Assembly of actin filaments and microtubules in Nwk F-BAR-induced membrane deformations

Abstract

F-BAR domains form crescent-shaped dimers that bind to and deform lipid bilayers, and play a role in many cellular processes requiring membrane remodeling, including endocytosis and cell morphogenesis. Nervous Wreck (NWK) encodes an F-BAR/SH3 protein that regulates synapse growth in Drosophila. Unlike conventional F-BAR proteins that assemble tip-to-tip into filaments and helical arrays around membrane tubules, the Nwk F-BAR domain instead assembles into zigzags, creating ridges and periodic scallops on membranes in vitro. In cells, this membrane deforming activity generates small buds, which can lengthen into extensive protrusions upon actin cytoskeleton polymerization. Here, we show that Nwk-induced cellular protrusions contain dynamic microtubules, distinguishing them from conventional filopodia, and further do not depend on actin filaments or microtubules for their maintenance. Our results indicate new ways in which close cooperation between the membrane remodeling and cytoskeletal machinery underlies large-scale changes in cellular morphology.

Keywords: F-BAR; Nwk; actin; membrane; microtubule.

Figure 1 (See previous page).

Nwk-induced cellular protrusions do not depend on actin or microtubules for stability once formed. (A) Time-lapse imaging of S2 cells co-transfected with Lifeact-mCherry and either EGFP (left) or Nwk1-428-EGFP (right), and treated with Lat B after spreading on ConA. F-actin disassembles upon treatment, but the cellular protrusions remain. Images show 2D projections of confocal stacks. Scale bar, 10 µm. Time scale is in minutes. (B) Confocal microscopy of fixed S2 cells expressing EGFP or Nwk^1-428-EGFP, and mCherry-tubulin. Nwk and tubulin localize to protrusions. Images show 2D projections of confocal stacks. Scale bar, 10 µm. Scale bar in magnified image is 5 µm. (C) Electron microscopy of Nwk^1-428-EGFP–expressing S2 cells treated with Lat B to disrupt the actin cytoskeleton. Microtubules can be observed throughout the protrusion (arrows). Scale bar, 100 nm. (D) Live cell imaging of S2 cells expressing EB1-GFP with mCherry (top) or Nwk^1-428-mCherry (bottom). EB1, a plus end microtubule binding protein, can be seen throughout protrusions (arrows, see also Movies 1 and 2). Scale bar, 10 µm. (E) Nwk-induced protrusions remain stable after microtubule depolymerization. Live cell imaging of S2 cells expressing Nwk^1-428-mCherry and treated with Nocodazole, a microtubule-depolymerizing drug, or DMSO control. Images show 2D projections of confocal stacks taken before (t = 0), and 15 minutes after (t = 15) treatment. (F) Quantification of cell shape before and after treatment. Protrusion index is represented by cell perimeter divided by the square root of cell area (P/√A).

Figure 2.

Model for formation of Nwk-induced protrusions at S2 cell plasma membranes. Higher-order assembly of Nwk in zigzags on the lipid bilayer results in membrane ridging and resultant inter-ridge buds or scallops (left), which are amplified by the cytoskeleton to form large-scale actin- and microtubule-filled cellular projections (center). In contrast, canonical F-BAR domains oligomerize into filaments and helical scaffolds to induce intracellular tubules (right).