W.M. Keck Dynamic Image Analysis Facility, Department of Biological Sciences, The University of Iowa, Iowa City, IA 52242, USA
* Author for correspondence (e-mail: david-soll{at}uiowa.edu)
Accepted 21 February 2005
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Summary |
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Key words: 3D-DIAS reconstruction, Cell migration, Filopodia, Myosin II heavy chain phosphorylation, Dictyostelium discoideum
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Introduction |
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To study the role filopodia play in locomotion and chemotaxis, we developed a computer-assisted dynamic image analysis system, DIAS 4.0, which provides 3D reconstructions of the cell surface, nucleus, pseudopodia and filopodia of migrating cells at 4-second intervals. Using this new technology, we analyzed the formation of filopodia by Dictyostelium amoebae migrating in buffer, moving in a directed fashion up a spatial gradient of chemoattractant towards an aggregation stream and responding to the rapid addition of chemoattractant (Soll et al., 2002). Through mutant analysis, we then tested whether myosin II heavy chain (MHC) phosphorylation-dephosphorylation plays a role in filopod formation, as it had been demonstrated to play a role in cell locomotion, responsiveness to chemotactic signals and, most importantly, pseudopod formation (Stites et al., 1998
; Heid et al., 2004
). The three mutant cell lines were HS1, an MHC-null mutant (Manstein et al., 1989
), 3XALA, in which the three threonine phosphorylation sites of MHC are replaced with alanine so that it mimics the constitutively unphosphorylated state (Egelhoff et al., 1993
; Egelhoff et al., 1996
), and 3XASP, in which the three threonine phosphorylation sites of MHC are replaced with aspartate residues so that it mimics the constitutively phosphorylated state (Egelhoff et al., 1993
). Our results provide insights into the origin, stability, dynamics and possible function of filopodia, demonstrate that receptor occupancy regulates filopod formation and retraction, and indicate that MHC phosphorylation-dephosphorylation plays a role in the regulation of filopod formation.
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Materials and Methods |
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For experimental purposes, JH10 cells were grown in liquid HL-5 medium (Cocucci and Sussman, 1970) supplemented with 100 µg/ml thymidine. All other strains were grown first in HL-5 medium plus 10 µg/ml G418 for selection and then in HL-5 medium lacking the drug for experimental purposes. Cells were harvested from plates at the confluent monolayer stage, washed with buffered salts solution (Sussman, 1987
) and distributed on filter pads saturated with buffered salts solution, as previously described (Heid et al., 2004
). Cells were then washed from pads with buffered salts solution at the ripple stage, which represents the onset of aggregation (Soll, 1979
) and the time at which mutant and wild-type cells achieved maximum velocity (Varnum et al., 1986
; Heid et al., 2004
). For analysis in buffer in the absence of chemoattractant, cells were distributed on the glass wall of a Sykes-Moore perfusion chamber (Bellco Glass, Vineland, NJ). After 5 minutes of incubation, the chamber was perfused with buffered salts solution at a rate that turned over one equivalent chamber volume every 15 seconds (Falk et al., 2003
; Wessels et al., 1989
; Zhang et al., 2002
). Perfusion ensured that cells did not condition their microenvironment with the chemoattractant cAMP. A modification of a previously described aggregation chamber (Wessels et al., 1992
) was used for the analysis of cells in response to a gradient of chemoattractant released from an aggregation stream. In brief, a hole 17.5 mm in diameter was drilled through the top of a 35 mm plastic Petri dish. The hole was then covered with plastic wrap and sealed with a ring of petroleum jelly followed by a ring of glue. The plastic wrap provided a clear surface suitable for short working distance imaging with high resolution DIC optics. An aliquot of 1.4x106 cells in 0.5 ml BSS was deposited on the plastic wrap and incubated for 30 minutes to allow adherence. An aliquot of 1.5 ml buffered salts solution was then added to the chamber to prevent dehydration during image capture. For responses to the rapid addition of 106 M cAMP, cells were distributed on the wall of a Dvorak-Stotler chamber (Lucas-Highland, Chantilly, VA) and perfused with buffer alone for 5 minutes. Perfusion solution was then switched to buffer containing 106 M cAMP, as previously described (Wessels et al., 1989
).
3D reconstruction
Cells were imaged through a 63x oil immersion objective (NA 1.4) of a Zeiss ICM405 inverted microscope equipped with DIC optics. The shaft of the focusing knob was attached to an Empire Magnetics (Rohret, CA) IN-23P stepping motor. A Burst Electronics (Corrales, NM) MCG-2 micro character generator was inserted into the video path of a Hitachi KP-M3AN CCD camera to provide synchronization (up/down/pause) information corresponding to motor position. Both motor and character generator were controlled by a customized program written in Linux on a dedicated Intel platform. Only the up-scans were used for reconstructing cells. 60 sections were obtained through the z-axis, beginning at the substratum, in a 2-second period. The distance between sections was 0.33 µm, and the total distance for the set of sections 20 µm. The time it took to return to the substratum was 2 seconds. The sectioning process was repeated every 4 seconds. A Sony DV-G1000 digital video recorder was used to convert the live analog camera signal to digital video captured directly onto an iMac using iMovie. The iMovie capture screen allowed the experimenter to adjust the quality of the image and lighting via direct feedback. 1 hour of recording required approximately 15 GB of hard disk space. A customized program then converted the iMovie video to a QuickTime movie, which in turn was connected to a DIAS 4.0 movie (Soll, 1995; Soll and Voss, 1998
; Soll et al., 2000
; Heid et al., 2002
), retaining only the required up-scans. The resulting movies were transferred through a 100 base-T network to a high-performance Windows XP machine equipped with a 3.2 GHz Pentium 4 processor for DIAS reconstruction. Video compression was avoided because of the very fine nature of the filopodia. DIAS 4.0, the successor to the Macintosh-based 3D-DIAS program (Soll, 1995
; Soll and Voss, 1998
; Soll et al., 2000
), was used for dynamic 3D reconstruction. DIAS 4.0 is written in the Java programming language allowing multi-platform support, automatic memory management and the powerful `Swing' graphical user interface. Sun Microsystems JVM 1.4 and IBM WebSphere implementations of the Java virtual machine provided the necessary speed. The Java interface allowed `drag-and-drop', `on-the-fly' processing by simply dragging a movie into a `processing station'. As the diameters of filopodia were less than 0.1 µm, and because of their length and complex 3D trajectory, automated outlining did not accurately trace filopodia. Therefore, the cell surface, filpodia and cell compartments (nucleus, pseudopodia) were manually outlined with DIAS software. The in-focus edge of the cell body containing particulate cytoplasm, the regions of the cell containing non-particulate cytoplasm (pseudopodial regions), the nucleus, filopodia emanating from the general cell body and pseudopodia, and filopodia (tail fibers) emanating from the uropod, were color-coded green, blue, purple, red and yellow, respectively. The first 12 outlined optical sections through 3.67 µm in the z-axis of a representative cell under reconstruction are presented in Fig. 1A. The traces of cell body (green), pseudopodia (blue) and nucleus (red) in each optical section were placed in individual `trace slots'. The cell body, pseudopodia and nucleus were traced as closed curves (Fig. 1A). Each closed outline was converted to a beta-spline representation (Barsky, 1988
) and the result cleaned of spurious pixels. Filopodia, on the other hand, were traced as a series of short line segments in each optical section (Fig. 1A). Outlines of in-focus filopodial line segments are noted with arrows in select optical sections in Fig. 1A. Dilation was used to thicken the filopodial segments to achieve continuity and erosion was used to restore the outline to a more realistic width. Filopodia formed in pseudopodia and the general cell body, other than the uropod, were color-coded red, whereas filopodia from the uropod (`tail fibers') were color-coded yellow.
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Results |
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The representative cell in Fig. 3, and four additional cells analyzed in the same fashion (data not shown) turned into an adjacent stream by forming a lateral pseudopod towards the stream and turning into it. For each cell, the temporal and spatial dynamics of lateral pseudopod and filopodia could be separated into three phases, as will be demonstrated for pseudopod c in Fig. 4. The phases were of similar duration for the five analyzed cells.
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Phase two
In the second phase (20-40 seconds), pseudopod c did not extend along the substratum or retract towards the stream, but it did continue to change shape (Fig. 3A,B). At 40 seconds, it transiently fragmented into two portions (Fig. 4). During phase two, the number of filopodia remained at roughly six per pseudopod (Fig. 5A). Through phase two, pseudopod c and its filopodia remained in contact with the substratum (Fig. 4).
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Behavior in the three delineated phases suggests that a cell may make a decision, probably in the second phase, to either extend a lateral pseudopod and turn into it, or to retract it back into the cell body. In that putative decision-making phase, the majority of filopodia emanating from the pseudopod contact the substratum. If phase two indeed involves that decision, then when a pseudopod is formed in the wrong direction (i.e. away from the stream), it should undergo the first two phases in a manner similar to a pseudopod formed in the correct direction, but then retract. It was of interest to know the behavior of associated filopodia in retracting pseudopods. We, therefore, reconstructed pseudopodia formed at a 90° to 180° angle to the direction of the deduced chemotactic gradient (i.e. towards the stream). In Fig. 6, we have reconstructed a frontal view of a pseudopod that forms and then retracts, in this case pseudopod b of the cell reconstructed in Figs 3 and 4. This pseudopod had formed at a 90° angle to the deduced chemotactic gradient emanating from the stream. It grew to maximum size between 0 and 16 seconds (phase one), remained relatively constant between 16 and 36 seconds (phase two), and then was retracted back into the main cell body between 36 and 60 seconds (phase three) (Fig. 6). Clearly this pseudopod underwent an initial phase of pseudopod growth, then a second phase involving shape changes without growth, like a pseudopod that formed in the correct direction. However, in the third phase, the pseudopod was retracted, in marked contrast to a pseudopod that had formed in the correct direction. The majority of filopodia that extended from the pseudopod in phase two contacted the substratum, as was the case for pseudopodia formed in the correct direction. During pseudopod retraction, however, filopodia were retained and remained in contact with the substratum (Fig. 6), in contrast to the loss of filopodia during pseudopod extension in the correct direction. Similar filopodial dynamics were observed for three additional pseudopodia formed in the wrong direction by cells aligned with a stream.
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Chemoattractant regulates filopod formation
A comparison of the representative cell migrating in buffer in Fig. 2 and the cell responding to a spatial gradient of chemoattractant in Fig. 3 suggests that the latter possess more filopodia than the former. Indeed, the mean number of filopodia on cells in buffer (5.1±1.6; n=24) was less than half that in a spatial gradient of chemoattractant (11.2±3.1; n=10). Pseudopodia that formed in buffer did so in a more continuous fashion than those formed by cells in spatial gradients of chemoattractant, with little indication of three phases. These results suggest that filopod formation is regulated by the chemotactic receptor and are consistent with earlier reports that cells extend increased numbers of filopodia when treated with the chemoattractant cAMP (Kobilinsky et al., 1976; De Chastellier and Ryter, 1980
; Choi and Siu, 1987
) or, in the case of vegetative cells, folic acid (Rifkin and Isik, 1984
). To analyze this response at the single cell level, cells were perfused in buffer for 5 minutes and then in buffer containing 106 M cAMP, the concentration of chemoattractant attained at the peak of the natural chemotactic wave (Tomchik and Devreotes, 1981
). This treatment results in a rapid receptor-mediated response within 25 seconds that has been demonstrated to include doubling of total F-actin, dismantling of F-actin enriched pseudopodia and a dramatic increase in F-actin and myosin II in the cell cortex, a dramatic reduction in the rate of cellular translocation, a dramatic decrease in cytoplasmic particle movement, rounding up of the cell body and blebbing from the cell surface (Hall et al., 1989
; Wessels et al., 1989
; Levi et al., 2002
). After 2 minutes of perfusion with 106 M cAMP, cells partially adapt by reforming pseudopodia, but they remain relatively apolar, extend pseudopodia in random directions and do not translocate in a persistent fashion in any one direction (Wessels et al., 1989
).
Within 25 seconds of addition of 106 M cAMP, cells dismantled pseudopodia, formed blebs around their periphery (color-coded purple) and retracted the great majority of their filopodia (Fig. 7A,B). After 4 minutes in 106 M cAMP, cells had partially adapted. They had stopped blebbing, extended pseudopodia again and reformed filopodia, remained relatively apolar and did not translocate in a persistent fashion (Fig. 7C). Although cells migrating in buffer possessed 5.1±1.6 (n=24) filopodia per cell, cells that had been treated with 106 M cAMP for 25 seconds possessed 0.3±1.0 (n=20) filopodia per cell, a 17-fold reduction. After 4 minutes in 106 M cAMP, cells had reformed filopodia, averaging 10±3 (n=20) per cell, twice the average number before treatment. Hence, the rapid addition of 106 M cAMP first caused filopod retraction, which occurred in parallel with the dismantling of pseudopodia and transient blebbing, and then overproduction of filopodia, which coincided with the reformation of pseudopodia. These results support the conclusion that filopodia formation is regulated by the chemotactic signal.
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Myosin phosphorylation regulates filopod formation
It was recently demonstrated (Heid et al., 2004) that the phosphorylation-dephosphorylation of MHC plays a role in the stability of the anterior pseudopod, the regulation of lateral pseudopod formation, the efficiency of chemotaxis in a spatial gradient of cAMP and the capacity to enter aggregation streams. We, therefore, tested whether it played a role in filopod formation by analyzing the mutants, HS1, (Manstein et al., 1989
), 3XALA, which contains mutant MHC that mimics the constitutively unphosphorylated state (Egelhoff et al., 1993
; Egelhoff et al., 1996
), and 3XASP, which contains mutant MHC that mimics the constitutively phosphorylated state (Egelhoff et al., 1993
). We first compared the total number of filopodia on migrating control (JH10, HS1-rescue) and mutant cells perfused with buffer in the absence of chemoattractant. The mean number of filopodia per cell (±s.d.) for JH10, the control for HS1 and for the HS1-rescue, the control for 3XALA and 3XASP, was 5.1±1.6 and 5.2±0.9, respectively (Fig. 8A). The number per cell for the mutant HS1 was similar (5.1±3.3), but the s.d. was twice that of control cells, reflecting far greater variability, as is evident in the histogram in Fig. 8A. The number of filopodia per cell in the mutant 3XALA, however, was 1.2±1.1, approximately one-quarter that of control cells, whereas that of the mutant 3XASP was 11.0±3.4, more than twice that of control cells (Fig. 8A). An analysis of the number of filopodia per pseudopod revealed that the mean for 3XALA cells was significantly lower and the mean for 3XASP cells significantly higher than that for control cells (Fig. 8B). An analysis of lengths further revealed that filopodia that formed on 3XALA cells were significantly shorter than those of control cells HS1-rescue, 5.4±3.3 µm (n=22); 3XALA, 3.1±1.8 µm (n=23); P=0.002). These results were confirmed qualitatively in multiple clones of control and mutant strains.
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Discussion |
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The general characteristics of filopod formation
Our results demonstrate that for cells migrating in buffer or turning in a spatial gradient of chemoattractant late in the aggregation process, the majority of filopodia originate from pseudopodia, which is consistent with previous findings that filopodia arise from pre-existing F-actin networks (Small et al., 1998; Svitkina et al., 2003
). A small minority of filopodia do originate on the cell body of Dictyostelium, but more commonly filopodia on the cell body originate on pseudopodia and relocate to the cell body through pseudopod retraction or extension. Our results also indicate that filopodia located on the uropod (tail fibers) are more stable and on average longer than filopodia located on pseudopodia and the rest of the cell body, and are replenished from filopodia that relocate from pseudopodia to the cell body. Different types of dendritic filopodia have been reported (Portera-Cailliau et al., 2003
), supporting the view that filopodia on the same cell can exhibit different characteristics and hence, may serve different functions.
Filopod dynamics and function during chemotaxis
Although filopodia formed during migration in buffer and in spatial gradients of chemoattractant exhibit similar general characteristics, the former have on average half the number of filopodia as the latter, supporting previous observations that the addition of chemoattractant to Dictyostelium causes an increase in filopodia (Kolilinsky et al., 1976; De Chastellier and Ryter, 1980; Choi and Siu, 1987
; Rifkin and Isik, 1984
). To characterize filopod dynamics during chemotaxis, we first analyzed cells turning towards multicellular aggregation streams through biased extension of a lateral pseudopod. Reconstructions of cells over time revealed that these turns were not mediated through physical contact of filopodia with cells in the stream, which appears to be the case when cells have entered the stream and are forming end-to-end contacts mediated by the adhesin gp80 (Choi and Siu, 1987
). These biased turns were mediated, instead, through assessment of the chemotactic signal released by the multicellular aggregation stream, which we assume is in the form of a spatial gradient. We distinguished three phases of lateral pseudopod behavior associated with a biased turn toward a stream, each phase approximately 20 seconds in duration. In phase one, the lateral pseudopod evaginates and grows to a relatively blunt protrusion filled with non-particulate cytoplasm. At the end of phase one, the pseudopod is in contact with the substratum. In phase two, the pseudopod continually changes shape, but does not extend towards the stream. Throughout phase two, the majority of filopodia remain in contact with the substratum. In phase three, the pseudopod expands towards the stream, drawing particulate cytoplasm into its proximal end. During lateral pseudopod extension in phase three, the number of filopodia on the pseudopod decreases dramatically. New filopodia that form on the extending pseudopod, do so transiently and do not contact the substratum. If a pseudopod forms on the flank of a cell away from a stream (i.e. in the wrong direction), it progresses through two behavioral phases similar to phase one and phase two of pseudopodia formed towards a stream (the right direction). However, in phase three, pseudopodia formed in the wrong direction are retracted back into the cell body. During retraction, a pseudopod retains its filopodia, which continue to contact the substratum. This is opposite the dynamics of filopodia on pseudopodia formed towards a stream. This sequence of behaviors suggests that during phase two, a cell decides whether it will extend the lateral pseudopod towards a stream or retract it. During this putative decision-making period, while the pseudopod continues to change shape, the majority of filopodia remain in contact with the substratum. When the decision is made to extend up the spatial gradient of chemoattractant, the number of filopodia on the pseudopod decreases dramatically, but when it makes a decision to retract, filopodia remain attached to the substratum. This sequence of events suggests that filopodia may play a role in repressing pseudopod extension and turning through interactions with the substratum.
The three phases displayed by lateral pseudopodia in chemotactic gradients rarely accompanied lateral pseudopod formation in buffer. Together with the observation that cells migrating in buffer possess on average half as many filopodia as cells undergoing chemotaxis, our results demonstrate that the natural chemotactic signal regulates filopod formation. Consistent with this suggestion, we found that the rapid addition of a high concentration of chemoattractant (106 M cAMP) to cells in buffer, in addition to inducing the transient retraction of pseudopodia, blebbing and apolarity (Wessels et al., 1988), also induces transient retraction of almost all filopodia, followed by reformation of filopodia at twice their original density during adaptation.
MHC phosphorylation-dephosphorylation regulates filopod formation
Both 3XALA cells, containing MHC that constitutively mimics the unphosphorylated state, and 3XASP cells, containing MHC that constitutively mimics the phosphorylated state, formed filopodia. However, although the former mutant possessed half as many filopodia, on average, as control cells, the latter possessed twice as many. Hence, the acts of phosphorylation or dephosphorylation of MHC are not essential for filopod formation. Rather, the state of MHC, which affects myosin II polymerization and the level of cortical tension, regulates the number of filopodia, suggesting that a balance of unphosphorylated and phosphorylated MHC in a cell must fine-tune the number of filopodia formed by pseudopodia. In addition, we show for the first time that unphosphorylated myosin is essential for the receptor-mediated rapid response of Dictyostelium amoebae to cAMP, which includes the retraction of almost all filopodia followed by re-formation at twice the original number. Together, these results suggest that the formation and retraction of filopodia associated with chemotaxis may be regulated by receptor-mediated changes in the phosphorylation-dephosphorylation of MHC.
Interestingly, 3XASP cells, which formed more than twice as many filopodia as control or 3XALA cells, neither retracted filopodia nor formed blebs as did control and 3XALA cells in response to the rapid addition of 106 M cAMP. These results suggest that cells must have myosin II-mediated cortical tension in order to retract filopodia and bleb. The latter two events may in turn represent a dependent sequence. Blebs may arise as a result of excess plasma membrane resulting from the rapid retraction of the F-actin cores of filopodia. Consistent with these observations, a high-throughput screening assay identified the small molecule blebbistatin, which blocked non-muscle myosin II-dependent processes including blebbing (hence its name) in vertebrate cells, implicating myosin II function in the blebbing process (Straight et al., 2003).
Other genes involved in filopod formation in Dictyostelium
Mutants of other genes implicated in the regulation of chemotaxis also exhibit defects in filopod formation. Dictyostelium null mutants of VASP exhibited a dramatic decease in filopod formation as well as a dramatic decrease in chemotactic efficiency (Han et al., 2002). Ddvasp mutant cells were far less capable of maintaining pseudopodia on the substratum in a gradient of chemoattractant (Han et al., 2003), which is consistent with a role for filopodia in stabilizing pseudopodia on a surface. Furthermore, there is evidence that the Rac1 GTPases regulate filopodia (Dumantier et al., 2000). Expression of constitutively activated Rac1A resulted in a dominant-negative effect on filopod formation (i.e. no filopodia), whereas expression of constitutively inactivated Rac1A resulted in the formation of abnormally greater numbers of short filopodia. The former mutant exhibited both motility and development defects, consistent with a defect in chemotaxis. Mutants of two other genes have also been demonstrated to affect filopod formation. The rasG mutant forms excess filopodia (Tuxworth et al., 1997
), whereas transformation of wild-type cells with a constitutively activated form of RasG reduces the number of filopodia (Chen and Katz, 2000
). Null mutants of two genes related to the mammalian phosphotidylinositide 3-kinases, ddpik1/ddpik2, also form excessive filopodia, although these mutants have been demonstrated to perform chemotaxis normally in a spatial gradient of cAMP (Buczynski et al., 1997
). Myosin VII has also been found in filopodia (Tuxworth et al., 2001
). It would appear that a number of different genes implicated in cell motility and chemotaxis play a role in the regulation of filopodia.
The results presented in this study demonstrate that filopodia originate on pseudopodia, are regulated by the chemotactic signal and appear to play a role in the decision by a cell to extend a lateral pseudopod and turn up a spatial gradient of chemoattractant. Their interactions with the substratum suggest that they play a role in stabilizing pseudopodia during the decision-making phase of lateral pseudopod formation, although we have in no way excluded other potential roles. The filopod reconstruction software we describe here for the first time should aid in elucidating the exact role played by filopodia through future analyses of chemotaxis mutants.
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Acknowledgments |
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