Defining the Phases of Actin Patch Assembly and Movement
For actin patches, the phases in their life have been defined by the presence of different components and by the character of their movement (). Phase I is characterized by the presence of WASp/Las17, a protein that targets active Arp2/3 complex (Wen and Rubenstein, 2005
). Phase I patches show a limited amount of motion, which is random in direction and restricted in range, as though the patch were in a corral or anchored by a tether on the plasma membrane.
To quantitatively analyze the motion of many patches under different conditions, we developed a computer-assisted method to track the position of patches over time in movies of living cells expressing GFP fusion proteins (Carlsson et al., 2002
). Movie 1 helps to illustrate the method. One useful way to analyze these data is to calculate the square of the distance of the patch from its starting point. The average of these values, the mean squared displacement (MSD), is then plotted versus time. Such plots reveal the character of the motion as well as quantitating its magnitude. Tethered random motion, characteristic of phase I, produces an MSD plot that is concave down, approaching a horizontal asymptote (corralled/tethered in ; Las17–3XGFP in ). Actin patches accumulate endocytic adaptors and actin-binding proteins over time. The tethered movement characteristic of phase I is also observed at the start of MSD plots with GFP labeling of the endocytic adaptors Sla1 and Sla2, and the actin-binding proteins Abp1, fimbrin (Sac6), and capping protein (Cap1/Cap2), which bind to actin filaments in different ways.
Phase II is the initial movement of the patch away from the membrane. This movement is slow and short, compared with subsequent movement in phase III. Phase II movement is observed with GFP labeling of the endocytic adaptors and the actin-binding proteins, but not with WASp/Las17 (). In MSD plots, phase II movement occurs as a relatively straight segment of low slope. Phase III consists of longer and faster movement directed away from the membrane, which shows in MSD plots as a concave-up segment of the curve after phase II. Phase III movement is only seen when tracking actin-binding proteins ().
Dependence of Movement on Actin Polymerization
In the current model relating actin assembly to endocytosis (Kaksonen et al., 2005
), the movements associated with phase II and phase III depend on actin polymerization, and we found this to be the case by using Abp1-GFP to track the patches. LatA at 50 μM partially inhibited patch movement, and 100–200 μM LatA provided complete inhibition, as seen in MSD plots (). The lifetime of the patches was increased in LatA, manifested in MSD plots by the curves extending to longer times. These plots were cut off when half the patches disappeared, indicating the median lifetime of the patches. The Abp1-GFP fluorescence intensity per patch decreased over time upon addition of LatA (our unpublished data), consistent with LatA inhibiting the progress of actin polymerization.
Patches in phase I do move, albeit relatively little. We asked whether this movement depended on actin polymerization by adding LatA to Las17-GFP cells. Patch tracking revealed that the movement was inhibited (). The lifetime of Las17-GFP patches was greatly prolonged by LatA, and the fluorescence intensity was not decreased (our unpublished data). The inhibition of movement may seem contradictory to the notion that actin polymerization is “downstream” of Las17 recruiting active Arp2/3 complex to the membrane. However, a locus of accumulated Las17 should be subject to forces coming from the actin filaments that polymerize around the site.
The Role of Capping Protein in Actin-based Motility
CP is proposed to play a central role in the dendritic nucleation model (Pollard and Borisy, 2003
), and CP is necessary to reconstitute the actin-based motility of Listeria
with pure proteins (Loisel et al., 1999
). We asked whether CP was important for actin-based motility in yeast actin patches.
First, we examined the assembly of CP on the actin patch with respect to other components. A Cap1-GFP fusion protein functioned normally, measured by rescue of polarization of the actin cytoskeleton. We compared Cap1-GFP with fimbrin/Sac6-GFP and Abp1-GFP by tracking actin patches in wild-type cells. The results, as MSD plots, were essentially the same (our unpublished data). We examined the order of assembly by dual-label imaging of Cap1-YFP and Abp1-CFP (). Following individual patches in wild-type cells, we found a nearly complete coincidence, in space and time, between the two labels, as shown in frames from a movie () and a graph of the fluorescence intensity over time (). Thus, the components of the actin network assemble in a concerted manner, suggesting that the assembly process is cooperative and rapid.
Figure 2. (A) Images of a single patch labeled with Cap1-YFP and Abp1-CFP, in YJC 4267. The time interval between frames is 1 s. The YFP and CFP images were collected for 0.5 s, consecutively, not simultaneously, so that the interval between columns is 1 s. The (more ...)
Next, we examined the effect of the loss of CP on actin patch assembly and early endocytosis by imaging and tracking actin patches in cells expressing Las17-GFP, Sla1-GFP, Abp1-GFP, or Sac6-GFP. Actin patches did form in CP mutants, but their movement away from the membrane was less, as seen in time-lapse images from movies ( and Movies 1 and 2). To analyze the data quantitatively, we tracked patches in movies of several independent isolates of each genotype. To examine the behavior of all the patches within a sample, we first produced plots of MSD versus time in which we aligned the data for individual patches on the left, at the start of the patch lifetime, and then averaged the results for all the patches from one isolate (, left). The results for the different isolates were similar to each other, and we show examples here to illustrate the degree of variation. We also aligned the data for individual patches on the right, at the end of the patch lifetime, to focus on motion that might have occurred at the end of the track.
Figure 3. Time-lapse microscopy of wild-type or cap1Δ cells expressing Abp1-GFP or Sla1-GFP. Note the different time intervals between frames in each column. In wild-type cells, the arrowhead indicates the starting position of a patch that moves, and the (more ...)
Figure 4. Quantitative analysis of patch motility in the presence and absence of capping protein. Patches labeled with GFP fusions of Abp1, Sac6, Sla1, or Las17, were tracked in wild-type, cap1Δ or cap2Δ cells. In the left panels, each curve is (more ...)
To examine phase I, we tracked Las17-GFP patches. There was no effect from the loss of CP, with patch tracks aligned at the start or the end of their lifetime before averaging to create MSD plots (). Next, we measured the time that patches spent at the membrane, from the time of their initial appearance until they either moved away from the membrane or disappeared, which can result from disassembly or z-axis movement (). In CP null mutants, Las17-GFP patches did spend slightly more time at the membrane, compared with wild-type (wt) cells, by an amount that was statistically significant ().
Figure 5. Mean time spent by patches in the tethered phase on the membrane. The time measured was the interval from the appearance of a patch until its movement away from the membrane or disappearance. The error bar is 1 s. Dimethyl sulfoxide. n = 24–50 (more ...)
Tracking the endocytic adaptor Sla1-GFP in CP null mutants, compared with wt cells, revealed substantial changes in the dynamics of patches in phase II. With MSD plots aligned on the left before averaging, cap1/2 cells showed much less motion for Sla1-GFP than did wt cells (, left). We considered two reasons for this difference. We thought that Sla1-GFP patches might persist at the membrane for longer and variable times or that they might move less after leaving the membrane. To test these possibilities, we first measured the lifetime of Sla1-GFP patches at the membrane and found it increased by a substantial amount, more than twofold (). Second, we aligned the MSD plots on the right-hand side before averaging them, i.e., at the end of their lifetime, to focus on the phase where inward movement normally exists. This analysis showed that the average movement away from the membrane was greatly inhibited by the loss of CP, albeit not completely (, right).
To examine phase III, we tracked patches using the actin-binding proteins Abp1-GFP and Sac6-GFP. Both markers gave similar results; only the Abp1-GFP results are shown here. With MSD plots aligned on the left, CP null mutant strains showed a substantial loss of the concave-up portion of the normal curve (, left). The lifetime of the patches at the membrane was increased, by approximately threefold (). With MSD plots aligned on the right-hand side, to focus on motion at the end of the track, CP null mutant cells showed a substantial decrease in average motion compared with wt cells (, right). The effect of the loss of CP here was less than the effect on patch lifetime or on MSD plots aligned on the left. In addition, the effect of the loss of CP on Abp1-GFP, with MSD plots aligned on the right, was less than what was seen for Sla1-GFP. To investigate further the movement of phase III, we tracked Abp1-GFP patches after they had moved away from the membrane, defined by a distance of 200 nm. The average movement was still decreased by the loss of CP, based on MSD plots aligned either on the left or the right before averaging ().
Figure 6. Average movement of Abp1-GFP patches after leaving the plasma membrane in wild-type, cap1Δ, or cap2Δ cells. Patches were tracked after they had moved 0.2 μm away from the plasma membrane. MSD plots on the left and right were aligned (more ...)
When actin-based motility of Listeria
was reconstituted from pure proteins in vitro, the curve describing how motility depends on the concentration of CP was bell-shaped, with motility falling off at high and low concentrations (Loisel et al., 1999
). This was also the case for ADF/cofilin and Arp2/3 complex. To test this prediction of the dendritic nucleation model in vivo, we overexpressed the two subunits of CP, Cap1 and Cap2, from a bidirectional promoter. We observed strong inhibition of movement, similar to the effect of the loss of CP, based on tracking of Abp1-GFP patches (). Thus, the concentration of CP in vivo is optimal for actin patch assembly and motility.
Figure 7. Movement of Abp1-GFP patches in cells overexpressing CP, compared with wild-type and cap1Δ cells. MSD plots were aligned on the left for averaging. Strain numbers are as follows: overexpression, YJC 4091; wild-type, YJC 3995; and cap1Δ, (more ...)
Patch Motility in the Absence of Actin Cables
Cells without CP are known to have a moderate defect in actin cable assembly and polarization (Karpova et al., 1998
), so we asked whether their cable defect could account for their decrease in patch movement. Actin cables are highly dynamic bundles of filaments, polymerizing at their ends near the bud tip on a rapid time scale comparable with that of actin patch assembly (Yang and Pon, 2002
). Actin patches are sometimes seen at the ends of cables and vice-versa (Karpova et al., 1998
). Some actin patches have been observed to move along actin cables, seeming to attach to the cable and move rearward as the cable treadmilled (Huckaba et al., 2004
). We reasoned that the decrease in the number of cables in CP mutant cells might be a contributing factor in the decrease in patch movement.
To test this hypothesis, we tracked actin patch assembly and movement with Abp1-GFP in conditional mutants known to lose actin cables completely and rapidly upon shift to restrictive temperature. We used two formin double mutants, bnr1
(Evangelista et al., 2002
). Formins nucleate and sustain the polymerization of the actin filaments in the cable, so that loss of formin function leads to rapid disappearance of cables. We confirmed that these mutants did lose cables rapidly at the restrictive temperature under our conditions, by rhodamine-phalloidin staining (our unpublished data). Surprisingly, tracking actin patches revealed a modest increase of patch motility upon loss of cables, not a decrease (). Thus, a decrease in cables cannot account for the decrease in patch movement in capping protein mutants.
Figure 8. Acute loss of actin cables increases patch motility. The motion of Abp1-GFP patches was tracked in wild-type, bnr1Δ, bnr1Δ bni1-11, and bnr1Δ bni1[hyphen]12 cells, at permissive (30°C) and restrictive temperatures (35°C). (more ...)
Endocytosis with Membrane Markers
Because loss of CP inhibited the movement of patches away from the membrane, and patches have been associated with endocytic vesicles as defined by FM4-64 labeling (Huckaba et al., 2004
), we asked whether the loss of CP delayed the transit of FM4-64 through the endosomal system in a pulse experiment (). The initial internalization step was delayed in the cap1
Δ mutant, to a degree similar to that seen for sla2
Δ, a positive control endocytosis mutant (Wesp et al., 1997
), based on the fraction of cells that persist in the initial pattern of fluorescence distribution (). This result confirmed our results with actin patch tracking in the capping protein mutants.
Figure 9. Endocytosis traffic assessed with FM4-64 pulse labeling. Cells were incubated with FM4-64 on ice, washed, and incubated at 30°. (A) Representative fluorescence images at different time points for wild-type, cap1Δ, and sla2Δ cells. (more ...)
In contrast, when examining the timing for the appearance of subsequent distribution patterns for the FM4-64 fluorescence, the cap1Δ curve was closer to the wild-type curve than it was to the sla2Δ curve (). This result suggested that movement of vesicles to the vacuole might be less affected by the loss of capping protein. We tested this hypothesis directly with time-lapse movies of living cells in which we examined the movement of FM4-64 vesicles in the cytoplasm at intermediate times during a pulse label experiment. In wild-type cells, vesicles that were free in the cytoplasm moved rapidly, with frequent sharp turns. In cap1Δ cells, the vesicle behavior was similar to that of wild-type cells. Tracking analysis and MSD plots showed that cap1Δ and wild-type vesicles were indistinguishable ( and Movies 3 and 4), supporting the hypothesis. The high frequency of turning caused the MSD plots to be linear, characteristic of random paths, as opposed to concave up, characteristic of directed motion.
Figure 10. Movement of FM4-64 and Ste2-GFP vesicles in the cytoplasm. Single vesicles, not adherent to a larger membrane, were tracked. MSD plots were aligned on the left for averaging. (A) FM4-64. n = 133 and 144 vesicles for wild-type (YJC 2588) and cap1Δ (more ...)
To test the hypothesis further, we asked whether the movement of endocytic vesicles in the cytoplasm labeled in another manner had a similar lack of dependence on CP. The membrane receptor Ste2 is constitutively endocytosed, and Ste2-GFP can be seen as vesicles moving rapidly though the cytoplasm on their way to the vacuole (Chang et al
). We tracked the movement of Ste2-GFP vesicles and found no difference between cap1
Δ and wild-type ( and Movies 5 and 6). Therefore, the movement of endocytic vesicles at a later phase in the endocytic pathway seemed not to depend on capping protein, whereas the movement of early endocytic vesicles did.
To explore further the idea that actin patches and Ste2 vesicles move with different mechanisms, we examined the dependence of their movement on Lsb6, a L
inding protein. Las17/WASp and Lsb6 were found to be important for the movement of Ste2-GFP vesicles in the cytoplasm (Chang et al
). Therefore, we tested actin patch motility in an lsb6
Δ null mutant, by Abp1-GFP tracking. We found no effect of the loss of Lsb6 (), supporting the conclusion that the mechanism of actin-based motility for actin patches differs from that for Ste2 vesicles.
Figure 11. Normal actin patch motility in the absence of Lsb6, based on Abp1-GFP patch tracking, with alignment of individual patch plots on the left before averaging. LatA treatment of the wt strain provides a positive control. Aligning the plots on the right also (more ...)