MHCK domain organization and MHCK C biochemical activity
The enzymes MHCK-A and MHCK-B have established roles in the control of D. discoideum myosin filament assembly both in vitro and in vivo [16, 17, 24], and Egelhoff, T. T., (unpublished studies). These enzymes have a conserved domain organization that includes a highly novel protein kinase catalytic domain unrelated to conventional kinases, and a carboxyl-terminal WD repeat domain that targets these enzymes to myosin II filaments (Figure 1). Genomic sequence corresponding to the related enzyme MHCK-C was deposited in GenBank by Loomis and colleagues (accession number AAC31918). MHCK-C differs from MHCK-A and MHCK-B in that it lacks any significant amino-terminal domain upstream of the catalytic domain; conserved catalytic domain residues are present within approximately 30 residues of the methionine start codon of MHCK-C (Figure 1). Of this set of enzymes, MHCK-C is also unique in that it contains a large central domain of approximately 30 kDa that consists of highly repetitive stretches rich in serine, asparagine, proline, and glutamine. However, the strong similarity of MHCK-C to MHCK-A and MHCK-B in the conserved catalytic domain and the carboxyl-terminal WD repeat domain suggested that it might represent a third myosin heavy chain kinase in this organism.
To test this possibility at the biochemical level, we engineered and expressed an epitope-tagged construct that contains an amino-terminal FLAG epitope fused to codon 2 of MHCK-C. FLAG-MHCK-C was purified using a combination of ammonium sulfate fractionation and FLAG-peptide affinity chromatography. Purified FLAG-MHCK-C was subjected to SDS-PAGE. Western blot analysis with anti-MHCK-C polyclonal antibodies (Figure 2A) and with anti-FLAG monoclonal antibodies (not shown), and Coomassie staining (Figure 2A) confirmed the purification of the expressed protein, migrating at the correct size for MHCK-C. The FLAG-MHCK-C protein consistently suffered proteolytic cleavage during purification. In initial lysates, the major immunoreactive species migrated at ~ 100 kDa, but during purification a major clipped species formed with a size of ~ 35 kDa (Figure 2A). This species reacted with both anti-MHCK-C antisera and with anti-FLAG epitope antibodies (not shown), indicating this fragment to be an amino-terminal domain of MHCK-C. Addition of protease inhibitors during purification reduced but did not eliminate formation of this clipped MHCK-C.
Purified FLAG-MHCK-C readily phosphorylated native D. discoideum myosin II on the myosin heavy chain (MHC) in vitro (Figure 2B and 2C). No incorporation was detected on autoradiographs at the mobility of myosin light chains, supporting the model that MHCK-C, like MHCK-A, modulates filament assembly and not myosin motor activity (data not shown). To determine whether phosphorylation by MHCK-C affected myosin II assembly properties, samples of purified D. discoideum myosin II were incubated with MHCK-C in the presence and absence of ATP. Samples were then subjected to centrifugation to sediment myosin II filaments, and the proportion of myosin II in the pellet in each sample was quantified using SDS-PAGE and Coomassie blue staining as a measure of filament assembly (Figure 3A). Incubation of myosin II with MHCK-C in the absence of ATP resulted in assembly levels typical for purified Dictyostelium myosin, with 82% of the myosin sedimenting in the current set of assays (Figure 3B). Incubation of myosin II with MHCK-C in the presence of ATP resulted in substantial filament disassembly, with only 32% of the myosin II sedimenting following phosphorylation. These results confirm that MHCK-C can phosphorylate myosin II, and that this phosphorylation is capable of driving filament disassembly in vitro.
Myosin II phosphorylation experiments revealed two additional features of MHCK-C biochemical behavior. First, FLAG-MHCK-C autophosphorylates during the course of in vitro phosphorylation reactions (Figure 2B). Second, the activity of FLAG-MHCK-C appears to be very low in the initial stages of in vitro phosphorylation reactions, but then rises after approximately 5 minutes (Figure 2C). These attributes are reminiscent of the behavior of MHCK-A, which upon purification exists in an unphosphorylated low activity state. In vitro autophosphorylation of MHCK-A was found to increase the Vmax of the enzyme 50-fold [25]. To test for similar autophosphorylation regulation of MHCK-C, we tested the activity of FLAG-MHCK-C with and without an initial autophosphorylation step, towards the peptide substrate MH-1 (a 16-residue peptide corresponding to one of the mapped MHC phosphorylation target sites for MHCK A in the myosin tail). If FLAG-MHCK-C was not subjected to a pre-autophosphorylation step, 32P incorporation into the peptide displayed a similar lag phase as observed for myosin II phosphorylation (Figure 4A and 4B, open symbols). If FLAG-MHCK-C was pretreated with Mg-ATP for 10 min at room temperature, the lag phase for peptide phosphorylation was eliminated (figure 4A and 4B, closed symbols). These results support the model that autophosphorylation activates MHCK-C. Another feature reported earlier for MHCK-A activation is that myosin II itself stimulates autophosphorylation [25]. To test whether MHCK-C autophosphorylation is accelerated in the presence of myosin II, the stoichiometry of FLAG-MHCK-C autophosphorylation was evaluated in the presence and absence of myosin II filaments. Under the assay conditions here, myosin II did not significantly stimulate the rate of FLAG-MHCK-C autophosphorylation (Figure 4C). This result suggests that MHCK-C may be regulated in vivo by mechanisms distinct from those that regulate the activity of MHCK-A.
MHCKs have different subcellular localizations in interphase cells
To gain insights into the relative cellular roles and localization of MHCK-A, MHCK-B, and MHCK-C, we have evaluated the behavior of GFP fusions corresponding to each of these enzymes. Distribution of GFP-labelled MHCKs (GFP-MHCK-A, -B and -C) was examined in live AX2 cells (containing an endogenous mhcA gene). These GFP-MHCK expressing cells were able to sporulate and grow in suspension, indicating that at the expression level of these clonal cell lines, the expression of GFP-MHCKs in the AX2 cells does not detectably change myosin II expression or function. The fluorescence distributions of these cells were compared with cells expressing GFP-myosin II, obtained by transforming myosin null cells with a plasmid that carries GFP-mhcA-containing plasmid p102 (Materials and Methods) designated as GFP-myosin II cells hereafter.
The localization pattern of the GFP-MHCKs in the presence of myosin II was first compared to the distribution of GFP-myosin II cells in interphase (Fig. 5). Many cells of each transformation were examined (n > 50) and examples of the distribution of GFP-MHCK-A (Fig. 5-A, top), GFP-MHCK-B (Fig. 5-B, top) and GFP-MHCK-C (Fig. 5-C, top) are shown. GFP-myosin II distributed in the cytoplasm and enriched in a cortical layer in interphase as has been described earlier [7] is shown in Fig. 5-M (top). GFP-labelled MHCK-A and B distributed in the cytoplasm, and appeared to be excluded from the area that corresponded to nucleus. In contrast to GFP-Myosin II, GFP-labelled MHCK-A and B did not concentrate in the cell cortex (Fig. 5-M, top). Pixel intensities on a line drawn through the center of the cells allow a more quantitative comparison of the enrichment of GFP-MHCKs. A cortical distribution shows a distinctively increased accumulation of GFP fluorescent intensity at the cell edges, displaying two peaks flanking the cell cross-section as seen in the case of the GFP-myosin II cells (Fig. 5-M, middle). Out of the three MHCKs, only GFP-MHCK-C appeared to be concentrated in the cell cortex (Fig. 5-C, top), and had the fluorescent profiles containing the two flanking peaks (Figure 5-C, middle). GFP-MHCK-C also appears to be excluded from the nucleus, similar to that seen in cells expressing GFP-myosin II.
In free-moving cells, GFP-MHCK-A was frequently transiently enriched in the protruding edge (Fig. 5-A, bottom), and hence results in rather noticeable pseudopods at the anterior region compared with that in the GFP-myosin II cells. A time-lapse movie in Quicktime format illustrating this behavior is available as an additional file (see additional file 1). GFP-MHCK-B, however, displayed no indication of transient enrichment in any part of the cells while moving; instead it distributes homogeneously within cells (Fig. 5-B, bottom). The cells expressing GFP-MHCK-B appeared to have smooth cell edges because the fluorescence did not label the dynamic pseudopods at the leading edge of the cell, compared with that in GFP-MHCK-A cells. In contrast to MHCK-A and MHCK-B distribution, GFP-MHCK-C was frequently enriched in the posterior cortex of the moving cells (Fig. 5-C, bottom), as seen also for GFP-myosin II (Fig. 5-D, bottom). GFP-MHCK-C occasionally displayed transient enrichment in pseudopodial extensions as well (data not shown).
Dynamic localization of GFP-myosin II and GFP-MHCK-C in the cortex of living D. discoideum cells
As shown above, in interphase GFP-myosin II and GFP-MHCK-C expressed in the presence of myosin II both concentrate in the cell cortex. The actin-rich cortex is estimated to be approximately 0.1–0.2 μm thick in D. discoideum cells [26], similar to the thickness in other eukaryotic cells [27]. This dimension makes total internal reflection fluorescence (TIRF) microscopy an attractive tool to examine cortical GFP-labelled proteins at the cell-surface contacts. Total internal reflection occurs when light travelling in a medium with high refractive index encounters a medium with low refractive index beyond the critical angle, determined by the ratio of the two refractive indices according to the Snell's law [28]. In our experiments, the coverslip and the cells represent the media with high and low refractive indices, respectively. Under this condition, there is still an exponentially-decayed, evanescent wave penetrating into the D. discoideum cells. The typical depth of the evanescent wave is in the range of 100–200 nm away from the coverslip, which is suitable for exciting cortical GFP-proteins in living D. discoideum cells.
As examined by in vitro electron microscopy, negatively stained purified wild-type myosin II molecules formed bipolar thick filaments 0.6–0.8 μm in length [29]. Filament structures of similar size have also been observed via immunofluorescent microscopic observation of fixed cells [30]. Examination of live, GFP-myosin II expressing cells with a TIRF microscope (Fig. 6A) shows an accumulation of fluorescent rod-like shapes with an estimated length to be approximately 0.64 μm (standard deviation = 0.10, n = 146). This dimension is consistent with myosin II thick filaments measured from the in vitro electron microscopy photographs [29]. Using TIRF microscopy, these structures, putative filaments, are not apparent in GFP-3xAsp myosin II cells in which the three potential Thr phosphorylation targets have been replaced with Asp residues (data not shown). As this MHC mutation inhibits filament assembly [11], the inability of GFP-3xASP myosin to form such structures suggests that these TIRF-resolved structures are discrete myosin II filaments. Our TIRF analysis furthermore indicated that a few of the GFP-myosin II thick filaments move dynamically. Some filaments moved a long distance across the field with a velocity up to approximately 0.4 μm/sec before stopping. Other filaments seemed to be confined to a small region and eventually disappeared.
GFP-MHCK-C expressing cells (in the presence of myosin II), display punctate aggregates of fluorescence, with the longer dimension approximately 0.30 μm (standard deviation = 0.05, n = 165, Fig. 6B). The shorter dimension is about 0.15–0.2 μm, similar to what was observed in myosin II thick filaments. The GFP-MHCK-C fluorescent aggregates showed dynamic motility similar to that of GFP-myosin II aggregates. These structures could not be detected when GFP-MHCK-C was expressed in myosin II null cells (data not shown), suggesting that the formation of the GFP-MHCK-C punctate aggregates is myosin II-dependent.
Differential localization of GFP-MHCKs during cytokinesis
At the early stage of cytokinesis, GFP-myosin II reorganizes and is concentrated into the region that flanks the furrow located at the equatorial region of the dividing cells (Fig. 7-M, top). As the two daughter cells continue to separate (denoted the late stage of cytokinesis), GFP-myosin II persists in this zone, which becomes the posterior region of each of the two forming daughter cells (Fig. 7-M, bottom). GFP-myosin II, on the other hand, was not enriched in the polar region and therefore the polar regions displayed a smooth contour in fluorescent images, as described earlier [7].
Neither GFP-MHCK-A (Fig. 7-A) or -B (Fig. 7-B) was observed to be concentrated in the furrow region during any stage of cytokinesis; nor did they localize to the posterior region of the two daughter cells at the late stage of cytokinesis. Instead, GFP-MHCK-A was enriched in the protrusions extending from the poles of the dividing cells, which resulted in a more prominent appearance of the ruffling polar pseudopods throughout the cytokinesis process. GFP-MHCK-B, however, stayed homogeneously cytoplasmic during cytokinesis without any sign of enrichment in any region. It was excluded from the polar protrusions, as seen by the smooth contour of the poles (Fig. 7-B). Interestingly, as the cells progressed from the early to late stages of cytokinesis, GFP-MHCK-B appeared to be excluded from the furrow (Fig 7-B), as compared to the appearance of GFP-MHCK-A or GFP-MHCK-C.
The localization pattern of GFP-MHCK-C was different from the other two kinases (Fig. 7-C). At the early stage of cytokinesis, GFP-MHCK-C generally displayed a cytosolic distribution with some uniform cortical enrichment, but showed no sign of furrow concentration. However, at the late stage of cytokinesis, as soon as the two daughter cells begin to separate from each other, GFP-MHCK-C became detectable at the newly formed posterior regions of the two daughter cells. The spatial localization of GFP-MHCK-C is similar to that observed for GFP-myosin II at the late stage of cytokinesis (Fig. 7-B and 7-M). In summary, these three GFP-MHCKs have distributions that are temporally and spatially different, as summarized in the sketches shown in Figure 7 bottom.
To further illustrate the differential temporal localization, images of two cells expressing GFP-MHCK-C are compared to a cell expressing GFP-myosin II from the interphase (I, Figure 8) to the fully divided daughter cells (D, Figure 8). During interphase, all three cells display cortical distribution of the GFP-labeled proteins. When the cells progress into the quiescence stage (Q; equivalent to mid-mitosis), GFP-MHCK-C loses its cortical enrichment while GFP-myosin II typically remains cortical. When the cells begin to elongate (E, Figure 8), GFP-myosin II already concentrates at the equatorial region and remains there through the early stage (Ce, Figure 8), the mid-stage (Cm, Figure 8), and the late stage of cytokinesis. GFP-MHCK-C, however, displays no sign of furrow localization until the late stage of cytokinesis, when it suddenly appears at the posterior region of the daughter cells and stays for the duration of cell division (D). Time lapse movies in Quicktime format corresponding to each series in figure 8 are available as additional files (see additional file 2, additional file 3, and additional file 4).
Localization of GFP-MHCKs in the absence of myosin II
To understand whether the differential distribution observed on GFP-MHCK-A, -B and -C cells depended on the existence of myosin II, we expressed these kinases in myosin II null cells and compared the localization patterns. GFP-MHCK-A and -B showed identical localization in both interphase and cytokinesis cells regardless of the presence of myosin II in the cells (data not shown). GFP-MHCK-C, however, failed to localize to the cortex in interphase cells (Fig. 9-C, M null, top), and the two characteristic peaks were missing in the linescan. During free movement in the absence of myosin II, GFP-MHCK-C was not enriched in the posterior region of the cells (Fig. 9-C, M null, bottom). At the early stage of cytokinesis in myosin II null cells, GFP-MHCK-C was not enriched the furrow region (figure 10, top), similar to what was observed in the presence of myosin II as shown in Figure 7-C, top. However, when myosin II null cells progressed to the late stage of cell separation, GFP-MHCK-C was never localized to the constricting furrow or to the forming posterior region of the two daughter cells (Fig. 10-C, M null, bottom).