A growth factor signaling cascade confined to circular ruffles in macrophages

Abstract

The formation of macropinosomes requires large-scale movements of membranes and the actin cytoskeleton. Over several minutes, actin-rich surface ruffles transform into 1-5 µm diameter circular ruffles, which close at their distal margins, creating endocytic vesicles. Previous studies using fluorescent reporters of phosphoinositides and Rho-family GTPases showed that signals generated by macrophages in response to the growth factor Macrophage Colony-Stimulating Factor (M-CSF) appeared transiently in domains of plasma membrane circumscribed by circular ruffles. To address the question of how signaling molecules are coordinated in such large domains of plasma membrane, this study analyzed the relative timing of growth factor-dependent signals as ruffles transformed into macropinosomes. Fluorescent protein chimeras expressed in macrophages were imaged by microscopy and quantified relative to circular ruffle formation and cup closure. The large size of macropinocytic cups allowed temporal resolution of the transitions in phosphoinositides and associated enzyme activities that organize cup closure. Circular ruffles contained transient and sequential spikes of phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)), phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), diacylglycerol, PI(3,4)P(2), PI(3)P and the activities of protein kinase C-α, Rac1, Ras and Rab5. The confinement of this signal cascade to circular ruffles indicated that diffusion barriers present in these transient structures focus feedback activation and deactivation of essential enzyme activities into restricted domains of plasma membrane.

Keywords: Macropinocytosis; Phosphoinositides; Signal transition.

Fig. 1.. Macropinosome closure precedes peak localization of Rab5.

(A) 4D reconstruction of Cherry-Rab5 localization during macropinocytosis. Through-focus z-stacks of CFP-MEM and mCherry-Rab5 at regular intervals during M-CSF-stimulated macropinocytosis, showing distribution of CFP-MEM (blue, top row), Cherry-Rab5 (red, middle row) and Cherry/CFP ratio (pseudocolor). Time points are separated by 60 s; “0 s” marks the point of cup closure. Rab5 localization increased after complete cup closure. (B–D) The timing of cup closure relative to Rab5 localization. (B) Macrophages expressing CFP-MEM and Citrine-Rab5 were imaged in 1 µg/ml FM4-64. Top row: phase-contrast; middle row: intensity of ratiometric FM4-64/CFP-MEM; bottom row: Citrine-Rab5/CFP-MEM ratio. Ruffle closure occurred at t = 0. FM4-64 photobleaching increased at t = 100 s, indicating cup closure. Scale bars  =  3.0 µm. (C) Plot of ratiometric FM4-64/CFP-MEM intensity during macropinosome formation; the inflection point occurred at t = 100 s. (D) Plot of Cit-Rab5/CFP-MEM ratios during macropinosome formation. Rab5 localization peaked at t = 180 s, after macropinosome closure. Error bars indicate standard deviation. n≥9 for each curve.

Fig. 2.. Fluorescence imaging of phosphoinositide localization during macropinocytosis.

(A–D) Macrophages expressing mCerulean, mCherry-Rab5, and Citrine-tagged probes for PIs were imaged during M-CSF-stimulated macropinocytosis. Top rows: phase-contrast; middle rows: Citrine-tagged fluorophore/mCerulean ratios; bottom rows: mCherry-Rab5/mCerulean ratios. Ruffle closure occurred at t = 0. Scale bars  =  3.0 µm. (A) Citrine-PLCδ1PH peaked at t = 20 s. (B) Citrine-BtkPH peaked at t = 80 s. (C) Citrine-Tapp1PH peaked at t = 100 s. (D) Citrine-FYVE peaked at t = 180 s. (E) Plot of compiled data comparing relative timing of peak labeling by different PI probes. Error bars indicate standard deviation. n≥9 for each condition.

Fig. 3.. Fluorescence imaging of the DAG pathway during macropinocytosis.

(A–C) Macrophages expressing mCerulean, mCherry-Rab5, and Citrine-tagged probes were imaged during macropinocytosis. For each panel: top row  =  phase-contrast, middle row  =  ratio of Citrine-tagged fluorophore/mCerulean, bottom row  =  ratio of mCherry-Rab5/mCerulean. Ruffle closure occurred at t = 0. Scale bars  =  3.0 µm. (A) Citrine-C1δ peaked at t = 60 s. (B) Citrine-PLCγ did not demonstrate differential localization during macropinocytosis. (C) Citrine-PKCα localization peaked at t = 180 s, resembling localization patterns of Rab5. (D) Relative timing of signaling in the DAG pathway. n≥9 for each curve. Error bars indicate standard deviation.

Fig. 4.. Imaging of GTPase activity during macropinocytosis.

(A,B) Macrophages expressing mCerulean, mCherry-Rab5, and either mCitrine-PBD, a probe for Rac1-GTP, or mCitrine-RBD, a probe for Ras-GTP, were imaged during macropinocytosis. Top rows: phase-contrast; middle rows: Citrine-tagged fluorophore/mCerulean ratio; bottom rows: mCherry-Rab5/mCerulean ratio. t = 0 indicates ruffle closure. Scale bars  =  3.0 µm. Color bars indicate relative intensities of ratio images. (A) Citrine-PBD localization peaked at t = 80 s. (B) Citrine-RBD peaked at t = 180 s. (C) Quantitation showed that peak activation of Rac1 occurred at t = 80 s. Ras reached maximal activation at t = 180 s. n≥9 for each condition.

Fig. 5.. Summary of the signal cascade in circular ruffles.

The sequence of chemical transformations occurring between the formation of a fully circular ruffle and the closure of the circular ruffle into the cell as a macropinosome begins with an increase in PI(4,5)P₂. (A) Chemical changes in cups. Solid lines with small arrowheads indicate precursor-product relationships. Hatched lines with large arrowheads indicate activation pathways enhanced inside circular ruffles. The spike of PI(4,5)P₂ is followed by increases of PIP₃, DAG and the activity of Rac1 (Node 1), and later by a transient increase of PI(3,4)P₂. The levels of PI(3)P and the activities of Ras, Rab5 and PKCα (Node 2) increase continuously in the cup, peaking after cup closure and the transient increases of PI(4,5)P₂, PIP₃, DAG and PI(3,4)P₂. (B) The timing of signals in cups relative to Ruffle Closure and Cup Closure.