.1001200.t001
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Figure 5. Levels of UNC-104 motor in cargo binding variant alleles depends on ubiquitination. Ubiquitination is reduced using the
mutant uba-1(it129ts) at 22uC. (A1,A2) Anti-UNC-104 western blots. (A1) Total UNC-104 levels increase in unc-104(e1265) upon reducing
ubiquitination. The same western blot with two different exposures has been provided for unc-104(e1265). (A2) UNC-104 levels increase in unc-
104(e1265tb107) and in unc-104(e1265tb120) upon reducing ubiquitination. (B) Anti-UNC-104 immunoreactivity in all unc-104 cargo-binding variants
in uba-1(it129ts) background. Upon reducing ubiquitination, UNC-104 levels increase in unc-104(e1265), unc-104(e1265tb107) and unc-104(e1265tb120)
in synapse rich regions of the nerve ring (arrow) and ventral cord (arrowhead). (C) Localization of SNB-1::GFP cargo markers in neuromuscular
junctions observed using juIs1 in unc-104 cargo binding variant alleles. Upon reducing ubiquitination, cargo marked by synaptobrevin increases at
synapses in unc-104(e1265tb107) and in unc-104(e1265tb120). Arrowhead points to ventral cord motor neuron synaptic puncta. Arrow points to the
cell body. (D) Mean fluorescence intensity of neuromuscular junction synaptic puncta marked by synaptobrevin::GFP. The intensity of synaptic puncta
in unc-104(e1265tb107) and unc-104(e1265tb120) increases upon blocking ubiquitination. (n = 10 animals and *p,0.05). Data represented as mean 6
SEM. (E) Aldicarb resistance assay in the UNC-104 PH domain variant alleles. Time for aldicarb induced paralysis decreases in unc-104(e1265tb107) and
in unc-104(e1265tb120) upon block in ubiquitination. n = 30 animals, done three times independently. Data represented as mean 6 SEM (*p = 0.005).
The alleles unc-104(e1265), unc-104(e1265tb107) and unc-104(e1265tb120) are labeled in the figure by the respective protein changes they encode,
namely D1497N, D1497N R1501Q and D1497N M1540I.
doi:10.1371/journal.pgen.1001200.g005
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are degraded at least partially through the ubiquitin pathway in
synapse rich regions of the nerve ring and ventral cord.
Transgenic variants, except UNC-104(D1497N), restore
function and motor protein levels
To provide further support for the observed loss of the UNC-104
motor upon lack of PI(4,5)P
2
binding, we made low copy number
transgenic lines of several UNC-104 variants by bombardment into
unc-104(e1265) animals. The UNC-104 motor variant transgenic
lines were made using wild type UNC-104, UNC-104(D1497N),
UNC-104(D1497N R1501Q), UNC-104(D1497N M1540I) with
and without a GFP fused to the C-terminus expressed under the
control of the unc-104 promoter. The GFP containing and GFP
lacking transgenic lines behaved identically in both locomotion and
aldicarb resistance assays, suggesting that addition of GFP did not
alter the function of the UNC-104 variants (Figure S3B1, S3B2,
S3C1, S3C2). All variants except UNC-104(D1497N) provided full
or partial rescue of the localization of GFP::RAB-3 in a pattern
similar to that observed in wild type animals (Figure S4E). All
transgenic lines except UNC-104(D1497N)::GFP provide signifi-
cant restoration of both locomotion and synaptic transmission as
assayed by aldicarb sensitivity (Figure 6B, 6C). All transgenic lines
except UNC-104(D1497N)::GFP express GFP in a pattern similar
to the pattern of immunoreactivity seen in wild type animals
(Figure 6A). However none of the three independently generated
UNC-104(D1497N)::GFP transgenic lines express GFP (Figure 6A:
c1,c2). Further, injecting the UNC-104(D1497N)::GFP construct at
high DNA concentrations (,200ng/
ml) did result in motor-GFP
expression in a pattern similar to high copy number UNC-
104::GFP transgenic lines (Figure 6A: b1,b2,d1,d2; Figure S4C). We
think that this expression in very high copy number transgenic
UNC-104(D1497N)::GFP lines is likely due to saturation of the
endogenous degradation machinery. These transgenic animals
clearly demonstrate that some fusion protein expressing GFP could
be produced by this construct when sufficiently high copy numbers
of the encoding DNA are provided but this expression still does not
provide behavioural rescue (data not shown, all transgenic data are
summarized in Table 1). However when expressed at levels closer to
endogenous levels, the UNC-104(D1497N) variant is not detectable,
possibly due to being targeted for degradation. The most
parsimonious conclusion is that specific binding to PI(4,5)P
2
molecules present on cargo vesicles is essential for maintaining the
levels of the UNC-104 motor.
Mutating the conserved W1549 in UNC-104(D1497N
M1540I) abolishes preferential PI(4,5)P
2
binding, leading
to loss of motor
To further confirm that the ability to maintain preferential
PI(4,5)P
2
binding is co-related to in vivo motor levels, we mutated the
W1549 to A. The intragenic suppressor M1540I carries out its
suppression indirectly. This residue is ,2.4 A
˚
away from the highly
conserved Trptophan at 1549. In the homology model, I1540
orients its b carbon methyl group towards W1549, which in turn lies
close to KK1463/4 (,7.8 A
˚
/3.2 A
˚
respectively) (Figure 3A). We
predicted that the W1549 residue would mediate the suppression of
M1540I through interaction with the classical KK1463/4 residues.
KK1463/4 are known to play important roles in vitro in binding
PI(4,5)P
2
[12]. Since the side chain of isoleucine is bulkier than
methionine, M1540I mutation might be amenable for better
interaction with the conserved W1549. This may directly cause a
change in the binding site for better presentation to the ligand.
Thus, changing the W1549 to A1549 is likely to reduce the
presumptive interaction from I1540 to the KK1463/4.
Therefore, we tested the in vitro lipid binding specificity of an
UNC-104 PH domain carrying (D1497N M1540I W1549A)
mutations and observed that this triple mutation abolishes the
preferential PI(4,5)P
2
binding in vitro while increasing binding to
PC and PI, and behaves similarly to the D1497N mutation alone
(Figure 3B, 3C). As predicted, this triple mutation abrogates the
ability of the M1540I to suppress the deleterious effects of the
D1497N lesion.
We also made low copy number integrated transgenic lines
using bombardment with both UNC-104(W1549A)::GFP and
UNC-104(D1497N M1540I W1549A)::GFP into unc-104(e1265).
The UNC-104(W1549A)::GFP lines exhibit wild type locomotory
behaviour, sensitivity to aldicarb and motor expression levels and
localization (Figure 6A: j1,j2), suggesting that a motor with
W1549A does not materially alter function in vivo (Figure 6B, 6C,
Figure S3B1, S3B2). However the UNC-104(D1497N M1540I
W1549A)::GFP does not show any expression of GFP in any of the
transgenic lines generated (Figure 6A:f1,f2). Nor does it exhibit
normal locomotion and moreover the unc-104(e1265) animals
carrying this transgene continue to be resistant to aldicarb
(Figure 6B, 6C). Thus all analyzed mutations that reduced the
preferential PI(4,5)P
2
binding also show reduced UNC-104 motor
protein levels.
Discussion
We provide the first evidence that the C. elegans UNC-104/
KIF1A motor does not return from the synapse, is degraded in vivo
through the ubiquitin pathway and that the degradation takes
place in synaptic regions. To study a possible mechanism
underlying this degradation, we developed an allelic series of
mutants that either strongly or moderately affect the ability of the
UNC-104 motor to bind PI(4,5)P
2
and hence pre-synaptic vesicles,
leading to corresponding failure of pre-synaptic vesicles to reach
synaptic regions. In these mutants, levels of the UNC-104 motor
depend on its ability to bind cargo and moreover the motor is
degraded in synapse rich regions of the nervous system. These data
together provide support to the hypothesis that the UNC-104
motor is degraded at synaptic regions upon releasing its synaptic
vesicle cargo.
Directionality of anterograde transport
Failure of UNC-104 to return from synaptic regions in C. elegans
neurons (Figure 2 A-C) corroborates prior reports showing, via
axon ligation assays, that motors such as the mammalian KIF1A,
Kinesin-1 and KIF3A/B do not get retrogradely transported back
to the cell body [6,21,22]. Degradation at synapses can explain this
apparent macroscopic directionality of anterograde transport.
Such degradation could have consequences for cargo transport, for
instance, by providing a mechanism for preventing tug-of-war with
a retrograde motor or return of retrogradely directed cargo back
to the synapse.
Possible mechanism for motor degradation
That UNC-104 is degraded near synaptic regions is demon-
strated by increase in motor levels at synapses in mechanosensory
neurons upon blocking ubiquitin-mediated degradation
(Figure 1A:e1,e4). Together with the observed in vivo ubiquitina-
tion of UNC-104 (Figure 2D), this suggests that degradation of the
motor at synapses is mediated directly or indirectly by ubiquitina-
tion. The degradation of UNC-104 through the ubiquitin pathway
is likely to require the PH domain since a transgenic motor::GFP
fusion protein lacking the PH domain has been shown to be highly
expressed [12] (Figure S4C). This explanation is also consistent
UNC-104 Degrades upon Loss of Cargo Binding
PLoS Genetics | www.plosgenetics.org 12 November 2010 | Volume 6 | Issue 11 | e1001200
with the observed direct interaction of ubiquitin with a split PH
domain that shares significant homology to the UNC-104 PH
domain [39]. Further, the UNC-104 PH domain has 70%
similarity to a 43 amino acid ubiquitin-mediated degradation
sequence found in kinesin Kip1p [40]. Moreover several lysine
residues are present in the PH domain, including three in the PH
domain that may be targets for attaching ubiquitin to the UNC-
104 motor (Figure S1B). While these facts suggest that the
Figure 6. Localization and behavioural rescue of UNC-104::GFP transgenes with different PH domain mutations. (A) Expression and
localization of UNC-104::GFP PH domain variants in unc-104(e1265) background. All transgenic animals except those with the D1497N and D1497N
M1540I W1549A mutations in the PH domain express GFP at high levels. Over-expression of UNC-104(D1497N)::GFP shows expression and
localization of the protein similar to high copy number UNC-104::GFP transgenic animals. In all transgenes that show expression, the localization of
UNC-104 is similar to wild type UNC-104::GFP. Scale bar: 10
mm. (B) Locomotory behaviour of unc-104(e1265) animals carrying various UNC-104::GFP
transgenes. All transgenes except UNC-104(D1497N)::GFP (*p = 10
-31
) and UNC-104(D1497N M1540I W1549A)::GFP (*p = 10
-29
) rescue locomotry
behaviour. While UNC-104(D1497N R1501Q)::GFP and UNC-104(D1497N M1540I)::GFP also provide rescue, they are significantly different from wild
type (*p = 10
-7
,10
-6
). UNC-104(D1497N) does rescue viability of the unc-104(rh142), a null allele (data not shown). All data represented as mean 6
SEM. (C) Aldicarb resistance of unc-104(e1265) animals carrying various UNC-104::GFP transgenes. UNC-104(D1497N)::GFP (*p = 0.001) and UNC-
104(D1497N M1540I W1549A)::GFP (*p = 0.007) animals are highly resistant to aldicarb and do not provide functional rescue. All other transgenes
confer aldicarb resistance similar to wild type and provide functional rescue. All data represented as mean 6 SEM.
doi:10.1371/journal.pgen.1001200.g006
UNC-104 Degrades upon Loss of Cargo Binding
PLoS Genetics | www.plosgenetics.org 13 November 2010 | Volume 6 | Issue 11 | e1001200
degradation of UNC-104 is likely to occur via ubiquitin
interactions with the PH domain of the motor, we cannot rule
out other degradation pathways, for instance involving a more
indirect role for ubiquitination and/or a role for phosphoinositides
[41].
Observed characteristics in the allelic series of UNC-104
mutants
In the allelic series consisting of wild type, unc-104(e1265) and its
two intragenic suppressors, the ability to bind PI(4,5)P
2
determines
the levels of motors on pre-synaptic vesicles in vivo, the extent of
transport of synaptic vesicle proteins to the synapse, and hence the
extent of locomotion and of synaptic transmission. We think that
the primary defect in these unc-104 mutants is differential
abrogation of cargo binding ability, rather than other effects such
as altered localization, motility, folding or stability of the mutant
motor. The fact that over-expressed UNC-104(D1497N)::GFP
and over-expressed UNC-104::GFP localize and move similarly in
vivo (Figure 6A, Figure 4G) argues against localization and motility
being affected. We cannot currently exclude the possibility that
protein folding or stability is changed in vivo. We discuss this in the
next section.
We also found the levels of UNC-104 in all alleles to be directly
related to their cargo binding ability. Further the mutant motors
undergo ubiquitin-mediated degradation in synapse rich regions of
the animal, as seen by the small increase (see the next paragraph)
in UNC-104 expression in these regions after blocking ubiquitin-
mediated degradation (Figure 5B). The D1497N lesion in itself is
unlikely to cause the mutant UNC-104 motor to be targeted for
degradation since the D1497N residue is not a direct target for
ubiquitin conjugation, and hence UNC-104(D1497N) is unlikely
to generate a new site for poly-ubiquitin attachment.
The likely reason why only a small increase is observed in
mutant UNC-104 levels upon blocking ubiquitination is that uba-1
is a mild temperature sensitive mutant providing sufficient function
for viability of the uba-1 animals. This is also the probable reason
behind the apparent lack of change seen in endogenous UNC-104
levels in uba-1 mutants alone in these assays (Figure 5A1, 5B: i,m).
One would expect to see such a change in view of the
independently established degradation of the UNC-104 motor in
mechanosensory neurons (Figure 1A:e1,e4). But since endogenous
wild type UNC-104 is present in all neurons in large amounts, we
think that the small change caused by uba-1 is difficult to detect. It
may be possible to see more robust effects, including on
endogenous wild type UNC-104, if one identifies a specific E3
ubiquitin ligase, rather than using a general block of degradation
provided by uba-1.
A proposed relationship between cargo release and
UNC-104 degradation
The observed degradation patterns of UNC-104 in the animals
in the allelic series, coupled with the direct relationship between
UNC-104 levels in these animals and cargo binding ability of the
mutant motors, provide support to the following hypothesis. The
endogenous UNC-104 motor carrying synaptic vesicles goes to
synaptic regions and is degraded there upon cargo release.
At present we cannot rule out potential instability of the mutant
UNC-104 motor as the primary factor leading to its degradation
and hence to loss of cargo binding and other ensuing phenotypes.
However this explanation is considerably less parsimonious since it
leaves unexplained the following localization and movement
patterns of mutant motors. Observed steady state localization of
all three mutant motors is confined to synapse rich regions, as is the
increase upon blocking ubiquitination (Figure 5B). This suggests
that the mutant motors can get transported to synaptic regions.
Further, the microscopic movements of over-expressed UNC-
104(D1497N)::GFP suggests that at least some mutant motors are
able to fold and move correctly (Figure 4G). Moreover, the nearly
identical CD melting spectra of both the wild type and D1497N PH
domains imply their structural similarity (data not shown).
The fate of UNC-104 motors not carrying synaptic vesicles is
less clear. In case of the mammalian KIF1A, motors unbound to
cargo have recently been shown to be held in an auto-inhibited
state preventing transport to neurite tips [17]. However there are
reported differences between UNC-104 and KIF1A, e.g., UNC-
104 appears to exist as a monomer and is thought to dimerize on
the surface of the cargo [14], whereas KIF1A has been reported to
move as a monomer [42] and recently it is reported to be held as a
dimer [17]. Moreover UNC-104 has been previously reported to
enter axons even after deletion of its cargo binding PH domain,
demonstrating that cargo binding is not necessary for movement of
the motor (Figure S4C) [12]. In all our allelic variants including
wild type, we find almost no UNC-104 present in most neuronal
cell bodies, even in the uba-1 background (Figure 5B: e, m, o, p
contrast with Figure 4C: h). One possible explanation is that most
motors enter the axon very quickly with mutant versions
conceivably carrying other lipids or even no cargo and upon
reaching synaptic regions and after losing binding to cargo, the
motor is rapidly targeted for degradation. Other mechanisms, such
as degradation of motor as soon as the motor-cargo complex
reaches the synapse or only after inactivation of the motor, cannot
be excluded. However, our work suggests that a plausible
mechanism is one in which release of the motor from its cargo
may expose free motors to degradation at or near the synapse.
Materials and Methods
Modeling of UNC-104 PH domain
A BLAST search of the UNC-104 PH domain sequence against
the RSCB protein data bank identifies DAPP1/PHISH (Dual
adaptor of phosphotyrosine and 3-phosphoinositides, from Homo
sapiens, PDB code 1FB8) as the closest homolog. The two
sequences were then aligned using CLUSTAL W (EBI server)
and carefully adjusted using manual intervention, to ensure
maximum conservation of motifs and minimal gap regions. A
homology model was then generated using MODELLER v7.0
[43]. Output structure was relaxed with 500 steps of energy
minimization (Steepest Descent) using SYBYL (Tripos Associates,
Inc.). The energy-minimized structure was then used as input for
docking PI(4,5)P
2
using GRAMM [34].
Bacterial protein expression and purification for in vitro
lipid binding assays
The starting constructs for all was an UNC-104 PH domain
fused in frame to GFP [25]. Various point mutations (D1497N,
D1497N M1540I, D1497N R1501Q, D1497N W1549A M1540I)
in the PH domain were generated using site directed mutagenesis
using the Stratagene QuickChange protocol with TaKaRa Ex Taq.
PH domain constructs were cloned into pET17b vector and all
constructs were verified by DNA sequencing. The proteins were
expressed in Rosetta bacterial cells (Invitrogen), purified by Ni-
NTA chromatography (QIAGEN) and kept frozen in 10mM Tris
pH 8.0, 4mM EGTA, 5% sucrose.
Liposome preparation
The followings lipids were purchased from Avanti Polar lipids.
Egg PC (Cat. no. 840051), PI(4)P (Cat. no. 840045), PI(4,5)P
2
UNC-104 Degrades upon Loss of Cargo Binding
PLoS Genetics | www.plosgenetics.org 14 November 2010 | Volume 6 | Issue 11 | e1001200
(Cat. no. 840046), PA (Cat. no. 840101), PI (Cat. no. 840044) and
BL (Cat. no. 131101). Composition of brain lipids (BL) contains
Phosphatidylethanolamine (16.7%), Phosphatidylserine (10.6%),
Phosphatidylcholine (9.6%), Phosphatidic acid (2.8%), Phosphati-
dylinositol (1.6%) and others (58.7%).
5
mM concentration of the desired lipids was used to prepare
liposomes in the following ratio: 10% desired lipid and 90% carrier
lipids. Phosphatidylcholine (PC) was used as a carrier lipid and the
remaining 10% of the lipids used were either Phosphatidylinositol
(PI), Phosphatidylinositol-4-phosphate PI(4)P, Phosphatidylinositol-
4,5-bisphosphate PI(4,5)P
2
or brain lipid (BL) in chloroform. After
mixing the desired and carrier lipids, chloroform was evaporated
under a constant Nitrogen gas stream. Once the lipid film was dried
completely, lipids were rehydrated by the addition of LB buffer (30
mM tris, 4 mM EGTA, pH 8.0). These lipids were sonicated
(ultrasonic bath) for 30 seconds to break up the lipid aggregates and
were then extruded through a 100 nm pore polycarbonate filter
(Avestin, Ottawa, Canada) using a miniextruder from Avanti polar
lipids. The liposomes were stored in the dark at 4uC and used within
a week of preparation.
Liposome binding assay
Liposomes were prepared as previously described [14]. Briefly,
liposomes (5
mM total lipid concentration) were prepared in LB
buffer (30 mM tris, 4 mM EGTA, pH 8.0). 100
ml of freshly
prepared liposomes were mixed with about 1
mg protein and
incubated on ice for 30 min. The incubation reaction mixtures
were centrifuged at 50000g
av
(4uC) for 45 min in a TLS-120 rotor
(Beckman). After centrifugation, fractions from the pellet that
contains liposome bound protein and supernatant that contains
unbound protein were collected. Samples were dissolved in 20
ml
LB buffer and analyzed by SDS-PAGE followed by Coomassie
staining. Gels were digitized on a flatbed scanner and protein
bands were quantified using ImageJ (version 1.37, NIH). Binding
specificity was determined by normalizing binding observed with
PI(4,5)P
2
and brain lipid compared to binding observed using PC
alone carrier liposomes.
unc-104(e1265) suppressor screen
L4 unc-104(e1265) worms were washed with M9 buffer, using
sterile glass pipettes. Washed worms were transferred into a tube of
1x PBS containing ethyl methanesulfonate (Sigma) at a final
concentration of 50mM. Tubes were kept in a rotary shaker at 20uC
for 4 hours. After mutagenesis, 3-4 worms were transferred each 60
mm Petri plate. F1 and F2 progeny were regularly examined under
a Nikon SMZ645 dissecting microscope for improved locomotion in
a non-clonal screen of approximately 60,000 haploid genomes.
Intragenic suppressors were identified in genetic crosses that
mapped them close to the unc-104 locus. Intragenic suppressors of
unc-104(e1265) isolated were unc-104(e1265tb107) and unc-104
(e1265tb120). Throughout the paper, proteins encoded by these
alleles are referred to as UNC-104(D1497N), UNC-104(D1497N
R1501Q) and UNC-104(D1497N M1540I) respectively.
Worm motility assays
1 day adult hermaphrodites were transferred on to a fresh NGM
agar plate, allowing them to acclimatize for 1 hour. Movement
was recorded on a Nikon SMZ800 dissecting microscope at 1 to
1.3 frames per sec (100061000 pixels) for 2-3 min with a cooled
monochrome camera (Evolution Qei, Media Cybernetics). Move-
ment was tracked manually using ImageJ (version 1.37, NIH)
software. Worms that moved for a minimum of 10 frames were
tracked. Worm velocities were obtained by calculating the straight
line distance between the centroid positions of the worm in a given
interval.
Aldicarb assays
Aldicarb plates were prepared by adding aldicarb (Chemical
Service, Westchester, PA) solution (in 70% ethanol) to NGM agar.
These plates were seeded with OP50 bacteria. All assays were
performed on 1 day old adult hermaphrodites at room
temperature (21-23uC). 30 individuals were incubated for 6-8 hr
on aldicarb plates of defined concentration. At 30 min intervals
each worm was touched with a platinum wire and was checked for
paralysis [36]. Aldicarb inhibits acetylcholine esterase causing the
neurotransmitter acetylcholine to persist longer at the synapse and
hyperstimulate the post-synaptic sites. This leads to loss of co-
ordinated motion and finally paralysis. Faster paralysis indicates
more acetylcholine release at synapses. In our experimental
context wild type paralyzes the fastest while mutants that do not
have vesicles to release paralyze the slowest. Any reduction in
paralysis time indicates more vesicles present at synapses for
release.
Constructs
A wild type UNC-104::GFP construct was provided by Jon
Scholey [25]. This construct harbours the unc-104 promoter
driving the combination of intronless and genomic region of unc-
104 and provides the entire open reading frame of the protein.
Mutations were introduced using site directed mutagenesis using
the Stratagene QuickChange protocol with TaKaRa Ex Taq.
Various point mutations (D1497N; D1497N M1540I, M1540I,
D1497N R1501Q, R1501Q, W1549A, D1497N M1540
W1549A) were generated. All constructs were verified by DNA
sequencing. GFP was deleted from UNC-104::GFP, UNC-
104::GFP(D1497N), UNC-104::GFP(D1497N R1501Q) and
UNC-104::GFP(D1497N M1540I) using the restriction enzymes
Apa1 and Kpn1. After T4 DNA polymerase treatment, ligation
was done using T4 DNA ligase.
C. elegans strains
Worms were grown at 20uC on NGM agar plates seeded with
E.coli Strain OP50 under standard laboratory conditions (Brenner,
1974). Strains used in the study, provided by the Caenorhabditis
Genetics Center (CGC), are as follows: wild type N2, unc-
104(e1265), unc-104(rh43), unc-104(rh142).
juIs1(
p
unc-25-SNB-1::GFP) a transgenic strain expressing green
fluorescent protein (GFP)-tagged synaptobrevin-1 in GABA motor
neurons [24,37]
jsIs1 (
p
snb-1::snb-1::GFP) a transgenic strain that expresses SNB-
1::GFP in all neurons [44].
zdIs5(
p
mec4::GFP) a transgeneic strain expressing soluble GFP in
mechanosensory neurons [29]
jsIs821(
p
mec7::GFP::RAB-3) a transgenic strain expressing GFP
tagged RAB-3 in mechanosensory neurons [27]
trIs25 (him-4p::MB::YFP, F25B3.3P::DsRed2) has Membrane–
anchored yellow-fluorescent protein expressed in body wall
muscles [38].
jsIs682(
p
rab-3::gfp::rab-3) a transgenic strain that expresses
GFP::RAB-3 pan-neurally [27].
gqIs125(
p
rab-3::ppk-1) a transgenic strain that over-expresses the
PI(4,5)P
2
biosynthetic enzyme Type I PIP kinase ppk-1 in all
neurons [35]
uba-1(it129ts) is a temperature sensitive mutant allele in the E1
ubiquitin activating enzyme [26]
js1111 (
p
mec4::UNC-104::GFP) a transgenic strain that expresses
UNC-104::GFP only in mechanosensory neurons.
UNC-104 Degrades upon Loss of Cargo Binding
PLoS Genetics | www.plosgenetics.org 15 November 2010 | Volume 6 | Issue 11 | e1001200
UNC-104 transgenes are all expressed pan-neurally under
its endogenous promoter
UNC-104- 5 transgenic lines- tbIs183
UNC-104::GFP- 5 transgenic lines-
tbIs147
UNC-104(D1497N)- 3 transgenic lines-
tbIs188. This transgene
rescues unc-104(rh142), the lethal null allele
UNC-104(D1497N)::GFP- 3 transgenic lines-
tbIs149. This
transgene provides viability to unc-104(rh142), the lethal null allele
UNC-104(D1497N R1501Q) - 3 transgenic lines-
tbIs194
UNC-104(D1497N R1501Q)::GFP- 3 transgenic lines-
tbIs152
UNC-104(D1497N M1501I)- 4 transgenic lines-
tbIs191
UNC-104(D1497N M1501I)::GFP- 3 transgenic lines-
tbIs156
UNC-104(M1540I)::GFP-13 transgenic lines-
tbIs157
UNC-104(R1501Q)::GFP- 4 transgenic lines-
tbIs170
UNC-104(D1497N M1540I W1549A)::GFP- 5 transgenic
lines-
tbIs199
UNC-104(W1549A)::GFP- 2 transgenic lines
tbIs181
Underlined strain was most commonly used, at least one other
transgenic strain was assayed in all assays and no co-injection
marker was used to make the above transgenic animals.
Transgenic development
Micro particle bombardment of C. elegans unc-104(e1265)
hermaphrodites was carried out using a BioRad Biolistic PDS-
1000/HE particle delivery system (Bio-Rad Laboratories,
Hercules, CA, USA) [45]. For each bombardment, 5-6
mg
plasmid DNA was fixed to 0.5mg of 1.0
mm micro carrier
tungsten particles, as described in the PDS-1000/HE user’s
manual, and bombarded on to a monolayer of unc-104(e1265)
L4. Worms were allowed to recover for 0.5 to 1 hr after
bombardment and were then transferred on to 100mm seeded
Na22 plates and grown at 20uC. After 8-12 days worms were
screened for improved movement and/or GFP expression as
examined using a Zeiss fluorescence microscope. Individual
animals were cloned. Homozygous stable lines were identified by
thecompleteabsenceofunc-104(e1265) mutant progeny over
several generations [45]. We used unc-104(e1265) as the
background for bombardment since this was the healthiest
hypomorphic allele of unc-10 4 available.
Image acquisition and analysis
For quantitation of SNB-1::GFP puncta at motor neuron
synapses synaptic, unsaturated images of immobilized worms were
taken in the linear range of exposure and quantified using ImageJ
(NIH) similar to what has been described in [46].
For in vivo live imaging, young adult hermaphrodites were
immobilized with 3-5mM levamisole (Sigma-Aldrich) in M9
and mounted on a 2% agarose pad. Time-lapse images of
anterior mechanosensory neurons expressing GFP::RAB-3
were obtained with OLYMPUS IX81 using 100X/1.4 NA
plain Apochromat objective attached with spinning disk
confocal head (YOKOGAWA CSU22) equipped with EMCCD
camera (ANDORiXon-897EMCCD). Time-lapse images (5126
512 pixels) were taken at a constant frame rate of 6-7 frames per
second. Image analysis was done using Image J (version 1.37, NIH).
Kymographs were obtained from lines that were drawn along the
axon from cell body towards synapse. Flux analysis was carried out
within a range of 15-20
mm along the axon length, at a distance of
15-25
mm away from the cell body. Flux was calculated as number
of anterogradely moving particles in a movie. Any particle static for
3 frames or with velocity less than 0.3
mm/s was considered as
stationary. Pause frequency was calculated as the number of pauses
taken by a particle for unit distance traveled (number of pauses/total
distance traveled).
Statistical analysis
All significance was calculated using pair-wise comparisons
using the Student’s T-test with unequal variance. p values less than
0.05 were considered as significant.
Monoclonal antibody generation
The protein region of UNC-104 (amino acid 740-1117) was
cloned into pRSETA vector (Invitrogen) using standard tech-
niques. Protein was expressed in BL21 cells (Invitrogen), and
purified using Ni-NTA chromatography (QIAGEN). Purified
protein was given to Bioklone, Chennai, India to generate
monoclonal antibodies. Specificity of the antibodies was tested
by immunostaining unc-104(rh142), a null allele. All monoclonal
antibodies tested showed pan-neural staining in wild type animals
and no staining in the unc-104(rh142) animals (Figure S4D).
Immunostaining and western blots
Animals were fixed with 2% paraformaldehyde for 10 minutes
at 4
o
C and freeze-thawed using liquid nitrogen and fixed for an
additional 10 minutes at 4u C. Following this 4-5 washes with
0.5% BT buffer (20mM H
3
BO
3,
0.5% TritonX-100, pH 9.5) and
then 5-6 washes (1 hour each) with 0.5%BTB (BT with 2%
mercaptoethanol) were carried out. Blocking was done with PBST
(phosphate buffered saline, 0.5%BSA, 0.5% TritonX-100, 10mM
sodium azide). Samples were incubated two overnights with
monoclonal anti-UNC-104 antibody (1:5), washed for 4-5 times
with PBST (each of 15 minutes) before mounting. Rabbit anti-
syntaxin was used at 1:10,000 [47]. Appropriate secondary
antibody (1:200) incubations (anti-mouse Alexa 488, Alexa 568)
were done for two overnights at 4u C. Images were captured using
Zeiss Axiovert inverted microscope. Images were processed with
Adobe Photoshop Version 9.0.
Western sample of worms were prepared by sonication. After
sonication, worm lysates were boiled with SDS lysis buffer and
proteins were separated on SDS PAGE (8% acrylamide). Proteins
were transferred to a nitrocellulose membrane (Amersham),
probed with a mouse serum or a mouse monoclonal antibody of
anti-UNC-104 (1:60), rabbit anti-tubulin (1:1,000) (Thermo-
scientific), rabbit anti-synaptobrevin (1:5000) [44] and rabbit
anti-ubiquitin (1:500) (Sigma-Aldrich) followed by HRP based
chemiluminescence detection (Pierce). Exposure time was varied
from 30 seconds to 5 minutes, scanned and intensities quantitated
using ImageJ. These intensities were pooled from multiple
experiments and graphed and the exposure time chosen was
determined to be in the linear range for all genotypes.
FRAP experiments and analysis
Worms of respective genotypes were anesthesized in 5mM
levamisole. Photobleaching experiments were done on confocal
Zeiss LSM-5 Live (line scanner) equipped with a 63X objective (oil
immersion, 1.4 NA) with a 488 nm solid state laser. Images were
acquired on a CCD camera at the frame rate of 4 Hz. 35-40
mmof
the axon was bleached across the synaptic branch. Fluorescence
recovery was quantified from the distance covered by the UNC-
104::GFP signal in bleached axons at fixed times after bleaching.
The fluorescent recovery along the anterograde and retrograde
directions was represented as velocity in both anterograde
(recovery from cell body) and retrograde (recovery from synapse)
directions. All the analysis was done using ImageJ version1.41
(NIH).
UNC-104 Degrades upon Loss of Cargo Binding
PLoS Genetics | www.plosgenetics.org 16 November 2010 | Volume 6 | Issue 11 | e1001200
Immunoprecipitation and sucrose gradient
sedimentation
N2 worms were used for immunoprecipitation. For sedimenta-
tion assays we used jsIs1 and various unc-104 mutants in the jsIs1
background. The worms and various mutants were grown on 10-
15 large plates until food was exhausted. Worms were mechan-
ically homogenized in homogenization buffer (15mM HEPES-
NaOH pH 7.4, 10 mM KCl, 1.5 mM MgCl
2,
0.1 mM EDTA, 0.5
mM EGTA 0.05 M sucrose and protease inhibitors (Roche) and
mildly sonicated at 4uC. The final supernatant was centrifuged at
50,000g for 40 min in a TLA 100.3 rotor to clear debris and heavy
membrane fractions. The supernatant was collected again and
centrifuged at 175,000g in TLA100.3 rotor for 150 min. The final
pellet was resuspended in homogenization buffer or IP buffer (20
mM HEPES, 40 mM KCl, 5 mM EGTA, 0.1m M EDTA, 5 mM
MgCl
2
with protease inhibitors) as needed.
For immunoprecipitation the high speed re-suspended pellet
was incubated with specific antibody for 5-6 hrs at 4uC. Final
concentration of UNC-104 antibody used was 1:10 and ubiquitin
antibody (Sigma-Aldrich) used was 1:10. Protein A agarose beads
were added to the antigen-antibody mixture and incubated for 3-4
hours at 4uC. The beads were centrigufed, washed with IP buffer
then analyzed by western blotting. A Western analysis was carried
out on immunoprecipitated material using the anti-UNC-104
antibody and anti-ubiquitin antibody. The blot was first probed
for UNC-104 and then stripped (no signal was observed after
stripping) and re-probed for ubiquitin (1:500) (Sigma-Aldrich).
The anti-ubiquitin antibody recognized the same band detected by
anti-UNC-104. A Western analysis was carried out on immuno-
precipitate obtained using the anti-ubiquitin antibody. This blot
was probed using anti-UNC-104 and a band that migrates at the
same size as endogenous UNC-104 was observed.
For sucrose gradient density, the resuspended high speed pellets
were loaded on a discontinuous sucrose gradient centrifugation
(0.05 M, 0.6 M, 1 M and 1.5 M) and centrifuged in a SW41 rotor
at 60,000g for 120 min. Fractions were collected from top of the
gradient up to the first layer (between 0.05M-0.6M). The last two
fractions collected were below the formed layer where no synaptic
vesicle proteins were detected. Western blot analysis with exposure
maintained in the linear range was carried out on the fractions
collected.
Supporting Information
Figure S1 (A) A schematic domain representation (drawn to
scale) of C.elegans (CeUNC-104). The different domains of C. elegans
UNC-104 (as indicated from left to right in figure) are: Motor
domain (aa 1-354), fork head-homology (FHA) domain (aa 463-
592), homologous to liprin binding (LBD) region (aa 589-1267)
and pleckstrin homology (PH) domain (aa 1460-1558). Details of
mutations in the various alleles of unc-104 are shown in the table
below and their relative positions have been marked in the
schematic representation. The intragenic suppressor that encodes
UNC-104(D1497N M1540I) was isolated three independent times
and named sup1, tb101 and tb120. Of these the nucleotide change
in tb120 differs from those in sup1 and tb101 although the aa
change is identical. (B) Primary sequence alignment of the PH
domains of the following UNC-104 family members C. elegans
(CeUNC-104), Drosophila melanogaster (DmUNC-104/imac), H.
sapiens (HsATSV), Mus musculus (MmKIF1A) and Dictostylium
discoidum (DdUNC-104). The D1497N residue mutated in unc-
104(e1265) is highly conserved. The two intragenic suppressors
unc-104(e1265tb107) and unc-104(e1265tb120) have two compen-
satory mutations M1540I and R1501Q respectively. The R1501 is
well conserved while the M1540 varies but is still maintained as an
acidic/neutral residue. Other residues demonstrated to be
important for PI(4,5)P
2
binding, KK1463/4, R1496 are also
highlighted. In addition, another highly conserved residue W1549
that mediates the suppression of M1540I on N1497 has also been
marked. (C) The RSCB protein data bank identifies DAPP1/
PHISH (Dual adaptor of phosphotyrosine and 3-phosphoinosi-
tides, from Homo sapiens, PDB code 1FB8) as the closest homolog
with an E-value of 3.4 [33]. The sequence identity and similarity
between the query and templates were 22% and 38% respectively.
* indicates identical amino acids, : indicates highly similar amino
acids and . indicates similar amino acids between the DAPP1/
PHISH and UNC-104 PH domains.
Found at: doi:10.1371/journal.pgen.1001200.s001 (4.41 MB TIF)
Figure S2 (A) Muscle arms are visualized using trIs25. Muscle
arm number is altered in unc-104(e1265) as well as its suppressors.
Muscle arm number is significantly decreased in unc-104(e1265)
shown in (b) as compared to wild type (a) and partially restored in
intragenic suppressors unc-104(e1265tb107) (c) and unc-104
(e1265tb120) (d). The 9
th
to 11
th
muscles in the dorsal right
quadrant are shown in all panels. Arrow points to muscles arms.
Scale bar: 20
mm. (B) Quantitation of muscle arm numbers.
Muscle arms are significantly reduced in unc-104(e1265), but are
partially restored in intragenic suppressors unc-104(e1265tb107)
and unc-104(e1265tb120). Data represented as mean 6 SEM.
*p,0.05 (C) Aldicarb paralysis assays of wild type, unc-104(e1265),
unc-104(e1265tb107) and unc-104(e1265tb120) showing all time
points assayed. (D) GFP::RAB-3 distribution in mechanosensory
neurons using the transgenic line jsIs821. GFP::RAB-3 (pre-
synaptic vesicle marker) distribution in NR and process of
posterior lateral mechanosensory neuron (PLM process) shown
respectively in wild type (a,b), unc-104(e1265) (c,d), unc-
104(e1265tb107) (e,f), unc-104(e1265tb120) (g,h). When compared
to unc-104(e1265) animals, increased signal resulting from greater
transport was observed both in the NR and PLM processes of the
suppressors. In (a, c, e, g) arrow points to the nerve ring and in
PLM axon, the arrowhead and arrow mark the cell body and axon
respectively. Scale bar: 10 mm. The alleles unc-104(e1265), unc-
104(e1265tb107) and unc-104(e1265tb120) are labeled in the figure
by the respective protein changes they encode, namely D1497N,
D1497N R1501Q and D1497N M1540I.
Found at: doi:10.1371/journal.pgen.1001200.s002 (3.08 MB TIF)
Figure S3 (A1,A2) Aldicarb paralysis/resistance assays in
different mutant backgrounds that over-express ppk-1 in neurons
resulting in 40% increase in in vivo PI(4,5)P
2
levels. (B1 and B2)
Different transgenic variants of UNC-104::GFP (wild type, D/N,
D/N R/Q, D/N M/I, D/N M/I W/A, M/I, R/Q, W/A) in an
unc-104(e1265) background were tested for aldicarb analysis. We
have shown data for two independently isolated transgenic lines
for each UNC-104::GFP variant construct. (C1, C2) Different
transgenic variants of UNC-104 lacking GFP (wt, D/N, D/N R/
Q, D/N M/I) in an unc-104(e1265) background were tested for
aldicarb analysis and locomotion . UNC-104 transgenes with and
without GFP behave identically in these assays. (D) Quantitation
of real time unc-104 RNA levels in wild type and unc-104(e1265).
(n = 3 in duplicate). (E) Over expression of ppk-1 using gqIs125 also
decreases the paralysis time in UNC-104(R1496A) and UNC-
104(KK1463/4AA) transgenic lines. This demonstrates that the
motors encoded by these transgenes are responsive to changes in
PIP
2
levels in vivo like unc-104(e1265tb107) and unc-
104(e1265tb120). Data represented as (mean 6 SEM) time taken
to paralyze the 50% of the worms. (n = 30). (F) Western blot
analysis using anti-UNC-104 antibody of intragenic suppressors
UNC-104 Degrades upon Loss of Cargo Binding
PLoS Genetics | www.plosgenetics.org 17 November 2010 | Volume 6 | Issue 11 | e1001200
with and without gqIs25 over expressing PI(4,5)P
2
in neurons.
Control for protein loading is done using an anti-tubulin antibody.
The alleles unc-104(e1265), unc-104(e1265tb107) and unc-
104(e1265tb120) are labeled in the figure by the respective protein
changes they encode, namely D1497N, D1497N R1501Q and
D1497N M1540I.
Found at: doi:10.1371/journal.pgen.1001200.s003 (1.17 MB TIF)
Figure S4 Immunostaining of UNC-104 in wild type as well as
different UNC-104 mutant alleles (A, B, D). (A) Immunostaining
with anti-UNC-104 polyclonal antibody shows high immunore-
activity in (a) wild type as well as (b) unc-104(rh43) in nerve ring
(shown by arrow) as compared to (c) unc-104(e1265). Arrow in B
points to the cell body. Scale bar, 10
mm. (B) Distribution of UNC-
104 in wild type worms is pan-neurally expressed in synapse rich
regions of the ventral cord and nerve ring (arrows), some
commissural process and a few cell bodies near the nerve ring
(arrowhead). Scale bar 10
mm. (C) Expression of UNC-104::GFP
with various PH domain mutations in the ventral cord, sub-lateral
cords, commisures and dorsal cord. The UNC-104 motor with
deletion of the PH domain sometimes lacks signal in the
commisures (arrowhead). (D) In unc-104(rh142); jsIs682 (unc-104
null mutant expressing GFP::RAB-3 pan-neurally) worms on
which specificity of 25H11MAb against UNC-104 was tested.
UNC-104 immunoreactivity was absent in (a) whereas immuno-
reactivity for GFP from GFP::RAB-3 was present (b) in worms of
the same background. Scale bar 15
mm. (E) Distribution of cargo
(tagged with GFP::RAB-3) in the nerve ring and neuronal process
of the PLM expressing UNC-104 PH domain variants lacking
GFP in transgenic lines made in unc-104(e1265); jsIs821 back-
ground. UNC-104 wild type protein (a, b). Intragenic mutant
UNC-104 protein versions restore transport (e-h) while the UNC-
104(D1497N) expressing transgene does not (c,d). Arrow indicates
nerve ring (a,c,e,g) and PLM neuronal process (b,d,f,h). Arrow-
head marks cell body of PLM neurons (b,d,f,h). Scale bar: 25
mm.
(F) Ratio of Mean fluorescence intensity (cell body/ synapse) of
UNC-104::GFP with and without uba-1(it129ts) grown at 16 uC
(permissive) and 22 uC (restrictive). (G, H) Ratio of fluorescent
intensities in various parts of the PLM neuron in animals
expressing UNC-104::GFP (jsIs1111), soluble GFP (zdIs5)in
mechanosensory neurons. (I) anti-UNC-104 immunoreactivity in
UNC-104 PH domain variant alleles over-expressing PI(4,5)P
2
using the gqIs25 transgene. Upon over-expressing PI(4,5)P
2
UNC-
104 levels consistently increase in the synapse-rich regions of the
ventral cord of unc-104(e1265tb107) and unc-104(e1265tb120).An
occasional inconsistent increase was observed in unc-104(e1265)
and no gross changes in a wild type UNC-104 background were
observed. The alleles unc-104(e1265), unc-104(e1265tb107) and unc-
104(e1265tb120) are labeled in the figure by the respective protein
changes they encode, namely D1497N, D1497N R1501Q and
D1497N M1540I.
Found at: doi:10.1371/journal.pgen.1001200.s004 (1.99 MB TIF)
Video S1 Transport of GFP::RAB-3 in the anterior mechano-
sensory neurons of different genotypes. Movement of
GFP::RAB-3 in wild type neurons.
Found at: doi:10.1371/journal.pgen.1001200.s005 (3.09 MB
MOV)
Video S2 Movement of GFP::RAB-3 marked vesicles in unc-
104(e1265).
Found at: doi:10.1371/journal.pgen.1001200.s006 (1.58 MB
MOV)
Video S3 Movement of GFP::RAB-3 in unc-104(e1265tb120).
Found at: doi:10.1371/journal.pgen.1001200.s007 (1.08 MB
MOV)
Acknowledgments
We thank Dr. Smith, Dr. Schuske, Dr. Roy, and CGC, respectively, for
uba-1(it129ts), gqIs25, trIs25, and unc-104 mutant strains. We thank Dr.
Subramanian, IIT-Kanpur for access to his gene bombardment equip-
ment, Sunder Naganathan for maintaining strains, and Sucheta Kulkarni
for laser axotomy. We thank Dr. Krishna and CIFF at NCBS for use of
confocal microscopes.
Author Contributions
Conceived and designed the experiments: SPK. Performed the experi-
ments: JK BCC SPK. Analyzed the data: SPK. Contributed reagents/
materials/analysis tools: RM QZ MLN SR DRK SPK. Wrote the paper:
SPK.
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UNC-104 Degrades upon Loss of Cargo Binding
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    • "For steady state imaging of mitochondrial marker Pitr- 1:TOM-20 1-54aa ::yfp, we used Nikon microscope equipped with Andor Spinning Disc Setup and EM-CCD camera (Andor iXon 3). For live imaging of GFP tagged mitochondria, worms of appropriate age (day-1 and day- 3 old adult) were anesthetized in 2-3 mM of levamisole (Biomol) [58] and mounted on 2 % agarose pads. Time lapse (3fps) images of mitochondria tagged with GFP in mechanosensory neurons were acquired at 63x with a Zeiss epifluorescence microscope equipped with a CCD (Photometrics) camera. "
    [Show abstract] [Hide abstract] ABSTRACT: Background A certain number of mutations in the Microtubule-Associated Protein Tau (MAPT) gene have been identified in individuals with high risk to develop neurodegenerative diseases, collectively called tauopathies. The mutation A152TMAPT was recently identified in patients diagnosed with frontotemporal spectrum disorders, including Progressive Supranuclear Palsy (PSP), Frontotemporal Dementia (FTD), Corticobasal Degeneration (CBD), and Alzheimer disease (AD). The A152TMAPT mutation is unusual since it lies within the N-terminal region of Tau protein, far outside the repeat domain that is responsible for physiological Tau-microtubule interactions and pathological Tau aggregation. How A152TMAPT causes neurodegeneration remains elusive. Results To understand the pathological consequences of this mutation, here we present a new Caenorhabditis elegans model expressing the mutant A152TMAPT in neurons. While expression of full-length wild-type human tau (Tauwt, 2N4R) in C. elegans neurons induces a progressive mild uncoordinated locomotion in a dose-dependent manner, mutant tau (TauA152T, 2N4R) induces a severe paralysis accompanied by acute neuronal dysfunction. Mutant TauA152T worms display morphological changes in neurons reminiscent of neuronal aging and a shortened life-span. Moreover, mutant A152T overexpressing neurons show mislocalization of pre-synaptic proteins as well as distorted mitochondrial distribution and trafficking. Strikingly, mutant tau-transgenic worms do not accumulate insoluble tau aggregates, although soluble oligomeric tau was detected. In addition, the full-length A152T-tau remains in a pathological conformation that accounts for its toxicity. Moreover, the N-terminal region of tau is not toxic per se, despite the fact that it harbours the A152T mutation, but requires the C-terminal region including the repeat domain to move into the neuronal processes in order to execute the pathology. Conclusion In summary, we show that the mutant TauA152T induces neuronal dysfunction, morphological alterations in neurons akin to aging phenotype and reduced life-span independently of aggregation. This comprehensive description of the pathology due to TauA152T opens up multiple possibilities to identify cellular targets involved in the Tau-dependent pathology for a potential therapeutic intervention. Electronic supplementary material The online version of this article (doi:10.1186/s13024-016-0096-1) contains supplementary material, which is available to authorized users.
    Full-text · Article · Dec 2016
    • "The rhythmic pharyngeal contractions per minute were counted to express pharyngeal pumping rate. Pharyngeal pumping and locomotion rate were calculated on day 5 and 10 post transfer of L4 animals to PC plates and assayed for duration of 30 s (Kumar et al. 2010). The assay was performed on population of both PC-treated (100 μg mL −1 ) and untreated worms. "
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    • "(not significant) are comparisons of each strain to wild type. a decrease in anterograde velocity (Kumar et al. 2010; Maeder et al. 2014). Since the ce782 mutation is in the motor domain, which is on the opposite end of the protein from the cargo binding domain, it is possible that the ce782 mutation also indirectly affects dynein motor activity in these bidirectionally moving vesicles. "
    [Show abstract] [Hide abstract] ABSTRACT: The functional integrity of neurons requires the bidirectional active transport of synaptic vesicles (SVs) in axons. The kinesin motor KIF1A transports SVs from somas to stable SV clusters at synapses, while dynein moves them in the opposite direction. However, it is unclear how SV transport is regulated and how SVs at clusters interact with motor proteins. We addressed these questions by isolating a rare temperature-sensitive allele of Caenorhabditis elegans unc-104 (KIF1A) that allowed us to manipulate SV levels in axons and dendrites. Growth at 20° and 14° resulted in locomotion rates that were 3 and 50% of wild type, respectively, with similar effects on axonal SV levels. Corresponding with the loss of SVs from axons, mutants grown at 14° and 20° showed a 10-and 24-fold dynein-dependent accumulation of SVs in their dendrites. Mutants grown at 14° and switched to 25° showed an abrupt irreversible 50% decrease in locomotion and a 50% loss of SVs from the synaptic region 12-hr post-shift, with no further decreases at later time points, suggesting that the remaining clustered SVs are stable and resistant to retrograde removal by dynein. The data further showed that the synapse-assembly proteins SYD-1, SYD-2, and SAD-1 protected SV clusters from degradation by motor proteins. In syd-1, syd-2, and sad-1 mutants, SVs accumulate in an UNC-104-dependent manner in the distal axon region that normally lacks SVs. In addition to their roles in SV cluster stability, all three proteins also regulate SV transport.
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