Plectin, a major organizing element of IF cytoarchitecture in keratinocytes
To investigate whether plectin plays a role in IF network organization, we first compared the general appearance of such networks in plectin wild-type (ple [+/+]) and plectin-deficient (ple [−/−]) basal keratinocytes using immunofluorescence microscopy. In interior cytoplasmic regions, both primary and p53-deficient ple (+/+) keratinocytes exhibited dense networks of keratin filaments. The filaments showed partial colocalization with discontinuous plectin-positive structures, which in superimposed images often resembled beads on a string (). However, at the cell margins, hardly any IFs were found, leaving a peripheral ring–shaped, filament-free zone densely packed with plectin structures. Keratin filaments extended to the inner circumference of this ring (, A and G, arrows), as if peripheral plectin acted in an inhibitory manner on filament extension. In support of this view, the keratin network system of wild-type cells seemed to be more densely packed around the cell center compared with that of ple (−/−) cells, where it extended further to the cell periphery (, compare B and H with E and K, respectively). This phenotype was noticed independent of whether the anti-pan keratins K5, K6, and K18 or anti-K5 or K6 antibodies alone were used (; unpublished data). Furthermore, particularly in subconfluent cell cultures (~60% confluence), ple (−/−) cells exhibited significantly enlarged mesh size of keratin networks at their periphery compared with wild-type cells (, compare B with E and H with K, along with their corresponding magnifications). A quantitative analysis of subconfluent cell populations revealed that >70% of ple (−/−), but only <8% of ple (+/+), keratinocytes displayed such a phenotype. Regarding expression levels of different keratins, no differences were observed between ple (+/+) and (−/−) keratinocytes, as revealed by immunoblotting analysis (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200605172/DC1
). Interestingly, the vimentin filament network of plectin-deficient fibroblasts showed similar characteristics. It appeared more bundled and less delicate compared with wild-type cells, creating a network with wider spaces between the filaments (unpublished data).
Figure 1. Cytoarchitecture of keratin filament networks in wild-type and plectin-deficient basal mouse keratinocytes. Cells were double immunolabeled using antiserum to plectin and mAbs to pan-keratin (A–L), or single-labeled as indicated (M–R). (more ...)
In both ple (+/+) and (−/−) cells, actin filaments exhibited a subcortical organization typical of subconfluent keratinocyte cultures (, M and P; Vasioukhin et al., 2000
). However, ple (−/−) keratinocytes showed a slight increase in the number of stress fibers and, moreover, were extending small lamellipodia (, arrows), which is indicative of an ongoing transition from a stationary to a migratory phenotype. Furthermore, no gross differences were observed between ple (+/+) and (−/−) cells in the organization of microtubules (). Peripheral integrin (INT) α6 clusters were formed in both cell types (), and in ple (+/+) cells, partial colocalization with plectin was observed (Fig. S2, A–C, available at http://www.jcb.org/cgi/content/full/jcb.200605172/DC1
). In addition, keratin staining partially overlapped with the ring formed by integrin clusters (Fig. S2, D–F).
Transmission electron microscopy revealed a prominent lateral bundling of keratin filaments in peripheral regions of ple (−/−) cells, resulting in larger spaces between individual and bundled filaments (). In some regions, filaments showed a complete lateral collapse, appearing as one massive filamentous bundle (, arrow). Bundling to such an extent was never observed in ple (+/+) cells, where corresponding areas were dominated by numerous fine, and apparently short, filamentous structures, with orthogonal cross-bridges filling the space between the filament bundles ().
Figure 2. Plectin reduces the mesh size of IF networks. (A) Keratinocytes were subjected to transmission electron microscopy, as described in the text. Boxed areas in a and b are shown as 7×-magnified images in c and d, respectively. Arrow in b marks massively (more ...)
To demonstrate that the observed changes in keratin network organization were directly connected to plectin deficiency, the phenotypic rescue potential of plectin was examined by transient transfection of ple (−/−) keratinocytes with an expression plasmid encoding full-length mouse plectin isoform 1a, which is the major isoform normally expressed in this type of cells (Andrä et al., 2003
). Similar to endogenous plectin of corresponding wild-type cells (), reexpressed plectin 1a showed a punctated distribution pattern (). A quantitative analysis revealed that 90% of ple (−/−) cells expressing plectin 1a at significantly high levels displayed keratin networks with mesh sizes as small as those of ple (+/+) cells (). This strong rescue potential of plectin 1a clearly demonstrated a direct link between the absence of plectin and the observed alterations of the keratin network cytoarchitecture.
Keratin filaments of ple (−/−) keratinocytes are more sensitive to hypoosmotic shock
Osmotic and heat-shock assays serve as useful tools in monitoring keratin network properties and their alterations in EBS caused by keratin mutations (Morley et al., 1995
; D'Alessandro et al., 2002
). Therefore, we examined whether the altered keratin filament cytoarchitecture of ple (−/−) keratinocytes affected their response to changes in osmolarity and temperature. Upon urea treatment, INTα6-positive retraction fibers, which are a characteristic feature of migrating and mitotic cells (Geuijen and Sonnenberg, 2002
), became very pronounced at the rear of both cell types (). Most probably, the general shrinkage of the cells, which started shortly after exposure to urea (D'Alessandro et al., 2002
), triggered the elevated formation of these structures. INTα6-positive retraction fibers of ple (−/−) cells, however, were significantly longer than those of ple (+/+) cells, possibly reflecting a stronger shrinkage of plectin-deficient cells (). In accordance with recent studies showing increased urea-induced bundling of keratin filaments (Werner et al., 2004
), the IF network appeared less filamentous after urea treatment and accumulated around the cell center, indicating that it had collapsed onto the nuclei (). Interestingly, at the leading edge of cells, which is an area devoid of retraction fibers, keratin bundles of ple (+/+) cells displayed a regular form and were closely associated with peripheral integrin clusters, whereas in ple (−/−) cells filament bundles were conspicuously tangled and distant from the cell periphery (). The appearance of such “torn” filaments in ple (−/−) cells suggested a reduction in their INTα6β4 anchorage. This would be consistent with the reduced attachment of keratin filaments to HDs reported for EBS patients (McMillan et al., 1998
Figure 3. Keratin filaments of ple (−/−) keratinocytes are more sensitive to hypoosmotic shock and show reduced association with integrin α6/β4. (A) Osmotic shock–treated keratinocytes were fixed and immunolabeled for INTα6 (more ...)
To address this issue biochemically, we prepared cytokeratin-enriched cell fractions from ple (+/+) and (−/−) keratinocytes grown on collagen I and compared their INTα6β4 content using antibodies to INTβ4. As expected, such cell fractions were highly enriched in keratins 5 (unpublished data) and 14 () and, in the case of wild-type cells, contained considerable amounts of plectin. INTβ4 was, however, completely absent from cytokeratin fractions of ple (−/−) cells. The absence of INTβ4 from the cytokeratin fraction of ple (−/−) keratinocytes correlated well with the reduced number of HD-like structures found in these cells (, compare O with R; Andrä et al., 2003
Collectively, these data supported the notion that the increased susceptibility of ple (−/−) keratinocytes to urea-induced deformation (as revealed by the conspicuous tangling of filaments and dramatic increase in the length of INTα6β4 retraction fibers), was caused by a lack of filament attachment to integrin clusters. On the other hand, the response of ple (+/+) and (−/−) cells to elevated temperatures in heat-shock assays was very similar, leading to a partial granulation of keratin filaments in both cases (unpublished data).
Plectin regulates IF disassembly dynamics
If, in the absence of plectin, the rigidity of IF networks is reduced and filaments are more loosely bound, or not bound at all, to the outer membrane, one might expect the network to be disassembled more readily in ple (−/−) compared with ple (+/+) cells. To test this, we monitored by immunofluorescence microscopy the kinetics of IF disassembly upon treatment of ple (+/+) and (−/−) cells with the serine/threonine phosphatase inhibitor okadaic acid (OA), which is known to selectively cause the disruption of IFs (Strnad et al., 2001
). After a 2-h treatment of keratinocytes, the well-spread keratin network had formed thick bundles of filaments that seemed to be retracting toward the nucleus (, arrowheads). At later time points (4 and 6 h), a progressive breakdown of filaments was observed, with keratin granules forming first at the cell periphery (, arrowheads), followed by their collapse into a dense perinuclear ring (, arrow) that eventually became fragmented into numerous granules of various sizes (, arrow). The initial bundling of filaments appeared to occur more efficiently in ple (−/−) cells, as indicated by the increased mesh size of keratin networks visualized in the majority of these cells compared with ple (+/+) cells (, arrows). Moreover, at the 4- and 6-h time points, the proportions of cells with keratin granules were significantly higher in ple (−/−) compared with ple (+/+) cells (). For a statistical analysis of keratin filament disassembly, cells were classified into three categories (1–3), where category 1 represented cells with no granules, category 2 represented cells with granules and residual filaments (, cell marked with asterisk), and category 3 represented cells in which complete granulation, including that of the perinuclear ring structure, had occurred (, cell marked with arrow). At the 2-h time point, ~15% of ple (−/−) cells already fell into category 2, whereas only a mere 4% of ple (+/+) cells did (). After 4 h, the majority of the ple (−/−) cell population had their keratin network either partially (30%) or completely (50%) disassembled, whereas ~70% of ple (+/+) cells were still without any granules (). After 6 h, the difference between ple (+/+) and (−/−) cells became less pronounced, but ~30% of ple (+/+) cells still did not show any granules, versus only ~7% in the case of ple (−/−) cells (). Based on this, we concluded that the disassembly of IFs upon OA treatment is significantly accelerated in ple (−/−) compared with wild-type keratinocytes. In agreement with a previous study (Strnad et al., 2001
), even after 6 h of OA treatment, no disruption of microtubules or microfilaments was observed. In fact, actin stress fiber formation was found to be significantly increased in ple (−/−) cells, and to some extent, also in ple (+/+) cells (unpublished data).
Figure 4. Faster OA-induced keratin filament disruption and soluble keratin pool elevation in plectin-deficient cells. (A) Keratinocytes treated with OA for 2 (a and d), 4 (b and e), and 6 h (c and f) were immunolabeled using mAbs to pan-keratin. (a and d) Arrows, (more ...)
Plectin itself appeared to dissociate from IFs upon OA treatment of keratinocytes. Immunofluorescence microscopy revealed a strong increase in nonfilamentous (diffuse) plectin staining in keratinocytes at the 2-h time point (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200605172/DC1
), whereas no colocalization with residual filaments nor with keratin granules was observed at later time points (Strnad et al., 2001
; unpublished data). Consistent with this, in cell fractionation experiments, plectin was no longer detectable in the cytokeratin fraction beyond the 2-h time point (Fig. S3 B).
Monitoring detergent-soluble keratin pools during OA treatment of keratinocytes, we found the level of soluble keratin proteins to already be elevated by approximately twofold in plectin-negative compared with wild-type cells before drug treatment (, 0 h). This difference further increased to approximately threefold within 2 h of drug treatment. Thereafter, keratin solubility in ple (−/−) cells stayed about level, while that in ple (+/+) cells further increased, approaching a level similar of that of ple (−/−) cells (, 4 h).
Plectin deficiency affects stress-activated p38 and Erk1/2 MAP kinases
Previous studies have shown stress-activated p38 MAP kinase to be one of the major candidates for mediating the effects of OA on vimentin and keratins (Cheng and Lai, 1998
; Toivola et al., 2002
). Therefore, we examined whether the OA-induced changes in network appearance and solubility of keratins were paralleled by changes in p38 activity. Using anti–phospho-p38 antibodies to monitor the activation status of p38 kinase, we found no significant differences between ple (+/+) and (−/−) keratinocytes under basal conditions (). Upon addition of OA to ple (−/−) cells, a moderate increase in p38 activity during the first hour was followed by a steep increase during the second hour and a sharp decline thereafter. In contrast, p38 kinase activity levels in ple (+/+) keratinocytes showed only a modest increase during the first 2 h, staying constant thereafter. Thus, the maximum of p38 activity measured in plectin-negative cells was more than twice as high as in wild-type cells.
Figure 5. Plectin deficiency is linked to accelerated OA-induced activation of p38 MAP kinase and alterations in Erk1/2 basal activity. (A and C) Keratinocytes were either left untreated or treated with OA for the times indicated. Lysates from these cells were (more ...)
To assess whether the elevated response of p38 kinase to OA in ple (−/−) cells was specific, the activity of another MAP kinase, Erk1/2, was monitored in a similar fashion. Unexpectedly, we found the basal phosphorylation of Erk1/2 kinases to be already significantly elevated in ple (−/−) keratinocytes () compared with ple (+/+) cells. Erk1/2 activities in ple (+/+) and (−/−) cells showed a similar response to OA treatment, however, reaching maxima after 1 h, followed by a decrease to levels below those observed before the treatment (). Hence, the accelerated OA-induced activation of p38 kinase in plectin-deficient, compared with wild-type, keratinocytes seemed to be specific for this MAP kinase, correlating with the observed tendency for faster keratin solubilization in these cells.
Faster in vitro migration of plectin-deficient compared with wild-type keratinocytes
Plectin-mediated attachment of the keratin cytoskeleton to INTα6β4 has been shown to play a crucial role in stabilizing adhesion of keratinocytes to the matrix, thereby inhibiting cell migration (Geuijen and Sonnenberg, 2002
). Plectin-deficient keratinocytes, showing no association of INTα6β4 with keratins (), together with their up-regulation of Erk1/2 (see previous section), which is a kinase that positively regulates keratinocyte migration (Huang et al., 2004
), prompted us to compare the migratory potentials of ple (+/+) and (−/−) keratinocytes using an in vitro wound-healing assay. Average migration distances measured for ple (−/−) cells were almost twice as long as those of ple (+/+) cells (). Interestingly, the mesh size of the keratin network in ple (−/−) keratinocytes along the wound edge was much larger compared with that of cells at a distance from the wound and, in these regions, differences to the keratin network of ple (+/+) cells became most prominent (). This was consistent with our finding that an increased keratin network mesh-size characteristic of ple (−/−) keratinocytes was particularly evident in subconfluent cell cultures (). Furthermore, in migrating wound-edge keratinocytes, plectin's localization changed from basal integrin cluster– to keratin filament–associated (), highlighting the importance of plectin in organizing keratin cytoarchitecture during cell migration.
Figure 6. Faster migration of ple (−/−) keratinocytes in scratch wound closure assays. (A) Cells were stained using mAbs to actin (false gray color). The lines drawn in the images represent reference lines marking the width of the scratch when it (more ...)
Because we had observed a correlation between the enhanced migration of ple (−/−) keratinocytes and the up-regulation of Erk1/2, we asked whether pharmacological inhibition of MEK1/2, which are the upstream kinases of Erk1/2, would decrease migration of ple (−/−) keratinocytes. As shown in , when ple (−/−) keratinocytes were exposed to PD98059, which is a specific inhibitor of MEK1/2 activities, their migration distances were reduced by almost a factor of two, bringing their level close to that of untreated wild-type cells. Wild-type cells showed a similar drug response. The observed decrease in migration of drug-treated cells directly correlated with inhibition of Erk1/2 activities, as demonstrated by analysis of Erk1/2 phosphorylation (). A similar analysis showed that other MAP kinases, in particular JNK, were unaffected under these conditions (unpublished data).
Importantly, although MEK1/2 inhibition decreased the migration rate of ple (−/−) keratinocytes, it had no effect on their aberrant keratin network organization (), clearly placing plectin in the MAP kinase cascade upstream of Erk1/2. These data established a causal relationship between plectin deficiency and accelerated migration of keratinocytes, showing hyperactivation of Erk1/2 to be a consequence of plectin deficiency.
PKCδ and c-Src both have been suggested as major players in signaling pathways responsible for migration of keratinocytes (Yamada et al., 2000
; Li et al., 2002
), and both have been shown to be upstream activators of Erk1/2 (Miranti et al., 1999
; Gagnoux-Palacios et al., 2003
). Therefore, we next investigated activation of these kinases in membrane and cytosolic fractions of ple (+/+) and (−/−) keratinocytes. As shown in , both, PKCδ and c-Src kinase, exhibited increased phosphorylation (corresponding to higher activities) in the membrane fraction of ple (−/−) keratinocytes compared with wild-type cells. Although total c-Src levels in membrane and cytosolic fractions from both cell types were comparable, those of PKCδ were lower in the membrane fraction of ple (−/−) compared with ple (+/+) cells. In the cytosolic fractions, total PKCδ signals were hardly detectable in any of the two cell types.
Figure 7. Up-regulation of c-Src and PKCδ in ple (−/−) keratinocytes and increased migration potential of RACE1-overexpressing cells. Measurement of various protein kinase activities (A–C) and time-lapse microscopy of migrating keratinocytes (more ...)
To assess whether up-regulation of c-Src was related to enhanced Erk1/2-dependent migration of ple (−/−) keratinocytes, cells were treated with the Src family kinase inhibitor PP2, a suppressor of cell motility and Erk activation (Matsubayashi et al., 2004
), before scratch wound assays. As expected, this treatment led to suppression of cell motility (unpublished data) and a dose-dependent decrease in phosphorylation (activity) of Erk1/2 ().
Forced expression of the plectin-binding protein RACK1 in keratinocytes leads to increased motility
In a previous study, we revealed a role of plectin as a cytoskeletal regulator of PKC signaling and possibly other signaling events (Osmanagic-Myers and Wiche, 2004
). We proposed that plectin sequesters RACK1, which is a receptor and scaffolding protein of activated PKC and a direct binding partner of plectin, to the cytoskeleton when PKC is inactive. Because of the lack of its cytoskeletal docking site in the absence of plectin, in plectin-deficient cells RACK1 accumulates (together with PKC) at the periphery of cells, similar to the situation in wild-type cells after activation of PKC. According to this model, one may expect that the forced expression of RACK1 in wild-type keratinocytes mimics the situation in ple (−/−) cells, leading to their characteristic phenotypes. To test this we analyzed the migration potential of keratinocytes expressing an EGFP-RACK1 fusion protein using time-lapse video microscopy.
In accordance with migration distances measured in scratch wound closure assays (), ple (−/−) keratinocytes displayed a migration velocity (1.58 μm/min) approximately two times as high as that of ple (+/+) cells (0.82 μm/min), when observed 2–6 h after plating (not depicted). As shown in (controls), 14–18 h after plating, ple (−/−) cells migrated three times as fast as ple (+/+) cells (1.53 vs. 0.49 μm/min). Expression of EGFP-RACK1 led to an increase in the average migration rates of both cell types. Transfected wild-type cells (1.30 μm/min) migrated 2.6 times faster than untransfected control cells, reaching 85% of the speed of untransfected ple (−/−) cells, whereas transfected ple (−/−) cells (2.02 μm/min) migrated 1.3 times faster than their untransfected counterparts. Similar experiments were performed with the cytoplasmic nonreceptor tyrosine kinase Fer, which, like RACK1, directly binds to plectin and thereby is inhibited in its activity (Lunter and Wiche, 2002
). In this case the speed of wild-type cells was increased by approximately twofold (unpublished data).
In contrast, expression of an EGFP-plectin isoform 1a (full-length) fusion protein in ple (−/−) cells led to a significant slowdown of the cells, reducing their average speed to 0.98 μm/min (). This was equivalent to a slightly >50% rescue potential of the fusion protein, taking the values of control ple (+/+) and (−/−) cells into account. The lower rescue potential of plectin 1a in this assay compared with restoration of keratin network cytoarchitecture () may reflect the requirement of other major isoforms expressed in keratinocytes, such as plectin 1c and 1 (Andrä et al., 2003
), for full phenotype restoration. Thus, whereas overexpression of plectin-controlled signaling proteins, such as RACK1, led to downstream mechanisms boosting cell motility (), reexpression of a major plectin isoform in ple (−/−) cells led to the partial reversal of their aberrant migration.