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Cell Biology. in the Article ‘‘Dictyostelium Myosin II Null September 2, 1997, of Proc Correction Proc. Natl. Acad. Sci. USA 95 (1998) 405 Cell Biology. In the article ‘‘Dictyostelium myosin II null September 2, 1997, of Proc. Natl. Acad. Sci. USA (94, 9684– mutant can still cap Con A receptors’’ by Carmen Aguado- 9686), the authors request that Figs. 1 and 2 be reprinted with Velasco and Mark S. Bretscher, which appeared in number 18, higher contrast. The figures and their legends are shown below. FIG. 2. Con A is capped more efficiently when two additional layers of cross-linking are added. Micrographs are as in Fig. 1. (a) Wild-type Ax2 cell. (b) mhcA2 (HS2206) cell. (c) Histogram showing the extent of capping Con A receptors (as defined in Fig. 1e; about 300 of each cell type were scored). Note that no substantial endocytosis of fluorescence occurs in these experiments. FIG. 1. Con A distribution on the surfaces of amoebae after a 30-min incubation (Nomarski images on left, Con A fluorescence on right). (a) Wild-type Ax2 cell. (b–d) mhcA2 (HS2206) cells showing '10% clearance from two regions at opposite ends of the cell (b), '30% clearance (c) and '50% clearance (d). (e) Histogram of the extent of capping Con A receptors in which all the cells (about 200) in a field were scored. The cartooned amoebae above the histogram indicate the extent of capping in each case: 0 signifies an uncapped amoeba, 1 ' 10% capped, 3 ' 30% capped, and so on. Note also that the cells in a–d all have some internalized fluorescent Con A. (Scale bar in d is 5 mm.) Downloaded by guest on September 24, 2021 Proc. Natl. Acad. Sci. USA Vol. 94, pp. 9684–9686, September 1997 Cell Biology Dictyostelium myosin II null mutant can still cap Con A receptors CARMEN AGUADO-VELASCO AND MARK S. BRETSCHER* Division of Cell Biology, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom Communicated by David D. Sabatini, New York University School of Medicine, New York, NY, July 11, 1997 (received for review April 1, 1997) ABSTRACT Cross-linked antigens on the surface of a METHODS motile cell cap at the trailing end of the cell. In Dictyostelium mhcA2 Dictyostelium discoideum, myosin II null mutants have previously been [strain HS2206, from D. Manstein and J. Spudich (11)], its parent Ax2, and a new mhcA2 line derived reported to be unable to cap Con A receptors, although they from DH1 (strain AC12, kindly provided by Terry O’Halloran are able to locomote. This finding implicated myosin II as an and Arturo De Lozanne, Duke University Medical Center, essential component of the capping mechanism, although not Durham, NC) were grown in HL5 medium (1% proteose of the machinery for locomotion. Here we show that myosin II peptoney0.5% yeast extracty1% glucosey2.4 mM Na HPO y null mutants do cap Con A receptors, albeit less efficiently 2 4 8.8 mM KH2PO4, pH 6.5) on plates. Cells were washed and than does wild type. This shows that cap formation is not allowed to attach to wells on glass slides (precoated with 0.1 absolutely dependent on myosin II and that a close mecha- mgyml BSA) for 10 min in KK2 buffer (20 mM potassium nistic relationship between capping, particle movement, and phosphate, pH 6.2, 2 mM MgSO4) supplemented with 1 mM cell migration may still exist. CaCl2 (KC buffer). They were then either (i) incubated in 25 mgyml fluorescent Con A (Vector) in KC containing 1 mgyml When cell surface receptors on motile cells are cross-linked by BSA (KCB) for 1 min, washed, and held in KC for various an appropriate antibody or lectin, they move away from the times before fixation in 2% formaldehyde in KK2 buffer for 30 front of the cell and collect at its tail to form a ‘‘cap’’ (1). min, or (ii) incubated in 25 mgyml biotinylated Con A (Vector) Capping is not induced by the binding of ligands to cell surface in KCB for 1 min, washed, incubated in rabbit anti-biotin proteins per se—non-cross-linking antibodies or lectins do not antiserum (1:500, kindly provided by Tim Mitchison, Univer- work—but rather results when surface proteins are cross- sity of California, San Francisco) in KCB for 3 min, washed, linked to form two-dimensional precipitates or patches. The labeled in 1:50 fluoresceinated donkey anti-rabbit Ig (Amer- molecular mechanism underlying the process in which patches sham) in KCB for 3 min, and finally washed in KCB before are driven toward the tail of the cell is uncertain, but two incubation in KC, all operations being carried out at about general classes of model exist: the motive force for capping is 23°C. Fixed cells were mounted in diazabicyclo[2.2.2]octane provided either by the cytoskeleton or by membrane flow (see, (DABCO) and viewed in a Zeiss microscope (403 objective) for example, refs. 2–6). and photographed using Kodak Technical Pan film (for No- A similar rearward movement of surface proteins occurs marski) or T-Max 400 film (for fluorescence). during cell migration. The feet of the cell are surface pro- teins—usually integrins—which bind to the substratum and RESULTS are effectively cross-linked by it. When the cell migrates, these Wild-type (Ax2) and mutant (HS2206 or AC12) amoebae were attached receptors are driven toward the rear of the cell. If the placed on glass slides, briefly labeled with fluorescein-labeled cell’s feet behave as do other cross-linked surface receptors, Con A, washed, and incubated at room temperature (about they would undergo capping and then drive the cell forward 23°C) for different times; they were then fixed prior to because they are immobilized by the substratum. Thus, in both observation. This showed that, after 30 min at 23°C, many capping and cell locomotion there is a movement of cross- wild-type cells had formed a cap of fluorescent Con A, whereas linked proteins toward the rear of the cell. This common mhcA2 cells had not, as reported (7, 8). However, careful feature implies that movement and capping may share a inspection of the surface fluorescence of individual mhcA2 common mechanism; if so, this would allow that mechanism to cells, compared with their Nomarski images, showed that most be investigated through experiments on capping, which are in of them had cleared the Con A receptors from at least a part some cases much easier and more revealing. of the cell surface. To quantitate these effects, fields of cells The connection between capping and cell locomotion has, were photographed and the fluorescence and Nomarski im- however, been challenged (7, 8). It had previously been found ages of each cell were compared: the proportion of each cell that there is only a single myosin II heavy chain gene in surface cleared of Con A receptors was judged by eye. Fig. 1a Dictyostelium discoideum (9). Cells lacking this myosin II gene, shows a typical capped wild-type amoeba; Fig. 1 b–d shows mhcA2, are still able to migrate (10), yet they are reported to mhcA2 cells in which regions of '10%, '30%, and '50% of be unable to cap cell surface Con A receptors (7, 8). These the cell surface, respectively, have been cleared of Con A results have been interpreted to demonstrate that capping and receptors after a 30-min incubation; mhcA2 and wild-type cells cell locomotion are mechanistically distinct processes. that were fixed before incubation with Con A displayed only Here we show that mhcA2 Dictyostelium cells in fact retain uniform fluorescence (not shown). A histogram of the pro- some capping activity. Therefore models invoking a relation- portion of cells displaying a particular degree of clearance is ship between capping and the rearward movement of a cell’s presented in Fig. 1e, which also shows that the two independent feet as it migrates remain tenable. mhcA2 amoeba strains behave similarly: both are able to cap their Con A receptors, but do so poorly compared with wild The publication costs of this article were defrayed in part by page charge type. We also examined those cells which had been labeled payment. This article must therefore be hereby marked ‘‘advertisement’’ in with Con A, but incubated for different times before fixation. accordance with 18 U.S.C. §1734 solely to indicate this fact. This showed that the extent of clearance of Con A increased © 1997 by The National Academy of Sciences 0027-8424y97y949684-3$2.00y0 PNAS is available online at http:yywww.pnas.org. *To whom reprint requests should be addressed. 9684 Cell Biology: Aguado-Velasco and Bretscher Proc. Natl. Acad. Sci. USA 94 (1997) 9685 FIG. 2. Con A is capped more efficiently when two additional layers of cross-linking are added. Micrographs are as in Fig. 1. (a) Wild-type Ax2 cell. (b) mhcA2 (HS2206) cell. (c) Histogram showing the extent of capping Con A receptors (as defined in Fig. 1e; about 300 of each cell type were scored). Note that no substantial endocytosis of fluorescence occurs in these experiments. no intracellular label is seen when three layers are used, even after an extended incubation of an hour. DISCUSSION Our studies, which have been carried out with two independent 2 2 FIG. 1. Con A distribution on the surfaces of amoebae after a mhcA cell lines with similar results, show that mhcA cells are 30-min incubation (Nomarski images on left, Con A fluorescence on able to cap Con A receptors. Previous experiments, using Con right). (a) Wild-type Ax2 cell. (b–d) mhcA2 (HS2206) cells showing A alone, had detected a large difference between wild-type and '10% clearance from two regions at opposite ends of the cell (b), mhcA2 amoebae in their abilities to cap Con A—which we '30% clearance (c) and '50% clearance (d).
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