Methods
Strains
Wild-type animals were C. elegans variety Bristol, N2. The transgenic
strains and alleles used in this work are described in Supplementary
Table 5. Genetic crosses were made to transfer transgenes to the appro-
priate genetic background. The presence of the mutant allele was veri-
fied by polymerase chain reaction (PCR). Nematodes were grown and
handled following standard procedures, under uncrowded conditions,
at 15 °C, on NGM (Nematode Growth Medium) agar plates seeded with
Escherichia coli strain OP50. Eggs or parents to lay eggs were trans-
ferred to 20 °C or 25 °C at the start of the experiment. Experiments
were conducted with hermaphrodites at 20 °C (or at 25 °C to obtain
sterile DCD130, CF512 animals or to accelerate LBP-2 aggregation, as
indicated in the figure legends). Day 1 of adulthood is defined as 24 h
after the last larval stage L4.
Cloning and strain generation
Cloning was carried out using the Gateway system (Life Technolo-
gies). The myo3 promoter was provided by B. Lee. The lbp-2 and lys-7
promoters were amplified from N2 total DNA extract. lbp-2, lys-7 and
lys-3 genes were amplified from cDNA from N2. C36C5.5, F56B6.6
and clec-1 genes were amplified from N2 total DNA extract. All con-
structs contain the unc-54 3′ UTR. The tagRFP vector was obtained
from Evrogen (AXXORA). mVenus was generated by targeted muta-
tion of the YFP gene and C-terminal HisTag (RGSH6) was added. Con-
structs were sequenced at each step. plbp-2::lbp-2::tagRFP, pmyo
-3::F56B6.6::mVenus::hisTag, pmyo-3::C36C5.5::mVenus::hisTag,
pmyo-3::clec-1::mVenus::hisTag, pmyo-3::lys-3::mVenus::hisTag were
injected at 100 ng μl−1 (whole plasmid injected). plys-7::lys-7::tagRFP
was injected at 50 ng μl−1 (whole plasmid) with the co-injection marker
podr-1::CFP at 30 ng μl−1. Constructs were injected into N2 animals,
except C36C5.5, F56B6.6, clec-1 and lys-3 injected in DCD23 animals.
plbp-2::lbp-2::tagRFP transgene was integrated using UV irradiation
and was backcrossed four times into the wild-type N2 strain. Animals
overexpressing pmyo-3::C36C5.5::mVenus::hisTag incorporated the
transgene into their genome during culture.
Aggregation quantification in vivo
Starting with a population of approximately 100 synchronized worms,
aggregation levels were determined between days 2 and 12 as reported
in the figure legends using a Leica fluorescence microscope M165 FC
with a Planapo 2.0× objective. The number of fluorescent-labelled
LBP-2 or LYS-7 puncta was manually counted only in the head region
(or tail region as indicated in figure legend) of the worms and animals
were classified into three categories: animals with no puncta, animals
with up to ten and more than ten.
RNAi screen
Using SignalP v.4.0 (http://www.cbs.dtu.dk/services/SignalP/)^31 , we
selected all genes which contain predicted signal peptides. To exclude
membrane proteins that remain in the ER–Golgi or that are localized
to the plasma membrane, we removed all genes with predicted trans-
membrane domains using the TMHMM bioinformatics resource
(http://www.cbs.dtu.dk/services/TMHMM)^32. RNAi clones matching
these criteria and tested for changes in LBP-2 aggregation are found in
Supplementary Table 6. All RNAi clones were obtained from the Marc
Vidal RNAi feeding library or the Julie Ahringer RNAi feeding library
(Source BioScience) and RNAi clones for top 13 ECRs were sequenced.
The empty vector L4440 was used as control. RNAi by feeding was
performed as previously described^33. To collect large numbers of ani-
mals for the screen, 200,000 synchronized DCD130 transgenic worms
were cultured at 25 °C in liquid culture with OP50-1 resistant against
streptomycin until L4 stage as described previously^34. After reaching
the L4 stage, animals were repeatedly washed with sterile M9 to remove
bacteria and 30 worms were placed on each RNAi NGM plate (triplicate
plates for each RNAi clone). Plates were kept at 25 °C and the number of
puncta was counted at day 6 of adulthood. RNAi clones were considered
positive, when two out of three plates displayed higher numbers of fluo-
rescent puncta in the head region of the worms compared to negative
control (empty vector L4440). 162 positive clones were found in total.
For the validation screen, hits from the whole screen were retested in
quadruplicate. The repeat was performed with the similar procedures
as for the whole screen. Fifty-seven RNAi clones induced higher number
of fluorescent puncta in the head region of the worms in two out of
four plates when compared to negative control (empty vector L4440).
Thirteen RNAi clones induced higher LBP-2 puncta formation in all
plates (seven out of seven) of the whole and repeat screens at 25 °C and
in repeats at 20 °C with animals treated with RNAi during development.
Of note, knockdown of several ER resident proteins with signal peptide
involved in the ER unfolded protein response including key chaperones
(h sp -3, hsp-4 and dnj-7) and protein disulfide isomerases (pdi-1, pdi-6
and C14B9.2)^35 did not accelerate LBP-2 aggregation, perhaps because
misfolded proteins would be removed by ER assisted degradation.Evaluation of endocytosis
Endocytosis was determined by evaluating the uptake of secreted GFP
by coelomocytes. Animals were grouped into two categories: animals
with or without GFP-labelled coelomocytes. For this, GS1912 transgenic
animals, synchronized at larval stage L1, were placed on RNAi plates
at 25 °C. The number of animals with or without fluorescent-labelled
coelomocytes was counted at day 1 of adulthood.Imaging
For microscopy, worms were immobilized in levamisole 100 mM
(Sigma-Aldrich) on 2% agar pads. Using a Zeiss Axio Observer Z1 micro-
scope and software ZEN 2.6 (2.6.76.000000), whole worm micrographs
were taken with a 10× objective (EC Plan-NEOFLUAR 10×/0.3) and
coelomocyte micrograph with a 63× oil objective (Plan-Apochromat
63×/1.40). For confocal analysis with a Leica SP8 confocal and software
Leica Application Suite X (3.5.2.18963), worms were examined either
with 63× glycerol objective or 40× oil objective (HC PL APO CS2 63×/1.30
or 40×/1.30). The tagRFP was detected using 555 nm as excitation and
an emission range from 565 to 650 nm, mVenus using 515 nm as excita-
tion and an emission range from 521 to 551 nm, GFP using 488 nm as
excitation and an emission range from 500 to 550 nm. To visualize the
muscle structures for confocal analysis, worms were collected at day 4
and fixed in 4% paraformaldehyde (PFA) for 10 min at room tempera-
ture. Worms were stained with Phalloidin-iFluor488 conjugate (1:50,
Biomol) to visualize F-actin as described^36. Phalloidin was visualized by
excitation at 488 nm and with an emission window between 506 and 551
nm. Representative confocal images are displayed as maximum z-stack
projection or single planes as described in the legends.Insoluble protein extraction and western blot
Culture and protein extraction of C. elegans were performed as previ-
ously described^34. To test the LBP-2 protein level with ageing, DCD130
were grown as a synchronized population at 25 °C in liquid culture.
Worms were collected at days 2 and 10 of adulthood and separated
from bacteria and dead worms by sucrose separation. Before freez-
ing in liquid nitrogen, worms were resuspended 1:1 in RAB buffer
(0.1 M MES, 1 mM EGTA, 0.1 mM EDTA, 0.5 mM MgSO 4 , 0.75 M NaCl,
0.02 M NaF, Roche Complete Inhibitors 2x). For total protein extraction,
ground worms were homogenized in 8 M urea, 2% SDS, 50 mM DTT,
50 mM Tris pH 7.4 at room temperature. For soluble and insoluble
protein extraction, ground worms were homogenized in RIPA buffer
(50 mM Tris pH 8, 150 mM NaCl, 5 mM EDTA, 0.5% SDS, 0.5%
SDO, 1% NP-40, 1 mM PMSF, Roche Complete Inhibitors 1x). The
detergent-soluble supernatant was collected after centrifugation for
20 min at 18,400g. The detergent-insoluble pellet was washed once