1. An Elmo-like Protein Associated with Myosin II Restricts Spurious F-Actin Events to Coordinate Phagocytosis and Chemotaxis 
Nilgun Isik, Joseph A. Brzostowski, Tian Jin 
Developmental Cell 
Volume 15, Issue 4, Pages (October 2008)
DOI: /j.devcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
ElmoA Functions in Development and Phagocytosis in D. discoideum
(A) Developmental phenotypes of wild-type (WT), elmoA−, and elmoA− cells expressing ElmoA-GFP. Cells were plated on nonnutrient DB agar. In 24 hr, WT cells completed development and formed fruiting bodies, elmoA− cells had a delayed phenotype, and ElmoA-GFP rescued the phenotype of elmoA− cells.
(B). ElmoA-GFP proteins were detected at the predicted size by immunoblotting with anti-GFP antibodies.
(C) Actin-mediated phagocytosis in WT and elmoA− cells. WT and elmoA− cells expressing ΔlimE-RFP (red), a marker of F-actin, were fed with FITC-labeled, head-killed yeast (green). Individual cells were imaged simultaneously for RFP and FITC fluorescence during a time course by confocal microscopy. Arrows indicate the yeast that were internalized. See Movies S1–S4. Scale bar is 10 μm.
(D) Percentage of wild-type and elmoA− cells that internalize 1, 2, or 3 yeast at any given time (n = 20).
(E) Quantitative analyses of phagocytosis. Cells were incubated with TRITC-labeled, heat-killed yeast at room temperature to allow internalization. Samples were collected at the indicated times (min) and treated with Trypan blue to quench fluorescence of the noninternalized yeast. Fluorescent intensity, which reflects the number of internalized yeast, was measured by FACS. Means and SD (n = 3) are shown for cells of WT, elmoA−, and elmoA− expressing ElmoA-GFP.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

3. Figure 2 ElmoA Plays a Role in Chemotaxis and Cell Polarity
(A) elmoA− cells are able to generate PHCrac-GFP polarization in response to a cAMP gradient. elmoA− cells expressing PHCrac-GFP, a fluorescence probe that monitors the PIP3 level around the cell membrane, were placed in a cAMP gradient.
(B) elmoA− cells were defective in chemotaxis toward cAMP in an under-agar assay. Developed wild-type (WT), elmoA−, and elmoA− expressing ElmoA-GFP cells were plated into wells on either side of a well containing 5 μM cAMP and were allowed to migrate under 0.5% agar for 60 min. Low-magnification images were captured on a stereomicroscope to include all wells. Data are representative of at least of four independent experiments.
(C) Cell shape analysis of developed wild-type and elmoA− cells migrating toward a well filled with 5 μM cAMP under 0.5% agar. The stacked images of migrating cells are shown. Individual cells were traced and processed from a time-lapsed series using 2D-DIAS software.
(D) Quantitation of pseudopod number as described in (G [below]) for cells migrating under 0.5% agar is shown (n = 5). Error bars represent SD.
(E) Average chemotactic index for cells migrating under 0.5% agar is shown (n = 5). Error bars represent SD.
(F) Morphology of developed wild-type and elmoA− cells.
(G) elmoA− cells exhibit defects in motile behaviors and pseudopod formation in the absence of chemoattractant. Developed WT and elmoA− cells were plated in a one-well chamber in DB buffer and images were recorded in 20 s intervals. After 3 min (indicated by filled arrows), cells were exposed to a steep cAMP gradient via micropipette filled with 1 μM. Black dots indicate the position of the micropipette. Cell shapes were traced and constructed with 2D-DIAS software. The extending pseudopods are colored in green and the trailing regions are red. Note the extension of multiple lateral pseudopods around elmoA− cells, especially before exposure to the cAMP gradient at 3 min.
(H) Quantitation of pseudopod number in WT (gray) and elmoA− cells (black) at the indicated time points. The arrow shows introduction of cAMP point source. Means and SD are shown (n = 5).
(I) Quantitation of the directional index of WT (gray) and elmoA− cells (black) at different time points. The arrow shows introduction of cAMP point source. Mean and SD are shown (n = 10). Significant differences are indicated by ∗p < 0.01, ∗∗p < 0.03 as determined by a Student's t test.
(J) Cell shape analysis of developed wild-type and elmoA− cells migrating to a micropipette filled with 1 μM cAMP as in (C). Scale bars are 10 μm.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

4. Figure 3 ElmoA Affects F-Actin Levels in Cells
(A) Immunoblot analysis shows that wild-type, elmoA−, and elmoA− expressing ElmoA-GFP cells have a similar levels of total actin.
(B) Quantitation of the G-actin and F-actin ratio in wild-type, elmoA−, and elmoA− expressing ElmoA-GFP cells. Means and SD are shown from three independent experiments. ∗p < by Student's t test comparing F-actin levels between WT and elmoA− cells.
(C) Visualization of F-actin in wild-type and elmoA− cells. Cells were fixed, permeablized, and stained with rhodamine-phalloidin and imaged using a confocal microscope. Scale bar is 5 μm.
(D) Quantitation of relative F-actin in wild-type (gray), elmoA− (black), and elmoA− expressing ElmoA-GFP (green) cells using FACS analyses. Curves represent rhodamine-phalloidin fluorescence levels in a population of cells.
(E) Actin polymerization events imaged by TIR-FM in live cells. Wild-type and elmoA− cells transformed with ΔlimE-RFP were allowed to adhere to glass coverslips in neighboring wells and were imaged under identical conditions to visualize the relative difference in actin polymerization. Intensity images (grayscale) were thresholded (red) with the same scale to calculate the area of actin polymerization events.
(F) Thirty cells for each strain were imaged, and the ratio of the area of actin polymerization to total cell area was calculated and plotted. ∗p < by Student's t test.
(G) Actin polymerization in response to cAMP stimulation. Developed cells were stimulated at time 0, and samples were removed at indicated time points and fixed with TRITC-phalloidin. Labeled actin was extracted with MeOH, and fluorescence intensity was measured in a spectrofluorometer. Means and SD are shown from five independent experiments.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

5. Figure 4
ElmoA Association with F-Actin and the Myosin II Heavy Chain Is Facilitated by an Actin-Binding Domain
(A) Lysates (lanes 1, 5, and 9) of wild-type (negative control) and elmoA− cells expressing either ElmoA-GFP or ElmoAΔ-GFP were incubated with beads coupled to anti-GFP antibodies. Elutes were subjected to SDS gel electrophoresis and stained with Coomassie blue. The bands (a–h) were excised from the gel and identified using a mass spectrometry. a and e are the myosin heavy chain; b is ElmoA-GFP and f is ElmoAΔ-GFP; c and g are contaminating IgG heavy chain; d and h are actin.
(B) Activation of cAR1 induces association between ElmoA and F-actin. elmoA− cells expressing ElmoA-GFP or ElmoAΔ-GFP were stimulated with 5 μM cAMP, and samples were lysed at 0 s (lanes 2), 10 s (lanes 3), 60 s (lanes 4), and 120 s (lanes 5). Lysates were incubated with beads coupled with anti-GFP, and elutes were analyzed by immunoblotting to detect actin. Wild-type cells were used as a control. Lane 1 for each sample contains total proteins to monitor the input actin levels.
(C) Quantitation of cAR1-induced association of actin between ElmoA-GFP and ElmoAΔ-GFP. The intensity of each actin band shown in (B) was quantified using Image J software. The relative actin intensity levels associated with ElmoA-GFP (gray) or ElmoAΔ-GFP (white) were normalized by subtracting intensity measured in corresponding lanes in WT samples. Means and SD from three independent experiments are shown.
(D) Elutes of lysates of wild-type (lane 1), elmoA− cells expressing ElmoA-GFP (lane 2), myoII− cells expressing ElmoA-GFP (lane 3), elmoA− cells expressing ElmoAΔ-GFP (lane 4), and myoII− cells expressing ElmoAΔ-GFP (lane 5) were incubated with beads coupled to anti-GFP antibodies, subjected to SDS gel electrophoresis, and stained with Coomassie blue. As expected, myosin II (MyoII) is not present in lanes 3 and 5. Arrows point to ElmoA-GFP and ElmoAΔ-GFP.
(E) After electrophoresis, the corresponding elutes in (D) were blotted and probed with an anti-actin antibody.
(F) The level of actin was quantified as in (C) for three experiments performed in (E). The strain is indicated below the bars, and the expressed GFP-tagged protein is indicated above. ∗p < 0.01 for ElmoA-GFP and ∗p < 0.05 for ElmoAΔ-GFP by Student's t test.
(G) Elutes of lysates of wild-type negative control (lane 1), wild-type expressing ElmoA-GFP (lane 2), elmoA− cells expressing ElmoA-GFP (lane 3), myoII− cells expressing ElmoA-GFP (lane 4), elmoA− cells expressing ElmoAΔ-GFP (lane 5), and myoII− cells expressing ElmoAΔ-GFP (lane 6) were incubated with beads coupled to anti-myosin II antibody antibodies, subjected to SDS gel electrophoresis, and stained with Coomassie blue. The myosin II band is indicated.
(H) After electrophoresis, the corresponding elutes in (G) were blotted and probed with an anti-GFP antibody. ElmoA proteins are indicated.
(I) Elutes of lysates of myoII− cells expressing MyoII-GFP (lane 1), wild-type expressing MyoII-GFP (lane 2), and elmoA− cells expressing MyoII-GFP (lane 3), were incubated with beads coupled to anti-GFP antibody antibodies and were subjected to SDS gel electrophoresis and stained with Coomassie blue. Coprecipitated actin and endogenous myosin II and GFP-tagged myosin II are indicated. Molecular weight marker (M).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

6. Figure 5
Myosin II Localizes to the Sides and Rear of Chemotaxing elmoA− Cells
(A) Chemotactically competent wild-type and elmoA− cells expressing myosin II fused to GFP (MyoII-GFP) were recorded by time-lapse confocal microscopy moving up a cAMP gradient.
(B) An inverse relationship between ElmoA and F-actin is observed in membrane protrusions in vegetative cells. Shown is a still from a time-lapse movie (see Movie S7) of an elmoA− cell expressing ElmoA-GFP (green) and the F-actin (red) marker limEΔ-RFP.
(C) The relative intensity of each signal over the line drawn in the composite is graphed.
(D) An inverse relationship between ElmoA and F-actin is observed at the leading edge of chemotaxing cells. elmoA− cells expressing ElmoA-GFP and the F-actin marker limEΔ-RFP were subjected to a chemoattractant gradient under agar and imaged by TIR-FM (see Experimental Procedures for details). Shown are TIR-FM images for the green and red channels of a cell in a stream.
(E) Shown is a subregion of the leading edge that was enlarged and rotated. The bracketed area is 12 pixels from top to bottom. An average intensity value was calculated across (left to right) for each pixel position.
(F) The values were normalized to MIN and MAX to scale on the graph shown.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

7. Figure 6
Actin Polymerization Events Correlate with a Dispersion of ElmoA-GFP and MyoII-GFP Proximal to the Plasma Membrane as Visualized by TIR-FM
(A) An elmoA− cell expressing ElmoA-GFP (ElmoA) and ΔlimE-RFP (F-actin) was imaged by TIR-FM in two channels simultaneously for over 3 min. The images shown (left panels) were captured at 108 s. The boxed subregion shows an area where actin periodically polymerizes and depolymerizes over the course of the image sequence. This image sequence and other examples can be viewed in Movies S8 to S10. Specific time points of the boxed subregion are shown (right panels) to highlight the dips of ElmoA-GFP intensity and the corresponding peaks of F-actin. The mean intensity values for both channels in the boxed subregion were normalized to MIN and MAX and graphed versus time. The dashed vertical lines are drawn through the actin peak intensity to visually emphasize the rightward phase shift (indicated by the arrow) of the ElmoA-GFP intensity plot for each event.
(B) The mean delay and SD in seconds of the peak minimum intensity (y axis) of ElmoA-GFP or MyoII-GFP from the peak maximum intensity of ΔlimE-RFP (F-actin) were obtained from plots of at least 12 actin polymerization events from four cells (see [A], [F], [G], and [H] for examples). The strain is indicated under each plot. The first two strains express ElmoA-GFP and the last two MyoII-GFP (indicated). ∗∗p < indicates a statistical difference relative to elmoA− cells expressing ElmoA-GFP by ANOVA.
(C) Stimulation of elmoA− cells expressing ElmoA-GFP and ΔlimE-RFP (F-actin) with 10 uM cAMP (arrow) causes a rapid, transient increase in F-actin and concomitant decrease in ElmoA-GFP intensity as monitored by TIR-FM for the entire cell area (n = 5). Due to the range of raw intensity values, the data were normalized to MIN and MAX before plotting. Error bars represent SD.
(D) Treatment of elmoA− cells expressing ElmoA-GFP and ΔlimE-RFP (F-actin) with 1 uM Latrunculin B causes a rapid decrease in F-actin and a concomitant increase in ElmoA-GFP intensity as monitored by TIR-FM for the entire cell area (n = 4). The data were normalized to MIN and MAX before plotting. Note that no intensity changes were observed with DMSO-treated control cells (not shown). Error bars represent SD.
(E–H) Cells were imaged and presented similarly as described in (A). (E) An elmoA− cell expressing ElmoAΔ-GFP and ΔlimE-RFP is shown. No dispersion of ElmoAΔ-GFP is observed. (F) A WT cell expressing MyoII-GFP and ΔlimE-RFP is shown. (G) An elmoA− cell expressing MyoII-GFP and ΔlimE-RFP is shown. (H) A myoII− cell expressing ElmoA-GFP and ΔlimE-RFP is shown.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions