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Volume 111, Issue 12, Pages (December 2016)

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Presentation on theme: "Volume 111, Issue 12, Pages (December 2016)"— Presentation transcript:

1 Volume 111, Issue 12, Pages 2698-2710 (December 2016)
Frustrated Phagocytic Spreading of J774A-1 Macrophages Ends in Myosin II- Dependent Contraction  Daniel T. Kovari, Wenbin Wei, Patrick Chang, Jan-Simon Toro, Ruth Fogg Beach, Dwight Chambers, Karen Porter, Doyeon Koo, Jennifer E. Curtis  Biophysical Journal  Volume 111, Issue 12, Pages (December 2016) DOI: /j.bpj Copyright © 2016 Biophysical Society Terms and Conditions

2 Figure 1 J774A.1 macrophages spreading on surfaces opsonized with 15 μg/mL of anti-BSA serum. Each colored time trace depicts the spreading behavior of an individual cell. (a) Example area-time traces for 13 nonspreading cells (complete data set not shown). (b) RICM images of cell-surface contact for a cell corresponding to the black trace in (a). (c) Example traces for 13 spreading cells (complete data set not shown). Spreading is characterized by a rapid increase in contact area within the first 5 min of FP. After the spreading phase, the cells often contract. (d) RICM images of a spreading and contracting cell (black trace in c). Lower right: the enlargement shows retraction tethers that formed during the contraction phase. For a video of the example cell, see Movie S4. Note: the yellow line drawn around the cells corresponds to a hull line around the binary contact map determined using the intensity threshold algorithm described in Materials and Methods. To see this figure in color, go online. Biophysical Journal  , DOI: ( /j.bpj ) Copyright © 2016 Biophysical Society Terms and Conditions

3 Figure 2 FP response to opsonin density. Surfaces were opsonized in 0.15, 1.5, 15, 150 μg/mL of anti-BSA serum. Controls (PBS only) are indicated as BSA. (a) Average peak-contact areas. With each surface treatment, a fraction of cells did not spread (red triangles). Nonspreading cells have contact areas that remain below 500 μm2. Blue squares correspond to spreading cells and indicate that the spreading area is not significantly affected by the opsonin density. Error bars indicate 95% CI. Contact areas are not statistically different (F(4,115) = 1.85, p = 0.125). (b) Fraction of cells that spread rapidly. Error bars indicate 95% CI for a binomial fit. To see this figure in color, go online. Biophysical Journal  , DOI: ( /j.bpj ) Copyright © 2016 Biophysical Society Terms and Conditions

4 Figure 3 Traction force results. (a) Color map indicating the magnitude of traction stresses during FP. Vector arrows have been omitted to facilitate plotting; however, at all times, forces point toward the cell body (see Movie S1). (b) Strain energy, average strain energy density, and projected cell area (measured manually) for the cell depicted in (a). The blue line indicates the half-maximum points used to calculate the contraction peak duration. (c) Histogram of peak strain energy for all cells measured. (d) Histogram of contraction duration, measured as the time between the initial rise and the fall to 50% peak strain energy. (e) Histogram of contraction onset, defined as the time corresponding to the initial rise to 50% peak strain energy. (f) Histogram of the ratio of average strain energy during the spreading and contraction phases. Ratios were calculated by averaging the strain energy during the period in which each cell was spreading divided by the average strain energy during the period in which the cell was noticeably contracting. To see this figure in color, go online. Biophysical Journal  , DOI: ( /j.bpj ) Copyright © 2016 Biophysical Society Terms and Conditions

5 Figure 4 SIM images of F-actin cytoskeletal reorganization during FP. Cells were fixed at different times and labeled with phalloidin-488 (green) and DAPI (blue). Each image is a different cell. At 5 min (the spreading phase), F-actin forms a dense band near the edge. This is consistent with the F-actin band seen during lamellipodial extension in migrating cells. At 10 min (the spreading-contraction transition), banding near the edge begins to fade and actin bundles begin to form throughout the cell body. Areas that are beginning to contract show retraction-tether formation. (a–c) At 15 min (the contraction phase), F-actin forms bundles that surround the perimeter of the cell. As shown in insets (b) and (c), these bundled fibers are often strongest near edges that are retracting, as indicated by retraction tethers. To see this figure in color, go online. Biophysical Journal  , DOI: ( /j.bpj ) Copyright © 2016 Biophysical Society Terms and Conditions

6 Figure 5 Blebbistatin-treated cell fragmenting during FP. Isolated spreading fronts eventually pull the cell into two separate lobes connected by a thin tether. The protrusion velocity of each lobe gradually decreases from 0.06 μm/s to 0.02 μm/s as the cell is pulled apart. See Supporting Materials and Methods for information about the cell edge analysis routine. To see this figure in color, go online. Biophysical Journal  , DOI: ( /j.bpj ) Copyright © 2016 Biophysical Society Terms and Conditions

7 Figure 6 Maximal area of FP under different conditions. WT corresponds to untreated cells spreading on 150 μg/mL anti-BSA substrates (n = 32 cells), 150 mM corresponds to cells exposed to hypertonic buffer (n = 20 cells), DMSO indicates cells exposed to 0.012% v/v DMSO (n = 40 cells), and Bleb indicates cells exposed to 2 μM blebbistatin (n = 35 cells). Box plots follow same convention listed in Fig. 2. The p-values for each treatment are listed below figure. Blebbistatin-treated cells were compared with both untreated (WT) and DMSO-treated cells. To see this figure in color, go online. Biophysical Journal  , DOI: ( /j.bpj ) Copyright © 2016 Biophysical Society Terms and Conditions

8 Figure 7 Schematic of F-actin reorganization during FP. During the spreading phase, a dense ARP2/3 branched actin network drives spreading via the actin-treadmill mechanism. As the cell’s membrane reservoir is depleted, tension rises. Two general schemes might explain the onset of contraction. Membrane tension sensors may signal actin remodelers to stop actin polymerization. Alternatively, tension may stall outward protrusion mechanically. With protrusion stopped, myosin working from the cell interior toward the edge would cause actin filaments to bundle into contractile fibers. With the filaments bundled, myosin motors would exert tension along the perimeter bundles, causing the cell to contract. To see this figure in color, go online. Biophysical Journal  , DOI: ( /j.bpj ) Copyright © 2016 Biophysical Society Terms and Conditions


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