DYNAMIN AT RUFFLES

The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes.

We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin-SH3 domain associates with the proline-rich domain (PRD) of dynamin.

Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)DPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology.

This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.

J Cell Biol. 2000 October 2; 151 (1): 187-198
Regulated Interactions between Dynamin and the Actin-binding Protein Cortactin Modulate Cell Shape
Mark A. McNiven, Leung Kim, Eugene W. Krueger, James D. Orth, Hong Cao, and Tai Wai Wong

The SH3 domain of cortactin binds dynamin. a, A cytosolic extract of NIH/3T3 cells was incubated with GST or GST-cortactin SH3 domain that was immobilized on glutathione Sepharose beads (lanes 1 and 2) or immunoprecipitated with the Dyn 2-specific antibody (lane 3). The bound proteins were fractionated and analyzed by blotting with an antidynamin mAb.

As shown by the prominent dynamin band in lane 2, a substantial amount of dynamin was retained on the GST-cortactin SH3 domain beads as compared with the GST beads alone (lane 1). b, The GST pull-down assay was carried out in the absence or presence of one of the five synthetic peptides (P1-P5) at 0.1- or 1-mM concentrations. Binding of dynamin to the immobilized GST-cortactin SH3 domain was measured by blotting with an antidynamin mAb.

Note that the P2 and P1 peptides were the most effective in blocking dynamin binding to the GST-SH3 domain beads (c). As a control the GST pull-down assay was carried out using a GST fusion protein containing the SH3 domain of PLCg-1, in the absence (lane 1) or presence (lanes 2-6) of 1 mM of each of the five peptides. Binding of dynamin by the PLCg-1 SH3 domain was assayed by blotting with antidynamin antibodies. As for the cortactin-SH3 domain-associated beads, peptides P2 and P1 inhibited dynamin binding, and some inhibition was demonstrated by the P4 peptide. d, Peptide sequence of the Dyn 2 COOH terminus. The four proline rich sequences are underlined. The bottom shows the sequences of the five synthetic peptides used to test the inhibition of SH3- dynamin interactions. The consensus sequence PXXPSRP was found to inhibit Dyn 2 binding to the GST-cortactin SH3 domain. Click here for a larger image.

Dynamin colocalizes with cortactin in the lamellipodia of growth factor-stimulated fibroblasts. Immunofluorescence localization of dynamin and cortactin in quiescent, and PDGF-treated NIH/3T3 cells. Resting cells were fixed and double-labeled with antibodies to dynamin (MC63) and cortactin (a-a''), and displayed a nonpolarized phenotype with cortactin localized around the cell cortex. Upon stimulation with PDGF, there was a marked recruitment of both Dyn 2 (b) and cortactin (b') into large cortical ruffles (arrows) situated at the leading edges of the actively migrating cells. Bars, 10 mm. Click here for a larger image.

Expression of a truncated dynamin (Dyn 2(aa)DPRD) induces profound changes in cell shape with a concomitant proliferation of actin stress fibers. Fluorescence micrographs of cultured clone 9 cells expressing either wt Dyn 2(aa)-GFP (a) or Dyn 2(aa)DPRD-GFP (b-e).

Cells expressing the wt protein (a) display a normal discoidal shape and a punctate distribution of dynamin at both the plasma membrane and Golgi apparatus, identical to that of untransfected cells. In strong contrast, cells expressing the Dyn 2(aa)DPRD-GFP became elongated, sprouting long peculiar neurite-like appendages (b-e).

Morphological measurements of >200 wt and mutant cells confirmed this shape change showing a six- to sevenfold increase in width versus length (f). Shape changes are most prevalent in sparsely plated cells (b-e). Bars, 10 mm. Click here for a larger image.