Latrunculin B

To study particle binding, phagocytes at confluence were challenged with 10 particles per cell in growth medium (RPE) or serum-free growth medium (other cells) for the duration of the experiment, washed three times with PBS containing 1 mM MgCl₂ and 0.2 mM CaCl₂ (PBS-CM), and fixed in ice-cold methanol. OS were covalently labeled with FITC or Texas red, and thus could be observed directly. Apoptotic cells were visualized by TUNEL staining or by granulocyte-specific myeloperoxidase immunofluorescence staining using FITC- or Texas red–conjugated secondary antibodies. Nuclei were counterstained with DAPI or propidium iodide at 1 ng/ml in PBS-CM. For competition experiments, OS and apoptotic cells were labeled with different fluorochromes. Particle labeling with either fluorochrome yielded identical results. Unlabeled particles competed with fluorescence-labeled particles for binding and phagocytosis by all cell types. For inhibition experiments, GRGDSP or GRADSP peptides (Calbiochem) were used at 1 mg/ml. Effects of peptides and antibodies were concentration dependent as established previously 26, and maximal effective concentrations of antibodies, 20–50 μg/ml, were used throughout this study. Concentrations of pharmacological reagents were as follows: calphostin C at 100 nM (light activated), cytochalasin D (Cyt D) at 5–20 μM, Gö6976 at 10 nM, hypericin at 5 μM, and latrunculin B at 1 μM. PMA was routinely used at 50 nM; 16–160 nM gave similar results. Cells were pretreated for times indicated in the figure legends with activators or inhibitors before challenge with OS or apoptotic granulocytes in the continuous presence of reagent. Cell viability and morphology remained unchanged, and none of the cell types initiated detectable apoptosis over the course of the experiments. Since experiments were performed on confluent cells, the spreading effect of PMA on macrophages was negligible.
Phagocytosis or binding of OS was quantified by fluorescence scanning of fixed samples as described 26. Samples were scanned with a STORM 860 Imager, at 950 V (blue or red fluorescence setup; Molecular Dynamics). Areas representing the binding by 1–2 × 10⁵ phagocytes were selected, and the fluorescent signals were quantified with ImageQuant 1.2 (Molecular Dynamics). Within one experiment, particle counts directly correlated with particle binding. To compare particle binding of different cell types, as RPE cells and macrophages, the fluorescence of propidium iodide (nuclei, red) and the particle-derived FITC fluorescence were both measured in each field. The binding plus internalization index or the binding index (bound particles at early times of phagocytic challenge) were calculated dividing particle counts by nuclei counts, thereby normalizing for phagocyte numbers. Quenching of fluorescence derived from externally bound particles using trypan blue 2641 allowed determination of the internalization index. Phagocytosis or binding of apoptotic cells was quantified following the same procedure based on TUNEL or myeloperoxidase immunostaining fluorescence emissions; binding indices were determined dividing by nuclei counts of control fields, so as not to count apoptotic nuclei. Microscopic observation revealed that 75% of RPE-J cells and >90% of J774 cells had bound or phagocytosed an average of 5 ± 1 OS after 2 h. Using the double fluorescence scanning method on the same samples, this translated into a mean index combining binding plus internalization of 7.6 ± 0.9 for macrophages and 6.3 ± 0.9 for RPE. After 30 min, 75% of macrophages had bound multiple OS; this translated to a mean binding index of 3.8 ± 0.4. At this time point, <20% of particles had been internalized by macrophages, similar to the 2-h time point of RPE 2641.

We have studied how the latrunculin B treatment can influence the cellular force generation. For this purpose, ~1×10⁶/mL HeLa cells were first pretreated with 5–60 μM of latrunculin B for 60 min at 37 °C inside a cell culture incubator. After that, cells were separated and dispersed in cell culture media, ~1×10⁵ cells was then casted onto the surface of the TGT sensor and incubated for 75 min. After washing with 0.1 M phosphate buffer (pH 7.4) for several times, the SWV of 5 mM [Fe(CN)₆]⁴⁻ on the electrode was recorded using the above-mentioned parameters: step potential, 20 mV; pulse amplitude, 50 mV; and frequency, 20 Hz.

When imaging FRB protein recruitment without any additional inhibitors (Fig. 1D–I, Fig. 4, Fig. S4), 50 µl of 50 µM rapamycin was added to a well containing 450 µl of buffer at the indicated time for a final concentration of 5 µM. Similarly, in experiments with CK666 alone (Fig. 2A–B, Fig. S3, Fig. 3A–B), 50 µl of 1 mM CK666 was added to a well containing 450 µl of buffer at the indicated time for a final concentration of 100 µM. For experiments with rapamycin and CK666 (Fig. 3C–E, Fig. S4A–C) or CK666 and latrunculin (Fig. 2C–H), 50 µl of each of the drugs was added to a well containing 400 µl of buffer at the indicated times for a final concentration of 5 µM Rapamycin, 100 µM CK666, and 5 µM latrunculin. In Fig. 2C–E, cells were pre-incubated with 3 mM caffeine, and all drugs were diluted in buffer containing 3 mM Caffeine.
For cAMP stimulation experiments, cells were pre-treated in 400 µl DB containing rapamycin (Fig. S2C–D), CK666, or CK666 and latrunculin at the concentrations indicated above for 30 minutes. Then, 50 µl of 10 nM cAMP was added to the well during imaging followed at least a minute later by 50 µl of 1 µM cAMP for final concentrations of 1 nM and 100 nM respectively.
In Fig. S2A–B, the + rap population was treated with 5 µM rapamycin at least 30 minutes prior to imaging. For cells pre-treated with CK666 in Fig. 2F–G and Fig. 3C–E, cells were treated with 100 µM CK666 at least 30 minutes prior to imaging.