Sequential fluorescence images of an Ax2 cell with an extending protrusion induced by quinine. Cells had been stained with RH-795 10 minutes before quinine application. Time=0, 3.1, 6.2, 9.2, 15.4, 21.5, 27.7, 40.0 and 52.3 seconds from left to right. The estimated area of the apical membrane region with the fluorescence intensity higher than that of the original cell membrane was almost constant throughout the period of elongation. For this calculation, it was assumed that the protrusion is in the shape of a hemisphere attached to a cylinder and that the sections are on the horizontal median plane of the protrusion. In the presence of quinine, the difference in the fluorescence intensity between the contractile vacuole membrane and plasma membrane was usually not as large as without quinine, partly because the contractile vacuole membrane is integrated in the plasma membrane upon its expulsion, as shown here and Fig. 6C. Bar, 10 μm. Movies of this and other sequential images are available at http://www.biologists.com/JCS/movies/jcs1984.html).

Nomarski contrast (left column) and immunofluorescence (right column) images of control cells (A,B,E,F) and quinine-treated cells (C,D,G,H) stained with an antibody against the 100 kDa subunit of V-ATPase (A,B,C,D) or an anti-calmodulin antibody (E,F,G,H). Cells were fixed after a 5 minute incubation with 2 mM quinine. Insets in C and D show an enlarged contractile vacuole in a quinine-treated cell. Bar, 5 μm.

Sequential images showing concanavalin A-tagged fluorescent beads attached to the cell surface during elongation of protrusion. Fluorescence is shown in red and Nomarski images in green. Intervals between successive frames are 19.4 seconds. Bar, 10 μm.

Dependence of the rate of protrusion elongation on the sorbitol concentration in buffer. Vertical bars indicate means±s.e.m. (n=31, 26, 26, 22, 19, from left to right, respectively).

(A, B) Phase-contrast images of mhc⁻ cells 1 minute after exchange of the medium to buffer without (A) or with (B) 2 mM quinine. Bar, 10 μm. (C) Sequential fluorescence images of an mhc⁻ cell showing expulsion of a contractile vacuole. The cell had been stained with RH-795 10 minutes before quinine application. A patch of fluorescence stayed on the cell membrane at the site of expulsion of one of the contractile vacuoles. Intervals between successive frames are 13.7 seconds in the upper panel. The intervals in the lower panel are 0.55 seconds to show the moment of discharge at shorter time intervals. Bar, 5 μm.

Changes in the distribution of GFP-myosin II during the formation, elongation and retraction of protrusions in the presence of quinine. (A) Fluorescence is shown in red and Nomarski images in green. Time=0, 5.0, 10.0, 15.0, 20.1, 25.1, 70.9, 142.5 seconds. (B) A cell with a retracting protrusion and a large protrusion that is about to stop extending. The small protrusion is entirely covered with a layer of GFP-myosin, whereas the large protrusion has a constriction at its base, from which a band of GFP-myosin II extends towards the tip. Intervals between successive frames are 47.7 seconds. Bars, 10 μm.

The distribution of the distance over which the mass of solid cellular materials protruded from lysed cells in the presence of quinine. The buffer contained 1% methyl cellulose to increase the viscosity. The distance between the tip and the base of the protruded solid materials was measured (indicated with the arrows in the insets). (A) Ax2 cells; (B), mhc⁻ cells. Bar, 5 μm. Mean±s.e.m. is 5.8±0.4 μm, n=62 (A), 3.4±0.2 μm, n=70 (B), with the difference being significant (P<0.001).

Nomarski (left) and fluorescence (right) images of cells stained with fluorescent-phalloidin. (A,B) control, (C,D,E,F) quinine, (G,H) quinine and cytochalasin A. The image shown in F is a projection of 30 confocal sections at 0.4 μm intervals covering the entire depth of the cell. Insets in F show the sections on the distal and proximal sides of the protrusion to show that the stripes seen near the tip of the protrusion in F make up a spiral. Bar, 5 μm.

Sequential images of GFP-ABD cells exposed to quinine (A,B) and fluctuation of the velocity of protrusion elongation accompanying detachment of GFP-ABD layers from the apical membrane of the protrusion (C,D). Fluorescence is shown in red and Nomarski in green. (A) A cell showing successive extension and retraction of protrusions. (B) A cell in locomotion by continuous elongation of a large protrusion. Intervals between successive frames are 10 seconds in A and B. The correlation between the protrusion and a contractile vacuole can be seen in B and for the first two protrusions in A, whereas no contractile vacuole is visible that would have triggered the third protrusion in A. This may be because either it is out of focus or the protrusion was formed independently of a contractile vacuole. In B, a particle (arrowhead) that was accidentally attached or located very close to the cell surface can be seen to move along with the elongating protrusion, which may be indicative of the presence of membrane components that flow forwards. Coexistence of membrane components showing rearward (c.f. Fig. 3) and forward movements have been demonstrated in amoebae and mammalian cells (Grębecki, 1986; Sheetz et al., 1989). Bars, 10 μm. (C,D) Confocal images of another GFP-ABD-expressing cell were taken at intervals of 0.89 seconds, and the images within the thin window (width 1 μm, shown by rectangle in D) on successive frames are arranged from left to right to illustrate the progression of the leading front of the protrusion, the accumulation and rearward propagation of the fluorescence, and their temporal relationship (D). For clarity, D has been expanded twofold in the vertical direction. Horizontal bar in C and vertical bar in D, 5 μm; horizontal bar in D, 10 seconds.

Bleb formation in the presence of quinine and cytochalasin A. Fluorescence of GFP-myosin II is shown in red, Nomarski in green. Bar, 5 μm. Intervals between successive frames are 2.7 seconds.

(A) Sequential phase-contrast images of a mechanically-dissociated slug cell that shows continual locomotion in a thin layer of phosphate buffer without quinine. Intervals between successive frames are 8.0 seconds. The mean velocity of the cell front is 0.76 μm/second. (B) Nomarski (top) and fluorescence (bottom) images of a dissociated slug cell with an elongated protrusion stained with fluorescent phalloidin. Bar, 10 μm.