Filopodia, microspikes, and retraction fibers
Crawling cells elaborate filopodia, microspikes, and retraction fibers in the course of cycles of protrusion and withdrawal. These have been considered as distinct entities, but because of their structural similarities, we investigated whether they were truly distinct or interconvertible. Determining whether they were functionally related was important to define the scope of our study.
We followed the kinetics of peripheral actin bundles by phase contrast or fluorescence microscopy in untransfected or GFP-actin–expressing cells, respectively. We observed many examples of transition between filopodia, microspikes, and retraction fibers (). The predominant order of transitions was from microspike to filopodium to retraction fiber. Transitions in the opposite direction were also observed. For each type of structure, the filament bundle was able to protrude, suggesting that the actin polymerizing machinery was functional in each morphological state. The protrusive activity of the surrounding lamellipodium seemed to be an important factor determining the transitions between these organelles. Depending on whether the lamellipodium advanced as fast as or slower than an actin bundle elongated, the bundle appeared as a microspike or a filopodium. If the lamellipodium withdrew while the actin bundle remained stable or elongated, the bundle appeared as a retraction fiber. Increased net protrusion of an actin bundle also contributed to the transition from microspikes to filopodia, especially after fusion of two microspikes. Thus, filopodia, microspikes, and retraction fibers are interconvertible organelles, which may transform one into another because of a disparity in the protrusion velocity of the bundles themselves and of the surrounding lamellipodium. Therefore, in this paper, we will consider these types of peripheral actin bundles together and will refer to them collectively as “filopodia,” because this is the most commonly used term.
Figure 1. Interconversion between microspikes, filopodia, and retraction fibers. Time-lapse sequences of untransfected (A, phase contrast) or GFP-actin expressing (B and C, fluorescence) cells. Time in seconds. (A) Lamellipodium containing several microspikes (triple (more ...)
Kinetics of filopodia initiation
To approach the central question of the mechanism of filopodia initiation, we first investigated the kinetics of spontaneous filopodia initiation using GFP-tagged structural proteins. If filopodia were initiated by an Arp2/3-containing nucleation center, one would expect a nascent filopodium to arise from a distinct fluorescent dot of actin or Arp2/3 complex, whereas the rearrangement model predicts a gradual condensation of actin fluorescence into a filopodial bundle.
Filopodia in GFP-actin–expressing cells displayed a broad range of lengths and fluorescence intensities. Histories of large filopodia revealed that they were formed by fusion of smaller filopodia, which in turn were the result of fusion at an even finer scale ( A). Events of true filopodia initiation were recognized as the appearance of thin, faint nascent filopodia contained within the lamellipodial network. In most cases (81%, n = 124), they arose from fishtail-shaped actin densities within the lamellipodium (). These densities, which we will call Λ-precursors because of their shape, were just slightly brighter than the surrounding lamellipodium at their vertices, but gradually diffused into a lamellipodial network at their bases ( D). Although hardly distinguishable from the rest of the lamellipodial network, Λ-precursors could be consistently recognized, after contrast enhancement, by tracing back in time the sequences of newly formed filopodia. In the remaining cases of filopodial initiation (19%), Λ-precursors were not visible, possibly because of insufficient temporal resolution or contrast. Nascent filopodia subsequently fused with each other () or with other filopodia, and thus increased in size. Fusion produced Λ-configurations of filopodial bundles reminiscent of the shape of Λ-precursors, but with more distinct individual branches. Over time, these Λ-shaped bundles treadmilled backward at the root of the fused filopodium ( C) until they disappeared in the course of depolymerization. The observed actin kinetics appears more consistent with the idea of network reorganization as a mechanism of filopodia initiation.
Figure 2. Actin kinetics during filopodia initiation. (A–C) Time-lapse sequences of GFP-actin–expressing B16F1 cells. Time in seconds; individual features marked by arrows and arrowheads. Nascent filopodia are marked starting from the frame (more ...)
The Arp2/3 complex is predicted to be enriched in the hypothetical filopodial nucleation center. Therefore, we performed kinetic analysis of GFP-Arp3–expressing cells ( A). Because filopodial bundles were invisible in GFP-Arp3 images, we acquired phase contrast images at the beginning and at the end of the sequence to detect nascent filopodia initiated during the sequence. Filopodia were observed to appear by phase microscopy and GFP-Arp3 was present throughout the lamellipodium, but no increase in GFP-Arp3 intensity was detected to spatially and temporally correlate with filopodial initiation. The essentially uniform distribution of the GFP-Arp3 signal does not support the hypothesis of an Arp2/3-based nucleation center for filopodial initiation.
Figure 3. Kinetics of marker proteins during filopodia initiation. Time-lapse sequences of GFP-Arp3 (A), GFP-fascin (B and C), or GFP-VASP (D–F). (A) Two flanking phase contrast frames (0 and 164 s) demonstrate formation of a new filopodium during the sequence. (more ...)
To obtain insight into the actual mechanism of filopodia initiation, we next analyzed the kinetics of proteins enriched in filopodia, i.e., fascin and VASP. In GFP-fascin–expressing cells, a majority of nascent filopodia (66%, n
= 207) first appeared as a bright dot or short rod on a dark background (). In other cases (34%), a bright dot of GFP-fascin rather suddenly appeared at the tip of a very faint Λ-shaped density in lamellipodia ( C, inset in 16 s frame). Both kinds of nascent fascin dots subsequently elongated to form a filopodium. Fusion of mature fascin-containing filopodia was also frequently seen ( C). Because fascin is present in lamellipodia, albeit at much lower concentration than in filopodia, the faint Λ-shaped fascin densities might correspond to Λ-precursors, suggesting that in the course of filopodia initiation, fascin initially appears at the tips of the Λ-precursors (see next section). In GFP-VASP sequences, we followed the formation of brighter dots corresponding to filopodial tips among the weaker fluorescence of the lamellipodial edge. The major pathway for filopodia initiation (, D–F) was a gradual coalescence of VASP to ultimately produce a bright dot. The first sign detected was a slight elevation of GFP-VASP fluorescence intensity within a small domain (2–4 μm) along the leading edge. Then, this region gradually shrank into a dot concurrently increasing its intensity. In some cases, the condensing region became discontinuous during shrinkage ( F), suggesting intermediate formation of smaller filopodia. Because Ena/VASP proteins bind barbed ends and protect them from capping (Bear et al., 2002
), these data suggest that elongating barbed ends from the lamellipodial network gradually segregate into a small region, which becomes a filopodial tip.
Protein composition of Λ-precursors
Our kinetic analysis identified Λ-precursors as intermediates in filopodial initiation. Next, we examined whether molecular markers that discriminate between lamellipodia and filopodia are present in Λ-precursors (). Expression of GFP-actin or staining with labeled phalloidin was used to visualize actin. Putative Λ-precursors were identified in the actin channel based on their characteristic shape, and slightly increased actin density. Immunostaining or expression of GFP-tagged proteins was used to localize the second protein.
Figure 4. Localization of filopodial and lamellipodial markers in Λ-precursors. Left column; actin revealed by Texas Red-phalloidin (A, B, and F) or by GFP-actin expression (C–E). Central column; actin-binding proteins (as indicated) (more ...)
Fascin and VASP were used as filopodial markers. In GFP-fascin–expressing cells, putative Λ-precursors either did not contain significant amount of fascin ( A, arrows; also see ), or they had fascin enriched only at the tip ( A, arrowheads), suggesting that fascin was recruited to the tips of the preformed Λ-precursors. In GFP-VASP–expressing cells ( B), VASP fluorescence at the leading edge became less uniform after extraction, perhaps revealing more strongly associated protein. The brightest VASP dots corresponded to established filopodia. The majority of Λ-precursors contained a distinct VASP dot at their vertex ( B, arrowheads). Weak VASP dots were also evident without recognizable Λ-precursors ( B, arrows). These may represent fluctuations in density of actin filament barbed ends within the lamellipodium or Λ-precursors not detected in the actin channel.
Figure 6. Actin filament organization in Λ-precursors. Correlative fluorescence and EM of the same cell. First row; fluorescence microscopy of the leading edge showing Texas Red-phalloidin labeled actin (left), GFP-fascin (middle), and merged image (more ...)
As lamellipodial markers, we used Arp2/3 complex, cortactin, and capping protein. Previously, we have shown that Arp2/3 complex is excluded from filopodial bundles (Svitkina and Borisy, 1999a
). Here, we report that cortactin and capping protein are also excluded from filopodia (). In Λ-precursors, these proteins were partially depleted, especially close to the vertex, but not completely absent (, C–E), suggesting that gradual depletion of lamellipodial proteins occurs during formation of Λ-precursors and filopodia initiation. No enrichment of Arp2/3 complex was detected at filopodial roots.
We also investigated the ability of α-actinin to discriminate between filopodia and lamellipodia. Previously, α-actinin has been shown to localize to lamellipodia (Langanger et al., 1984
), but its localization in filopodia is unclear. GFP-tagged α-actinin associated with both lamellipodia and filopodia ( F), but appeared in these organelles with delay and, consequently, localized to the base of the fast-protruding lamellipodia and filopodia. Thus, α-actinin is a late marker for actin bundling in filopodia and is apparently not involved in filopodia initiation.
Together, the molecular marker analysis suggests that Λ-precursors represent a transitional structure displaying enrichment of filopodial markers and partial depletion of lamellipodial proteins. Dual-channel correlation of actin distribution with that of filopodial markers suggested that VASP accumulation occurred early in the process of formation of Λ-precursors, and that fascin appeared at the tips of established Λ-precursors.
Structure of Λ-precursors
Light microscopic analysis suggested a gradual reorganization of the lamellipodial network into bundles through intermediate formation of Λ-precursors. This hypothesis was analyzed using a higher resolution technique; platinum replica electron microscopy (EM). Treadmilling behavior of filopodia has a remarkable consequence in that the history of the actin array is imprinted in its structure (Katoh et al., 1999a
), so that moving from the leading edge in a proximal direction in space is analogous to traveling back in time. To understand the mechanism of filopodia initiation, we first focused on the analysis of filopodial roots. In this work, we were most interested in analyzing young filopodia, which are usually thin and short according to our kinetic study.
The majority of apparently young filopodial bundles were splayed apart at their roots into smaller bundles or individual filaments (), suggesting that the bundles were formed by convergence of the composing elements. Filopodial roots consisting of two or more smaller bundles are consistent with an event of filopodial fusion in the recent history of that filopodium ( B). More importantly, we observed many examples of filopodial bundles whose roots suggested the convergence of individual filaments originating from distant places in the surrounding lamellipodial network and entering the bundle at different levels. In some cases, it was possible to track filaments back from the bundle toward their origin as a branch on another filament in the surrounding network ( C). These findings suggest that filaments comprising filopodial bundles were asynchronously recruited from the dendritic network. Remarkably, filaments entering filopodial bundles were long compared with the branched network near the leading edge (, inset in A). Older filopodia, which could be recognized by their length and thickness, either had their actin bundles rooted deeply in the cytoplasm, which impeded visualization, or had tapered (not splayed) roots. This is consistent with depolymerization from the pointed ends of the composing filaments causing progressive elimination of the original splayed roots.
Figure 5. Filopodial filaments originate from the surrounding dendritic network. Platinum replica EM. (A) Filopodium contains a tight bundle of actin filaments that splays apart at its root and becomes an integral part of the surrounding network. Filaments in the (more ...)
Splayed filopodial roots apparently corresponded to aged Λ-precursors that treadmilled backward during filopodium growth. To identify Λ-precursors at a stage when they had not yet produced a filopodium, we performed correlative light microscopy and EM (). Putative Λ-precursors were identified in cells by fluorescence microscopy and relocalized after EM processing of the same cells. For these experiments, we used cells expressing GFP-fascin, which allowed us to compare parts of the Λ-precursor containing and not containing fascin.
Λ-precursors lacking fascin clearly displayed features of dendritic organization, such as short filaments, branching filaments, and numerous free filament ends (). Also, consistent with the idea of the transitional character of Λ-precursors, we found many rather long filaments within Λ-precursors, whereas long filaments were not frequent in the dendritic network outside Λ-precursors (). These long filaments apparently became enriched during transition of Λ-precursors into splayed filopodial roots, perhaps because of faster depolymerization of short filaments. The actin array in fascin-positive parts ( B) had a clearly bundled organization with densely packed filaments. The more proximal parts of actin bundles were not significantly enriched in fascin and displayed long, loosely aligned filaments ( B), suggesting that fascin-mediated bundling was delayed compared with accumulation of long filaments in the forming bundle. Thus, structural analysis of Λ-precursors and filopodial roots demonstrated enrichment of long filaments in these structures that apparently occurred before fascin-mediated bundling.
Structural organization of filopodia with known history
Because not every Λ-precursor produced a filopodium in kinetic studies, we performed correlative EM for cells with known history. For this purpose, we acquired time-lapse sequences of GFP-actin–expressing cells. After extraction and fixation, we prepared those cells for EM and analyzed filopodia formed in the course of the sequence (). A illustrates the correlation between the last live image of one such cell, the image of the lysed cell, and the EM image of the same cell taken at low magnification comparable with that of light microscopy. During the 19-s interval between the last live image and the image of the lysed cell, the lamellipodium protruded ~0.9 μm, which is evident in the superimposed image. The subsequent processing for EM did not introduce significant distortions into the structure of the lamellipodium because extracted light and low power EM images could be almost perfectly overlapped. Coincidence of light and EM features could also be seen at higher magnification, where brighter areas in fluorescence corresponded to denser actin arrays in EM ( B).
Figure 7. Structural organization of filopodia with known history. Correlative live imaging and EM. (A) Overview of the cell lamellipodium at different stages of sample processing. GFP-actin fluorescence images taken just before (live, 16 s) and immediately after (more ...)
Several filopodia were spontaneously formed during the total period of 35 s of this sequence. C illustrates the history of three nascent filopodia of different age, which are color coded in yellow, blue, and green for convenience of description. The filopodium marked with a yellow arrow was the oldest one. This filopodium was formed in the course of fusion of two faint, converging linear densities existing at the beginning of the sequence. A bright spot of actin fluorescence, which appeared in the filopodium shaft in the second frame, allows one to recognize treadmilling and retrograde flow in this filopodium. The filopodium marked with the blue arrow was not visible in the first frame; at 4 s, it appeared as a Λ-precursor, which at 8 s could be seen near the edge, and which produced a faint filopodium by 12 s. The filopodium marked with the green arrow is the youngest one. It was formed from a Λ-precursor first visible at 12 s. Tips of all three filopodia converged by the end of the sequence, suggesting that they began fusing at the moment of extraction.
The detailed structural organization of the region containing all three filopodia is shown in D. The root of the “yellow” filopodium consisted of two thin, fusing bundles corresponding to two converging lines in the first frame of the sequence. Each of these sub-bundles, when followed backward, splayed into individual filaments originating from the surrounding dendritic network (not depicted). The Λ-precursor of the “blue” filopodium that treadmilled backward during the sequence was identified with the splayed root of the blue filopodium in the EM image, indicating that splayed roots of filopodia indeed represent former Λ-precursors. Like in other EM images, filaments were collected into the bundle of the blue filopodium from the wide surrounding area ( D). In fluorescence images ( C), the splayed root became invisible already in 12 s frame, probably because of low fluorescence intensity. The root of the youngest “green” filopodium displayed many features of the dendritic organization of Λ-precursors. It had relatively high network density, many short filaments, frequent branching, and numerous free barbed ends (, inset 2 in D). Some filaments originating as a branch on another filament could be seen to enter the bundle of the green filopodium. These data support the idea that Λ-precursors initially represent a part of the dendritic network, but lose short filaments with age. An interesting feature of the green filopodium was that it was barely recognizable in the EM image because its filaments, although long, were not well-aligned, suggesting that filament cross-linking is not an early event during filopodial formation, in agreement with delayed recruitment of fascin to Λ-precursors.
In contrast to delayed bundling, the filament barbed ends at filopodial tips were in register, suggesting they were interacting with each other, even though they supposedly encountered each other just a few seconds before extraction. A substantial amount of granular material was associated with the tip of the fused filopodium (, inset 1 in D). The presence of tip-associated material may play an important role in filopodial formation, and we therefore investigated it in more detail.
Filopodial tip complex
In EM images, many filopodial tips were associated with a distinct structure, which had a rough granular surface and variable shape and size ( A). To test whether this tip complex was involved in physical association of filopodial barbed ends with each other, we incubated lysed cells overnight in phalloidin-containing buffer. Phalloidin prevented depolymerization of actin filaments during incubation, whereas dissociation of other proteins was allowed. We monitored dissociation of fascin or VASP using cells expressing GFP-fusion proteins. Although lysis removed soluble and weakly bound proteins, lysed cells initially retained most of filopodia-associated fascin and VASP. However, fascin was completely gone after incubation, whereas VASP remained ( B). EM of incubated cells revealed that filopodial bundles became loose, consistent with the loss of fascin, but the tip complexes were mostly preserved, consistent with retention of VASP, and filament barbed ends remained associated with each other and with the filopodial tip complex ( C). These results suggest that the tip complex physically links barbed ends in filopodia independently of fascin. Occasionally, the tip complex detached partially or completely during incubation. In such cases, released filopodial filaments completely splayed apart.
Figure 8. Filopodial tip complex. (A) Structure of tip complex in three filopodia seen by replica EM. Lower magnification, left; higher magnification, right. (B) Incubation of lysed cells in buffer causes dissociation of fascin (top) but not VASP (bottom) (more ...)
The next question we addressed was the origin of the tip complex. Because filopodia formation seemed to occur by gradual convergence of filaments from the dendritic network, we wondered whether smaller tip complexes existed in lamellipodia. Careful examination of the leading edge of lamellipodia indeed revealed junctions between barbed ends of two or more lamellipodial filaments ( D). Occasionally, additional material could be seen at the junction points, which may correspond to the tip complex of established filopodia. Association between filament-barbed ends was retained even after overnight incubation in the phalloidin-containing buffer ( D).