In cultured ARPE-19 epithelial cells, myo6 is found associated with uncoated endocytic vesicles located in peripheral actin-rich regions (Aschenbrenner et al., 2003
). These vesicles lack both clathrin and the early endosome marker, EEA1 (; Aschenbrenner et al., 2003
). GFP-tagged myo6 (GFP-M6; ) targets to these uncoated vesicles (), which were monitored by time-lapse fluorescence digital microscopy at 25°C (; Supplementary Movies 1 and 2).
Figure 1. Myo6 and GFP-tagged myo6 constructs localize to peripheral uncoated vesicles. (A) Schematic of GFP-myo6 constructs. The motor domain point mutation, K157R, is indicated. (B) Staining of ARPE-19 cells using rabbit anti-myo6 antibodies (endogenous M6) or (more ...)
GFP-M6–decorated vesicles exhibited complex trajectories within cell peripheries, with combinations of long unidirectional movements either laterally or toward more central regions interspersed with pauses (). Vesicle velocity was variable over time (). Statistical analysis of >250 vesicles revealed that the distribution of instantaneous velocities was broad, averaging 38.4 ± 16.6 nm/s (). Although the GFP-M6–associated vesicles appeared to take a very circuitous route through cell peripheries (), vector analysis, which compared the start and end points of vesicle tracks, revealed that all vesicles tracked exhibited a net vector inward into the cell (). The average net distance traveled was 1.8 ± 1.4 μm (), with distances ranging between 0.3 and 5.5 μm. This variation was expected, as the distance required for transport would differ depending on the vesicle's initial position and the position of the presumed destination early endosome. Overall, the distance traveled was consistent with the width of the actin mesh seen in ARPE-19 cells () and suggests that myo6 was recruited to move vesicles out of actin-rich regions and not further into the cell.
Pulse-chase experiments following transferrin trafficking had revealed a 2–10-min delay between formation of a new clathrin-coated vesicle and delivery of the vesicle contents to the endosome (Hopkins, 1983
; Hanover et al., 1984
; Eskelinen et al., 1991
; Trischler et al., 1999
). Although the actual time varied between cell lines, these results suggested that uncoated vesicles in all cell types had a relatively short lifetime of a few minutes. We calculated the lifetime of >250 GFP-M6–decorated vesicles by comparing the timing of their first appearance within the cell (when GFP-M6 was recruited upon vesicle uncoating) to their disappearance (when GFP-M6 departs, an event we correlate with fusion with the early endosome as myo6 is not present on this destination compartment). Taking into account vesicles with short lifetimes (<40 s) and the fact that the final movement of each uncoated vesicle, which culminated in fusion with the early endosome, was missed in our analysis, we calculated an average lifetime of 4.5 ± 2.7 min (). This lifetime is consistent with their identity as uncoated vesicles.
Our analysis of individual myo6-associated uncoated vesicles illuminated several types of membrane dynamics not previously described for this compartment. The most commonly visualized event was fusion between GFP-M6–associated vesicles. Fusion was characterized by the apparent meeting of two vesicles, producing a single vesicle with approximately twice the apparent fluorescent intensity that remained as a single entity for the remainder of the vesicle's lifetime (; Movies 1 and 2). In a 250-μm2 area an average of 1.4 ± 1.3 fusion events were observed per min, with the number of vesicle fusions ranging from 0 to 4 events per min. Overall, approximately 1 in 10 vesicles participated in a fusion event, suggesting that myo6-based transport serves two purposes: not only does it allow transport through actin-rich regions, but it also uses its association with the actin cytoskeleton to enable vesicle fusion.
Vesicle separation or fission events were not seen; however, we noted multiple occurrences of vesicle stretching, characterized by pulling of a vesicle to produce an elongated shape, followed by what appeared to be separation into two less-fluorescent daughter vesicles (). In all cases these fission events were unsuccessful, and the stretched vesicle returned to its original shape (). An average of 0.75 ± 0.5 stretching events were seen per min within a 250-μm2 area.
Uncoated Vesicle Movement Requires myo6 Motor Activity
In all cells analyzed by time lapse, GFP-M6–decorated vesicles appeared, exhibited movements and then disappeared (see Supplementary Movies 1 and 2). We hypothesized that the observed movements were due to myo6 motor activity. To test this we used a construct that fused GFP to the globular cargo-binding tail of myo6 but lacked a motor domain (GFP-M6tail; ). When expressed in ARPE-19 epithelial cells, GFP-M6tail targeted to peripherally located vesicles (). Time-lapse microscopy revealed that the GFP-M6tail–associated vesicles exhibited short, slow movements interspersed with pauses (; Supplementary Movies 3 and 4). The lack of directionality suggested that the vesicles were exhibiting Brownian-like motion. Quantitation of 250 vesicles revealed an average maximal instantaneous velocity of 13.3 ± 6.9 nm/s () with an average of 0.7 ± 0.5 μm traveled (), both significantly depressed from that seen for GFP-M6–expressing cells (p < 0.001). Therefore, myo6 motor activity is important for uncoated vesicle movement.
Figure 4. Analysis of GFP-M6tail–associated vesicles reveals a requirement for the myo6 motor domain for vesicle movement. (A) Images of a GFP-M6tail–expressing ARPE-19 cell monitored by time lapse, presented as a phase contrast (a) and a fluorescence (more ...)
The vesicle stretching events noted in GFP-M6–expressing cells, were not detected in GFP-M6tail–expressing cells, confirming that myo6 motors and not other motor proteins, are generating force during these events. When vesicle fusion was evaluated in GFP-M6tail–expressing cells, however, it was evident that, although trapped in cell peripheries, the uncoated vesicles did still exhibit fusion (). To quantify these events, we monitored ~120 vesicles over a 13.3-min period and 16.6% of the GFP-M6tail–associated vesicles exhibited vesicle fusion; however, this high percentage is misleading because of the long vesicle lifetime (see below). The overall fusion rate was 0.37 fusion events per min per 250 μm2, a level significantly lower than that seen in GFP-M6–expressing cells, suggesting that myo6 motor activity modulates or accelerates the fusion rate.
Disruption of myo6 Motor Function Increases the Uncoated Vesicle Lifetime
Previous studies had shown that overexpression of GFP-M6tail led to a 15-min delay in transferrin trafficking to the early endosome (Aschenbrenner et al., 2003
). Consistent with this delay, GFP-M6tail–associated vesicles monitored by time-lapse microscopy exhibited a dramatic increase in vesicle lifetime, averaging 15.4 ± 5.6 min (). No difference in lifetime was noted based on vesicle position within the cell (unpublished data). This lifetime is likely an underestimate because cells were recorded for 20–22 min, and of 250 vesicles monitored the majority (65–85%) were still present at the movie's end. Therefore only a small percentage of vesicles disappeared as a result of early endosome fusion, suggesting that the defect in trafficking to the early endosome increases the apparent vesicle lifetime.
Vector analysis monitoring the start and end position of each vesicle confirmed that the GFP-M6tail–associated vesicles were not moving great distances (). However, analysis of many cells revealed that over the long vesicle lifetime there was a trend of net movement inward for many vesicles tracked. Inspection of phase-contrast images suggested that this was not due to general cell contraction. GFP-M6tail expression in epithelial cells causes a delay, not a block, in transferrin trafficking; given 30 min, the contents of the uncoated vesicles can be delivered to the early endosome (Aschenbrenner et al., 2003
). We therefore hypothesized that the inward movement of GFP-M6tail–associated uncoated vesicles could reflect an alternate mechanism for slow vesicle trafficking in the absence of myo6.
Uncoated Vesicle Movement Is Not Linked to Retrograde Actin Flow
One explanation for the apparent slow inward movement of GFP-M6tail–associated vesicles could be the action of residual endogenous myo6 associated with these vesicles. However, previous studies have shown that this construct effectively displaces endogenous myo6 off of the surface uncoated vesicles, even when expressed at low levels (Aschenbrenner et al., 2003
). As all GFP-M6tail–expressing cells analyzed, regardless of expression level, exhibited this slow inward flow, this simple explanation seemed insufficient to explain this trend.
The GFP-M6tail–associated vesicles exhibited Brownian-like motion, with periods of nonmovement interspersed with short randomly oriented ~10-nm/s movements (), suggesting some freedom of motion. Therefore, the net vesicle movement could be due to diffusion coupled to endosome capture. We theorized that any vesicle that exited the actin meshwork would immediately fuse with an early endosome because these endosomes are abundant outside the actin mesh. Therefore, this fusion event could serve as a mechanism to bias diffusion thereby producing a net inward vesicle flow.
Alternatively, because the cell region that accumulated uncoated vesicles in GFP-M6tail–expressing cells was highly actin-rich, we hypothesized that the slow net vesicle influx could be due to vesicle coupling to retrograde actin flow. Retrograde actin flow describes the process whereby actin polymerization at the plasma membrane is coupled to myosin II–dependent force production, resulting in a net inward flow of actin and associated components (reviewed in Cramer, 1997
). Retrograde actin flow is not commonly observed in nonmotile epithelial cells, but retrograde flow in motile fibroblasts and nerves can generate inward transport of actin-associated components at a rate of 1–7 μm/min (16–116 nm/s; Cramer, 1997
). This rate is similar to the maximal velocity of GFP-M6tail–associated vesicles, making retrograde flow a reasonable explanation for the apparent inward movement.
To distinguish between these two theories, retrograde flow vs. vesicle diffusion and capture, we created a myo6 mutant predicted to bind irreversibly to F-actin. GFP-M6(K157R), contains a point mutation in the ATP-binding site (). The K157R mutation is homologous to the P-loop K185R mutation shown in the Dictyostelium
myosin II system to fail to complement myosin II null mutations (Ruppel and Spudich, 1996
). In myosin II, the K185R mutation caused a defect in myosin ATP binding. Furthermore, the mutated myosin exhibited no ATPase activity and was defective in actin filament sliding due to rigor-like actin binding (Ruppel and Spudich, 1996
). We predicted that by introducing the K157R mutation into myo6, cells expressing this mutated version would exhibit actin-bound uncoated endocytic vesicles, allowing us to evaluate the importance of retrograde flow and free diffusion on vesicle trafficking.
When expressed in ARPE-19 cells, GFP-M6(K157R) targeted to peripherally located vesicles distinct from early endosomes, a subset of which contained the transferrin receptor (; Supplemental Figure 1A). These vesicles were uncoated, as they did not contain clathrin (percent overlap 4.4 ± 0.4%; Supplemental Figure 1B). This localization to uncoated vesicles was expected, as the tail domain, and not the motor domain, is required for targeting (Aschenbrenner et al., 2003
Time-lapse video microscopy revealed that GFP-M6(K157R)–associated uncoated vesicles displayed essentially no movement (; Supplementary Movies 5 and 6). These vesicles also exhibited no fusion events (200 vesicles monitored), nor was vesicle stretching seen in GFP-M6(K157R)–expressing cells (Movies 5 and 6). Analysis of 250 vesicles revealed few instances of directional movement as judged by velocity measurements (average maximal velocity = 10.6 ± 10.9 nm/s; ) and the average distance traveled was 0.5 ± 0.4 μm. (). Both measurements are statistically lower than that seen for GFP-M6tail–associated vesicles (p < 0.001), suggesting that the K157R mutation produced vesicles with rigor-like strong attachment to the peripheral actin.
To directly evaluate the effects of GFP-M6(K157R) expression on vesicle diffusion, we computed the mean squared displacement (MSD) for GFP-M6tail– and GFP-M6(K157R)–associated vesicles and plotted them as a function of time to determine the apparent two-dimensional diffusion coefficients associated with these vesicle populations (200 vesicles analyzed). In both cases, the majority of the vesicles exhibited plots with a downward curved parabolic curve, indicating confined motion. Seventy-one vesicles for both constructs exhibited a more linear correspondence between MSD and time, allowing us to calculate their apparent diffusion coefficients (). GFP-M6tail–associated vesicles had a diffusion coefficient of 1.42 ± 1.24 × 10–12
, a value similar to the three-dimensional diffusion coefficient described for chromaffin granules docked on the actin cytoskeleton (Oheim and Stuhmer, 2000
). GFP-M6(K157R)–associated vesicles exhibited a significantly depressed diffusion coefficient (p < 0.001) of 0.76 ± 0.65 × 10–12
. Therefore, GFP-M6(K157R)-associated vesicles exhibited an apparent decrease in two-dimensional diffusion.
We predicted that if retrograde flow was a mechanism for vesicle influx, then the actin bound GFP-M6(K157R)–associated vesicles should exhibit an inward flow similar to that seen for GFP-M6tail–expressing cells. Remarkably, vector analysis of the GFP-M6(K157R)–associated vesicles over their lifetime revealed no net inward vesicle flow (). Therefore retrograde actin flow does not serve as a mechanism for vesicle influx, supporting the model that diffusion followed by endosome capture is responsible for the vesicle movements seen in GFP-M6tail–expressing cells.
GFP-M6(K157R)–associated vesicles exhibited long lifetimes in our time-lapse analysis (average lifetime = 16.4 ± 5.6 min; ). Essentially all vesicles were still present at the movie's end (85–92%; see Supplementary Movies 5 and 6, movie lengths 20–22 min), making 16 min an underestimate. Therefore tight association with the peripheral actin also lengthened vesicle lifetime, suggesting a block in trafficking.
An Actin-bound myo6 Motor Mutant Blocks Transferrin Trafficking to the Early Endosome at the Uncoated Vesicle Stage
The block in transferrin trafficking seen in GFP-M6tail–expressing cells can be overcome by extending the incubation time to 30 min (; Supplementary Figure 2; Aschenbrenner et al., 2003
). We theorized that, if rescue was due to diffusion of GFP-M6tail–associated vesicles followed by their early endosome capture, then GFP-M6(K157R)–expressing cells, with their actin-bound vesicles, would not be sensitive to this type of rescue.
Figure 6. Expression GFP-M6(K157R) blocks transferrin trafficking and it cannot be rescued by extending uptake time. GFP-, GFP-M6–, GFP-M6tail–, and GFP-M6(K157R)–transfected cultures were incubated with rhodamine-conjugated transferrin (more ...)
ARPE-19 cells were transfected with the GFP constructs shown in , incubated with rhodamine-conjugated transferrin (R-Tsfn) for 15 or 30 min and then scored for transferrin accumulation in the pericentriolar early endosome compartment. Seventy to 80% of control transfected and untransfected cells exhibited a prominent pericentriolar accumulation of R-Tsfn after 15-min uptake (, Suplementary Figure 2; Aschenbrenner et al., 2003
). In contrast, GFP-M6(K157R) overexpression caused a drastic decrease in steady state R-Tsfn uptake at 15 min, with only 19.6 ± 3.3% of cells exhibiting a pericentriolar accumulation (; Supplementary Figure 2). These results are similar to those reported for cells expressing GFP-M6tail (22.0 ± 2.3%), confirming that GFP-M6(K157R) expression disrupts transferrin trafficking. Pulse-chase experiments comparing the location of the endocytosed R-Tsfn to GFP-M6(K157R) confirmed that the block in trafficking was at the uncoated vesicle stage (Supplementary Figure 3 and associated text). As predicted, the block in trafficking was not rescued by extending the time period; 25.6 ± 1.2% of GFP-M6(K157R)–transfected cells exhibited a pericentriolar accumulation of R-Tsfn after 30-min uptake (; Supplementary Figure 2). This lends further support to the diffusion and capture model for vesicle trafficking out of the actin mesh in the absence of myo6 motor activity.
Actin Is a Barrier to Uncoated Vesicle Trafficking
Our analysis of myo6 mutants suggested that actin was a barrier slowing uncoated vesicle trafficking to the early endosome. If this were the case, we predicted that actin depolymerization should accelerate the rate of transferrin delivery to the early endosome. We used the F-actin–depolymerizing drug latrunculin A (LatA) (Spector et al., 1983
), which sequester G-actin monomers. Titration experiments revealed that nanomolar LatA concentrations were sufficient to remove the peripheral actin meshwork from ARPE-19 epithelial cells without significantly altering cell attachment or cell shape (). Treatment with 0.015 μM LatA had no effect on the transferrin endocytosis rate in ARPE-19 cells, as judged by an ELISA-based assay that quantified biotinylated transferrin uptake (). In addition, pulse-chase experiments following R-Tsfn and comparing its location to clathrin confirmed that treatment with LatA had no effect on the rate of transferrin exit from clathrin-coated pits and vesicles (). Therefore, depolymerizing the peripheral actin cytoskeleton had no effect clathrin-coated vesicle formation or uncoating, allowing us to directly test the effects of LatA on the next step, the delivery of components to the early endosome.
Figure 7. Removal of the actin barrier in ARPE-19 cells accelerates trafficking to the early endosome and rescues the trafficking defects seen upon GFP-M6tail and GFP-M6(K157R) expression. (A) Fluorescence staining of F-actin in cells treated for 30 min with DMSO (more ...)
To determine early endosome delivery kinetics, we allowed cells treated with DMSO or LatA to endocytose R-Tsfn for fixed time periods ranging from 1 to 15 min at 37°C and then quantified the percent of cells where R-Tsfn had reached the pericentriolar early endosome (see MATERIALS AND METHODS). In DMSO-treated cells, R-Tsfn maximally reached the early endosome after 5–7 min (). In LatA-treated cells, R-Tsfn delivery to the early endosome was accelerated, reaching the pericentriolar region within 2–3 min (p < 0.001). A similar acceleration was observed in LatA-treated cells when the location of endocytosed transferrin was compared with the early endosome marker, EEA1 (unpublished data). This acceleration suggests that actin is a barrier to uncoated vesicle trafficking to the early endosome and that actin's presence causes a 3–5-min delay in endosome delivery equivalent to the normal uncoated vesicle lifetime.
Transferrin-containing vesicles accumulate in peripheral actin-rich regions of GFP-M6tail– and GFP-M6(K157R)–expressing cells, thereby blocking delivery to the early endosome. We hypothesized that F-actin depolymerization should rescue this phenotype. We first evaluated the effects of actin depolymerization on cells transfected with GFP and GFP-M6. These control experiments revealed that the presence of DMSO coupled with the exposure to transfection reagent resulted in a slower net rate of steady state transferrin uptake compared with untransfected controls. After 15 min of uptake in the presence of DMSO or LatA, 60–75% of GFP- or GFP-M6–transfected cells exhibited a pericentriolar R-Tsfn accumulation, a number significantly less than the ~95% seen for untransfected cells after 15 min (). Although trafficking was slower, in the presence of DMSO, cells transfected with GFP-M6tail and GFP-M6(K157R) still exhibited a significant block in transferrin trafficking, with only 32.7 ± 7.1 and 34.1 ± 4.5% of cells exhibiting a pericentriolar accumulation, respectively. After treatment with LatA, however, 57.0 ± 7.3% of GFP-M6tail–transfected cells and 57.9 ± 7.3% of GFP-M6(K157R)–transfected cells exhibited pericentriolar transferrin accumulation, levels equivalent to those seen in controls. Therefore the block in trafficking seen upon GFP-M6tail and GFP-M6(K157R) overexpression is due to trapping in the actin cytoskeleton, and this block can be released by removing the F-actin barrier.