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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Research Article
Collective cell migration of primary zebrafish keratocytes Jose L. Rapanana, Kimbal E. Coopera, Kathryn J. Leyvab, Elizabeth E. Hulla,n a
Biomedical Sciences Program, College of Health Sciences, Midwestern University, Glendale, AZ 85308, United States Department of Microbiology & Immunology, Arizona College of Osteopathic Medicine, Midwestern University, Glendale, AZ 85308, United States b
article information
abstract
Article Chronology:
Fish keratocytes are an established model in single cell motility but little is known about their
Received 26 November 2013
collective migration. Initially, sheets migrate from the scale at 145 μm/h but over the course of
Received in revised form
24 h the rate of leading edge advance decreases to 23 μm/h. During this period, leader cells
14 June 2014
retain their ability to migrate rapidly when released from the sheet and follower cell area
Accepted 17 June 2014
increases. After the addition of RGD peptide, leader cell lamellae are lost, altering migratory
Available online 26 June 2014
forces within the sheet, resulting in rapid retraction. Leader and follower cell states interconvert
Keywords:
within minutes with changes in cell–cell adhesions. Leader cells migrate as single cells when they
Collective cell migration
detach from the leading edge and single cells appear to become leader cells if they rejoin the
Rho associated kinase
sheet. Follower cells rapidly establish leader cell morphology during closing of holes formed
Fish keratocyte
during sheet expansion and revert to follower cell morphology after hole-closure. Inhibition of
Primary culture
Rho associated kinase releases leader cells and halts advancement of the leading edge suggesting an important role for the intercellular actomyosin cable at the leading edge. In addition, the presence of the stationary scale orients direction of sheet migration which is characterized by a more uniform advance of the leading edge than in some cell line systems. These data establish fish keratocyte explant cultures as a collective cell migration system and suggest that cell–cell interactions determine the role of keratocytes within the migrating sheet. & 2014 Elsevier Inc. All rights reserved.
Introduction Zebrafish keratocytes rapidly and collectively migrate when established in explant cultures. Although the mechanisms of motility of keratocytes as single cells have been extensively studied [1–6], little is known about the mechanisms which direct the migration of keratocytes as cell sheets. This experimental system is particularly compelling in vitro model for collective cell migration during reepithelialization in response to wounding for several reasons. First, establishment of explant cultures promptly initiates an n
Corresponding author.
http://dx.doi.org/10.1016/j.yexcr.2014.06.011 0014-4827/& 2014 Elsevier Inc. All rights reserved.
epithelial to mesenchymal transition (EMT), which rapidly progresses over the first seven days of explant culture as assessed by changes in gene expression including a switch from E-cadherin to N-cadherin expression, morphological changes, and cytoskeletal rearrangement [7,8]. As changes in gene expression consistent with inflammation and wound healing occur concurrently with EMT, this system has been characterized as a wound healing model [7]. Second, as explant cultures, keratocytes are not transformed and have not undergone the changes in gene expression associated with transformation or passage of primary cultures [9–13]. Thus, the
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Video 1 Migration of keratocytes from underneath a stationary scale in an explant established 8 h prior to filming. The elapsed time is 5 h with frames taken 5 min apart. A video clip is available online. Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.yexcr.2014.06.011.
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model allows rapid collective cell motility assays (complete within 24 h of initial treatment) in untransformed cells in the context of a defined wound healing process. During the re-epithelialization stage of wound healing, large sheets of epithelial cells collectively migrate to reestablish the intact epidermis. Key to this process are the alterations in quiescent, pre-wound epithelial cells to form a polarized sheet with the formation of leader cells at the wound edge (reviewed in [14]) and an epithelial to mesenchymal transition which typically promotes motility. Although the common hallmarks of collective cell migration have been established, including the presence of leader and follower cells and intact cell junctions, many of the mechanisms guiding collective cell migration are yet to be determined. Due to its compelling characteristics, including rapid motility assays, the zebrafish keratocyte system has promise to shed light on mechanisms of collective cell migration. The data presented here establish the migration of zebrafish keratocyte cell sheets as a collective cell migration system through 1) characterization of distinct leader and follower cells in 24 h sheets and 2) demonstration of the presence of cell–cell junctions that coordinate an actomyosin cable between adjacent
Fig. 1 – Actin, tubulin, vinculin, and phosphomyosin light chain in single, leader, and follower keratocytes. Untreated 24 h explant cultures were fixed and stained with primary antibody to tubulin, vinculin, or phosphorylated myosin light chain (red) and counterstained with phalloidin (green), and DAPI (blue). Vinculin localization in follower cells is presented without phalloidin counterstain as display of actin obscures vinculin localization. Single cells resemble cells at the leading edge while follower cells (photographed in regions with a single cell layer) are not polarized. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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cells which is necessary for advance of the leading edge of the sheet. Next, the mechanism of collective cell migration is investigated using soluble RGD peptides to disrupt adhesion formation and Rho associated kinase inhibition to alter actomyosin contractility. The data suggest that cell–cell interactions determine the role of keratocytes within the sheet and that changes in forces acting on the advancing sheet result in a slowing of migration.
Results Keratocyte explants are established when scales removed from anesthetized zebrafish are incubated in culture medium. Within minutes, epithelial cells that were removed along with the scale rapidly migrate collectively from underneath the scale, while the scale itself does not move relative to the substrata. An example
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of this collective migration is shown in Video 1. The video demonstrates how cells migrate away from the adherent scale as a collective unit, or sheet. Also note that cells do break away from the sheet periodically; in the initial frames, an individual cell that has broken away from the leading edge of the sheet can be observed migrating rapidly out of the frame of the video. This explant was established 8 h prior to filming but similar collective migration is observed in cultures from the initial emergence of the cell sheet approximately 1 h after removal of the scale until the ongoing EMT process leads to fragmentation of the sheet [7]. To establish the zebrafish keratocyte system as a model for collective cell migration, we first used fluorescence microscopy to characterize the leader and follower cells within the keratocyte sheet. As shown in Fig. 1, there are distinct differences between leader and follower cells after 24 h in explant culture. Leader cells resemble single cells in that they are polarized as shown by the
Fig. 2 – Leader and follower localization of E-cadherin and phosphorylated forms of E-cadherin and β-catenin. Untreated 24 h explant cultures were fixed and stained with primary antibody to unphosphorylated E-cadherin (green), E-cadherin phosphorylated on serines 838 and 840 (red), and β-catenin phosphorylated on tyrosine 654 (red) and counterstained with DAPI (blue). Evidence for disassembly of cell junctions is seen in the large amount of intracellular staining with anti-phosphorylated E-cadherin and an increased intranuclear staining with anti-phosphorylated β-catenin. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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presence of an extensive actin-rich lamella and an asymmetric distribution of β-tubulin around the nucleus. In contrast, follower cells are not polarized, maintain cell–cell junctions on all sides, and exhibit a cortical actin cytoskeleton. In addition, leader cells have adhesions that label with vinculin and contain contractile cables perpendicular to the direction of motion similar to single cells. Evidence for actomyosin contractility is seen in the presence of phosphorylated myosin light chain (pMLC) which co-localizes with actin cables that are coupled via cell–cell junctions in adjacent leader cells. Interestingly, the pattern of pMLC in follower cells is variable and is sometimes localized at (or near) the cortical actin cytoskeleton but with no staining seen in other regions. Unlike single cells, leader cells retain E-cadherin and β-catenin cell–cell junctions and these junctions appear to link the actin cable in adjacent cells (Fig. 2). Although a polyclonal antibody to E-cadherin yields diffuse staining within the leader cells, this staining appears absent in the lamella while the same antibody localizes Ecadherin to cell–cell junctions in follower cells. Given that the cell sheets are undergoing an epithelial to mesenchymal transition with consequent down-regulation of E-cadherin [7], we next asked if part of the diffuse staining was due to ongoing disassembly of cell–cell junctions. We used an anti-phospho-E-cadherin antibody (pSer838 and pSer840 in human) as phosphorylation at multiple sites in this region of E-cadherin which is associated with altered cell–cell adhesion and intracellular localization [15–18]. Staining with this antibody yields reduced localization at cell–cell junctions (Fig. 2). In leader cells, there is a large amount of staining in what appears to be
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vesicular structures inside the cell. This pattern is replicated, to a lesser extent, in follower cells. Consistent with this implied disassembly of cell–cell junctions, the phosphorylated form of β-catenin (pTyr654) associated with altered cell adhesion [19] localizes in the nucleus of leader cells but in the cell–cell junctions of follower cells. As shown in Fig. 3A, during explant culture, the rate of advance of the leading edge slows from 143.872.9 μm/h (seen between 1 and 2 h) to 23.570.76 μm/h (seen between 24 and 25 To investigate this slowing rate, we asked if leader cells lost their ability to rapidly migrate over time by disrupting the cell–cell junctions with a brief, mild EDTA treatment. This treatment does not visibly affect the sheet but, after incubation with complete media for 30 to 60 min, increased numbers of leader cells are seen breaking away from the sheet. As shown in Fig. 3B, leader cells released from the sheet migrate at rates typically seen in single cells that naturally break away from the sheet (425.8731.2 vs 433.7724.1 μm/h). Thus, without adhesion to the cell sheet, cells retain their ability to migrate rapidly and, when released from the slower moving follower cells, slowly moving leader cells migrate rapidly as single cells. As the sheet moves forward, we hypothesized that follower cell spreading might result. We focused on follower cell spreading as measured by changes in internuclear distance and cell area comparing 4 h sheets, the earliest time point which we could reproducibly fix and stain, and 24 h cultures. As shown in Fig. 3, there is 1.8 fold increase in internuclear distance (Fig. 3C) and 3.1 fold increase in cell area (Fig. 3D).
Fig. 3 – Mechanisms of keratocyte sheet migration. (A) Rate of leading edge advance decreases with time in explant culture (p¼3.5 10 25). (B) Leading edge cells released by disruption of cell–cell junctions with mild EDTA treatment migrate at rates equivalent to single cells (p¼1.9 10 12). (C) Internuclear distance increases approximately 1.8 fold between 4 and 24 h of culture (p¼ 3.3 10 5). (D) Follower cell area increases 3.1 fold between 4 and 24 h of culture (p¼ 3.4 10 6). Each graph represents three independent experiments in triplicate with error bars as SEM.
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Fig. 4 – Retraction of sheet with addition of soluble RGD peptide. Addition of a soluble RGD peptide to 4 h cultures (A and D) leads to a small retraction and loss of lamellae (B and E) within 10 s and an extensive retraction of the sheet by 40 s (C). The average area of three sheets treated with RGD peptide, compared to sheets treated with control peptide, is plotted in F, showing no effect of control peptide.
Fig. 5 – Conversions of individual, leader, and follower keratocytes. In untreated, 24 h sheets, individual to leader and leader to individual conversions occur with the breaking or forming of cell–cell adhesions. During the closure of holes formed within the sheet, follower cells become leaders. Fluorescence microscopy demonstrates the presence of actin rich lamellae (green) during hole closure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Evidence for changes in forces within the sheet is seen in the rapid recoil of the entire sheet after the addition of soluble peptides containing an RGD sequence to inhibit adhesion
formation in 4 h cultures (Fig. 4). Panel A shows the entire untreated sheet after 4 h of culture with Panel D showing an enlarged area of that same sheet, illustrating the extensive
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Video 2 Dynamics of hole formation and closure in an explant established 24 h prior to filming. A hole in the sheet is visible in the initial frames near the indentation of the leading edge, which begins to close shortly after the start of filming. As this hole closes, another hole begins to open (left of the closing hole). The elapsed time is 90 min with frames taken 1 min apart. A video clip is available online. Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.yexcr. 2014.06.011.
lamellae present at the leading edge. Ten seconds after the addition of soluble RGD peptide, there is a slight retraction of the leading edge (Panel B) and these leader cells have lost their lamellae (Panel E). Twenty seconds after addition of the peptide, the sheet is completely retracted and folded adjacent to the scale (Panels C and F). No response is seen with the addition of control peptide, as indicated by no change in cell sheet areas (Panel F). At 24 h, a similar but less dramatic recoil is observed and has also been reported elsewhere [20]. Although infrequent, spontaneous dissociations of leader cells to become single migrating cells are observed. As shown in Fig. 5 (top panel), leader cells can be seen breaking away from the sheet within minutes. Similarly, individually migrating cells in the culture may re-join the cell sheet (middle panel) and become morphologically indistinguishable from leader cells. The rate of migration of spontaneously formed single cells is comparable to those seen with brief, mild EDTA treatment (Fig. 3B). As migration depends on adhesion strength in a variety of experimental and theoretical studies [21–24], these data alleviated concerns that the use of mild EDTA treatment to release cells from the sheet might weaken adhesion and increase cell speed. An interesting example of follower to leader conversion is seen in holes that form during sheet migration (Fig. 5, bottom panel). As holes form and enlarge, cells lining the perimeter of the hole retain follower cell morphology. However, as holes begin to close, these cells lining the hole develop lamellae characteristic of leader cells, a morphological transformation that is confirmed by fluorescent visualization of the actin cytoskeleton. Once cell edges meet, after a variable period of time, cells regain the morphology consistent with those of follower cells. As shown in Video 2, holes tend to form in areas of the sheet that contain spreading follower cells, in this case between the leading edge and an area containing multiple cell layers adjacent to the scale. During the course of the video, the initial hole, near the
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Fig. 6 – Treatment with Y-27632 disrupts actomyosin contractility. (A) Evidence for intercellular actomyosin contractility can be seen in the presence of a coordinated actin cable (green) which co-localizes with pMLC (red). (B) After treatment, both actin and pMLC relocalize and the leading edge of the sheet fragments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
indentation of the leading edge, decreases in size while other holes open in adjacent areas (to the left of the original hole). The presence of an actin cable, apparently coordinated in adjacent cells through anchoring at cell–cell junctions and which co-localizes with phosphorylated myosin light chain, is suggestive of actomyosin contractility at the leading edge of the cell sheet (Fig. 6A). As intercellular actomyosin contractility is a defining feature of collective cell migration [25], we inhibited contractility with Y-27632, a Rho-associated kinase (ROCK) inhibitor [26], and assessed sheet migration. Treatment with Y-27632 leads to a redistribution of actin from the intercellular actin cable to newly formed lamellae and cytoplasmic staining of pMLC (Fig. 6B). Treatment of 4 h sheets with 20 mM Y-27632 for 2 h led to an apparent flattening of cells at the leading edge (Fig. 7A) consistent with the loss of intercellular actomyosin contractility. Longer treatments (Fig. 7B and C) fragmented the leading edge of the sheet and caused a slight net retraction of the leading edge, possibly due to dissociation of leader cells (suggested by the increased number of individually migrating cells which apparently detach from the leading edge). During this time, the leading edge of an untreated sheet would normally advance 125 mm (black arrow, Panel C). The presence of single cells which migrate at rates similar to untreated cells (425.8731.2 μm/h vs 399.7727.4 μm/h), suggest that a higher level of contractility is necessary for the advancement of the leading edge of the sheet than migration in individual cells (see Video 3). As suggested by Video 4, the advance of cells within the first few rows of cells at the leading edge of the keratocyte sheet is
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Fig. 7 – Sheet advance halts after treatment with Y-27632 releases leader cells from the sheet. (A) 2 h treatment with 20 lM Y-27632 of 4 h sheets leads to flattening of cells at the leading edge. (B) 3 h treatment leads to release of cells from the leading edge. (C) At 4 h treatment, the leading edge has not advanced when untreated sheets would typically advance 125 lm (black arrow). Cells in the follower region fail to advance between panels B and C (red arrowheads). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
during establishment in explant culture). However, unlike collective cell migration in cell line systems, keratocyte sheets appear to migrate away from a stationary scale. To investigate the effects of the immobile scale on sheet migration, we mechanically detached the scale from the substrata and followed its migration. In the example shown in Fig. 9 and Video 5, part of the sheet was simultaneously separated from the scale. Without attachment to the scale, the new free edges in the cell island (on the right) rapidly adheres to the substrata and those cells adopt the morphology characteristic of leader cells. With leader cells on all edges of this island, the collection of cells ceases to migrate with the rapid directionality characteristic of keratocyte sheets and appears more random. Surprisingly, the keratocytes still attached to the sheet (on the left) are able to move the scale relative to the substrata.
Discussion
Video 3 Effect on sheet migration after treatment with 20 lM ROCK inhibitor Y-27632 in an explant culture established 4 h prior to filming. The elapsed time is 3 h with frames taken 3 min apart. A video clip is available online. Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j. yexcr.2014.06.011.
relatively uniform with most leader cells extending extensive lamellae. This is in contrast to the cellular “fingers” at the leading edge of the sheet seen by others [27,28]. To analyze this motion more closely, we have traced the position of the nuclei in the first 2 to 3 rows of cells at the leading edge, presenting the motion path of these cells (Fig. 8). Our data show that cells near the leading edge follow similar motion paths, suggesting that similar net forces are likely generated in these cells within the observed timeframe. This result suggests that the intercellular actin cable may allow these neighboring cells to join forces to assist in the coordination of movement of the cell sheet, as also demonstrated by Tambe et al. [29]. As with other systems, the free edge of keratocyte sheets migrate to fill empty space created by wounding (or in this case,
Our data provide evidence that the movement of zebrafish keratocyte sheets in explant culture is an example of collective cell migration. Collective cell migration is defined as a group of cells maintaining cell–cell junctions that exhibit directional movement. This type of collective cell migration has been seen in a variety of systems (reviewed in [30,31]). One of the noteworthy characteristics of these systems is a distinction between leader and follower cells [32]. Cells at the leading edge of the sheet exhibit polarity while follower cells are morphologically unpolarized cells. Video 1 demonstrates the coordinated motion of a keratocyte sheet, and examination of this migrating complex shows definite polarity in the leader cells (in a manner resembling individually migrating cells) while this polarization is absent in follower cells. Fish keratocytes are known for their rapid motility and, as single cells, may achieve speeds up to 0.5 μm/sec. Consistent with the usually rapid rate of migration of individual cells, the initial rates of 143.872.9 μm/h in collective migration of keratocyte sheets is substantially faster than 6–60 μm/h typically reported for other systems (reviewed in [25]). Given the well-established relationship between adhesion strength and migration speed [21,23,24], this rapid migration of individual cells may depend on the use of close contacts as cell substratum adhesions which are weaker than fully developed focal adhesions [33]. As a similar distribution of adhesion
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Video 4 Migration of a cell sheet, focused onto several cells within the leading edge, from an explant established 24 h prior to filming (see Fig. 8). The elapsed time is 80 min with frames taken 1 min apart. A video clip is available online. Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.yexcr.2014.06.011.
Fig. 8 – Migration of cells within the leading edge of a 24 h explant. Top: Cells within the leading edge of the sheet have well-developed lamellae and appear to be moving forward as a collective unit. Bottom: Motion paths of cells within the first 2–3 rows at the leading edge were mapped by plotting the change in nuclear position every 10 frames for the length of the video (see Video 4).
proteins (β1 integrin and vinculin) is seen in both leader and individual cells (Fig. 1 and [20,33]), close contacts may be essential part of the rapid migration of keratocyte sheet and may make keratocytes more susceptible to disruption by an RGD peptide than cells with stronger focal adhesions [20]. A unique feature of the keratocyte explant model is the scale, which appears to help provide directionality to the sheet movement. The cell sheet lacks a free edge on the side attached to the scale so that leader cells develop and orient away from the scale. Tambe et al. [29] concluded that the direction of collective cellular migration followed the local orientation of maximal principle stress. Our data show cells within the leading edge all move in the same approximate direction (Fig. 8). This suggests that the cells near the leading edge experience similar net forces in the observed timeframe. Given
the reduced directional persistence of the cell island in Video 5, we suggest that this polarity is an essential part of the rapid motility of keratocyte sheets. Thus, the geometry imposed by the scale may be, at least in part, responsible for coordinating the movement of the keratocyte sheet [27–29]. Further examination of leader cells within the sheet documents extensive lamellae at the free edge. In addition, actomyosin cables are observed behind the lamellae and adherens junctions link these cables between adjacent cells; these adherens junctions do transmit contractile stresses between cells [29,reviewed in 34]. Within a migrating sheet, the collective forces within and between cells must be in balance so that the sheet moves as a unit at a constant velocity. As demonstrated in other systems, cooperative intercellular forces participate in propelling the sheet forward [29,34–38]. In comparison, treatment of a migrating sheet with a Rho kinase inhibitor visibly disrupts the actomyosin cable and causes the release of leader cells from the sheet (Figs. 6 and 7). The net result is a halt of the advance of the keratocyte sheet, thus supporting a role for actomyosin contractility in collective cell migration [38,39]. As suggested by p-E-cadherin and p-β-catenin stainings (Fig. 2), these junctions are apparently preferentially disassembled at the leading edge, a process which continues during explant culture and ultimately leads to the fragmentation of the sheet [7,20]. Given the importance of these junctions in maintaining the intercellular actomyosin cable at the leading edge, we suggest that this disassembly should be interpreted in the context of the ongoing EMT process that is occurring within the explant culture [7]. Our data also support an argument that leader cells may play a more important role in the advancement of the leading edge than the follower cells [39]. When soluble RGD peptide is added to a migrating sheet, the sheet rapidly recoils (Fig. 4A–C), resulting in a dramatic decrease in sheet area within seconds (Fig. 4F). RGD peptide treatment also visibly reduces the lamellae size (Fig. 4D and E). Given the well documented role that lamellar extension plays in migration, these data support the hypothesis that RGD peptide reduces pulling by leader cells, disrupting the steady state and effectively removing a force which opposes the contraction of the actomyosin cable. This may be partly responsible for the rapid recoil of the sheet. In addition, there is an evidence of crosstalk between cell–cell adhesions and integrin-mediated attachments that control cell adhesion strength and motility [40]. Follower cell spreading (Fig. 3C and D) may contribute significantly to the
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Fig. 9 – Migration of two segments of a 24 h cell sheet after mechanical separation. Mechanical separation of the scale from the substrata resulted in a small segment of the sheet still attached to the scale (top panel) and an “island” of cells (bottom panel). Cells still attached to the scale advance, pulling the scale forward. The segment of the sheet separated from the scale forms lamellae on the side previously attached to the scale. The resulting cell “island”, with lamellae along the entire perimeter, exhibits more random movement.
Video 5 Migration of two segments of 24 h cell sheet after mechanical separation (see Fig. 9). Mechanical separation of the sheet was performed just prior to filming. The elapsed time is 215 min with frames taken 5 min apart. A video clip is available online. Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.yexcr.2014.06.011.
The rapid interconversion of keratocytes among leader, follower, and single cells (Fig. 5) suggests that cell interactions are crucial for understanding the role of an individual keratocyte within a cell sheet. Given the speed at which these transitions occur, we suggest that signaling downstream of adhesion formation may be determining the mode of motility each cell assumes as substantial changes in gene expression are unlikely to be involved. An aspect of explant cultures not addressed in this work is the presence of multiple layers of cells in the follower regions. For clarity, the data on follower cells presented here avoids these regions as multiple layers are hard to visualize. The role of a second layer of cells within the follower region is unclear as this second layer is not always present. Sharma et al. [42] have reported that the second layer of cells is in contact with the substratum and, thus cells in the second layer have cell-substratum adhesions necessary to provide traction and stabilize tension within the sheet . Further work is needed to determine the interplay among keratocytes within a multilayer migrating sheet.
Summary expansion of the sheet as spreading has been shown to lead to monolayer expansion [41]. Alternatively, as adhesion is necessary for force generation, the weaker adhesions in the keratocyte sheet [33] may limit the contribution of follower cells to migration. Lastly, it may be possible that follower cell force generation is also sensitive to Rho-associated kinase inhibition. An interesting observation is the decreasing rate of the advance of the leading edge (Fig. 3A). This could represent decreased force generation by the leader cells. However, the rapid motility of leader cells released from the sheet (Fig. 3B) suggests that the decreased rate of advance is not due to leader cells losing the ability to move rapidly.
In conclusion, in collective migration of zebrafish keratocytes, the advancement of the sheet is stopped when leader cells are dissociated by treatment with the Rho-associated kinase inhibitor Y-27632. The contribution of follower cells to the forward movement of the sheet is unclear. Single cell-layer follower cells are not polarized, but it is clear that during the forward movement of the sheet, the follower cells spread. Although forward movement slows over time in culture, the leader cells maintain their capacity to migrate rapidly when released from the sheet. Future experiments will be aimed at the role of cell adhesions in determining the behavior of cells within the sheet.
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Statistical analysis
Materials and methods Cell culture and live cell microscopy Explant cultures were established from anesthetized AB wild-type zebrafish (Zebrafish International Resource Center, Eugene OR, USA) according to published procedures [7]. Cultures were observed using either a Meiji TC-5400 or an Olympus IX-51 phase contrast microscope and photographed using an XM-10 camera (Olympus, Center Valley PA, USA) operated using CellSens software (Olympus, Center Valley PA, USA) and analyzed using NIH Image J software. Cell sheet velocity was determined by measuring the advancement of the leading edge of the sheet relative to the edge of the scale between the indicated times. Cell displacements were determined by measuring the coordinates of the center of the nucleus every 10 frames. To release leader cells from the sheet, cultures were treated with 0.5 mM EDTA in 1 PBS for 3–5 min and immediately replaced with complete media and incubated for 30–60 min to allow leader cells to detach from the sheet. For treatments, culture media was replaced with media containing 20 μM Rho-associated protein kinase inhibitor Y-27632 (Calbiochem, Billerica, MA, USA), 500 mg/ml negative control (GRADSP) or adhesion disrupting RGD (GRGDNP) peptides (Enzo Life Sciences, Farmingdale, NY, USA) and photographed as indicated in the text to observe response of cell sheets to treatment.
Immunocytochemistry Immunofluorescence microscopy using rabbit polyclonal antibodies characterized for human antigens (Abcam, Cambridge MA, USA) and phalloidin (Invitrogen, Carlsbad CA, USA) was performed following previously published procedures [7]. Briefly, cultures were fixed with paraformaldehyde, permeabilized, blocked, and incubated with primary rabbit polyclonal antibody (Abcam), Alexafluor conjugated secondary antibody (Invitrogen) and counterstained with phalloidin (Invitrogen) and mounted with Prolong antifade containing DAPI (Invitrogen). All stained slides were viewed using a Zeiss Axiovert Apotome microscope with 63 objective (NA 1.4) under oil immersion and photographed using a Axiocam MRm Rev3 camera (Zeiss, Thornwood NY, USA). Internuclear distance and cell area were measured using an NIH Image J.
1o Antibody
Catalog #
Classification
β-Tubulin Vinculin pMyosin light chain E-cadherin
ab11309 ab73412 ab2480
Mouse mAb [TUB 2.1] Rabbit pAb to C-terminal peptide Rabbit pAb, phospho-S20 specific
ab53033
Rabbit pAb to synthetic peptide antigen Rabbit mAb [EP913(2)Y], phospho S838þS840 spedific Mouse mAb [1B11] specific for phospho Y654
pEcadherin pβ-catenin
ab76319 ab24925
When comparing levels of a dichotomous independent variable, an unpaired t-test was used (Microsoft Excel). All other means comparisons were performed using a one-way independent measures ANOVA (SPSS version 19, IBM). All tests were twotailed and the significance level was set at 0.05.
Acknowledgments We thank Agnes Pascual for technical assistance. This work was supported by funds from Midwestern University College of Health Sciences Research Facilitation Grant awarded to EEH and Midwestern University Office of Research and Sponsored Programs Intramural Grant awarded to KJL.
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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Research Article
Collective cell migration of primary zebrafish keratocytes Jose L. Rapanana, Kimbal E. Coopera, Kathryn J. Leyvab, Elizabeth E. Hulla,n a
Biomedical Sciences Program, College of Health Sciences, Midwestern University, Glendale, AZ 85308, United States Department of Microbiology & Immunology, Arizona College of Osteopathic Medicine, Midwestern University, Glendale, AZ 85308, United States b
article information
abstract
Article Chronology:
Fish keratocytes are an established model in single cell motility but little is known about their
Received 26 November 2013
collective migration. Initially, sheets migrate from the scale at 145 μm/h but over the course of
Received in revised form
24 h the rate of leading edge advance decreases to 23 μm/h. During this period, leader cells
14 June 2014
retain their ability to migrate rapidly when released from the sheet and follower cell area
Accepted 17 June 2014
increases. After the addition of RGD peptide, leader cell lamellae are lost, altering migratory
Available online 26 June 2014
forces within the sheet, resulting in rapid retraction. Leader and follower cell states interconvert
Keywords:
within minutes with changes in cell–cell adhesions. Leader cells migrate as single cells when they
Collective cell migration
detach from the leading edge and single cells appear to become leader cells if they rejoin the
Rho associated kinase
sheet. Follower cells rapidly establish leader cell morphology during closing of holes formed
Fish keratocyte
during sheet expansion and revert to follower cell morphology after hole-closure. Inhibition of
Primary culture
Rho associated kinase releases leader cells and halts advancement of the leading edge suggesting an important role for the intercellular actomyosin cable at the leading edge. In addition, the presence of the stationary scale orients direction of sheet migration which is characterized by a more uniform advance of the leading edge than in some cell line systems. These data establish fish keratocyte explant cultures as a collective cell migration system and suggest that cell–cell interactions determine the role of keratocytes within the migrating sheet. & 2014 Elsevier Inc. All rights reserved.
Introduction Zebrafish keratocytes rapidly and collectively migrate when established in explant cultures. Although the mechanisms of motility of keratocytes as single cells have been extensively studied [1–6], little is known about the mechanisms which direct the migration of keratocytes as cell sheets. This experimental system is particularly compelling in vitro model for collective cell migration during reepithelialization in response to wounding for several reasons. First, establishment of explant cultures promptly initiates an n
Corresponding author.
http://dx.doi.org/10.1016/j.yexcr.2014.06.011 0014-4827/& 2014 Elsevier Inc. All rights reserved.
epithelial to mesenchymal transition (EMT), which rapidly progresses over the first seven days of explant culture as assessed by changes in gene expression including a switch from E-cadherin to N-cadherin expression, morphological changes, and cytoskeletal rearrangement [7,8]. As changes in gene expression consistent with inflammation and wound healing occur concurrently with EMT, this system has been characterized as a wound healing model [7]. Second, as explant cultures, keratocytes are not transformed and have not undergone the changes in gene expression associated with transformation or passage of primary cultures [9–13]. Thus, the
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Video 1 Migration of keratocytes from underneath a stationary scale in an explant established 8 h prior to filming. The elapsed time is 5 h with frames taken 5 min apart. A video clip is available online. Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.yexcr.2014.06.011.
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model allows rapid collective cell motility assays (complete within 24 h of initial treatment) in untransformed cells in the context of a defined wound healing process. During the re-epithelialization stage of wound healing, large sheets of epithelial cells collectively migrate to reestablish the intact epidermis. Key to this process are the alterations in quiescent, pre-wound epithelial cells to form a polarized sheet with the formation of leader cells at the wound edge (reviewed in [14]) and an epithelial to mesenchymal transition which typically promotes motility. Although the common hallmarks of collective cell migration have been established, including the presence of leader and follower cells and intact cell junctions, many of the mechanisms guiding collective cell migration are yet to be determined. Due to its compelling characteristics, including rapid motility assays, the zebrafish keratocyte system has promise to shed light on mechanisms of collective cell migration. The data presented here establish the migration of zebrafish keratocyte cell sheets as a collective cell migration system through 1) characterization of distinct leader and follower cells in 24 h sheets and 2) demonstration of the presence of cell–cell junctions that coordinate an actomyosin cable between adjacent
Fig. 1 – Actin, tubulin, vinculin, and phosphomyosin light chain in single, leader, and follower keratocytes. Untreated 24 h explant cultures were fixed and stained with primary antibody to tubulin, vinculin, or phosphorylated myosin light chain (red) and counterstained with phalloidin (green), and DAPI (blue). Vinculin localization in follower cells is presented without phalloidin counterstain as display of actin obscures vinculin localization. Single cells resemble cells at the leading edge while follower cells (photographed in regions with a single cell layer) are not polarized. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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cells which is necessary for advance of the leading edge of the sheet. Next, the mechanism of collective cell migration is investigated using soluble RGD peptides to disrupt adhesion formation and Rho associated kinase inhibition to alter actomyosin contractility. The data suggest that cell–cell interactions determine the role of keratocytes within the sheet and that changes in forces acting on the advancing sheet result in a slowing of migration.
Results Keratocyte explants are established when scales removed from anesthetized zebrafish are incubated in culture medium. Within minutes, epithelial cells that were removed along with the scale rapidly migrate collectively from underneath the scale, while the scale itself does not move relative to the substrata. An example
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of this collective migration is shown in Video 1. The video demonstrates how cells migrate away from the adherent scale as a collective unit, or sheet. Also note that cells do break away from the sheet periodically; in the initial frames, an individual cell that has broken away from the leading edge of the sheet can be observed migrating rapidly out of the frame of the video. This explant was established 8 h prior to filming but similar collective migration is observed in cultures from the initial emergence of the cell sheet approximately 1 h after removal of the scale until the ongoing EMT process leads to fragmentation of the sheet [7]. To establish the zebrafish keratocyte system as a model for collective cell migration, we first used fluorescence microscopy to characterize the leader and follower cells within the keratocyte sheet. As shown in Fig. 1, there are distinct differences between leader and follower cells after 24 h in explant culture. Leader cells resemble single cells in that they are polarized as shown by the
Fig. 2 – Leader and follower localization of E-cadherin and phosphorylated forms of E-cadherin and β-catenin. Untreated 24 h explant cultures were fixed and stained with primary antibody to unphosphorylated E-cadherin (green), E-cadherin phosphorylated on serines 838 and 840 (red), and β-catenin phosphorylated on tyrosine 654 (red) and counterstained with DAPI (blue). Evidence for disassembly of cell junctions is seen in the large amount of intracellular staining with anti-phosphorylated E-cadherin and an increased intranuclear staining with anti-phosphorylated β-catenin. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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presence of an extensive actin-rich lamella and an asymmetric distribution of β-tubulin around the nucleus. In contrast, follower cells are not polarized, maintain cell–cell junctions on all sides, and exhibit a cortical actin cytoskeleton. In addition, leader cells have adhesions that label with vinculin and contain contractile cables perpendicular to the direction of motion similar to single cells. Evidence for actomyosin contractility is seen in the presence of phosphorylated myosin light chain (pMLC) which co-localizes with actin cables that are coupled via cell–cell junctions in adjacent leader cells. Interestingly, the pattern of pMLC in follower cells is variable and is sometimes localized at (or near) the cortical actin cytoskeleton but with no staining seen in other regions. Unlike single cells, leader cells retain E-cadherin and β-catenin cell–cell junctions and these junctions appear to link the actin cable in adjacent cells (Fig. 2). Although a polyclonal antibody to E-cadherin yields diffuse staining within the leader cells, this staining appears absent in the lamella while the same antibody localizes Ecadherin to cell–cell junctions in follower cells. Given that the cell sheets are undergoing an epithelial to mesenchymal transition with consequent down-regulation of E-cadherin [7], we next asked if part of the diffuse staining was due to ongoing disassembly of cell–cell junctions. We used an anti-phospho-E-cadherin antibody (pSer838 and pSer840 in human) as phosphorylation at multiple sites in this region of E-cadherin which is associated with altered cell–cell adhesion and intracellular localization [15–18]. Staining with this antibody yields reduced localization at cell–cell junctions (Fig. 2). In leader cells, there is a large amount of staining in what appears to be
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vesicular structures inside the cell. This pattern is replicated, to a lesser extent, in follower cells. Consistent with this implied disassembly of cell–cell junctions, the phosphorylated form of β-catenin (pTyr654) associated with altered cell adhesion [19] localizes in the nucleus of leader cells but in the cell–cell junctions of follower cells. As shown in Fig. 3A, during explant culture, the rate of advance of the leading edge slows from 143.872.9 μm/h (seen between 1 and 2 h) to 23.570.76 μm/h (seen between 24 and 25 To investigate this slowing rate, we asked if leader cells lost their ability to rapidly migrate over time by disrupting the cell–cell junctions with a brief, mild EDTA treatment. This treatment does not visibly affect the sheet but, after incubation with complete media for 30 to 60 min, increased numbers of leader cells are seen breaking away from the sheet. As shown in Fig. 3B, leader cells released from the sheet migrate at rates typically seen in single cells that naturally break away from the sheet (425.8731.2 vs 433.7724.1 μm/h). Thus, without adhesion to the cell sheet, cells retain their ability to migrate rapidly and, when released from the slower moving follower cells, slowly moving leader cells migrate rapidly as single cells. As the sheet moves forward, we hypothesized that follower cell spreading might result. We focused on follower cell spreading as measured by changes in internuclear distance and cell area comparing 4 h sheets, the earliest time point which we could reproducibly fix and stain, and 24 h cultures. As shown in Fig. 3, there is 1.8 fold increase in internuclear distance (Fig. 3C) and 3.1 fold increase in cell area (Fig. 3D).
Fig. 3 – Mechanisms of keratocyte sheet migration. (A) Rate of leading edge advance decreases with time in explant culture (p¼3.5 10 25). (B) Leading edge cells released by disruption of cell–cell junctions with mild EDTA treatment migrate at rates equivalent to single cells (p¼1.9 10 12). (C) Internuclear distance increases approximately 1.8 fold between 4 and 24 h of culture (p¼ 3.3 10 5). (D) Follower cell area increases 3.1 fold between 4 and 24 h of culture (p¼ 3.4 10 6). Each graph represents three independent experiments in triplicate with error bars as SEM.
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Fig. 4 – Retraction of sheet with addition of soluble RGD peptide. Addition of a soluble RGD peptide to 4 h cultures (A and D) leads to a small retraction and loss of lamellae (B and E) within 10 s and an extensive retraction of the sheet by 40 s (C). The average area of three sheets treated with RGD peptide, compared to sheets treated with control peptide, is plotted in F, showing no effect of control peptide.
Fig. 5 – Conversions of individual, leader, and follower keratocytes. In untreated, 24 h sheets, individual to leader and leader to individual conversions occur with the breaking or forming of cell–cell adhesions. During the closure of holes formed within the sheet, follower cells become leaders. Fluorescence microscopy demonstrates the presence of actin rich lamellae (green) during hole closure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Evidence for changes in forces within the sheet is seen in the rapid recoil of the entire sheet after the addition of soluble peptides containing an RGD sequence to inhibit adhesion
formation in 4 h cultures (Fig. 4). Panel A shows the entire untreated sheet after 4 h of culture with Panel D showing an enlarged area of that same sheet, illustrating the extensive
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Video 2 Dynamics of hole formation and closure in an explant established 24 h prior to filming. A hole in the sheet is visible in the initial frames near the indentation of the leading edge, which begins to close shortly after the start of filming. As this hole closes, another hole begins to open (left of the closing hole). The elapsed time is 90 min with frames taken 1 min apart. A video clip is available online. Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.yexcr. 2014.06.011.
lamellae present at the leading edge. Ten seconds after the addition of soluble RGD peptide, there is a slight retraction of the leading edge (Panel B) and these leader cells have lost their lamellae (Panel E). Twenty seconds after addition of the peptide, the sheet is completely retracted and folded adjacent to the scale (Panels C and F). No response is seen with the addition of control peptide, as indicated by no change in cell sheet areas (Panel F). At 24 h, a similar but less dramatic recoil is observed and has also been reported elsewhere [20]. Although infrequent, spontaneous dissociations of leader cells to become single migrating cells are observed. As shown in Fig. 5 (top panel), leader cells can be seen breaking away from the sheet within minutes. Similarly, individually migrating cells in the culture may re-join the cell sheet (middle panel) and become morphologically indistinguishable from leader cells. The rate of migration of spontaneously formed single cells is comparable to those seen with brief, mild EDTA treatment (Fig. 3B). As migration depends on adhesion strength in a variety of experimental and theoretical studies [21–24], these data alleviated concerns that the use of mild EDTA treatment to release cells from the sheet might weaken adhesion and increase cell speed. An interesting example of follower to leader conversion is seen in holes that form during sheet migration (Fig. 5, bottom panel). As holes form and enlarge, cells lining the perimeter of the hole retain follower cell morphology. However, as holes begin to close, these cells lining the hole develop lamellae characteristic of leader cells, a morphological transformation that is confirmed by fluorescent visualization of the actin cytoskeleton. Once cell edges meet, after a variable period of time, cells regain the morphology consistent with those of follower cells. As shown in Video 2, holes tend to form in areas of the sheet that contain spreading follower cells, in this case between the leading edge and an area containing multiple cell layers adjacent to the scale. During the course of the video, the initial hole, near the
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Fig. 6 – Treatment with Y-27632 disrupts actomyosin contractility. (A) Evidence for intercellular actomyosin contractility can be seen in the presence of a coordinated actin cable (green) which co-localizes with pMLC (red). (B) After treatment, both actin and pMLC relocalize and the leading edge of the sheet fragments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
indentation of the leading edge, decreases in size while other holes open in adjacent areas (to the left of the original hole). The presence of an actin cable, apparently coordinated in adjacent cells through anchoring at cell–cell junctions and which co-localizes with phosphorylated myosin light chain, is suggestive of actomyosin contractility at the leading edge of the cell sheet (Fig. 6A). As intercellular actomyosin contractility is a defining feature of collective cell migration [25], we inhibited contractility with Y-27632, a Rho-associated kinase (ROCK) inhibitor [26], and assessed sheet migration. Treatment with Y-27632 leads to a redistribution of actin from the intercellular actin cable to newly formed lamellae and cytoplasmic staining of pMLC (Fig. 6B). Treatment of 4 h sheets with 20 mM Y-27632 for 2 h led to an apparent flattening of cells at the leading edge (Fig. 7A) consistent with the loss of intercellular actomyosin contractility. Longer treatments (Fig. 7B and C) fragmented the leading edge of the sheet and caused a slight net retraction of the leading edge, possibly due to dissociation of leader cells (suggested by the increased number of individually migrating cells which apparently detach from the leading edge). During this time, the leading edge of an untreated sheet would normally advance 125 mm (black arrow, Panel C). The presence of single cells which migrate at rates similar to untreated cells (425.8731.2 μm/h vs 399.7727.4 μm/h), suggest that a higher level of contractility is necessary for the advancement of the leading edge of the sheet than migration in individual cells (see Video 3). As suggested by Video 4, the advance of cells within the first few rows of cells at the leading edge of the keratocyte sheet is
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Fig. 7 – Sheet advance halts after treatment with Y-27632 releases leader cells from the sheet. (A) 2 h treatment with 20 lM Y-27632 of 4 h sheets leads to flattening of cells at the leading edge. (B) 3 h treatment leads to release of cells from the leading edge. (C) At 4 h treatment, the leading edge has not advanced when untreated sheets would typically advance 125 lm (black arrow). Cells in the follower region fail to advance between panels B and C (red arrowheads). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
during establishment in explant culture). However, unlike collective cell migration in cell line systems, keratocyte sheets appear to migrate away from a stationary scale. To investigate the effects of the immobile scale on sheet migration, we mechanically detached the scale from the substrata and followed its migration. In the example shown in Fig. 9 and Video 5, part of the sheet was simultaneously separated from the scale. Without attachment to the scale, the new free edges in the cell island (on the right) rapidly adheres to the substrata and those cells adopt the morphology characteristic of leader cells. With leader cells on all edges of this island, the collection of cells ceases to migrate with the rapid directionality characteristic of keratocyte sheets and appears more random. Surprisingly, the keratocytes still attached to the sheet (on the left) are able to move the scale relative to the substrata.
Discussion
Video 3 Effect on sheet migration after treatment with 20 lM ROCK inhibitor Y-27632 in an explant culture established 4 h prior to filming. The elapsed time is 3 h with frames taken 3 min apart. A video clip is available online. Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j. yexcr.2014.06.011.
relatively uniform with most leader cells extending extensive lamellae. This is in contrast to the cellular “fingers” at the leading edge of the sheet seen by others [27,28]. To analyze this motion more closely, we have traced the position of the nuclei in the first 2 to 3 rows of cells at the leading edge, presenting the motion path of these cells (Fig. 8). Our data show that cells near the leading edge follow similar motion paths, suggesting that similar net forces are likely generated in these cells within the observed timeframe. This result suggests that the intercellular actin cable may allow these neighboring cells to join forces to assist in the coordination of movement of the cell sheet, as also demonstrated by Tambe et al. [29]. As with other systems, the free edge of keratocyte sheets migrate to fill empty space created by wounding (or in this case,
Our data provide evidence that the movement of zebrafish keratocyte sheets in explant culture is an example of collective cell migration. Collective cell migration is defined as a group of cells maintaining cell–cell junctions that exhibit directional movement. This type of collective cell migration has been seen in a variety of systems (reviewed in [30,31]). One of the noteworthy characteristics of these systems is a distinction between leader and follower cells [32]. Cells at the leading edge of the sheet exhibit polarity while follower cells are morphologically unpolarized cells. Video 1 demonstrates the coordinated motion of a keratocyte sheet, and examination of this migrating complex shows definite polarity in the leader cells (in a manner resembling individually migrating cells) while this polarization is absent in follower cells. Fish keratocytes are known for their rapid motility and, as single cells, may achieve speeds up to 0.5 μm/sec. Consistent with the usually rapid rate of migration of individual cells, the initial rates of 143.872.9 μm/h in collective migration of keratocyte sheets is substantially faster than 6–60 μm/h typically reported for other systems (reviewed in [25]). Given the well-established relationship between adhesion strength and migration speed [21,23,24], this rapid migration of individual cells may depend on the use of close contacts as cell substratum adhesions which are weaker than fully developed focal adhesions [33]. As a similar distribution of adhesion
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Video 4 Migration of a cell sheet, focused onto several cells within the leading edge, from an explant established 24 h prior to filming (see Fig. 8). The elapsed time is 80 min with frames taken 1 min apart. A video clip is available online. Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.yexcr.2014.06.011.
Fig. 8 – Migration of cells within the leading edge of a 24 h explant. Top: Cells within the leading edge of the sheet have well-developed lamellae and appear to be moving forward as a collective unit. Bottom: Motion paths of cells within the first 2–3 rows at the leading edge were mapped by plotting the change in nuclear position every 10 frames for the length of the video (see Video 4).
proteins (β1 integrin and vinculin) is seen in both leader and individual cells (Fig. 1 and [20,33]), close contacts may be essential part of the rapid migration of keratocyte sheet and may make keratocytes more susceptible to disruption by an RGD peptide than cells with stronger focal adhesions [20]. A unique feature of the keratocyte explant model is the scale, which appears to help provide directionality to the sheet movement. The cell sheet lacks a free edge on the side attached to the scale so that leader cells develop and orient away from the scale. Tambe et al. [29] concluded that the direction of collective cellular migration followed the local orientation of maximal principle stress. Our data show cells within the leading edge all move in the same approximate direction (Fig. 8). This suggests that the cells near the leading edge experience similar net forces in the observed timeframe. Given
the reduced directional persistence of the cell island in Video 5, we suggest that this polarity is an essential part of the rapid motility of keratocyte sheets. Thus, the geometry imposed by the scale may be, at least in part, responsible for coordinating the movement of the keratocyte sheet [27–29]. Further examination of leader cells within the sheet documents extensive lamellae at the free edge. In addition, actomyosin cables are observed behind the lamellae and adherens junctions link these cables between adjacent cells; these adherens junctions do transmit contractile stresses between cells [29,reviewed in 34]. Within a migrating sheet, the collective forces within and between cells must be in balance so that the sheet moves as a unit at a constant velocity. As demonstrated in other systems, cooperative intercellular forces participate in propelling the sheet forward [29,34–38]. In comparison, treatment of a migrating sheet with a Rho kinase inhibitor visibly disrupts the actomyosin cable and causes the release of leader cells from the sheet (Figs. 6 and 7). The net result is a halt of the advance of the keratocyte sheet, thus supporting a role for actomyosin contractility in collective cell migration [38,39]. As suggested by p-E-cadherin and p-β-catenin stainings (Fig. 2), these junctions are apparently preferentially disassembled at the leading edge, a process which continues during explant culture and ultimately leads to the fragmentation of the sheet [7,20]. Given the importance of these junctions in maintaining the intercellular actomyosin cable at the leading edge, we suggest that this disassembly should be interpreted in the context of the ongoing EMT process that is occurring within the explant culture [7]. Our data also support an argument that leader cells may play a more important role in the advancement of the leading edge than the follower cells [39]. When soluble RGD peptide is added to a migrating sheet, the sheet rapidly recoils (Fig. 4A–C), resulting in a dramatic decrease in sheet area within seconds (Fig. 4F). RGD peptide treatment also visibly reduces the lamellae size (Fig. 4D and E). Given the well documented role that lamellar extension plays in migration, these data support the hypothesis that RGD peptide reduces pulling by leader cells, disrupting the steady state and effectively removing a force which opposes the contraction of the actomyosin cable. This may be partly responsible for the rapid recoil of the sheet. In addition, there is an evidence of crosstalk between cell–cell adhesions and integrin-mediated attachments that control cell adhesion strength and motility [40]. Follower cell spreading (Fig. 3C and D) may contribute significantly to the
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Fig. 9 – Migration of two segments of a 24 h cell sheet after mechanical separation. Mechanical separation of the scale from the substrata resulted in a small segment of the sheet still attached to the scale (top panel) and an “island” of cells (bottom panel). Cells still attached to the scale advance, pulling the scale forward. The segment of the sheet separated from the scale forms lamellae on the side previously attached to the scale. The resulting cell “island”, with lamellae along the entire perimeter, exhibits more random movement.
Video 5 Migration of two segments of 24 h cell sheet after mechanical separation (see Fig. 9). Mechanical separation of the sheet was performed just prior to filming. The elapsed time is 215 min with frames taken 5 min apart. A video clip is available online. Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.yexcr.2014.06.011.
The rapid interconversion of keratocytes among leader, follower, and single cells (Fig. 5) suggests that cell interactions are crucial for understanding the role of an individual keratocyte within a cell sheet. Given the speed at which these transitions occur, we suggest that signaling downstream of adhesion formation may be determining the mode of motility each cell assumes as substantial changes in gene expression are unlikely to be involved. An aspect of explant cultures not addressed in this work is the presence of multiple layers of cells in the follower regions. For clarity, the data on follower cells presented here avoids these regions as multiple layers are hard to visualize. The role of a second layer of cells within the follower region is unclear as this second layer is not always present. Sharma et al. [42] have reported that the second layer of cells is in contact with the substratum and, thus cells in the second layer have cell-substratum adhesions necessary to provide traction and stabilize tension within the sheet . Further work is needed to determine the interplay among keratocytes within a multilayer migrating sheet.
Summary expansion of the sheet as spreading has been shown to lead to monolayer expansion [41]. Alternatively, as adhesion is necessary for force generation, the weaker adhesions in the keratocyte sheet [33] may limit the contribution of follower cells to migration. Lastly, it may be possible that follower cell force generation is also sensitive to Rho-associated kinase inhibition. An interesting observation is the decreasing rate of the advance of the leading edge (Fig. 3A). This could represent decreased force generation by the leader cells. However, the rapid motility of leader cells released from the sheet (Fig. 3B) suggests that the decreased rate of advance is not due to leader cells losing the ability to move rapidly.
In conclusion, in collective migration of zebrafish keratocytes, the advancement of the sheet is stopped when leader cells are dissociated by treatment with the Rho-associated kinase inhibitor Y-27632. The contribution of follower cells to the forward movement of the sheet is unclear. Single cell-layer follower cells are not polarized, but it is clear that during the forward movement of the sheet, the follower cells spread. Although forward movement slows over time in culture, the leader cells maintain their capacity to migrate rapidly when released from the sheet. Future experiments will be aimed at the role of cell adhesions in determining the behavior of cells within the sheet.
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Statistical analysis
Materials and methods Cell culture and live cell microscopy Explant cultures were established from anesthetized AB wild-type zebrafish (Zebrafish International Resource Center, Eugene OR, USA) according to published procedures [7]. Cultures were observed using either a Meiji TC-5400 or an Olympus IX-51 phase contrast microscope and photographed using an XM-10 camera (Olympus, Center Valley PA, USA) operated using CellSens software (Olympus, Center Valley PA, USA) and analyzed using NIH Image J software. Cell sheet velocity was determined by measuring the advancement of the leading edge of the sheet relative to the edge of the scale between the indicated times. Cell displacements were determined by measuring the coordinates of the center of the nucleus every 10 frames. To release leader cells from the sheet, cultures were treated with 0.5 mM EDTA in 1 PBS for 3–5 min and immediately replaced with complete media and incubated for 30–60 min to allow leader cells to detach from the sheet. For treatments, culture media was replaced with media containing 20 μM Rho-associated protein kinase inhibitor Y-27632 (Calbiochem, Billerica, MA, USA), 500 mg/ml negative control (GRADSP) or adhesion disrupting RGD (GRGDNP) peptides (Enzo Life Sciences, Farmingdale, NY, USA) and photographed as indicated in the text to observe response of cell sheets to treatment.
Immunocytochemistry Immunofluorescence microscopy using rabbit polyclonal antibodies characterized for human antigens (Abcam, Cambridge MA, USA) and phalloidin (Invitrogen, Carlsbad CA, USA) was performed following previously published procedures [7]. Briefly, cultures were fixed with paraformaldehyde, permeabilized, blocked, and incubated with primary rabbit polyclonal antibody (Abcam), Alexafluor conjugated secondary antibody (Invitrogen) and counterstained with phalloidin (Invitrogen) and mounted with Prolong antifade containing DAPI (Invitrogen). All stained slides were viewed using a Zeiss Axiovert Apotome microscope with 63 objective (NA 1.4) under oil immersion and photographed using a Axiocam MRm Rev3 camera (Zeiss, Thornwood NY, USA). Internuclear distance and cell area were measured using an NIH Image J.
1o Antibody
Catalog #
Classification
β-Tubulin Vinculin pMyosin light chain E-cadherin
ab11309 ab73412 ab2480
Mouse mAb [TUB 2.1] Rabbit pAb to C-terminal peptide Rabbit pAb, phospho-S20 specific
ab53033
Rabbit pAb to synthetic peptide antigen Rabbit mAb [EP913(2)Y], phospho S838þS840 spedific Mouse mAb [1B11] specific for phospho Y654
pEcadherin pβ-catenin
ab76319 ab24925
When comparing levels of a dichotomous independent variable, an unpaired t-test was used (Microsoft Excel). All other means comparisons were performed using a one-way independent measures ANOVA (SPSS version 19, IBM). All tests were twotailed and the significance level was set at 0.05.
Acknowledgments We thank Agnes Pascual for technical assistance. This work was supported by funds from Midwestern University College of Health Sciences Research Facilitation Grant awarded to EEH and Midwestern University Office of Research and Sponsored Programs Intramural Grant awarded to KJL.
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