Cells of D. discoideum spreading on differently coated glass surfaces (A–F) and non-spreading cells on passivated surfaces (G and H). Structures in the cell cortex were visualized by TIRF microscopy. (A and B) cleaned glass; (C and D) glass coated with dichlorodimethylsilane; (E and F) coated with fluoralkane; (G and H) glass passivated with F-127 (G) or polyethyleneglycol, PEG 2000 (H). The actin network beneath the substrate-attached cell surface was labeled with GFP-LimEΔ, except for the control cell shown in (F), which expressed free GFP in the cytoplasm. Arrowheads in (A and D) indicate dense accumulation of actin in front regions of the cells. Exposure time 9 milliseconds. Bars, 5 µm.

Attachment and spreading of cells labeled with GFP-myosin-II and mRFP-LimEΔ The two time series of (A and B) exemplify the establishment of front-to-tail polarity in spreading cells. The red LimEΔ label serves as a front marker and the green filaments of myosin-II are accumulating at retracting tail regions. The cell in (A) touches the substrate first with a small area of its surface (16 seconds frame). While this area expands on the substrate, filopodia and broader protrusions separately contact the substrate (arrowheads in the 57 and 74 second frames). Multiple front regions are formed, from which myosin-II is eliminated (85 seconds frame). Subsequently, the cell becomes polarized into one actin-rich front and a myosin-II enriched tail region. The cell in (B) attaches first with filopodia and subsequently protrudes three front regions in contact with the substrate (23 seconds frame). This sequence illustrates the interplay of protruding and retracting regions. In the 66 seconds frame, two of the fronts are retracting. In the 105 seconds frame the third front, now also enriched in myosin-II, retracts while a new front is generated on its upper side. At 218 seconds the cell is in a state of overall contraction, before at 287 seconds a new, broad front region is formed. The glass surface was coated with fluoralkane. Cells were recorded by spinning-disc confocal microscopy with the plane of focus at the substrate-attached cell surface. Frame-to-frame interval was 1 second. Exposure time for GFP was 30, for mRFP 60 milliseconds. Time is indicated in seconds after the first contact of a cell with the substrate surface. Bars, 5 µm.

A spreading cell labeled with GFP-myosin-IB (green) and mRFP-LimEΔ (red). This cell, attaching first with filopodia, forms blebs in contact with the substrate surface (open arrowheads). These round protrusions contain no distinct structures enriched in myosin-IB or actin. Subsequently, both proteins are accumulating, the blebs attain an irregular shape (closed arrowheads), and get integrated into the spreading cell. In the bottom panels the merged image (left) is shown in line with the separate GFP (middle) and mRFP (right) images. The cell spreading on a fluoralkane surface was recorded by spinning disc microscopy at the substrate-attached cell surface with a frame-to-frame interval of 1 second and an exposure time of 100 milliseconds each for GFP and mRFP. Numbers indicate seconds after the first cell-substrate contact. Bar, 5 µm.

Spreading of a cell labeled with GFP-coronin and mRFP-LimEΔ. The cell touches the substrate with lamellipodial protrusions, which in the optical section show a red-green banding pattern: red for filamentous actin, green for coronin, and yellow for merge of the labels. Like the cells shown in Figure 3, this spreading cell turns from a multipolar into a bipolar shape. During the extension of lamellipodia on the substrate surface, a zone of coronin appears behind the actin-enriched front (open arrowhead in the 11 and 15 second frame). Behind a protrusion, red zones convert into green ones (open arrowhead in the 15 seconds frame). A macropinosome seen on the bottom of the cell exhibits a characteristic sequence of labeling patterns (closed arrowheads in the 15 to 62 second frames): (1) filamentous actin prevails during protrusion of the macropinocytic cup (15 to 30 second frames), (2) actin dominates close to the membrane and coronin at the cytoplasmic face of the cup (46 seconds frame), (3) at late stages, when a vesicle is budded off (53 seconds frame) or when the cup regresses upon actin disassembly (62 seconds frame), coronin becomes the prevalent protein. The cell spreading on a cleaned glass surface is recorded by spinning disc microscopy slightly above the substrate surface with a frame-to-frame interval of 1 second and an exposure of 50 milliseconds each for GFP and mRFP. Numbers indicate seconds after beginning of the recording. Bar, 5 µm.

Clathrin-associated actin dynamics in a spreading cell. This cell expressing GFP-clathrin light chain (green) together with mRFP-LimEΔ (red) shows clathrin-dependent activities throughout spreading. Beginning with the first contact, clathrin-coated structures are present on the substrate-attached membrane area. Arrowheads point to the sporadic association of these structures with actin (color change from green to yellow) followed by their disappearance from the plane of focus. Clathrin-coated structures are excluded from the dense actin assemblies at leading edges and also from filopodia. Cells spreading on a fluoralkane surface were recorded by spinning disc microscopy with focus on the substrate-attached surface and an exposure time of 50 milliseconds for GFP and 100 milliseconds for mRFP. Time is indicated in seconds after first contact of the cell with the substrate. Bar, 5 µm.

Fluctuations in the area of cell-to-substrate contact on a non-adhesive (A), a hydrophilic (B), and a hydrophobic (C) surface. The time of first cell-substrate contact is set to zero seconds. (A) Two cells attempting to attach to a PEG 2000 coated glass surface. The cells adhered iteratively with small areas of their surface on the passivated glass but the adhesion was only temporary. (B) Fluctuations in contact area during cell spreading on cleaned glass surfaces. The contact areas of the five cells plotted indicate continued increases and decreases in area size over the entire 4 minutes of recording. (C) Fluctuations in contact area during cell spreading on fluoralkane. Three cells were recorded for up to 10 minutes in order to demonstrate that sizes of the contact area do not equilibrate after longer periods of time. Sizes of the contact area were determined on TIRF recordings of cells labeled with GFP-LimEΔ, using its distribution in the cytoplasm to identify the substrate area covered by a cell. The algorithm applied reveals the total contact area of a cell even if this area is dispersed. This means that filopodia or lamellipodia that touch the substrate remote from the compact cell body are included. Note differing scales on the ordinates. The insets in (A–C) zoom into the first 30 seconds of cell spreading.

Dynamics of protrusion and retraction in cells spreading on a fluoralkane-coated surface (A and B) or on a cleaned glass surface (C and D). Cells labeled with LimEΔ-GFP were recorded by TIRF microscopy at intervals of 1 second. (A and C) Sizes of the contact area (the time encoded in rainbow colors) are aligned with the local gain (red) and loss (green) of contact area and with the velocity of translocation of the cells' center of mass (black curves at the top). The local gain and loss was calculated by subtracting pixel-by-pixel and second-by-second the area in frame n from that in frame n + 10 (i.e., a frame recorded 10 seconds later). (B and D) Translocation of the cells' geometric center with time is encoded in the same rainbow colors as for the sizes of contact area. The crossing point of the inserted lines refers to the starting position of the center of mass. Closed arrowheads indicate simultaneous protrusion and retraction, open arrowheads protrusion accompanied by retraction in phases of negligible translocation of the cells.

A spreading cell labeled with GFP-Arp3 (green) and mRFP-LimEΔ (red). The label of the Arp2/3 complex is enriched in lamellipodia and in patches on the substrate-attached cell surface (yellow color indicating merge of the Arp3 and actin label). No enrichment is recognized in filopodia or in blebs (236 seconds frame). The cell spreading on cleaned glass was recorded by spinning disc microscopy at the substrate-attached cell surface. Exposure 100 milliseconds for GFP and 150 milliseconds for mRFP. Numbers indicate seconds after the first cell-substrate contact. Bar, 5 µm.

Spreading of two cells, labeled according to Figure 7 with GFP-coronin (green) and mRFP-LimEΔ (red). (A) Cell reverting its polarity. The coronin label persists longer than the actin label at the previous front during its conversion into a tail. (B) Cell showing fluctuations of the actin and coronin label at the front region in parallel with phases of protrusion and retraction. Patches on the substrate-attached surface turn from red to green, indicating that actin decreases while coronin is recruited (arrowhead in the 58 and 63 second frames). The cells spreading on a fluoralkane surface were recorded by spinning disc microscopy with the focus on the substrate-attached surface at an exposure time of 30 milliseconds each for GFP and mRFP. Numbers indicate seconds after first cell-substrate contact. Bar, 5 µm.

Recruitment of clathrin and actin-binding proteins to actin patches. Cells double-labeled with mRFP-LimEΔ for actin and with one of four GFP-tagged proteins were allowed to spread on fluoralkane surfaces. Cells were imaged by TIRF microscopy, and fluorescence intensities determined in a circular area of 1.4 µm diameter centered on stationary actin patches. The time of maximal intensity in the actin label (Lim) is set to zero on the time axis, and temporal changes in fluorescence intensities of the four other proteins are related to this peak: CLC, clathrin light chain; MyoB, myosin-IB; Arp3, subunit labeling the Arp2/3 complex; Cor, coronin. Fluorescence intensities before occurrence of the patches were set to zero and peak intensities were normalized to 1.