Cell culture and transfection
Swiss 3T3 cells and Swiss 3T3 cells stably expressing DsRed-clathrin were kindly provided by Wolfhard Almers (Oregon Health and Science University, Portland, OR). COS-7, HeLa, and SK-MEL-28 cells were purchased from the American Type Culture Collection (Manassas, VA). The cells were grown in DMEM containing 10% fetal bovine serum (FBS). Swiss 3T3 cells stably expressing DsRed-clathrin were cultured in DMEM containing 10% FBS and 0.4 mg/ml G-418. Swiss 3T3, HeLa, and SK-MEL-28 cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), and COS-7 cells were transfected using FuGENE-6 transfection reagent (Roche, Basel, Switzerland) following standard protocols.
DNA constructs, antibodies, and reagents
Full-length mouse and human Myo1E sequences were amplified from Swiss 3T3 and SK-MEL-28 cDNAs and cloned into the pEGFP-N1 vector (Clontech, Mountain View, CA). A Mito-Flag-GFP–tag empty vector with additional multiple cloning sites (pMito-Flag-GFP-JC3) was made from pCMV-Tag2B Mito-Flag-GFP-syndapin II plasmid (Kessels and Qualmann, 2006
). Mito-Flag-GFP–tagged rat dynamin 2aa was made by replacing the syndapin II sequence with the full-length dynamin 2aa sequence. Mito-GFP-dynamin 2aa ΔPRD was made by deleting the PRD domain in the construct. Mito-Flag-GFP–tagged mouse Myo1E was made by cloning the full-length sequence into the JC3 vector. Mito-Flag-GFP mouse Myo1E deletion constructs, ΔSH3 and SH3 domain alone, were made from pMito-Flag-GFP-JC3 mouse Myo1E. The pRSET-B dTomato and mCherry were kindly provided by Roger Tsien. The pdTomato-N1 and pmCherry-N1 vectors were made by replacing EGFP with the dTomato and mCherry sequences in the pEGFP-N1 vector. pEGFP-N1-rat dynamin 2aa was kindly provided by Mark McNiven. pmCherry N1-dynamin 2aa was made by subcloning rat dynamin 2aa into the pmCherry-N1 vector. pDsRed2-C1-human WIRE was kindly provided by Pontus Aspenström. The GFP-utrophin (amino acids 1–261) was kindly provided by William Bement. WIP-dTomato was made by subcloning WIP from CB6-GFP-WIP (kindly provided by Michael Way) into the pdTomato-N1 vector. The pdTomato-N1-N-WASP was made by subcloning the N-WASP sequence from emerald GFP-Flag-rat N-WASP (kindly provided by Matthew Welch) into the pdTomato-N1 vector. The pEGFP-C1-human clathrin LCa was kindly provided by Lois Greene.
The polyclonal goat anti-Myo1E antibody (N13) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal rabbit anti-human Sec23 antibody was kindly provided by Randy Schekman. The polyclonal sheep anti-human transferrin antibody (PC070) was purchased from The Binding Site (Birmingham, United Kingdom). The polyclonal rabbit anti-EEA1 antibody (2411) was purchased from Cell Signaling Technology (Danvers, MA). The MitoTracker Red CMXRos (M-7512), rhodamine–phalloidin (R415), Alexa Fluor 350–phalloidin (A22281), Alexa Fluor 488–conjugated human transferrin (T-13342), Alexa Fluor 568–conjugated donkey anti–rabbit immunoglobulin G (IgG; A10042), and CellLight BacMam early endosomes-GFP (C10586) were purchased from Invitrogen. The Odyssey blocking buffer (927-40000), IRDye 680 donkey anti–rabbit IgG (926-32223), and IRDye 800CW donkey anti–goat IgG (926-32214) were purchased from LI-COR Biosciences (Lincoln, NE). The polyclonal rabbit anti-Rab7 and anti-CHMP4 antibodies were kindly provided by Suzanne Pfeffer and Phyllis Hanson, respectively.
Live cell imaging and image analysis
In all experiments, cells were plated on precleaned borosilicate glass coverslips (25 mm, number 1; Thermo Fisher Scientific, Waltham, MA). Shortly before imaging, medium was replaced with an imaging buffer containing phenol red–free DMEM (Invitrogen), l-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Invitrogen) at pH 7.4, and 5% FBS. Cells were imaged in an environmental control system set to 37°C (Precision Control, Fall City, WA). Experiments were performed using a TIRF inverted microscope (IX81; Olympus, Tokyo, Japan) equipped with a 60×/1.45 numerical aperture Plan Apo Objective (Olympus, Center Valley, PA) and fully controlled by MetaMorph, version 7 Molecular Devices, Sunnyvale, CA). Two solid-state laser lines (488 and 568 nm; CVI Melles Griot, Albuquerque, NM) were coupled to a TIRF condenser through two optical fibers. The two channels were simultaneously imaged through a dual-view beam splitter (Photometrics, Tucson, AZ) to separate the green and red emission signals to two sides of the camera using a 565-nm dichroic mirror and 530/30 and 630/50 nm emission filters. Images were collected with a charge-coupled device camera OrcaER2 (1024 × 1024, 14-bit; Hamamatsu, Bridgewater, NJ). Cells were typically imaged at 0.5 Hz for 120 frames, without binning and using a time exposure between 0.4 and 0.9 s.
Analysis was performed using Imaris software, version 7.1 (Bitplane, Saint Paul, MN). CCSs were detected within a 100-μm2 region of interest (ROI) using the spot module. Object segmentation was performed using an estimated size of 350 nm and a manually adjusted quality filter. Objects detected were then tracked using the tracking module and Brownian motion algorithm. To avoid transient breaks in trajectory and lifetime, an estimated displacement of 500 nm and a gap closing of 7 frames were incorporated. Finally, the fidelity of the tracking results was assessed visually by overlapping detected tracks with the original image. Tracks touching the edges of the ROI were excluded from the analysis. Mean lifetime was calculated using the average of individual track lifetime.
Two-color TIRFM movies were also analyzed using ImageJ (National Institutes of Health, Bethesda, MD; Le Clainche et al., 2007
). The GFP and DsRed channels were aligned with an image of fluorescent beads. The two channels were then merged. The maximum fluorescence intensity of GFP or DsRed at each CCP was measured and plotted against time. The different CCP tracks were aligned at time zero when the fluorescence of DsRed-clathrin is at maximum before internalization occurs.
For the fluorescent transferrin-uptake assay, SK-MEL-28 cells were grown in six-well plates and treated with siRNAs as described. After 48 h, the cells were seeded on 25-mm round glass coverslips (0.17 mm in thickness) in six-well plates overnight. The cells were serum starved at 37°C for 1 h in starvation medium. They were then incubated with 25 μg/ml human transferrin conjugated to Alexa Fluor 488 in starvation medium (DMEM containing 20 mM HEPES, pH 7.4, and 5 mg/ml BSA) at 37°C for 15 min. The coverslips were then fixed in 4% paraformaldehyde (PFA) at room temperature for 20 min and mounted on glass slides using ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). The internalized fluorescent transferrin was quantified by measuring the average fluorescence intensity of the whole cell using ImageJ software.
For the quantitative ELISA-based transferrin-uptake assay, HeLa cells were grown in six-well plates and treated with siRNAs as described. After 72 h, the cells were washed two times with starvation medium and incubated at 37°C for 1 h. They were then put on ice in a 4°C cold room, and the medium was replaced with 4°C starvation medium containing 2 μg/ml biotinylated-human transferrin. The cells were incubated for 1 h on ice in the cold room. They were then washed two times with ice-cold starvation medium and moved immediately to a 31 or 37°C water bath for various durations (0, 4, 8, and 16 min) to allow transferrin internalization. To measure internalized transferrin, surface-bound transferrin was stripped by adding 2 ml of ice-cold stripping solution (10 mM HCl, 150 mM NaCl, pH 2.0) to the cells for 2 min, followed by a wash with 10 ml of ice-cold PBS. This stripping and washing steps were repeated once. To measure total transferrin bound, cells were washed in 10 ml of ice-cold PBS (Engqvist-Goldstein et al., 2004
). Cells were then lysed in lysis buffer (PBS containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, and 0.1% SDS) containing complete Mini Protease Inhibitor Cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride. An ELISA was used to quantify the amount of biotinylated transferrin in the cell lysate (Buss et al., 2001
). Briefly, ELISA plates were coated with anti-transferrin antibody (1:1000; The Binding Site) diluted in 50 mM NaHCO3
, pH 9.6, overnight at 4°C. The plates were washed twice with PBS and blocked at 37°C for 1 h with blocking solution (10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, and 0.2% BSA). Protein concentrations of cell lysates were measured using the BCA protein assay (Pierce, Thermo Scientific, Pittsburgh, PA) and diluted with the blocking solution. The diluted cell lysates were added to the ELISA plates and incubated at 4°C overnight. The plates were then washed two times with PBS and incubated with blocking solution at room temperature for 5 min. The ELISA plates were then incubated with streptavidin-conjugated horseradish peroxidase (1:1000; BioLegend, San Diego, CA) at room temperature for 1 h, washed three times with PBS, and incubated with 0.4 mg/ml o
-phenylenediamine dihydrochloride (Sigma-Aldrich, St. Louis, MO) in 0.1 M NaH2
, pH 5.0, containing 0.01% H2
at room temperature for 5–10 min. The absorbance was quantified at 492 nm using an ELISA plate reader.
Treatment of HeLa and SK-MEL-28 cells with siRNA
ON-TARGETplus siRNA pools against human Myo1E and nontargeting siRNA #4 (Dharmacon, Lafayette, CO) were diluted to 20 μM with 1× siRNA buffer (Dharmacon), aliquoted, and frozen at −20°C. HeLa or SK-MEL-28 cells were seeded in six-well plates 1 d before transfection. On the day of transfection, cells were ~30% confluent. Five microliters of siRNA solution (20 μM) was added to 250 μl of OptiMEM (Invitrogen) in tube 1. In tube 2, 10 μl of Lipofectamine RNAiMAX (Invitrogen) was added to 240 μl of OptiMEM. Tubes 1 and 2 were incubated at room temperature for 5 min before the two solutions were combined. The mixture was gently mixed and incubated at room temperature for another 25 min. It was then added to cells grown in 2 ml of DMEM with 10% FBS and incubated for 3 d.
Latrunculin A treatment
Latrunculin A powder (Invitrogen) was dissolved in DMSO at a stock concentration of 1 mM and stored at −80°C. SK-MEL-28 cells on coverslips were treated with 0.05–0.25 μM lat-A or equivalent concentration of DMSO diluted in starvation medium for 10 or 15 min. They were then analyzed by a fluorescent transferrin-uptake assay either in the absence or presence of lat-A. The coverslips were then fixed in 4% PFA at room temperature for 20 min and processed for immunofluorescence.
Transduction with CellLight early endosomes-GFP BacMam baculovirus
Ten thousand cells were grown overnight on coverslips in sic-well dishes. BacMam reagents were added (20 μl) to each of the well on the next day. The cells were incubated with the BacMam reagents overnight at 37°C and then processed for fixation and imaging.
Recycling transferrin assay
SK-MEL-28 cells were grown overnight on coverslips and serum starved at 37°C for 1 h in starvation medium. The cells were then incubated with 10 μg/ml human transferrin conjugated to Alexa Fluor 488 in starvation medium at 37°C for 1 h. The cells were washed with cold starvation buffer and cooled on ice. The cells were then washed with cold acidic buffer (DMEM containing 0.5% acetic acid and 500 mM NaCl) for 40 s to remove surface-bound fluorescent transferrin. The cells were washed five times with cold starvation buffer. They were then treated with either 0.01% DMSO or 0.1 μM lat-A for 15 min. The cells were acid washed again with the acid buffer and fixed with 4% PFA. To assay the recycling of internalized transferrin, the cells were incubated at 37°C for 15 min before the acid wash. The fixed cells were labeled with 6.6 nM rhodamine–phalloidin for 1 h and mounted on glass slide using ProLong Gold antifade reagent with DAPI.
Western blotting and quantitative analysis
For Western blotting, cells treated with siRNAs were washed once with PBS in six-well plates. The cells were dissociated by incubation in 1 ml of 0.5 mM EDTA for 5 min. The dissociated cells were transferred to 1.5-ml tubes and centrifuged briefly at 3000 rpm for 5 min. The supernatant was removed while leaving the cell pellet intact. To lyse the cells, 150 μl of boiling 2× protein sample buffer (125 mM Tris-HCl, pH 6.8, 10% glycerol, 10% SDS, 130 mM dithiothreitol, 0.05% bromophenol blue, 12.5% β-mercaptoethanol) was used to resuspend the pellet quickly by pipeting up and down. The tube was incubated in a 95°C hot block for 5 min. The cell lysates were loaded on SDS–PAGE and analyzed by immunoblotting.
For immunoblotting, protein samples on the gel were transferred to a polyvinylidene fluoride (PVDF) Immobilon-FL transfer membrane (IPFL-00010; Millipore, Billerica, MA) in transfer buffer (25 mM Trizma base, 200 mM glycine, 20% methanol, 0.025% SDS) at 50-V constant voltage in a 4°C cold room for 1 h. After the protein transfer, the PVDF membrane was rinsed briefly in TBS and then incubated in Odyssey blocking buffer (LI-COR Biosciences) diluted at 1:1 in PBS at room temperature for 1 h. All the incubation and washing steps were performed on a rotator. The membrane was incubated overnight at 4°C in primary antibodies, anti-Myo1E (1:200) and anti-Sec23 (1:200), diluted in Odyssey blocking buffer/PBS solution. After three 5-min washes with TBST (Tris-buffered saline with 0.1% Tween-20), the membrane was incubated with IRDye 680 or 800CW antibodies (LI-COR Biosciences) diluted at 1:5000 in Odyssey blocking buffer/PBS solution containing 0.1% Tween-20 at room temperature in the dark for 1 h. After three 5-min washes of TBST, the membrane was incubated in TBS and scanned in a LI-COR Odyssey infrared imaging system (LI-COR Biosciences). The protein expression levels were quantified using the Odyssey application software.
Quantitative transferrin-recycling assay and FACS analysis
Pulse labeling with 20 μg/ml Alexa Fluor 647–conjugated transferrin was performed at 37°C for 1 h in DMEM supplemented with 1% BSA (DMEM-BSA) to allow endocytosis. Cells were then transferred on ice, and surface-bound Alexa Fluor 647–transferrin was removed using ice-cold acid buffer (DMEM, 0.5% acetic acid, and 0.5 M NaCl) for 45 s, and the cells were then neutralized by extensive washes of DMEM-BSA. Next the cells were incubated on ice with DMEM-BSA and 0.1 μM lat-A (or DMSO as a control) for 20 min. To allow the efflux of internal Alexa Fluor 647–transferrin, cells were transferred to 37°C in DMEM-BSA in the presence of 0.1 μM lat–A (or DMSO) and nonfluorescent transferrin. The chase was performed using the following time course: 0, 20, and 40 min. Finally, the cells were transferred to ice, transferrin exposed extracellularly on the plasma membrane was removed using cold acid buffer, and cells were collected using trypsin-EDTA. After cold fixation in 1% PFA, intracellular fluorescence of Alexa Fluor 647–transferrin was quantified by FACS (Beckman-Coulter FC500). The results are expressed as a percentage of the intracellular fluorescence measured at time zero.
To measure the surface expression of transferrin receptor, cells were incubated on ice with DMEM-BSA and 0.1 μM lat-A (or DMSO as a control) for 20 min. Then, Alexa Fluor 647–conjugated transferrin (in the continuous presence of lat-A or DMSO) was provided for 40 min on ice. The cells were then washed extensively with PBS, collected, and fixed with 1% PFA. The fluorescence intensity was measured by FACS.