Download Report

Supporting Information
Chen et al. 10.1073/pnas.1408327111
SI Materials and Methods
General Methods and Strains. All C. elegans strains were derived
originally from the wild-type Bristol strain N2. Worm cultures,
genetic crosses, and other strain manipulation methods were essentially those described by Brenner (1). Worms were raised on
OP50 E. coli-seeded nematode growth medium (NGM) plates
at 20 °C. The wild-type C. elegans strain Bristol N2 was provided
by the Caenorhabditis Genetics Center (University of Minnesota,
Minneapolis). The sec-10(txu1) mutant strain has been described
(2). A complete list of strains used in this study can be found in
Table S1.
Plasmids and Generation of Transgenes. The transcriptional GFP
fusion Psec-10::GFP, which was used for expression analysis, was
described in detail (2). To construct N-terminal RFP fusion
transgenes for expression in the worm intestine, a skeleton vector was prepared by using the promoter region of the intestinespecific gene vha-6 cloned into Fire laboratory vector pPD49.26,
followed by TagRFP-T coding sequences. The cDNA sequences
of rab-10 and rme-1 were PCR amplified from plasmids Pvha-6::
gfp::rab-10 and Pvha-6::gfp::rme-1 (gifts from B. D. Grant,
Rutgers University, Piscataway, NJ), respectively, digested with
SmaI and KpnI, and individually cloned into the same site of
Pvha-6::TagRFP-T-vector to generate N-terminal fusions. rab10 Q68L and T23N mutants were generated by using standard
techniques of site-directed mutagenesis from the TagRFPT::rab-10 construct. To construct GLUT1::GFP and GLUT1::
TagRFP-T reporters, a 2.5-kb genomic fragment of R09B5.11
was amplified from wild-type worm genomic DNA, digested with
SmaI and AgeI, and inserted into the same site of the skeleton
vectors Pvha-6::gfp and Pvha-6::TagRFP-T, which were both
derived originally from pPD95.75. To generate the AMPH-1::
GFP reporter, a 3-kb genomic fragment was amplified and also
cloned into Pvha-6::gfp through SmaI-AgeI sites. Constructs were
verified by DNA sequencing. Plasmids were injected into wild-type
or mutant animals at concentrations of 10−30 ng/μL, with Pmyo-3::
mCherry at 5 ng/μL or pRF4, which contains the rol-6(su1006) allele, at 20 ng/μL as a coinjection marker, the total DNA concentration of injection mixtures was adjusted to 100 ng/μL by
adding PvuII-digested N2 genomic DNA fragments.
MosSCI lines were generated by the direct insertion method
as described (3). To obtain T7-tagged single copy insertion
transgenic strains, the sec-10 promoter sequence was cloned
into an AflII-SbfI digested MosSCI vector pCFJ151 (gift from
E. M. Jorgensen, University of Utah, Salt Lake City). The sec-10
genomic DNA fused by T7 with a linker sequence [(Gly4Ser)3] at
5′-terminal was amplified and cloned into pCFJ151-Psec−10 by
using SbfI and BsiWI sites to create fusion plasmid Psec-10::T7sec-10. A 700-bp sec-10 3′ UTR fragment was amplified from
genomic DNA, digested with BsiWI and BssHII, and inserted
into BsiWI-BssHII digested fusion plasmid to produce the single
copy insertion plasmid pCFJ151-Psec-10::T7-sec-10-3′ UTR.
Two independent single copy insertion transgenic lines were
made for the rescue examination. The T7-SEC-10 single copy
strains partially rescued the sec-10(txu1) developmental defect
phenotypes and perfectly restored the tubular network of hTACGFP. We also generated C-terminal T7 fusion single copy strain
SEC-10-T7, which failed to rescue the mutant phenotypes.
Yeast Two-Hybrid Assay. The Matchmaker GAL4 Two-Hybrid
System 3 (Clontech) was used according to the manufacturer’s
instructions. The yeast reporter strain AH109 with the bait
Chen et al. www.pnas.org/cgi/content/short/1408327111
plasmid pGBKT7 and the prey plasmid pGADT7 were used.
Prey and bait cDNAs, including C. elegans rab-10(Q68L and
T23N), sec-3, sec-5, sec-6, sec-8, sec-10, sec-15, exoc-7, and exoc-8,
were amplified from C. elegans cDNA. The prenylation motif for
membrane attachment at the C-terminal end of RAB-10 was deleted to improve entry of fusion proteins into the yeast nucleus.
Constructs were verified by DNA sequencing. Yeast transformants
were spread onto growth media lacking leucine and tryptophan for
plasmid selection. Interactions were tested on plates lacking leucine,
tryptophan, adenine, and histidine, containing X-α-Gal (5-bromo4-chloro-3-indolyl-α-D-galactoside) at 30 °C for 3–4 d. All fusion
proteins were tested for self-activation by using the appropriate
empty vector pGBKT7 or pGADT7, respectively. Murine p53
(binding domain) and SV40 large T-antigen (activation domain),
provided by the kit manufacturer, were used as a positive control.
Coimmunoprecipitation Analysis. HEK 293T cells transfected with
appropriate plasmids were lysed in lysis buffer [20 mM Tris·HCl
(pH 7.4), 150 mM NaCl, 0.5% Triton X-100, and 1× Roche
complete Protease Inhibitor Mixture]. The cell lysates were
centrifuged at 14,000 × g for 30 min at 4 °C and then incubated
for 1 h with anti-HA antibody (3F10; Roche), followed by incubation with protein-G (Life Technology) for 1 h at 4 °C. Beads
were washed with IP buffer three times. Precipitants were eluted
with SDS/PAGE sample buffer and subjected to immunoblotting
by using anti HA-Tag mouse monoclonal antibody (cwbiotech),
anti–α-tubulin antibody (DM1A; abcam) and anti-exocyst Sec15
subunit (15S2G6) monoclonal antibody (KeraFAST; ED2003).
Immunostaining in Intestines. Immunostaining of dissected intestines was performed as described (4, 5). Briefly, dissected worms
were fixed in −20 °C 100% methanol for 5 min. Subsequently,
samples were blocked in blocking solution [2% (wt/vol) BSA, 1×
PBS, 0.1% Tween-20, and 0.05% Na Azide] for 30 min at room
temperature. Mouse anti-exocyst complex Sec15 subunit monoclonal antibody (KeraFAST; ED2003) was used at 1:1,000 dilution
in incubation buffer (1% BSA, 1× PBS, 0.1% Tween-20, and 0.05%
Na Azide) overnight at 4 °C. Cy3-labeled goat anti-mouse IgG
(H+L) (Jackson ImmunoResearch) was used at a dilution of
1:600 and incubated for 2 h at room temperature. After extensive
washing, dissected worms were transferred to coverslips, which
were then adhered with 2% (wt/vol) agarose pads and sealed with
nail polish. The treated worms were used for confocal imaging.
RNA Interference. RNA interference was performed by the feeding
method, and all bacterial RNAi feeding strains were from the
Ahringer library (6, 7). The empty vector L4440 was used as the
control. As shown in Fig. S5C, RNAi of the individual exocyst
component efficiently knocked down the gene expression. The
actual knock-down efficiencies might be even higher locally in
the intestine, because the intestine is most sensitive to feeding
RNAi in C. elegans (8).
Quantitative Real-Time PCR Gene Expression Analysis. The F1 RNAiprogeny adult worms were collected and washed with 20 °C water
for the quantitative real-time PCR (qRT-PCR) analysis. Wholeanimal total RNA was extracted and purified with E.Z.N.A.
Total RNA Kit II (OMEGA) according to the manufacturer’s
instructions. The cDNA libraries were generated by using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). All
real-time PCR reactions were performed by using the Applied Biosystems 7900HT Real-Time PCR System with Roche FastStart
Universal SYBR Green Master (ROX). Each experimental
1 of 16
transcript was tested in quadruplicate and compared with an internal control gene act-1 and a control without template.
Endocytosis Assay in Intestines. To investigate basolateral endocytosis by the intestine, young adult hermaphrodites were microinjected with 0.5 mg/mL Rhod-Dex (Mr: 10 KDa; Sigma-Aldrich)
or 0.4 mM FM4-64 (Molecular Probes) dissolved in egg buffer
[118 mM NaCl, 48 mM KCl, 2 mM MgCl2, 2 mM CaCl2, and
25 mM Hepes, (pH 7.3)] into the pseudocoelom. To label acidic
compartments with LysoTracker, worms were prestained in a drop
of 2 μM LysoTracker Green DND-26 (Molecular Probes) in egg
buffer for 1 h without exposure to light before microinjection. The
injected worms were allowed to recover for another 1 h on seeded
NGM plates at 20 °C and followed by transferring to an ice-chilled
NGM plate to efficiently stop intracellular trafficking of endocytosed molecules before examination. For apical endocytosis assay,
wild-type and mutant animals were soaked in Rhod-Dex or FM464 for 1 h. The treated worms were used for confocal imaging.
Fluorescence Microscopy. Live worms were mounted on 2% (wt/vol)
agarose pads with 10 mM levamisole. Fluorescence imaging
was performed on a spinning-disk confocal imaging system
(CSU-X1 Nipkow; Yokogawa) equipped with an EM CCD camera
(DU897K; ANDOR iXon) and oil-immersion objectives (60×
N.A. 1.45, 100× N.A. 1.3, or 150× N.A. 1.45). Two 50 mW solidstate lasers (491 nm and 561 nm) coupled to an acoustic-optical
tunable filter (AOTF) were used to excite GFP/LysoTracker Green
and RFP/rhodamine dyes, respectively. A bandpass emission filter
(FF01-525/30-25; Semrock) were used to visualize broad-spectrum
intestinal autofluorescence caused by lysosome-related organelles
(9). For GFP channel, Semrock FF01-513/17 bandpass filter was
used to reduce interference from autofluorescence, because the
GFP fluorescence peak at 510 nm lacks significant contributions
from autofluorescent lysosomes (10). Z series of optical sections
were acquired at 0.2-μm step.
Nocodazole (50 μg/mL) or Latrunculin B (10 μM) was injected
into the pseudocoelom of young adult worms 1 h before imaging.
Drugs were diluted in DMSO and used at a final concentration
of 1% DMSO in egg buffer.
Imaging Analysis. Images were collected by using Andor IQ 2.0
software and analyzed in ImageJ 1.45m (Wayne Rasband, National Institutes of Health). Z-stack images were deconvolved by
using AutoQuant ×2 software (Media Cybernetics) and then
processed to yield maximum intensity projections, typically two
to three sections. The displayed range of the projection was set
1. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77(1):71–94.
2. Zhang L, et al. (2009) Sec-10 knockout increases the neuroactive-drug responses
without affecting function of the postsynaptic ionotropic receptors in neuromuscular
junctions. Prog Biochem Biophys 36(4):410–416.
3. Frøkjaer-Jensen C, et al. (2008) Single-copy insertion of transgenes in Caenorhabditis
elegans. Nat Genet 40(11):1375–1383.
4. Winter JF, et al. (2012) Caenorhabditis elegans screen reveals role of PAR-5 in RAB-11recycling endosome positioning and apicobasal cell polarity. Nat Cell Biol 14(7):666–676.
5. Duerr JS (2006) Immunohistochemistry. WormBook:1–61.
6. Fraser AG, et al. (2000) Functional genomic analysis of C. elegans chromosome I by
systematic RNA interference. Nature 408(6810):325–330.
Chen et al. www.pnas.org/cgi/content/short/1408327111
to the minimum and maximum intensities of the image. For
images of low signal-to-noise ratio, a median filter or a background subtraction method was used to decrease noise. Fluorescence intensities were quantified with ImageJ. Quantification
of the number or the occupied areas of endosomal structures was
performed by a wavelet transform algorithm with proper threshold
and analyzed by a customized Matlab program. Quantification of
ring-like structures was performed manually. Tubular structures
were identified by using ImageJ plugin “Tubeness” and analyzed
with ImageJ plugin “skeleton,” and the tubule length was obtained
by a customized Matlab program.
For the analysis of hTAC tubule dynamics, original images were
obtained from confocal live worm imaging for 100–200 s, with
exposures every 1 or 0.5 s. Brightness and contrast were adjusted
for the images, which were then analyzed frame by frame for
tethering/fusion events and for identifying the newly growing
hTAC tubules with RAB-10 residing at the leading edge. We
measured the number of hTAC tubule extension events occurring within a 100-μm2 square over a 10-min time period and
considered a tethering/fusion event successful if a tubule reached
out for another hTAC compartment and maintained for more
than 25 s.
Pearson’s Colocalization Coefficient. Images were deconvolved by
using AutoQuant, a 10-pixel-wide rolling-ball subtraction algorithm that was subsequently used to remove background noise.
For RFP-RME-1 transgenic worms, substacks were generated in
three sections spanning 0.4 μm near top layers, whereas RFPRAB-10 transgenic worms in six sections spanned 1.0 μm from
top to middle layers. The 3D Z-stack images were imported to
ImageJ for analysis. The Pearson’s coefficients were calculated
by using ImageJ plugin “Coloc 2.”
Statistical Analysis. When the data follow normal distribution,
Student’s t test or one-way ANOVA was used to evaluate the
statistical significance, otherwise Mann–Whitney rank sum test
was used. All data were presented as the mean value ± SEM.
Asterisks denote statistical significance compared with control,
with a P value less than 0.05 (*), 0.01 (**), and 0.001 (***). Each
displayed image was representative of at least three independent
experiments. No inclusion or exclusion criteria were applied
during analysis. The worms were selected randomly within the
same genotype. The investigators were not blinded to allocation
during experiments and outcome assessment.
7. Kamath RS, Ahringer J (2003) Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30(4):313–321.
8. Kamath RS, Martinez-Campos M, Zipperlen P, Fraser AG, Ahringer J (2001) Effectiveness of specific RNA-mediated interference through ingested double-stranded
RNA in Caenorhabditis elegans. Genome Biol 2(1):RESEARCH0002.
9. Hermann GJ, et al. (2005) Genetic analysis of lysosomal trafficking in Caenorhabditis
elegans. Mol Biol Cell 16(7):3273–3288.
10. Chen CC, et al. (2006) RAB-10 is required for endocytic recycling in the Caenorhabditis
elegans intestine. Mol Biol Cell 17(3):1286–1297.
2 of 16
Fig. S1. Structure of the sec-10 locus and the expression profile of SEC-10 in C. elegans. (A) Schematic representation of the genomic organization of the
sec-10 locus. Black boxes, coding regions; blank boxes, 5′ and 3′ untranslated regions. The bold line underneath the graph indicates the position of mutation in
the sec-10(txu1) allele in this study. A deletion of 140 bp and insertion of 5 bp in the sec-10(txu1) allele join exon 3 with intron 3, and introduce frameshift and
early stop codon. (B) Confocal images of adult hermaphrodites expressing GFP driven by the sec-10 promoter (5 kb upstream of the start codon). GFP is broadly
expressed in C. elegans, including coelomocytes, head neurons, hypodermis, intestine, body wall muscle, reproductive system, and vulva. (Scale bars: 20 μm.)
Chen et al. www.pnas.org/cgi/content/short/1408327111
3 of 16
Fig. S2. The basolateral recycling of fluid cargoes is blocked by sec-10 and sec-5 mutation. (A and B) Wild-type and sec-10(txu1) mutant worms exposed to
apical Rhod-Dex or FM4-64 showed normal intestinal uptake with a concomitant colocalization of dyes and autofluorescent gut granules. (C and E) WT, sec-10
(txu1), and sec-5(tm1443) mutant worms exposed to basolateral Rhod-Dex. Note that sec-10(txu1) and sec-5(tm1443) mutants accumulated abnormally RhodDex–positive puncta in intestinal cells, which colocalized with autofluorescent (C) or LysoTracker-stained LROs (E). It has been shown that autofluorescent gut
granules were identically stained with LysoTracker (1, 2). Arrowheads point to coelomocytes with internalized Rhod-Dex. (D and F) sec-10(txu1) and sec-5
(tm1443) mutant worms exposed to basolateral FM4-64 displayed similar intestinal uptake and LRO localization as WT worms. The right columns for each
locant are overlays of the corresponding green and red channels, magnified (3×) in Insets. The contours of intestines are outlined (dashed lines) in red channels.
(Scale bars: 20 μm.)
1. Hermann GJ, et al. (2005) Genetic analysis of lysosomal trafficking in Caenorhabditis elegans. Mol Biol Cell 16(7):3273–3288.
2. Clokey GV, Jacobson LA (1986) The autofluorescent “lipofuscin granules” in the intestinal cells of Caenorhabditis elegans are secondary lysosomes. Mech Ageing Dev 35(1):79–94.
Chen et al. www.pnas.org/cgi/content/short/1408327111
4 of 16
Fig. S3. Depletion of sec-15 or sec-10 affects endosomal structures in C. elegans intestines. (A and B) Knock down of sec-15 strongly increases the apical
enrichment of RAB-11 vesicles. (A) Representative confocal images of intestinal GFP-RAB-11 in control and sec-15 RNAi worms. Insets display magnified GFPRAB-11 signals at the apical PM. Asterisks depict the lumen of the intestine. (B) The average fluorescence intensity (FI) ratio of apical PM-associated RAB-11
signals relative to cytoplasma signals was quantified. n = 47/40 areas (control/sec-15 RNAi), 18 animals for each group. ***P < 0.001 (Student’s t test). (C–G)
RAB-5–positive early endosomes and RAB-7–positive late endosomes were accumulated in sec-10(txu1) intestines. (C and F) Representative confocal images of
intestinal GFP-RAB-5 (C) and GFP-RAB-7 (F) in WT and sec-10(txu1) mutants. (D and E) The average puncta number of RAB-5–positive endosomes from middle
and top sections was calculated. Middle: n = 19/12 areas (WT/sec-10), Top: n = 18/12 areas (WT/sec-10). ***P < 0.001 (Student’s t test). (G) Quantification of the
number of RAB-7–positive late endosomes. n = 21/28 areas (WT/sec-10). ***P < 0.001 (Student’s t test). (H) GFP-RME-1–labeled basolateral structures in control
and sec-15 RNAi worms. Arrowheads indicate the aberrant increase of RME-1 labeling of the apical domain. Asterisks depict the lumen of the intestine. (I)
Coverage percentage of the basolateral surface by GFP-RME-1, as the magnified regions shown in H, was quantified. n = 20 areas from 12 animals for each
genotype. ***P < 0.001 (Mann–Whitney rank sum test). (J) FI ratio of GFP-RME-1 between apical and basal sides. A 20-pixel-wide and a 10-pixel-wide region
along apical and basal PM, as shown in H, Lower, were selected for the quantification. n = 32/39 from 11/16 animals (control/sec-15 RNAi). ***P < 0.001 (Mann–
Whitney rank sum test). Error bars represent SEM. (Scale bars: 10 μm.)
Chen et al. www.pnas.org/cgi/content/short/1408327111
5 of 16
Fig. S4. The hTAC-containing tubular network is distinct from the ER. (A) Representative images illustrate no overlapping between hTAC-GFP and the ER
marker mCherry-TRAM in the WT intestine (Left, top layer; Right, middle layer). Outlined regions are shown at higher magnification (4×) in Insets. Asterisks
depict the lumen of the intestine. (B) Statistical analysis showed that the average diameter of ER tubular structures is twice larger than that of hTAC-containing
tubules. n = 208/176 tubules from 12 animals (hTAC/TRAM). ***P < 0.001 (Mann–Whitney rank sum test). (C and D) Linear profile analysis of the merged
channels showed that most of the hTAC-positive tubules did not colocalize with ER from beneath the plasma membrane (C, top layer) to the cytosol (D, middle
layer). Some weak hTAC signals were observed to colocalize with ER marker, suggesting low level of hTAC in the ER. (E and F) The morphology and average FI
of mCherry-TRAM-labeled ER in sec-10(txu1) mutants was identical to that of WT animals. n = 14/26 areas from 10/15 animals (WT/sec-10). P = 0.542 (Mann–
Whitney rank sum test). Error bars represent SEM. (Scale bars: 10 μm.)
Chen et al. www.pnas.org/cgi/content/short/1408327111
6 of 16
Fig. S5. Exocyst components are required for the hTAC-positive tubular network. (A and B) T7-SEC-10 MosSCI transgene rescues defective phenotypes of sec-10(txu1)
mutants. (A) Schematic of the construct Psec-10::T7-sec-10. Black boxes, coding regions; blank boxes, 5′ and 3′ untranslated regions; black arrowhead points to T7 tag;
wave line indicates linker. (B) T7-tagged SEC-10 MosSCI insertion strain driven by the sec-10 promoter was crossed into sec-10(txu1) mutant background, which partially
rescued the developmental defect phenotype and perfectly restored the tubular network of hTAC-GFP in intestines. Bright field micrographs (Top), high-magnification
confocal images of individual isolated embryos (Middle), and hTAC-GFP (Bottom) in WT, sec-10(txu1) mutant, and rescued worms are shown, respectively. (C–E) Depletion of exocyst components disrupts the hTAC-positive tubular network. (C) qRT-PCR quantification of the RNA silencing efficiency for the individual exocyst
component genes. The mRNA levels of controls were set as arbitrary unit 1. n = 3 independent experiments. (D) High-magnification confocal images provide detailed
architectures for hTAC-GFP signals, in F1 control and RNAi-progeny adult intestinal cells. (E) Quantification of the normalized average length of hTAC-positive tubules
in control and RNAi-treated worm intestines. The number of areas examined for each group is indicated in each bar. ***P < 0.001 (Student’s t test). Error bars represent
SEM. (Scale bars: 10 μm.)
Chen et al. www.pnas.org/cgi/content/short/1408327111
7 of 16
Fig. S6. The distribution and patterns of endogenous CIE cargo-containing endosomal tubules are altered in sec-10 mutants. (A) Colocalization images of
hTAC-GFP and GLUT1-TagRFP in WT intestines. Outlined regions are shown at higher magnification in Insets. (B–D) The endosomal tubular structures carrying
CIE cargoes were disrupted in sec-10(txu1) mutants. (B) Representative confocal images of intestinal GLUT1-GFP, DAF-4-GFP, and MIG-14-GFP in WT and
sec-10(txu1) mutants. Outlined regions are shown at higher magnification in Insets. (C ) The average length of GLUT1- or DAF-4-containing tubules as
shown in B was calculated. GLUT1: n = 43/40 areas (WT/sec-10), DAF-4: n = 24/31 areas (WT/sec-10). ***P < 0.001 (Student’s t test). (D) Quantification of the
number of MIG-14–containing puncta in B. n = 25 areas for both genotypes. Error bars represent SEM. (Scale bars: 10 μm.)
Chen et al. www.pnas.org/cgi/content/short/1408327111
8 of 16
Fig. S7. Mutations of rab-10 and rme-1 result in enlarged early endosomes and recycling endosomes, respectively. (A) Representative confocal images of
intestinal GFP-RAB-5 and GFP-RME-1 in WT and rab-10(q373) mutants. Large vacuoles in rab-10(q373) mutants were surrounded with early endosome marker
GFP-RAB-5. (B) Representative confocal images of intestinal GFP-RAB-5 and AMPH-1-GFP in rme-1(b1045) mutants. Large vacuoles in rme-1(b1045) mutants
were decorated with clusters of AMPH-1-GFP, which is a marker for basolateral recycling endosome and colocalizes with RME-1 (1). Outlined regions are shown
at higher magnification in Insets. Arrows indicate enlarged endosomes. Asterisks depict the lumen of the intestine. (Scale bars: 10 μm.)
1. Pant S, et al. (2009) AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling. Nat Cell Biol 11(12):1399–1410.
Chen et al. www.pnas.org/cgi/content/short/1408327111
9 of 16
Fig. S8. Verification of interaction between exocyst components and RAB-10. (A and B) Intestines extruded from hTAC-GFP transgene (A) or rab-10(dx2)
mutant (B) worms were fixed and stained with a mouse monoclonal anti–SEC-15 antibody. (A) Endogeneous SEC-15 partially colocalizes with hTAC-GFP tubules. Arrowheads indicate structures labeled by both hTAC and SEC-15. (B) SEC-15 was normally recruited to and associated on the vacuolar membranes in
rab-10(dx2) mutants. Arrowheads indicate vacuoles labeled by SEC-15. Asterisk depicts the lumen of the intestine. (Scale bars: 5 μm.) (C and D) Yeast two-hybrid
assay shows no direct interaction between C. elegans exocyst components and RAB-10. Lines and columns describe fusion forms of exocyst components and
RAB-10 used for transformation. pGBKT7-p53 and pGADT7-T-antigen were used as the positive control. (E) Coimmunoprecipitation experiments were performed in HEK 293T cells; HA-Rab10 consistently pulled down tubulin, but not Sec15, from cell lysates.
Chen et al. www.pnas.org/cgi/content/short/1408327111
10 of 16
Fig. S9. The tubular structures of hTAC-GFP depend on microtubule cytoskeleton. (A) Representative confocal images of worm intestines expressing hTAC-GFP
were treated with DMSO, Nocodazole (Noc, 50 μg/mL) or Latrunculin B (LatB, 10 μM) for 60 min before imaging. (B) Quantification of hTAC-GFP tubule length
in Noc or LatB treated intestines. n = 19/37/15 areas from 7/23/10 animals (DMSO/Noc/LatB). Error bars represent SEM. ***P < 0.001, *P = 0.021 (one-way
ANOVA). (C) Knockdown of actin gene (act-1) exerted no effect on the hTAC-positive tubules in intestines. The diminished oocyte-localized lifeact-GFP was
used as positive control. (D) Representative confocal images of worm intestines expressing hTfR-GFP were treated with DMSO, Nocodazole (Noc, 50 μg/mL), or
Latrunculin B (LatB, 10 μM) for 60 min before imaging.
Chen et al. www.pnas.org/cgi/content/short/1408327111
11 of 16
Table S1. Transgenic and mutant strains used in this study
N2*
rab-10(q373)*
rab-10(dx2)*
rme-1(b1045)*
pwIs72[Pvha-6::GFP::RAB-5]*
pwIs170[Pvha-6::GFP::RAB-7]*
pwIs69[Pvha-6::GFP::RAB-11]*
pwIs87[Pvha-6::GFP::RME-1]†
pwIs90[Pvha-6::hTfR::GFP]†
pwIs112[Pvha-6::hTAC::GFP]†
pwIs216[Pvha-6::RFP::RME-1]†
pwIs414[Pvha-6::RFP::RAB-10]†
pwIs922[Pvha-6::DAF-4::GFP]†
pwIs765[Pvha-6::MIG-14::GFP]†
qxIs162[Pges-1::mCherry::TRAM]‡
zbIs1[Ppie-1::lifeact::GFP]‡
sec-5(tm1443)§
EG4322: ttTi5605 II; unc-119(ed9) III{
sec-10(txu1)
txuIs4[Psec-10::SEC-10::T7] (this work)
txuIs5[Psec-10::T7::SEC-10] (this work)
txuEx10[Pvha-6::TagRFP-T::RAB-10] (this work)
txuEx11[Pvha-6::TagRFP-T::RAB-10(Q68L)] (this work)
txuEx12[Pvha-6::TagRFP-T::RAB-10(T23N)] (this work)
txuEx13[Pvha-6::TagRFP-T::RME-1] (this work)
txuEx21[Pvha-6::GLUT1::GFP] (this work)
txuEx22[Pvha-6::GLUT1::TagRFP-T] (this work)
txuEx23[Pvha-6::AMPH-1::GFP] (this work)
*These strains were provided by the Caenorhabditis Genetics Center (University
of Minnesota, Minneapolis, MN).
†
These strains were gifts from Barth D. Grant (Rutgers University, Piscataway, NJ).
‡
These strains were gifts from Xiaochen Wang (National Institute of Biological
Sciences, Beijing, China).
§
This strain was provided by the National BioResource Project (Tokyo Women’s
Medical University, Tokyo, Japan).
{
This strain was gift from Erik M. Jorgensen (University of Utah, Salt Lake City).
Chen et al. www.pnas.org/cgi/content/short/1408327111
12 of 16
Movie S1. SEC-10 serves for tethering RAB-11–positive apical recycling endosomes to the apical surface. Confocal live-worm movie of GFP-RAB-11 in wild-type
and sec-10(txu1) intestinal cells. Time interval between frames, 500 ms. Total time of acquisition, 50 s. Time reported in seconds. Playback is 10 frames per sec
(fps). Asterisks depict the lumen of the intestine. (Scale bar: 5 μm.) Refers to Fig. 2C.
Movie S1
Chen et al. www.pnas.org/cgi/content/short/1408327111
13 of 16
Movie S2. hTAC-positive endosomal tubules are dynamic. Confocal live-worm movie of hTAC-GFP in a wild-type intestinal cell. Time interval between frames, 1 s.
Total time of acquisition, 200 s. Time reported in seconds. Playback is 20 fps. (Scale bar: 5 μm.) Refers to Fig. 3C.
Movie S2
Movie S3. hTAC-positive endosomal tubules are fragmented and less dynamic in sec-10(txu1) mutants. Confocal live-worm movie of hTAC-GFP in sec-10(txu1)
mutant intestinal cells. Time interval between frames, 500 ms. Total time of acquisition, 100 s. Time reported in seconds. Playback is 20 fps. (Scale bar: 5 μm.)
Refers to Fig. 3B.
Movie S3
Chen et al. www.pnas.org/cgi/content/short/1408327111
14 of 16
Movie S4. RAB-10 leads the growing of hTAC-positive tubules. Confocal live-worm movie of hTAC-GFP and mRFP-RAB-10 in a wild-type intestinal cell. Arrows
mark the dynamic events. Time interval between frames, 2 s. Total time of acquisition, 160 s. Time reported in seconds. Playback is 10 fps. (Scale bar: 5 μm.)
Refers to Fig. 5B.
Movie S4
Movie S5. The tethering/fusion of RAB-10-guided hTAC tubules is reduced in sec-10(txu1) mutant worms. Confocal live-worm movie of hTAC-GFP and mRFPRAB-10 in sec-10(txu1) mutant intestinal cells. Arrows mark the dynamic events. Time interval between frames, 2 s. Total time of acquisition, 100 s. Time
reported in seconds. Playback is 10 fps. (Scale bar: 5 μm.) Refers to Fig. 5D.
Movie S5
Chen et al. www.pnas.org/cgi/content/short/1408327111
15 of 16
Movie S6. The stability of hTAC tubular structures depends on microtubule cytoskeleton. Intestinal cells expressing CIE cargo hTAC-GFP were imaged live by
using confocal fluorescence microscopy. Comparison of the morphology of hTAC-positive structures after treatment with 1% DMSO, nocodazole, and latrunculin B. (Scale bar: 5 μm.) Time interval between frames, 300 ms. Refers to Fig. S9A.
Movie S6
Chen et al. www.pnas.org/cgi/content/short/1408327111
16 of 16