Cell, Vol. 69, 951-962.

June

12. 1992, Copyright

0 1992 by Cell Press

The Dictyostelium Essential Light Chain Is Required for Myosin Function Richard S. Pollenz, Tung-Ling L. Chen, Leda Trivinos-Lagos, and Rex L. Chisholm Department of Cell, Molecular, and Structural Northwestern University Medical School Chicago, Illinois 60611

Biology

Summary A Dictyostelium mutant (7-i 7) that expresses less than 0.5% of wild-type levels of the myosin essential light chain (EMLC) has been created by overexpression of antisense RNA. Cells from 7-17 contain wild-type levels of the myosin heavy chain (MHC) and regulatory light chain (RMLC). Myosin isolated from 7-11 cells consists of the MHC with the RMLC associated in reduced stoichiometry, and binds to purified actin in an ATP-sensitive fashion. Purified 7-11 myosin displays calcium-activated ATPase activity with a V, about 15%-250/b of that of wild type, and a K, for ATP of 27 f 5 PM versus 83 f 30 PM for wild type. At actin concentrations as high as 17 FM, 7-77 myosin displays greatly reduced actin-activated ATPase activity. Phenotypically, 7-71 cells resemble MHC mutants, growing poorly in suspension and becoming large and multinucleate. When starved for multicellular development, 7-71 cells take several hours longer than wildtype cells to aggregate. Although multicellular aggregates eventually form, they fail to develop further. The cells are also unable to cap receptors in response to Con A treatment. Since cells expressing the EMLC are phenotypically similar to MHC null mutants, the EMLC appears necessary for myosin function, at least in part because it is required for normal actin-activated ATPase activity. Introduction Conventional two-headed myosin (myosin II) can be found in virtually all eukaryotic cells. In muscle it provides the force for contraction, while in nonmuscle cells it appears to be involved in cytokinesis (Fujiwara and Pollard, 1976; Mabuchi and Okuno, 1977; De Lozanne and Spudich, 1987; Knecht and Loomis, 1987) membrane capping (Pasternak et al., 1989b), and motility (Taylor and Condeelis, 1979; Wessels et al., 1988; Spudich, 1989). This myosin molecule is hexameric, consisting of two copies each of a heavy chain (MHC) and two different light chains. The MHC has two domains, a rod-shaped tail and a globular head region. The long a helical tail of the heavy chain is involved in both filament assembly (Huxley, 1963; De Lozanne et al., 1987) and localization of the myosin molecule within cells(Fukui etal., 1990; O’Halloranet al., 1990). The globular head domain has been shown to contain the sites for both ATP and actin binding (Adelstein and Eisenberg, 1980; Vibert and Cohen, 1988), as well as the light

chain binding sites. The site of light chain binding to the MHC has been localized to the head-rod junction by electron microscopy (Flicker et al., 1983; Tokunaga et al., 1987; Katoh and Lowey, 1989), biochemical techniques (Szentkiralyi, 1984; Sellers and Harvey, 1984; Mitchell et al., 1986), and molecular genetics (Mitchell et al., 1989; McNally et al., 1991). In skeletal muscle, the regulatory light chain (RMLC) can be removed by treatment with 5,5’dithiobis(2nitrobenzoic acid) (DTNB) without significantly affecting ATPase activity or actin binding (Weeds, 1969; Weeds and Lowey, 1971; Wagner and Weeds, 1977). In smooth muscle and nonmuscle systems, the stateof phosphorylation of the RMLC appears to control the actinactivated ATPase activity (Adelstein and Eisenberg, 1980; Kamm and Stull, 1985; Trybus, 1991), although in some systems there is evidence supporting a role for the tail in regulating ATPase activity (Collins and Korn, 1980; Kuczmarski and Spudich, 1980). A mutation in the RMLC of Drosophilacytoplasmic myosin, spaghetti-squash, causes a defect in cytokinesis and is lethal in the larval stage of development (Karess et al., 1991). The role of the other light chain in myosin function is less clear. Early studies of skeletal muscle myosin showed that these light chains could be removed by exposure to alkali (Weeds, 1967; Frederiksen and Holtzer, 1968; Gershman et al., 1969) leading to loss of actin binding and ATPase activity (Gershman and Dreizen, 1970; Dreizen and Gershman, 1970; Weeds and Lowey, 1971). Thus, the alkalisensitive light chains were named the “essential light chains” (EMLC). However, the requirement of light chains for actin binding and ATPase activity has been called into question by experiments demonstrating actin-activated ATPase in skeletal muscle myosin stripped of both light chains (Wagner and Giniger, 1981; Sivaramakrishnan and Burke, 1982). While the activity of myosin lacking the RMLC or both light chains has been studied, there are no reports of studies involving myosin lacking only the EMLC. Thus, the role of the EMLC in myosin function either in vitro or in vivo remains unknown. Dictyostelium has proven to be avery useful experimental system for the study of nonmuscle myosin because of the ability to obtain sufficient material for biochemical analysis, its ability to be manipulated by molecular genetic techniques (Crowley et al., 1985; De Lozanne and Spudich, 1987; Knecht and Loomis, 1987; Witke et al., 1987; Manstein et al., 1989; Jung and Hammer, 1990; Sun and Devreotes, 1991), and the availability of a variety of cellular assays to assess the motility properties of the cells (Wessels et al., 1988). These properties make it possible to correlate the phenotypes of the mutant cells with in vitro biochemical properties of the modified protein. In this paper we report the use of an EMLC cDNA (Chisholm et al., 1988; Pollenz and Chisholm, 1991) to generate a cell line deficient in EMLC expression. The mutant cells, produced by overexpression of an antisense EMLC mRNA, express less than 0.5% of the wild-type levels of the EMLC polypep-

Cell 952

A

about 20% of that of wild-type myosin, but fails to exhibit significant actin-activated ATPase. These results suggest that the Dictyostelium EMLC is required for myosin activity both in vivo and in vitro.

EMLC cDNA

Actin Promot

418 resistance gene

Results

1.8 kb 0.7

sense

Figure

1. Generation

of Dictyostelium

kb

antisense

Cell Line Deficient

in EMLC

(A) Schematic representation of the structure of the PAP-NEO-EX expression vector. The Dictyostelium actinl5 promoter was modified by oligonucleotide-mediated mutagenesis to insert a SamHI site 6 nt downstream of the transcription initiation site. The EMLC expression constructs were created by inserting a fragment of the EMLC cDNA in either the antisense (pRSP1) or sense (pRSP2) orientation into the SamHI siteof PAP-NEO-EX. The EMLC cDNAfragment used in these construct extends from the 5’end of the cDNA to a Hindlll site located 42 nt from the TAA termination codon. (6) Expression of EMLC mRNA in control and transformed Dictyostelium cells, RNA was harvested from Dictyostelium cells transformed with pRSP1 (“7-l 1”) and pRSP2 (‘sense”) and from untransformed control (“wildtype”) cells. Duplicate blots were hybridized to strandspecific EMLC probes as indicated. The 0.7 kb band represents the endogenous EMLC mRNA. The 1.6 kb band represents the RNA produced from the expression construct. (C) Expression of myosin subunits in wild-type, 7-11, and 7-11REV cells. Equal numbers of wild-type control AX4 cells, 7-7 7 cells, and the 7-l 7REV revertant of the 7-7 I antisense cells were dissolved in SDSPAGE sample buffer, separated on 12.5% polyacrylamide gels, transfered to nitrocellulose, and probed with NU-3 antibody, which detects the MHC, RMLC, and EMLC myosin subunit. Sound NU-3 antibody was detected with rz51]protein A and subsequent autoradiography. The regions of the autoradiogram corresponding to the MHC. RMLC, and EMLC are shown.

tide. The ceils show decreased motility and chemotaxis, fail to cap receptors, and exhibit a severe cytokinesis defect when grown in suspension. Myosin purified from these cells binds to actin and has a calcium-activated ATPase

Generation of Dictyostelium Ceil Lines Deficient in EMLC Thevector pRSP1 (Figure 1A) was transfected into Dictyostelium AX-4 cells and transformants selected in 20 uglml G418. Resistant colonies were plaque purified twice, then plated into plastic dishes and selected in increasing G418 concentrations to a final concentration of 250 uglml. This selection procedure ensured that the cells being analyzed represented clonal populationsof high copy number transformants. Cells that survived in high drug were split at least twice and then screened for decreased EMLC expression by Northern and Western blot analysis. Cell line 7-7 7 resulted from this selection and screening process. Northern blots of total RNA probed to detect sense strand RNA show high levels of the endogenous EMLC mFlNA (0.7 kb) in all samples except the antisense transformant 7-77 (Figure 18). The expression of the sense EMLC-CAT fusion RNA is evident as a band of 1.8 kb in size. The duplicate blot probed to detect antisense RNA shows a 1.8 kb band only in the 7-7 7 sample. Southern blot analysis using a 5’ flanking EMLC gene sequence probe shows the same pattern of hybridization to wild-type and 7-7 7 cell DNA. The observation of antisense mRNA but very little endogenous EMLC mRNA suggests that the endogenous mRNA is being degraded, most likely as a result of interaction with the antisense mRNA (Izant, 1989; Takayama and Inouye, 1990). Western immunoblots of whole cell protein showed greatly reduced levels of EMLC in the antisense sample (Figure 1C). Wild-type levels of RMLC and MHC are detected in all lanes, suggesting that the levels of these other myosin polypeptides are not significantly affected by reduced EMLC expression. The amount of EMLC peptide present in cells was estimated from the blots to be 0.5%2% of wild type. We do not know if this low level of expression is due to a small number of revertant cells in the population, or if all cells express very small amounts of the EMLC. However, despite the decreased EMLC expression, the MHC and RMLC were expressed at normal levels in 7-77 cells. To facilitate analysis of the in vivo consequences of reduced EMLC expression, we generated a phenotypic revertant of 7-7 7, which expresses nearly normal levels of EMLC. Cells from 7-77 were grown in the absence of G418 selection through four passages. Revertant clones showed levels of EMLC expression at least 75% of that seen in wild-typ-e cells (Figure 1C). To eliminate the possibility that some of the effects we observed were due to the high&e1 of G418 present in the growth medium, cell lines containing the pRSP2 sense expression construct were put through the same selection scheme that led to the identification of the 7-7 7 cell line. The pRSP2 transformed

Dictyostelium 953

EMLC

Mutant

7-l I AT,,

- - + + SPSPSPSP

wildtype - - +

+

B I

‘8

ii I

WlBIlr

Figure 2. 7-l 7 Myosin by ATP

Binds Actin Filaments

RMLC EMLC

and Binding

Is Inhibited

Actin and myosin were incubated together and pelleted with or without ATP. The composition of the resulting supernatant and pellet were analyzed by Coomassie blue-stained SDS-PAGE (A), and the stoichiometry of light chains was determined by probing Western blots with antibody that recognizes both light chains and the heavy chain (B). Myosin isolated from 7-l 1 cells and wild type sedimented with actin filaments in the absence of ATP, while in the presence of ATP, myosin appeared in the supernatant. The additional band in the supernatant of the minus ATP samples is hexokinase used to deplete ATP.

cells were phenotypically levels of EMLC.

126 (Figure 3A). The V,, of 7-7 7 myosin was consistently about 15%-25% of that observed for wild type. The K, values were 27 & 5 PM ATP for 7-77 myosin and 80 f 34 PM ATP for wild type, suggesting that 7-77 myosin interacted with ATP at least as well as wild type. This suggests that 7-7 7 has a functional active site, interacts with ATP as well as wild type, and is capable of hydrolyzing ATP at a rate 15%-25% of that of wild type. To eliminate the possibility that the calcium ATPase activity observed in 7-7 7 cells was due to some other calcium ATPase contaminating our myosin, we determined the level of calcium ATPase in cytoskeletons prepared from an MHC null cell line (Manstein et al., 1989). Since the preparations had no detectable calcium ATPase activity, myosin appears to be the primary cytoskeletal calcium ATPase in Dictyostelium cells. Actin activated the Mg*+ ATPase activity of wild-type myosin to 100 nmollminlmg at 5 uM actin, but less than 10 nmollminlmg for 7-77 myosin (Figure 3s). Even at 17 uM actin, 7-77 myosin showed activity of less than 10 nmollminlmg. Thus, the EMLC appears necessary for the actin-activated myosin ATPase of Dictyostelium myosin.

A

normal and expressed wild-type

Purified 7-71 Myosin Binds to Actin and Is Released by ATP Myosin from 7-77 cells, purified using standard procedures (Clarke and Spudich, 1974), carried 52% -c 8% of the wild-type levels of bound RMLC (Figure 26). Thus, the RMLC appears capable of associating with the MHC in the absence of the EM,LC, although the decreased stoichiometry suggests the association may not be entirely normal. When incubated with purified f-actin, the purified 7-77 myosin was pelleted, demonstrating the ability of 7-77 myosin to bind actin (Figure 2). ATP dissociated both 7-77 and wild-type myosin from actin filaments (lanes 3 and 7, Figure 28). These results show that myosin lacking the EMLC is capable of binding actin and can be released from actin by ATP. Since this experiment was performed at a ratio of 50 actin monomers to myosin, these experiments do not eliminate the possibility that 7-77 myosin could exhibit defective actin binding at lower actin concentrations, although the relative concentrations of actin to myosin in vivo are likely greater than 5O:l. 7-71 Myosin Exhibits Decreased Calcium-Activated ATPase and Lacks Actin-Activated ATPase Purified 7-77 myosin has a calcium ATPase activity with a V,, of 71 + 25 nmol of ATP hydrolyzed per minute per mg of protein, compared with the wild-type V,, of 323 f

0.12 0.1 0.06

.

0.06 0.04 0.02 0ir'l 0

Figure

3. ATPase

Activities

1

2 3 Actin (1M)

of Wild-Type

4

5

AX4 and 7-l 1 Myosin

Myosin was purified from wild-type (closed squares) and 7-l 1 cells (closed diamonds) and assayed for calcium-activated ATPase in the presence of varying ATP concentrations (A) and actin-activated ATPase (B). The 7-11 myosin exhibits reduced calcium-activated activity relative to wild type over ATP concentrations ranging from 2 uM to 1 mM. Double reciprocal plots of l/activity versus l/ATP concentration gave a V,, for wild type of 323 f 126 nmol/min/mg and 71 f 24 nmoll minlmg for 7-11 myosin. M, of the 7-l 1 mutant was 27 f 5 mM, while wild type was 60 f 36 mM. While wild-type myosin gave levels of actin-activated ATPase comparable to those previously reported (Clarke and Spudich, 1974; Griffith et al., 1967) significant actinactivated ATPase activity was not observed for 7-l 1 myosin.

Cdl 954

Figure 4. Growth Characteristics of Type, 7-77, and 7-77REV in Suspension

Wild

(A) Wild-type (closed star), 7-7 7REV (open circles), and 7-7 7 mutants (open diamonds) were inoculated into suspension culture and the number of cells per ml was determined with time in culture. The data are displayed as a semilog plot as an inset, (B-G) Samples of 7-7 7 (Band C), 7-77REV(D and E), and wild-type AX4 (F and G) cells were taken from the suspension cultures and examined by phase-contrast (B, D, and F) and DAPI immunofluorescence (C, E, and G). All cells were photographed at the same magnification. Note the large size of the multinucleate cells in (8) and (C).

0

10

20

30

40

60

60

70

HOURS

7-11 Cells Show Dramatically Decreased Growth Rates The parental, sense transformant, and revertant cell lines grew with doubling times of 13.3 f 1.4 hr, while the 7-7 7 mutant cells showed a greatly reduced doubling time of about 22.5 f 2.8 hr (Figure 4A). After about 4 days in suspension culture, thegrowth rateof 7-7 7 cells increased significantly, and they expressed increased levels of

EMLC. In contrast, when the 7-77 cells were grown on plastic, their doubling times were the same as wild type. When 7-77 cells were maintained in suspension, the cultures contained a large number of multinucleate cells, whic@ere 5-15 times the size of wild-type cells (Figures 46 and 4C), a phenotype similar to that observed for the MHC mutants (De Lozanne and Spudich, 1987; Knecht and Loomis, 1987). Revertant cells (Figure 4D, and Figure

Dictyostelium 955

Figure

EMLC

Mutant

5. lmmunofluorescence

Staining

of 7-l 7 and Wild-Type

Cells to Localize

Actin

and Tubulin

The 7-11 cells show an apparently normal cortical actin distribution (A and C) and normal interphase microtubule arrays (B and D) as compared with wild-type cells (E and F). In (D), all of the nuclei in a large, multinucleate cell appear to be undergoing mitosis synchronously.

4E) did not show the large multinucleate phenotype and were indistinguishable from the parental AX4 cells (Figures 4F and 4G). Similar to the MHC mutants, the 7-77 cells did not exhibit this large multinucleate phenotype when grown on a solid substrate. The 7-7 7 cells displayed a generally rounder shape and tended to form tighter colonies than did wild-type cells, suggesting that 7-77 cells are less motile than wild-type cells (Varnum and Soll, 1984). Organization of Cytoskeletal Components Is Apparently Normal in 7-77 Cells Figure 5 shows the actin (A, C, and E) and microtubule

(B,

D, and F) staining pattern of the mutant 7-77 (A-D) and wild-type AX-4 (E and F) cells grown in suspension. The large multinucleate 7-7 7 cells contain many microtubule organizing centers, each apparently associated with a nucleus. The 7-7 7 cells show an apparently normal cortical actin distribution (Figures 5A and 5C), and normal interphase microtubulearrays(compare Figures58 and 5F). Figure 5D shows a large multinucleate cell in which all of the nuclei appearto be undergoing mitosissynchronously. Since the micrographs in Figure 5 are at the same magnification, the differences in the size of the 7-77 mutants and the wild-type and revertant cells can be readily seen. Figure 6 shows the pattern of myosin (monoclonal anti-

Cdl 956

Figure

6. lmmunofluorescence

Staining

of 7-71,

Wild-Type,

and 7-TIREV

Cells to Localize

Myosin

Despite reduced EMLC expression, myosin localization in 7-l 7 ceils is cortical and appears normal compared with wild-type (C) and 7-7 7REV (D) cells. The 7-71 cells grown on plastic, which were apparently in the process of cell division, were examined by phase contrast (E and G) and immunofluorescence (F and H) to determine the pattern of myosin localization. Myosin localizes to an apparent cleavage furrow despite the absence of the EMLC.

body D-2) staining observed in 7-7 7 (A and B), wild-type (C), and revertant (D) cells grown in suspension. The localization of myosin is similar in all cells, displaying primarily cortical staining, with some faint cytoplasmic reactivity (Yumura et al., 1984; Yumura and Fukui, 1985; Nachmias et al., 1989). Cells from 7-7 7 grown on a solid substrate are smaller, and some appeared to have early cleavage furrows with myosin localization similar to that of wild-type Dictyostelium cells (Fukui and moue, 1991). However, we have not observed cells in which the cleavage furrow had contracted beyond the point shown.

Receptor Capping Is Defective in 7-71 Cells Like Dictyostelium cells lacking the MHC (Pasternak et al., 1989b; Fukui et al., 1990), 7-77 cells fail to cap their receptors when exposed to fluorescent concanavalin A (Con A). The Con A binds, but remains evenly distributed on the surface for at least 45 min of incubation (Figure 7). Cell Aggregation and Development Are Abnormal in EmC Mutants When starved to initiate multicellular differentiation, 7-7 7 cells aggregated much more slowly than wild type, consistent with the notion that 7-7 7 cells do not display normal

Dictyostelium 957

EMLC

Mutant

Figure

8. 7-l 1 Cells Never

Complete

Development

Wild-type AX4 (A) and 7-7 I(B) cells were developed on buffered agar. The morphology of the developing cells was recorded when cells had reached the terminal stage of development. While the AX4 cells produced normal-appearing fruiting bodies with spores located atop stalks, the 7-l 7 mutant never proceeded beyond the mound stage of development. In addition, aggregates wera smaller and more numerous. Insets show diagramatic side views of the terminal phenotype.

Figure

7. Receptor

Capping

Assay

in Wild-Type

and 7-77 Cells

Fluorescein-labeled Con A was incubated with cells for 45 min. Intensely labeled caps of Con A bound to lectin receptors appeared within 10 min of incubation in wild-type cells (A), but were absent in 7-71 cells incubated for 10 (6) or 45 (C) min.

motility and/or chemotaxis. The 7-77 mutant eventually formed multicellular aggregates typical of the mound stage of development (Figure 8) but failed to develop further. Migrating slugs or fruiting bodies were never observed. In contrast, the revertant strain aggregated normally and produced fruiting bodies indistinguishable from wild type. This behavior may reflect a number of different defects in locomotion or chemotaxis.

We have used overexpression

of an antisense

EMLC

mRNA to create a Dictyostelium cell line, 7-77, that expresses less than 0.5% of the wild-type level of the EMLC polypeptide and less than 5% of the wild-type level of mRNA. Since the cells express a low level of EMLC, it is possible that this low level of EMLC could provide sufficient activity for some critical cellular processes that do not appear to be significantly altered in the mutant. It is possible, for example, that the limited expression is somehow necessary for viability. Alternatively, the EMLC may not be required for growth under conditions of laboratory growth. The observation that 7-7 7 cells contain normal levels of both the RMLC and MHC suggests that myosin lacking the EMLC is reasonably stable. Studies of other cytoskeletal proteins such as tubulins (Burke et al., 1989) or keratins (Kulesh et al., 1989) suggest that altering the expression of one component of a multisubunit structure has dramatic effects on the level of the relevant partner proteins. In Drosophila, it has been shown that the stoichiometry of thick and thin filaments is critical for myofibril assembly (Beall et al., 1989). While little is known about the mechanism by which assembly of myosin subunits occurs, our observations make it seem unlikely that there is a stringent feedback mechanism that controls the levels of myosin subunit expression, since dramatically altering the level of one myosin subunit appears not to affect the level of the

Cell 958

other two. We have also overexpressed the EMLC in wildtype Dictyostelium cells and see no change in the levels of RMLC or MHC, despite a 5-fold higher level of EMLC (R. S. P. and R. L. C., unpublished data). Previous studies on the susceptibility of the EMLC to proteolysis (Stafford et al., 1979) or sulfhydryl reagents (Hardwicke et al., 1982) and cross-linking studies (Hardwicke et al., 1982, 1983) have suggested that the RMLC protects the EMLC, perhaps by binding to the MHC through interactions with the EMLC. In six different preparations of isolated 7-l 1 myosin, the amount of RMLC associated with MHC was 52% f 8% of that seen in wild type. While the EMLC does not appear to be required for association of the RMLC and MHC in vivo, the absence of EMLC results in decreased stability of the RMLC-MHC interaction. The ability to hydrolyze ATP and generate force is a hallmark of myosin’s enzymatic activity (Eisenberg and Hill, 1985). Because the 7-77 myosin exhibits a Ca2+activated ATPase with a V,, about 15%-25% of that seen in wild-type myosin preparations, it appears that the EMLC is not required for ATP hydrolysis. Since 7-7 7 myosin does have a small amount of EMLC, it is possible that some of the calcium ATPase activity is due to heads carrying both light chains. ltis unlikely, however, that a maximum of 5% of the heads could produce 15%-25% of the wild-type activity. Although our experiments do not rule out the possibility that 7-7 7 myosin could exhibit actin activation at higher actin concentrations, it is clear that, at a minimum, 7-77 myosin is less sensitive to actin activation than is wild-type Dictyostelium myosin. Since isolated 7-7 7 myosin carries only about 50% of the wild-type level of RMLC, it is difficult to determine if the decreased actin activation is dueonly to the absenceof the EMLC, or if the decreased RMLC levels also contribute to this result. However, since the reduction in actin-activated ATPase is much greater than 50%, it seems unlikely that the reduction seen is due only to loss of the RMLC. Indeed, the RMLC can be removed from skeletal muscle myosin without affecting actin-activated ATPase (Weeds, 1969; Weeds and Lowey, 1971; Wagner and Weeds, 1977). In smooth muscle, it has been suggested that actin-activated ATPase is suppressed by the RMLC, and that suppression is relieved by phosphorylation. Thus, removal of the RMLC might actually be expected to increase actin-activated ATPase (Rees and Frederiksen, 1981; Sellers et al., 1980; Hasegawa et al., 1990) although this has not been directly tested with Dictyostelium myosin. Since 7-77 myosin does not have significant levels of actin-activated ATPase under conditions where activation of the wild type is observed, the EMLC is necessary for normal levels of actin-activated ATPase. It has been shown (Lymn and Taylor, 1971; Jackson et al., 1986) that actin increases the ATPase activity by stimulating ADP and/or P, release following ATP hydrolysis, producing increased ATP turnover. Since the standard assays for myosin ATPase activity measure the release of inorganic phosphate, it is possible that the decreased calcium ATPase activity of 7-7 7 myosin could be due to either a reduced rate of hydrolysis or a decreased rate of

ADP and/or Pi release. Because we have not quantitatively determined the affinity of 7-7 7 myosin for actin, we cannot eliminate the possibility that an altered actin affinity contributes to the defective actin-activated ATPase. However, since 7-77 myosin can be pelleted in an ATP-sensitive fashion in an actin binding assay at levels of actin comparable to those used in the ATPase assays, we do not believe that a defect in actin binding is the most likely explanation for the defect in actin-activated ATPase. Studies on smooth muscle myosin suggest that the EMLC may be located near or involved in forming the active site (Okamoto and Sekine, 1986; Okamoto et al., 1986). It is tempting to speculate that the EMLC may be involved in facilitating product release, particularly in response to actin binding. Several lines of evidence suggest that the RMLC associates with the myosin head in close proximity to the EMLC (Hardwicke et al., 1983; Katoh and Lowey, 1989; Goodwin et al., 1990). Phosphorylation of the RMLC stimulates actin-activated ATPase activity of Dictyostelium myosin (Griffith et al., 1987). The reduced actin-activated ATPase in 7-7 7 myosin raises the possibility that the EMLC plays a role in transducing the regulatory signal generated by phosphorylation of the RMLC. Alternatively, the absence of an EMLCon the myosin head in 7-7 7 myosin could alter the conformation of the myosin head, affecting the ability of the RMLC to be phosphorylated. Griffith et al. (1987) have shown that myosin dephosphorylated by a partially purified myosin light chain phosphatase has dramatically reduced actin-activated ATPase. Based on preliminary experiments suggesting that the RMLC can be phosphorylated in 7-77 cells (R. S. P. and R. L. C., unpublished data), we favor the possibility that the EMLC plays a role in transmitting the regulatory signal from the RMLC to the active site, although the possibility exists that absence of the EMLC may affect the level of RMLC phosphorylation. MHC phosphorylation has also been shown to regulate ATPase activity in Dictyostelium (Kuczmarski and Spudich, 1980). We have not investigated the levels of MHC phosphorylation in the 7-7 7 mutant, and thus it is possible that the absence of the EMLC somehow affects MHC phosphorylation, which could in turn affect the levels of actin-activated ATPase. While the absence of EMLC appears to reduce myosin’s actin-activated ATPase, the mechanism by which this occurs remains to be elucidated. Cells from 7-7 7 exhibit many characteristics of the Dictyostelium MHC mutants, including defects in cytokinesis, motility, and receptor capping (Wessels et al., 1988; Pasternaket al., 1989b; Fukui et al., 1990). Like the MHC null mutants (Manstein et al., 1989) or cells expressing only heavy meromyosin (HMM) (De Lozanne and Spudich, 1987) in suspension cultures, the 7-7 7 cells grow poorly and form large multinucleate cells. These data are consistent with the idea that cells lacking the EMLC subunit are defective in cytpkinesis. It has been postulated that the MHCcuII mutants grown on a substrate divide by a mechanism Emed “traction-mediated cytofission” (Fukui et al., 1990). The formation of a presumptive cleavage furrow is consistentwith theobservationsof Fukuiand lnoue(1991), who have recently described two different components

Dictyostelium 959

EMLC

Mutant

involved in formation of the cleavage furrow. They suggest that the initial constriction is due to the formation of an actin-rich structure parallel to the orientation of the spindle. Subsequent addition of myosin completes the contractile ring, which then constricts in a myosin-dependent fashion. The localization of 7-7 7 myosin presented here is consistent with this idea. We suggest that the contractile ring forms normally in 7-77 cells. However, since the 7-77 myosin lacks actin-activated ATPase activity, it is unable to generate sufficient force to “contract” the contractile ring. Our studies of MHC localization show that 7-77 myosin localizes normally within the cell. This supports the results of O’Halloran et al. (1990) and Egelhoff et al. (1991) suggesting that the myosin tail and not ATPase activity is critical for localization of myosin. Because 7-77 myosin localizes normally to the cell cortex during interphase and to the cleavage furrow during cytokinesis without significant actin-activated ATPase activity, it seems unlikely that myosin provides the motive force responsible for its own movement. When plated at low density on plastic, the 7-77 cells form relatively small, tight colonies when compared with wild-type or revertant cell lines. In addition, the cells are generally less elongated in appearance than are wild type. When starved, 7-7 7 cells take several hours longer than wild-type cells to form multicellular aggregates, and then fail to progress further in multicellular development. These characteristics suggest a motility defect similar to the MHC mutants, which have been shown to exhibit defective translocation and chemotaxis (Wessels et al., 1988). Preliminary results from computer-assisted analysis of 7-7 7 motility suggests that these cells have decreased rates of movement and impaired chemotaxis (R. L. C., D. Wessels, and D. Ft. Soll, unpublished data). These defects do not result from a generalized disruption of the 7-7 7 cytoskeleton, as both the tubulin and actin staining patterns in 7-77 cells appear normal. This extends the previous observations of Knecht and Loomis (1987) and De Lozanne and Spudich (1987) that myosin was required for the morphogenesis that occurs during the multicellular phase of Dictyostelium development. Conclusions Myosin has been shown to play a central role in a number of motility processes in a variety of cell types. In Dictyostelium cells, MHC mutants have clearly established the requirement of myosin II for cytokinesis, translocation of cells at normal rates, normal chemotaxis, and receptor capping. The results presented here extend this work in two important ways. First, the somewhat controversiai contribution of the EMLC to myosin function has been clarified. At least for the nonmuscle myosin of Dictyostelium, myosin lacking the EMLC is similar to wild type with regard to ATP binding, actin binding, and ATP-mediated release from actin. Myosin lacking the EMLC can hydrolyze ATP to ADP and P,, but at a rate only 150/o-25% of that observed for Dictyostelium myosin consisting of all three subunits. In contrast, actin-activated ATPase activity is less than 10 nmollminlmg, suggesting that the EMLC is

necessary for the physiologically important ATPase activity of myosin. The effect of the EMLC on actin-activated ATPase could be either direct or indirect by affecting the ability of the RMLC or MHC to be phosphorylated. Second, the results presented here support the widely held notion that actin-activated myosin ATPase is required for cytokinesis, receptor capping, and multicellular development. While this point seems likely, based solely on our knowledge of myosin’s activities, experimental evidence to support this notion is limited. The work of Kiehart and Pollard (1984) correlated decreased actin-activated ATPase activity with decreased contraction of actomyosin gels. Since 7-7 7 myosin is apparently normal with regard to several myosin functions, our characterization of cells carrying myosin lacking actin-activated ATPase provides experimental support for the belief that actin-activated ATPase activity of myosin is critical for many of its functions in living cells. In conclusion, the data presented here support the notion that the EMLC is indeed essential for Dictyostelium myosin function. Experimental

Procedures

Cells and Growth Conditions Dictyostelium discoideum AX-2, AX-3, and AX4 strains were used. Cells were plaque purified by mixing 20-30 cells with Klebsiella aerogenes and spreading onto SM plates (Sussman, 1987). When plaques had grown to 1 cm, cells from the feeding zone were picked and replated into HL-5 media on plastic petri dishes. Cells grown in suspension were shaken at 250 rpm at constant temperature (22%). Construction of Antisense EMLC Expression Vector The Actin 15 promoter was isolated as a 300 nt Bglll-Hindlll fragment from pSC79 (Knecht et al., 1986) and cloned into Bluescript pSK. This fragment contains the first 6 aa of the acfin 15 gene and 280 nt of upstream promoter sequence. Site-directed oligonucleotide mutagenesis was used to replace all but 6 nt of the 5’ untranslated region and ATG translation initiation site with a BamHl site. Thevector pPAV-NE0 (Hopkinson et al., 1989) was cut at the Xhol and BamHl sites and the modified actin 15 promoter fragment was ligated to create the expression vector PAP-NEO-EX, shown in Figure 1, It contains the modified actin promoter, a promoterless CAT gene, and a G418 resistance cassette. To create a transformation plasmid that would produce antisense EMLC mRNA, the 484 nt BamHI-Hindlll fragment of EMLC cDNA (Pollenz and Chisholm, 1991) was cloned into the BamHl site of PAP-NEO-EX. Because the 3’region of the cDNA contained putative AATAAA polyadenylation signals when transcribed in the antisense direction, this region could not be included in theconstruct. Expression of this plasmid, designated pRSP1 (Figure l), should result in a 1.8 kb mRNA containing 484 nt of antisense EMLC sequence continuous with 1.3 kb of the CAT gene. Transformation Dictyostelium cells were transformed using calcium phosphate coprecipitation and a glycerol shock as described by Knecht and Loomis (1987). Cells were selected in G418 at concentrations indicated in the text. RNA Isolation and Northern Analysis RNA was isolated from cell pellets by phenol-chloroform extraction (Hopkinson et al., 1989). For Northern blot analysis, lo-20 ng of total RNA was separated electrophoretically on a 1% agarose gel containing 6% formaldehyde and transfered to nitrocellulose in 10 x SSC (1.5 M NaCI. 0.15 M sodium citrate). Blots were baked, prehybridized, and probed with high-specific-activity, strand-specific RNA probes. Western Blot Analysis Samples of total Dictyostelium 2-5 x lo5 cells, adding lo-20

protein were t~l of cracking

generated by pelleting buffer (10 mM Tris [pH

Cell 960

7.61. 5% SDS, 10% 5-mercaptoethanol, 25% glycerol), and boiling for 3 min. Protein samples were electrophoresed on pofyacrylamide gels and transferred to nitrocelluose. Blots were stained with a myosin antibody (NU-3) (Chisholm et al., 1988) and visualized by incubating stained blots with [‘251]protein A (gift of Dr. J. Battles). To determine the relative amount of myosin present in protein samples, autoradiographs were scanned with a laser densitometer (LKB) and the relative levels of MHC, RMLC, and EMLC weredetermined. To ensure that autoradiographic exposures were in the linear response range of the film, at least two different exposures of each autoradiograph were scanned. Antibodies Antibodies specific for Dictyostelium MHC were the generous gift of Drs. Y. Fukui (Northwestern University), David Knecht (University of Connecticut), and J. Condeelis (Einstein). Dr. Fukui also provided an actin-specific monoclonal antibody. For Western blot analysis, polyclonat antibodies generated against Dictyostelium myosin (NU-3) were used. In wild-type cells, NU-3 reacts with the MHC, RMLC, and EMLC in a 1:l:l ratio when detected with [‘251]protein A. Microtubules were stained using an antibody raised against a Dictyostelium a-tubulintrpE fusion protein produced in E. colt Morphological Analysis For tubulin, myosin, and actin staining, cells were harvested about 2 hr after being split. Cells were placed on uncoated coverslips and allowed to attach for 10 min. Cells were flattened by the agarose overlay technique and fixed using the two-step method described by Fukui et al. (1987). Following incubation with the appropriate primary and secondary antibodies, coverslips were mounted in Gelvatol containing 100 mglml DABCO, and photographed with T-MAX 400 film. DAPI staining was performed according to the manufacturer’s directions. Receptor capping assays were performed as described by Pasternak et al. (1989b) except that cells were fixed in 2% formaldehyde in MES buffer for 15 min, followed by 3 min in -20°C methanol.

ATPase Assays Calcium-activated ATPase was determined using 2 pg of purified myosin in a reaction mixture containing 10 mM Tris (pH 8.0) 0.5 M KCI, 10 mM CaC& or 1 mM EGTA and ATP to final concentrations ranging from 2 uM to 1 mM, with IZP]ATP in trace amounts. Inorganic phosphate was determined as described by Pollard and Korn (1973). To a 0.1 ml reaction, a 0.2 ml mixture of isopropanol and benzene (1:l) was added at the end of the reaction and followed by 0.05 ml of 4% silicotungstic acid and 3N sulphuric acid to denature the enzyme. The phosphate released by hydrolysis of ATP was then reacted with 0.02 ml of 10% ammonium molybdate, by vortexing. The phosphate was quantitatively extracted with isopropanol-benzene (1 :l). The amount of radioactive phosphate in the organic phase was determined by scintillation counting. Actin-activated ATPase was determined as described by Clarke and Spudich (1974). Actin Binding Assays Actin was purified from rabbit skeletal muscle as described by Pardee and Spudich (1982). The binding of myosin to actin filaments was assayed as described by Kieharl and Feghali (1986). G-actin (2.2 PM) was polymerized by the addition of salt (0.6 M KCI, 4 mM MgCI, in 20 mM imidazole-Cl [pH 7.01, 3 mM NaN3, 0.1 mM DTT) and incubated at 37OC for 30 min. Samples were either supplemented with 2 mM ATP or depleted of ATP by incubation with hexokinase (10 U/ml) for 5 min at room temperature. After chilling to O°C, the actin was mixed with myosin (39 nM for 7-11, 50 nM for wild type assuming a molecular mass of 480 kd), incubated for 10 min at 0°C layered onto a sucrose cushion (30% in actin polymerization buffer, with or without ATP), and then sedimented in a Beckman TL-100 ultracentrifuge for 20 min at 245,000 x g, The polypeptide composition of the supernatant and pellet were analyzed by Coomassie blue-stained SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the light chain stoichiometry was determined by Western blot analysis using the NU-3 antibody, visualized by ~*?]protein A. Acknowledgments

Myosin Purification Myosin was isolated by a modification of Clarke and Spudich (1974). Wild-type cells were grown in suspension to a density of 5 x 1 d cells/ ml. Cells from 7-7 7 were grown on 15 cm plastic dishes and harvested by scraping with a rubber policeman before they reached confluency. After washing with wash buffer (5 mM Na*HPO,, 5 mM KHIPO$, cells were suspended in Lysis buffer A (40 mM Na2Pz0,, 45% sucrose, 1 mM f+mercaptoethanol, 1 mM NaN3, 5 mM EDTA, 10 mM triethanolamine [pH 7.5],1 mM TLCK [Na-p-tosyl-L-lysine chloromethyl ketone], 1 mM TPCK IN-tosyl-L-phenylalanine chloromethyl ketone], 1 mM leupeptin) and sonicated to disrupt cells. The sonicated solution was centrifuged (30 min, 38,000 x g), and the resulting supernatant was centrifuged at high speed (2 hr, 150,000x g). The supernatant was recovered and dialyzed overnight (10 mM PIPES [pH 7.0],50 mM KCI, 5 mM EDTA, 0.5 mM 8-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM NaNa) to allow assembly of myosin filaments. The filamentswere pelleted bycentrifugation (30 min, 38,000 x g) and homogenized in 10 mM triethanolamine (pH 7.5) containing 50 mM KCI. Following addition of an equal volume of KI buffer (10 mM triethanolamine [pH 7.51, 1 mM EDTA, 1 mM dithiothreitol [DTT], 10 mM ATP, 10 mM MgCI,, 2 mM CaC&, 1.2 M KI), the mixture was homogenized again and centrifuged (30 min, 150,ooOx g). The supernatant was made 55% ammonium sulfate and stirred on ice for 20 min, and the precipitate was recovered by centrifugation (20 min, 38,000 x g). The pellet was resuspended in 10 mM triethanolamine (pH 7.5) 50 mM KCI, added to an equal volume of KI buffer, and clarified by centrifugation (30 min, 150,000x g). The supernatant, which consisted primarily of actin and myosin, was fractionated on S-500 (Pharmacia) equilibrated in buffer B (10 mM triethanolamine [pH 7.51, 1 mM EDTA, 0.6 M KCI, 1 mM DTT, 1 mM NaN3, 0.2 mM ATP). Fractions containing myosin were pooled and dialyzed overnight into 10 mM Tris-maleate (pH 6.5) 1 mM DTT, 50 mM KCI. Following dialysis, MgC& was added to 10 mM, incubated 1 hr, and centrifuged (30 min, 38,000x g). The pellet was resuspended by homogenizing in storage buffer (10 mM triethanolamine [pH 7.51, 10 mM EDTA, 0.5 M KCI, 1 mM EDTA, 1 mM NaN3) and clarifying by centrifugation (15 min, 25.000x g). Protein concentrations were determined by the BioRad Assay (BioRad) and confirmed by Western blot analysis.

We thank Dr. E. Kuzmarski for helpful comments and a generous gift of NU-3 antibody; Dr. J. Bartles for providing [‘251]protein A; Dr. Y. Fukui for many discussions, comments on the manuscript, and gifts of antibodies; Drs. J. Condeelis and D. Knecht for myosin antibodies; and Dr. K. Green for gifts of DAPI and rhodamine palloidin. We also thank an anonymous reviewer for significant scientific and editorial contributions to this paper. This work was supported by NIH grant GM39264 to R. L. C. The costs of publication of this paper were defrayed in part by the payment of page charges. This paper must therefore be hereby marked “adver?isemenY’ in accordance with 18 USC Section 1734 solely to indicate this fact. Received

August

5, 1991; revised

April 8, 1992.

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