Antibodies
Primary antibody concentrations ranged from 1–5 µg/ml for
immunoblotting and IF assays. Antibodies specific for clathrin isoforms produced
in our laboratory include mAb TD.1 against CHC17 (
Näthke et al., 1992), mAb X22 against CHC17 (
Brodsky, 1985a), rabbit pAb against CHC22
(
Vassilopoulos et al., 2009),
rabbit pAb against both CLCs (
Acton et al.,
1993), and mAb X16 against LCa (
Brodsky, 1985a). A rabbit pAb against ch-TOG used for IF assays
exclusively was a gift from L. Cassimeris (Lehigh University, Bethlehem, PA).
Commercially available antibodies were used to detect β-actin (mAb;
Sigma-Aldrich), γ-tubulin (mAb; Santa Cruz Biotechnology, Inc.),
α-tubulin (mAb from Sigma-Aldrich and rabbit polyclonal from Santa Cruz
Biotechnology, Inc.), PCNT2 (goat polyclonal; Santa Cruz Biotechnology, Inc.),
centrin-2 (mAb; Abcam), SNAP-tag (rabbit polyclonals from both GenScript and New
England Biolabs, Inc.), TACC3 (mAb; Santa Cruz Biotechnology, Inc.), ch-TOG
(rabbit polyclonal from AbCam and rabbit polyclonal from BioLegend), PCM1
(rabbit polyclonal; Cell Signaling Technology), glyceraldehyde 3-phosphate
dehydrogenase (GAPDH; mAb; EMD Millipore), TfR (mAb; BD), and EEA1 (mAb; BD).
Immunoblotting studies used secondary antibodies conjugated to HRP (Invitrogen)
at 1:10,000 or 1:20,000. Secondary antibodies for IF included Alexa Fluor
antibody conjugates 488, 555, and 647 (Molecular Probes or Invitrogen) and were
used at 1:400 or 1:500.
Plasmids
The cDNA encoding nonneuronal human uLCa was cloned into plasmid DNA such that
the SNAP-tag (New England Biolabs, Inc.) was incorporated at the N-terminal end
of uLCa. The cDNA vector for GFP-pericentrin (pLPx7-GFP-pericentrin) was
produced in the Doxsey laboratory at the University of Massachusetts Medical
School.
Cell culture and siRNA transfection
HeLa cells stably expressing either GFP–α-tubulin or
centrin-2–GFP were gifts from A. Straight (Stanford University, Stanford,
CA) and M.-F.B. Tsou (Sloan-Kettering Institute, New York, NY), respectively.
These cells were maintained in DME (Invitrogen) supplemented with 10% FBS, 100
U/ml penicillin, 100 µg/ml streptomycin, 10 mM Hepes, and 500
µg/ml G418 (Corning). Normal HeLa cells were maintained using the same
media without G418.
Duplex siRNAs were synthesized (QIAGEN) based on experimentally validated target
sequences for clathrin isoforms and subunits (
Huang et al., 2004;
Vassilopoulos
et al., 2009), γ-tubulin (Hs_TUBG1_5, catalog no. S102780750),
or nonsilencing siRNA (catalog no. 1027310). For RNAi treatments, cells were
seeded (21,000 cells/cm
2) in 6- or 24-well plates in Opti-MEM I
reduced serum media (Invitrogen) and then transfected with duplex siRNAs (20 nM
per treatment) complexed with HiPerFect (QIAGEN) in a 1:10 mixture
(siRNA/HiPerFect). Conditions targeting CHC17 and CLCs together used a
combination of 10 nM CHC17 and 10 nM CLC siRNA. CLC siRNA treatments included
two parts siRNA-targeting LCa combined with one part siRNA-targeting LCb. CHC22
depletion was achieved by combining two siRNAs (1:1), as previously described
(
Vassilopoulos et al., 2009). 6 h
after siRNA transfection, cells were returned to normal growth conditions and
then harvested for analysis 48 to 72 h later. Targeting and nontargeting siRNA
effects were confirmed by immunoblotting.
Generation of stable SNAP-uLCa HeLa cell clones
HeLa cells were seeded (50,000 cells/cm2) in 6-well plates 1 d before
plasmid transfection (5 h at 37°C in 5% CO2) using
Lipofectamine 2000 (2.0 µg plasmid in 5 µl Lipofectamine 2000) and
Opti-MEM I reduced serum media according to the manufacturer’s
instructions (Invitrogen). For 48 h after transfection, cells were cultured
under nonselective conditions, and then they were passaged in DME supplemented
with 10% FBS, 10 mM Hepes, 100 U/ml penicillin, 100 µg/ml streptomycin,
and 700 µg/ml G418 (G418 selection media). Single colonies were selected
1.5–2 wk after adding G418 selection media. Sterile glass cylinders
(Thermo Fisher Scientific) were stamped in autoclaved vacuum grease (Corning) to
trypsinize single resistant colonies for expansion. Clonal cell expression
SNAP-uLCa was validated by immunoblotting, IF, and IP.
IF and confocal imaging
IF protocols were modified from established methods (
Piehl and Cassimeris, 2003;
Straight et al., 2005). Cells grown on a 0.17-mm
coverglass (Corning) were fixed with 4% PFA (Electron Microscopy Sciences)
diluted in cytoskeleton stabilization buffer (10 mM MES, pH 6.1, 138 mM KCl, 3
mM MgCl, and 2 mM EGTA) for 20 min at room temperature. Fixation was quenched
with 25 mM glycine in PBS. To detect TACC3, ch-TOG, and clathrin at centrosomes,
cells were fixed in −20°C methanol with 1 mM EDTA (10 min at room
temperature;
Piehl and Cassimeris,
2003). All fixed cells were permeabilized (10 min at room temperature)
with TBS containing 0.5% Triton X-100, pH 7.4, washed two times with
TBS–0.1% Triton X-100, pH 7.4, and blocked in antibody dilution buffer
(Abdil; TBS containing 0.1% Triton X-100 and 2% wt/vol BSA, pH 7.4, for 10 min
at room temperature). Primary antibodies (1–5 µg/ml in Abdil) were
applied (2 h at room temperature), and, after washing with TBS containing 0.1%
Triton X-100, Alexa Fluor secondary antibodies suspended in Abdil were applied
(45 min at room temperature). Cells were then washed in TBS containing 0.1%
Triton X-100 followed by TBS alone. Where indicated, cells were incubated with
300 nM DAPI to stain DNA (10 min at room temperature) and then washed in TBS.
Cells were mounted for microscopy in 2.5% DABCO (Sigma-Aldrich), 50 mM TBS, pH
8.0, and 90% glycerol.
Cells processed by IF were analyzed by confocal laser-scanning microscopy using
an inverted system (DM1 6000 CS, SP5; Leica) with oil immersion objectives
(63× 1.4 NA and 100× 1.4 NA; both HCX Plan Apochromat; Leica) and
argon (488) and HeNe (543 and 633) lasers. Images were acquired using LAS AF SP5
software (Leica) in sequential scan mode with a 600-Hz scan rate, line averages
of four to six, and a 512 × 512– or 1,024 ×
1,024–pixel resolution. Raw images were processed using LAS AF SP5,
ImageJ (version 1.45c; National Institutes of Health), and Photoshop (CS3;
Adobe). To improve image quality, raw images were processed using a median
filter (LAS AF SP5) before any analysis was performed. Colocalization
measurements were performed using LAS AF SP5, and fluorescence distribution
measurements were performed on maximum projections of 3D images using the Radial
Profile Extended plug-in (version 1, by Philippe Carl) in ImageJ (version
1.45c). Adjustments to brightness and contrast of images were restricted to the
dynamic range of the fluorescent intensity profile for merged fluorophores,
applied to the whole image, and the same settings were maintained for all
samples within an experiment.
Time-lapse confocal microscopy imaging
Asynchronous cells were seeded (50,000 cells/cm2) in 35-mm culture
dishes 1 d before transfection with the GFP-pericentrin–encoding plasmid
pLPx7-GFP-pericentrin using Lipofectamine 2000 (0.8-µg plasmid in 2
µl Lipofectamine 2000) and Opti-MEM I reduced serum media according to
manufacturer’s instructions. After transfection, the Opti-MEM I
transfection media was removed, and cells were cultured in DME media
supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin,
and 10 mM Hepes for 48 h. Cells were then labeled with SNAP-TMR-Star (3
µM in phenol red–free DME with 10% FBS) for 30 min at 37°C
according to manufacturer’s instructions (New England Biolabs, Inc.).
Cells were then detached from culture dishes using PBS-based Cell Dissociation
Buffer (Invitrogen), pelleted (1,000 g for 5 min), resuspended
in phenol red–free DME with 10% FBS, and seeded in Lab-Tek II chambered
coverglass (0.17-mm glass thickness; Thermo Fisher Scientific). This transfer
step avoided residual SNAP-TMR-Star sticking to the coverglass surface. Cells
were incubated (37°C in 5% CO2 with 95% humidity) for 5 h
before initiating time-lapse imaging.
SNAP-TMR-Star–labeled cells transiently expressing GFP-pericentrin were
analyzed by confocal laser-scanning microscopy using an inverted system (DM1
6000 CS, SP5) with photomultiplier tube detection, an incubation chamber
(37°C in 5% CO2), an oil immersion objective (63× 1.4
NA; HCX Plan Apochromat), and argon (488 laser power, 2%) and HeNe (543 laser
power, 10%) lasers. Images were acquired using LAS AF SP5 in xyt-sequential scan
mode (10-s or 10-min scanning intervals), a 700-Hz scan rate, line averages of
two, and a 512 × 512–pixel resolution. Raw images were processed
using a median filter in ImageJ (version 1.45c). Adjustments to brightness and
contrast of images were performed in Photoshop (CS3), restricted to the dynamic
range of the fluorescent intensity profile for merged fluorophores, and applied
to the whole xyt image sequence series, and the same settings were maintained
for all samples within an experiment.
Immunoblot analysis
Cell lysates were resolved by SDS-PAGE (NuPage Bis-Tris 4–12%
SDS-acrylamide gels; Invitrogen), transferred to nitrocellulose membranes (EMD
Millipore), and exposed to primary (1–5 µg/ml) and
peroxidase-conjugated secondary antibodies. Labeled proteins were detected using
the ECL-Plus reagent (GE Healthcare). Molecular mass markers (Rainbow; GE
Healthcare) were used for calibration, and protein band intensities were
quantified using Quantity One (Bio-Rad Laboratories) or ImageJ (version 1.45c)
software. For detection of the SNAP-tag and CHC17 by blotting, the
anti–SNAP-tag antibody (New England Biolabs, Inc.) and TD.1 antibody were
used, respectively.
IP
The X22 mAb was used to immunoprecipitate CHC17 clathrin. IgG mAb (IgG1,
κ; BD) was used as a control. IP was performed from cell lysate prepared
in high-Tris IP lysis buffer (0.5 M Tris, pH 7.2, 1.0% Triton X-100, 20 mM EDTA,
5 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin
A, and 1 mM PMSF), conditions that disassemble clathrin coats (
Wilde and Brodsky, 1996). Lysates were
diluted in IP lysis buffer to a standard concentration of 2 mg/ml and 100
µl used for each specific IP, first preclearing with protein G Sepharose
(GE Healthcare), then incubating with 2 µg of primary antibody overnight
at 4°C and then with protein G for 1 h at 4°C followed by washing
(3×) with 500 µl of IP lysis buffer. Precleared and IP samples
were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed
by immunoblotting.
SNAP-tag labeling, fusion protein cross-linking, and Aurora A kinase
inhibition assays
For in vivo labeling of SNAP-tagged uLCa fusion proteins, live cells plated on
coverslips were incubated (30 min at 37°C in 5% CO2) with 3
µM SNAP-TMR-Star (New England Biolabs, Inc.) diluted in normal growth
media with serum. Cells were then washed three times with normal growth media
with serum, incubated (37°C in 5% CO2) an additional 30 min,
and then imaged using confocal laser-scanning microscopy, as detailed under
Time-lapse confocal microscopy imaging.
Covalent cross-linking of SNAP-uLCa was achieved using BG-GLA-BG, a SNAP-tag
homodimerizer shown in
Fig.
S5, synthesized at New England Biolabs, Inc. following
established methods (
Lemercier et al.,
2007). Stable HeLa-SNAP-uLCa clones were treated at specified
concentrations (5–10 µM) with BG-GLA-BG dissolved in DMSO and then
diluted in normal growth media with serum and incubated (2 h at 37°C in
5% CO
2).
For treatment with the Aurora A kinase inhibitor MLN8054 (Selleck Chemicals) and
comparison with BG-GLA-BG treatment, HeLa-SNAP-uLCa clone 3.3 cells were
synchronized to S phase by T/T and then treated at 0 h (T0) after T/T
with DMSO (mock) for 4 or 8 h, 10 µM BG-GLA-BG for 2 h, or 500 nM MLN8054
for 4 or 8 h. At 4 and 8 h, cells were fixed with methanol containing 1 mM EDTA
(10 min) and processed for IF.
Fluorescent transferrin internalization assay
Cells that were incubated in the presence or absence of homodimerizer were placed
on ice, washed three times with ice-cold HBSS (Invitrogen), and then exposed to
50 µg/ml Alexa Fluor 488–conjugated transferrin (Invitrogen) in
HBSS (30–60 min at 4°C). After washing (four times with HBSS at
4°C), prewarmed HBSS was added to cells, and cells were incubated
(37°C in 5% CO2) for 10 min to 1 h to allow for transferrin
internalization. Immediately after this incubation, the cells were fixed at room
temperature with 4% PFA and processed for IF.
Cell surface biotinylation and TfR internalization assay
Cell surface biotin labeling and receptor internalization assays were modified
from established methods (
Blagoveshchenskaya et
al., 2002). HeLa-SNAP-uLCa clone 3.3 cells were seeded in five 60-mm
dishes (80,000 cells/cm
2) 1 d before treating them with 5 µM
BG-GLA-BG or DMSO (mock) for 2 h at 37°C, 5% CO
2, and 95%
humidity. Cells were washed on ice with ice-cold PBS, pH 8.0, and then treated
with 10 mM sulfo-NHS-SS-biotin (Thermo Fisher Scientific) for 30 min at
4°C. Unreacted biotin was quenched by washing cells with ice-cold PBS, pH
7.2, containing 50 µM glycine. Two dishes representing 0 min (0*,
one to not be stripped of biotin, and 0, to be stripped of biotin) were kept on
ice while the other remaining three dishes were treated with prewarmed PBS
containing Ca
2+ and Mg
2+ and placed in a
cell incubator for 5–15 min at 37°C, and then the reaction was
stopped with 5 mg/ml iodoacetamide on ice. Cell lysates were prepared in
high-Tris IP lysis buffer after washing with ice-cold PBS and exposed to SA
agarose resin (Thermo Fisher Scientific) for 1 h at 4°C to pull down
internalized biotin-labeled receptors. SA-bound samples representing plasma
membrane–associated (0 min) and internalized (5, 10, and 15 min)
receptors were pelleted by centrifugation and then washed five times with 1%
NP-40 in PBS. SA-bound samples and total cell lysate for all time points were
immunoblotted for TfR and GAPDH. To quantify the extent of internalized TfR,
blotting signals were assessed by densitometry, and values defined for all time
points were expressed in arbitrary units using the following
equation:
TfR
int represents internalized SA-bound
TfR, plotted in . The symbol
t37 represents internalized SA-bound TfR from
lysates of cells treated at 5, 10, or 15 min at 37°C.
t0 represents residual biotin at the plasma
membrane of cells that were kept on ice and then stripped of biotin before
lysis.
t0* is plasma
membrane–associated, SA-bound TfR from lysates of cells that were kept on
ice and not stripped of biotin before lysis.
Cell synchronization
Cells were synchronized to the G1/S-phase boundary using the T/T method (
Bello, 1969). HeLa clones were seeded
(15,000 cells/cm
2) in 10-cm dishes 1 d before treatment with 2 mM
thymidine in normal G418 selection media for 19 h. Cells were released from this
first thymidine block by washing with PBS (two times) followed by incubation in
G418 selection media without thymidine for 9 h. A second thymidine block was
then performed for an additional 16 h before returning cells to G418 selection
media without thymidine. Cell synchronization was confirmed by flow cytometry
after labeling cells with propidium iodide (Fig. S4). Synchronized cells were
used in cross-linking experiments at varying time intervals after the second
thymidine release was initiated (2, 4, 6, 8, and 10 h).
Flow cytometry
Synchronized cells were harvested by trypsinization (0.05% trypsin-EDTA;
Invitrogen), centrifuged, and washed two times with ice-cold PBS. Cell pellets
were fixed by slowly adding 70% ethanol (chilled at −20°C) while
vortexing and then stored at 4°C. On the day of flow cytometry analysis,
fixed cells were pelleted and washed two times with ice-cold PBS to remove
ethanol. The supernatant was removed, and pellets were left to dry to avoid
ethanol contamination. Cell pellets were then resuspended in PBS containing 40
µg/ml propidium iodide and 40 µg/ml ribonuclease A and incubated
in the dark for 30–60 min at 37°C. Cells were analyzed for DNA
content using a flow cytometer (BD). Data were processed using FlowJo software
(version 9.4.11; Tree Star) to determine the proportion of cells in each phase
of the cell cycle (
Kawamoto et al.,
1979).
Statistical analysis
Data were statistically analyzed using GraphPad Prism software (GraphPad
Software). For nonparametric data, statistical analyses were performed using a
one-way analysis of variance Friedman’s matched pairs test and the
Dunn’s multiple comparison post-hoc test (90% confidence interval). A
regular one-way analysis of variance was performed for data that passed the
normality test, D’Agostino–Pearson omnibus test, and the post-hoc
test was Tukey’s multiple comparison test (95% confidence interval).
Online supplemental material
Fig. S1 shows quantification of the knockdown effects of all siRNA sequences used
in this study, demonstrating reproducible effects on different transfected HeLa
cell clones when compared with nonsilencing siRNA. Fig. S2 shows binding of the
SNAP-uLCa fusion protein to CHC17 in HeLa-SNAP-uLCa clones and colocalization
with endogenous CHC17 clathrin in cells representing all phases of the cell
cycle. Figs. S3 and S4 show that acute inactivation of clathrin by
SNAP-tag–mediated cross-linking in clone 3.3 cells did not significantly
affect CHC17, CHC22, or CLC levels, appearance of multinuclear cells within one
round of mitosis, or cell cycle progression but did induce a multipolar spindle
phenotype within one cell cycle. Fig. S5 shows the chemical structure of
BG-GLA-BG. Video 1 shows a live-cell image of clathrin colocalization with
GFP-pericentrin at an interphase centrosome and in local vesicular structures at
10-min intervals over 2 h. Video 2 shows a live-cell image of colocalized
clathrin and GFP-pericentrin in small structures moving toward the centrosome of
a G2/M-phase–transitioning cell at 10-s intervals over 2 min. Online
supplemental material is available at
http://www.jcb.org/cgi/content/full/jcb.201205116/DC1.
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