Latrunculin A (latA) and jasplakinolide were obtained from Sigma Chemical (St. Louis, MO) and resuspended in dimethyl sulfoxide (DMSO) at 10 and 1 mM concentrations, respectively. Oregon Green–labeled chicken skeletal muscle actin was prepared by covalent linkage of succinimidyl ester of Oregon Green (Molecular Probes, Eugene, OR) to lysine residues on purified skeletal muscle actin as described (Waterman-Storer, 2002
). Human diferric transferrin was biotinylated as previously described (Smythe et al., 1992
). All other reagents, unless otherwise noted, were obtained from Sigma.
Swiss 3T3 cells (mouse fibroblasts) stably expressing clathrin light-chain DsRed (kindly provided by W. Almers, Vollum Institute, Oregon Health and Sciences University) were grown in a humidified incubator at 37°C, 5% CO2 and maintained in DMEM containing 10% fetal bovine serum (Gemini, Woodland, CA) and 400 μl/ml geneticin (Invitrogen, Carlsbad, CA).
Cells, 1.2 × 106, were plated onto poly-l-lysine (PLL; 150,000–300,000 MW 0.1 mg/ml)-coated 22 × 22-mm glass no. 1.5 coverslips (Corning, Corning, NY; n = 1.523) in a 100-mm tissue culture dish. The following day cells were viewed with an inverted microscope (Nikon TE2000U, Garden City, NY) custom modified to allow for through-the-objective multispectral total-internal reflection fluorescence microscopy and wide-field epi-fluorescence (to be described in detail elsewhere) using a 100×, 1.45 NA objective (Nikon). The wavelength and intensity of light from a 50-mW KrAr air-cooled laser (Melles Griot, Rochester, NY) were controlled with a polychromatic acousto-optical modulator, the output of which was collected by a fiberoptic to transmit it to the microscope's custom modified, dual-input epi-illuminator. Lines at 568 and 488 nm were used for total internal reflection fluorescence microscopy (TIR-FM) excitation. Wide-field epi-fluorescence (WF-EF) light was generated by a 100-W Hg arc lamp and controlled with an electronic shutter/filter wheel device containing bandpass excitation filters for 488 ± 20 and 568 ± 20 nm (Sutter Instruments, Novato, CA). Laser and arc-lamp light were both focused at the aperture plane and directed to the coverslip by a custom dual dichromatic mirror (Chroma, Brattleboro, VT). The position of the beam was adjusted with a digital readout micrometer that moved the fiber-optic coupler at its input into the illuminator. We calibrated the micrometer to enable selection of specific beam angles. We used a penetration depth of ~100 nm of the evanescent field. Fluorescence emission was controlled with a filterwheel device (Sutter Instruments) containing narrow bandpass emission filters. Images were collected using a Hamamatsu Orca II-ERG camera (Bridgewater, NJ), operated in 14-bit mode. Control of all electronic hardware and camera acquisition was achieved with MetaMorph software (Universal Imaging, West Chester, PA). Stage temperature was controlled with a custom-modified stage incubator (20–20 Technologies, Wilmington, NC).
For imaging, coverslips of cells were preincubated for 30 min with imaging medium (DMEM [no phenol red], 10 mM HEPES, pH 7.5, 10% fetal bovine serum) containing either 5 μM latA, 1 μM jasp, or an equal volume of DMSO (0.1%) at 37°C. Coverslips were then mounted on a slide with two strips of double-sided tape spaced ~1 cm apart. The space between the strips of tape was filled with imaging medium additionally containing either 5 μM latA or 1 μM jasp, or DMSO. The coverslip was sealed with a 1:1:1 mixture of Vaseline, lanolin, and paraffin and the slide was transferred to a prewarmed microscope stage (37°C) for imaging. One or two cells were imaged per coverslip. Pairs of WF-EFM (100–200-ms exposures) and TIR-FM (200–500-ms exposures) images were taken for 10–15 min at 2-s intervals, with WF-EFM images taken first to minimize the time between WF-EFM and TIR-FM acquisitions.
For rescue experiments, cells were imaged in a custom built perfusion chamber (Gupton and Waterman-Storer, 2005
) to wash out latA during the course of imaging. After 10 min of imaging cells in the presence of 5 μM latA, cells were washed with >20 chamber volumes of DMEM (BioSource International, Camarillo, CA) +10% fetal calf serum + 10 mM HEPES, pH 7.4.
Cells were microinjected with Oregon Green G-actin (0.5 mg/ml) 4–7 h before imaging, by which time labeled actin was incorporated into the actin cytoskeleton. Cells were then imaged as above, except that culture medium was supplemented with 1.0 U oxyrase per ml (Oxyrase,) to reduce photodamage and photobleaching.
Swiss 3T3 cells stably expressing clathrin light-chain (CLC) EGFP were also analyzed for their sensitivities to drugs that disrupt the actin cytoskeletal dynamics. Similarly to the CLC-DsRed–expressing cells, we found that the CLC-EGFP dynamics were also dramatically halted when cells were treated with 5 μM latA or 1 μM jasp (unpublished data). Therefore, the effects that we see are not specific to the tag placed on the CLC.
Cells were grown on plastic culture dishes coated with PLL overnight, treated with 5 μM latA, 1 μM jasp, or DMSO for 30 min at 37°C, and then fixed in 2.5% glutaraldehyde in 0.1 M Na cacodylate buffer (pH 7.3) for 30 min at room temperature. Samples were washed three times in 0.1 M cacodylate buffer and treated with 1% osmium tetroxide in 0.1 M cacodylate buffer for 60 min. They were then washed three times in 0.1 M cacodylate treated with 0.5% tannic acid in 0.05M cacodylate for 30 min, rinsed in 1% Na2SO4 in 0.1 M cacodylate buffer for 10 min, and then rinsed in 0.1 M cacodylate buffer. Samples were dehydrated in graded ethanols, exchanged into HPMA (2-hydroxypropyl methacrylate), embedded in LX-112 (Ladd, Burlington, VT), removed from the plastic dish, and sectioned (transversely). Sections were stained with uranyl acetate followed by Pb citrate and viewed with a Philips CM100 electron microscope (FEI, Hillsbrough, OR). For quantification of CCS morphology and surface density, cells were randomly selected and imaged at low magnification (×1900). The cell perimeter was then examined at high magnification (×18,000) to identify, classify, and count CCSs. Deeply invaginated coated pits were distinguished from curved coated pits by the inward, negative curvature at the neck of the deeply invaginated coated pits. There exists no clear morphological correlate to constricted coated pits, which are defined biochemically. The linear surface examined was measured by tracing the cell surface in the low magnification images using Metamorph software (Universal Imaging).
To determine the degree of CCS internalization, CCSs were randomly identified, irrespective of size and shape, on the first frame of a 10-min time-lapse movie and analyzed to ascertain if the CCS or a portion of it was internalized. CCS internalization was detected as disappearance from TIR-FM before disappearance from WF-EFM. Internalization was also scored if a portion of a CCS separated and disappeared from TIR-FM before disappearing from WF-EFM. CCSs that disappeared simultaneously in both TIR-FM and WF-EFM were excluded from analysis. Forty to 150 CCSs were analyzed per cell. To determine the formation of CCSs, the number of new CCSs that appeared on a predefined region of the cell surface per 10-min interval was measured and presented as the fraction of CCSs that formed per unit area per 10 min relative to DMSO-treated controls. CCSs were classified as newly formed either if they appeared de novo (see ) or if they separated from a preexisting CCS and remained on the cell surface for at least 20 s. Surface density of CCSs was determined by averaging the number of CCS in a defined region on the first frame and last frame of a time-lapse movie and expressed as the average number of CCSs/μm2 surface area.
Figure 1. Assembled clathrin exhibits diverse behaviors. Images from time-lapse sequences showing the behavior of CLC-DsRed. TIR-FM (top panels) and WF-EFM (bottom panels). (A) Formation and internalization of CCV, Supplementary Video 1, (B) splitting and internalization (more ...)
To measure the lateral mobility of CCSs, the movement of CCSs in the TIR-FM time-lapse series were manually tracked using the track points function in Metamorph software (Universal Imaging). The distance moved in consecutive frames by CCS centroids were recorded for up to 3 min. If the CCSs did not persist in the TIR-FM image for the duration of the 3 min, then they were tracked until they could no longer be detected by TIR-FM. CCSs persisting for <1 min were excluded from the analyses. To calculate the average path-length that CCS moved in a 20-s interval, the distances (μm) that the CCS traveled in consecutive 20 intervals were averaged. A total of 66–94 CCSs were analyzed from three to four cells per condition. The average rate of CCS motility was determined from CCS displacements lasting for >3 consecutive frames.
To determine colocalization of actin and clathrin, cells were fixed with 3.7% formaldehyde, stained with Alexa-488 phalloidin as previously described (Cramer et al., 2002
), and mounted in phosphate-buffered saline and imaged by TIR-FM. TIR-FM images of actin and clathrin were pseudocolored and merged in Metamorph to identify by visual inspection CCSs that colocalized or partially colocalized with a local concentration of actin. The line scan function in Metamorph software was used to measure the average fluorescence intensity along a 3 pixel wide, 15 pixel long line drawn parallel to the longest clathrin, actin dimension. Data were collected from the individual actin and clathrin images. The signals were then normalized to the minimum signal along the line, and intensity was calculated as a percentage of the maximum signal along the line. CCSs and actin fluorescence were considered centered if their peak fluorescence intensities were <1 pixel (64 nm) or if their fluorescence profiles were fully overlapping. Otherwise, they were counted as spatially offset. Because the chromatic aberration of the microscope determined by measuring multispectral beads in different wavelengths was found to be less than one pixel, and because the overlap of the CLC-DsRed CCSs and the Oregon-Green–labeled actin or the Alexa-488 phalloidin were not found to be biased to one orientation (i.e., the green foci are not always to the right of the red foci, see Figures and ), we did not correct for this aberration. Spatially offset structures were subclassified as mutually exclusive if the peaks of fluorescence intensity were 4 or more pixels (256 nm) apart as prescribed by the Rayleigh limit. Structures that did not have a clear peak were excluded from the analyses.
Figure 2. Actin and clathrin associate during endocytosis. Images from time-lapse sequences showing the behaviors of CLC-DsRed and Oregon Green actin. TIR-FM CLC-DsRed (upper panels), corresponding Oregon Green actin colocalization (middle panels), and merged images (more ...)
Figure 3. Clathrin dynamics are perturbed after disruption of actin assembly/disassembly dynamics. (A–C) Merged TIR-FM images of Oregon Green actin (green) and CLC-DsRed (red) in (A) control cells, and cells treated with (B) 5 μM latA or (C) 1 μM (more ...)
Biochemical Internalization Assays
Transferrin endocytosis assays were modified from (Smythe et al., 1992
; Carter et al., 1993
). Swiss 3T3-CLC-DsRed–expressing cells (n = 1.2 × 106
) were plated onto PLL-coated, 12-mm circle coverslips (Fisher Scientific, Fairlawn, NJ) in 100-mm tissue culture dish (Nunc, Napierville, IL) and allowed to adhere overnight. Cells on coverslips were rinsed with SFM (DMEM + 0.2% bovine serum albumin + 10 mM HEPES, pH 7.5), transferred to four-well tissue culture dishes (Nunc; 2 dishes per time point), and incubated at 37°C with SFM containing either 5 μM latA, 1 μM jasp, or an equal concentration of DMSO as a control for 30 min. The time-course of internalization was initiated by adding SFM containing biotin-S-S-transferrin (BSST, 4 μg/ml) supplemented with drug or DMSO, and placing dishes on metal blocks in a 37°C water bath. Internalization was stopped by placing dishes on ice and washing wells three times with 0.5 ml SFM. Sequestration from avidin and cleavage of the biotin from the noninternalized transferrin by reduction of the disulfide bond was performed as previously described (Smythe et al., 1992
; Carter et al., 1993
), with the exception that Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), a more potent and stable reducing agent, was used instead of MesNa. After the internalization of BSST, 15 mM TCEP (Pierce, Rockford, IL) in reduction buffer (50 mM HEPES, 1 mM MgCl2
, 125 mM NaCl, 50 mM Tris, pH 8.0) was added to the wells and incubated at 4°C for 30 min. The reduction was quenched with iodoacetamide, and cells were solubilized in blocking buffer (1% TX-100, 0.1% SDS, 0.2% bovine serum albumin, 50 mM NaCl, 1 mM Tris, pH 7.4). Cell lysates were plated onto ELISA plates coated with anti-transferrin antibody and assayed for detectable BSST using HRP-labeled streptavidin as previously described (Smythe et al., 1992
; Carter et al., 1993
). Internalized BSST was expressed as the percent of total surface-bound at 4°C. The level of BSST internalization was normalized to the amount of internalization for DMSO-treated cells to account for day-to-day variation. All statistical analyses were performed using ANOVA analysis in Minitab software.