Allosteric control of dynamin-related protein 1 through a disordered C-terminal Short Linear Motif.

The mechanochemical GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial and peroxisomal fission, but the regulatory mechanisms remain ambiguous. Here we find that a conserved, intrinsically disordered, six-residue Short Linear Motif at the extreme Drp1 C-terminus, named CT-SLiM, constitutes a critical allosteric site that controls Drp1 structure and function in vitro and in vivo. Extension of the CT-SLiM by non-native residues, or its interaction with the protein partner GIPC-1, constrains Drp1 subunit conformational dynamics, alters self-assembly properties, and limits cooperative GTP hydrolysis, surprisingly leading to the fission of model membranes in vitro. In vivo, the involvement of the native CT-SLiM is critical for productive mitochondrial and peroxisomal fission, as both deletion and non-native extension of the CT-SLiM severely impair their progression. Thus, contrary to prevailing models, Drp1-catalyzed membrane fission relies on allosteric communication mediated by the CT-SLiM, deceleration of GTPase activity, and coupled changes in subunit architecture and assembly-disassembly dynamics.

Combining patch-clamping and fluorescence microscopy for quantitative reconstitution of cellular membrane processes with Giant Suspended Bilayers.

In vitro reconstitution and microscopic visualization of membrane processes is an indispensable source of information about a cellular function. Here we describe a conceptionally novel free-standing membrane template that facilitates such quantitative reconstitution of membrane remodelling at different scales. The Giant Suspended Bilayers (GSBs) spontaneously swell from lipid lamella reservoir deposited on microspheres. GSBs attached to the reservoir can be prepared from virtually any lipid composition following a fast procedure. Giant unilamellar vesicles can be further obtained by GSB detachment from the microspheres. The reservoir stabilizes GSB during deformations, mechanical micromanipulations, and fluorescence microscopy observations, while GSB-reservoir boundary enables the exchange of small solutes with GSB interior. These unique properties allow studying macro- and nano-scale membrane deformations, adding membrane-active compounds to both sides of GSB membrane and applying patch-clamp based approaches, thus making GSB a versatile tool for reconstitution and quantification of cellular membrane trafficking events.