DYNAMIN AT WAVES

The mechanisms by which mammalian cells remodel the actin cytoskeleton in response to motogenic stimuli are complex and a topic of intense study. Dynamin 2 (Dyn2) is a large GTPase that interacts directly with several actin binding proteins, including cortactin. In this study, we demonstrate that Dyn2 and cortactin function to mediate dynamic remodeling of the actin cytoskeleton in response to stimulation with the motogenic growth factor platelet-derived growth factor.

On stimulation, Dyn2 and cortactin coassemble into large, circular structures on the dorsal cell surface. These "waves" promote an active reorganization of actin filaments in the anterior cytoplasm and function to disassemble actin stress fibers. Importantly, inhibition of Dyn2 and cortactin function potently blocked the formation of waves and subsequent actin reorganization. These findings demonstrate that cortactin and Dyn2 function together in a supramolecular complex that assembles in response to growth factor stimulation and mediates the remodeling of actin to facilitate lamellipodial protrusion at the leading edge of migrating cells.

Mol Biol Cell. 2003 March; 14 : 1085-1096
A Dynamin-Cortactin-Arp2/3 Complex Mediates Actin Reorganization in Growth Factor-stimulated Cells
Eugene W. Krueger*, James D. Orth*, Hong Cao, and Mark A. McNiven
* denotes both authors contributed equally to this study

PDGF treatment induces the formation of Dyn2-cortactin-rich membrane waves. Immunofluorescence microscopy of NIH/3T3 cells, either resting or stimulated with 30 ng/ml PDGF-bb for 5 min and dual labeled with reagents to Dyn2 and cortactin. (A-A') In resting cells, both proteins associate modestly within the cell cortex and on internal membrane structures and cells show a flattened nonpolarized morphology. (B, B') In contrast, cells exposed to PDGF show dramatic changes in shape and in the distribution of Dyn2 and cortactin proteins. Cell staining with multiple antibody reagents show Dyn2 and cortactin seem to coassemble into punctate spots that coalesce into wave-like rings (arrows) marking the leading edge. (B') Higher magnification images of the waves show substantial overlap of Dyn2 and cortactin in the puncta of the wave structures. Bars, 10 mm.

Waves are dynamic and actively incorporate Dyn2-GFP in living cells. Time-lapse images of living human foreskin (phase contrast, A-E, and video 1) or NIH/3T3 (Dyn2-GFP, F-J, and video 2) fibroblasts stimulated with PDGF. (A) HFs display multiple small lamellae at the cell periphery just after stimulation. (B) Within 6 min after PDGF treatment, phase-dense puncta coalesced into concentric waves at the anterior end of the cell (arrows). (C-E) Within 40 min after stimulation the circular waves have constricted to a tight annulus and disappeared. (E) Concomitantly, the individual lamellipodia "fused" to form one large, rounded leading edge. (F-J) NIH/3T3 cells expressing Dyn2-GFP and stimulated with PDGF. (G and H, arrows)

Numerous Dyn2 puncta actively organized into wave structures within 5-10 min poststimulation. (H and I) Note the consistent size of the Dyn2 foci that seem to organize into linear chains that fuse together into larger spots or tubules. (J) Within 20 min, this wave has entirely disappeared. Bar, 10 mm. Click here for a larger image.

Loss of F-actin staining in stimulated cells corresponds with the sites of Dyn2-cortactin wave progression. Rhodamine-phalloidin staining of F-actin in resting vs. PDGF-stimulated cells. Three cell types are shown. (A) Resting cells display numerous, thick, brightly labeled stress fibers that traverse the long axis of the cell. (B-D) In contrast, stimulated cells display a dramatic loss of cortical actin and stress fibers that coincides with the advancing edge of the dorsal wave (arrows). Note the punctate organization of the actin within each wave structure and the marked difference of staining intensity on either side of the wave. (D) Actin fluorescence intensity was measured in the pre- (z) and postwave (x). (E) Fluorescence intensity values were normalized to cell thickness by expressing GFP alone and measuring the differences in intensity in the pre- (z) and postwave (x). (F) Quantitation of the average fluorescence intensity of actin for 125 waves. There is a 63% reduction of actin fluorescence in the anterior cytoplasm after wave migration. Bars, 10 mm. Click here for a larger image.

Actin nucleation and branching proteins p34 (Arp2/3) and N-WASp are prominent components of dorsal waves. PDGF stimulation induces formation of a multicomponent protein complex. (A and B) Immunocytochemical staining of stimulated cells with antibodies to the p34 subunit of the Arp2/3 complex and N-WASp in stimulated NIH/3T3 fibroblasts. (C) Dyn2 immunoprecipitation from resting vs. PDGF-stimulated NIH/3T3 cells. Under resting conditions, dynamin antibodies (MC65) pull down Dyn2, modest levels of N-WASp, and no detectable cortactin or small GTPases. In contrast, in stimulated cells substantial amounts of cortactin, N-WASp, p34, and Rac1 are coimmunoprecipitated from the extract. Note that RhoA and Cdc42 are not immunoprecipitated. All proteins migrated at their expected molecular mass and were aligned to facilitate comparison. Representative results are shown. -, no stimulation; +, stimulation with 30 ng/ml PDGF. Bars, 10 mm. Click here for a larger image.

Wave formation is required for active lamellipod protrusion. Phase microscopy of PDGF-stimulated HFs showing the position of waves (*) in relationship to newly formed lamellipod protrusions. (A-C) Cells that formed waves actively protruded membrane (shaded areas). Arrows indicate regions of the periphery distal to waves that subsequently did not protrude membrane. (D and E) Cells stimulated with PDGF that did not form waves exhibited only very modest protrusion (shaded areas). (F) Quantitation of the percentage of total cell area protruded in stimulated cells that formed waves (black bar) vs. stimulated cells that did not form waves (gray bar) compared with unstimulated cells (white bar). Note that wave formation led to more than a 2.5-fold increase in the lamellipod area protruded and that this occurred only along membrane adjacent to waves. Bar, 10 mm. Click here for a larger image.