Nicolas Chiaruttini

Membrane shape at the edge of the dynamin helix sets location and duration of the fission reaction.

The GTPase dynamin polymerizes into a helical coat that constricts membrane necks of endocytic pits to promote their fission. However, the dynamin mechanism is still debated because constriction is necessary but not sufficient for fission. Here, we show that fission occurs at the interface between the dynamin coat and the uncoated membrane. At this location, the considerable change in membrane curvature increases the local membrane elastic energy, reducing the energy barrier for fission. Fission kinetics depends on tension, bending rigidity, and the dynamin constriction torque. Indeed, we experimentally find that the fission rate depends on membrane tension in vitro and during endocytosis in vivo. By estimating the energy barrier from the increased elastic energy at the edge of dynamin and measuring the dynamin torque, we show that the mechanical energy spent on dynamin constriction can reduce the energy barrier for fission sufficiently to promote spontaneous fission. :

Relaxation of Loaded ESCRT-III Spiral Springs Drives Membrane Deformation

Summary ESCRT-III is required for lipid membrane remodeling in many cellular processes, from abscission to viral budding and multi-vesicular body biogenesis. However, how ESCRT-III polymerization generates membrane curvature remains debated. Here, we show that Snf7, the main component of ESCRT-III, polymerizes into spirals at the surface of lipid bilayers. When covering the entire membrane surface, these spirals stopped growing when densely packed: they had a polygonal shape, suggesting that lateral compression could deform them. We reasoned that Snf7 spirals could function as spiral springs. By measuring the polymerization energy and the rigidity of Snf7 filaments, we showed that they were deformed while growing in a confined area. Furthermore, we observed that the elastic expansion of compressed Snf7 spirals generated an area difference between the two sides of the membrane and thus curvature. This spring-like activity underlies the driving force by which ESCRT-III could mediate membrane deformation and fission.

Dynamic subunit turnover in ESCRT-III assemblies is regulated by Vps4 to mediate membrane remodelling during cytokinesis

The endosomal sorting complex required for transport (ESCRT)-III mediates membrane fission in fundamental cellular processes, including cytokinesis. ESCRT-III is thought to form persistent filaments that over time increase their curvature to constrict membranes. Unexpectedly, we found that ESCRT-III at the midbody of human cells rapidly turns over subunits with cytoplasmic pools while gradually forming larger assemblies. ESCRT-III turnover depended on the ATPase VPS4, which accumulated at the midbody simultaneously with ESCRT-III subunits, and was required for assembly of functional ESCRT-III structures. In vitro, the Vps2/Vps24 subunits of ESCRT-III formed side-by-side filaments with Snf7 and inhibited further polymerization, but the growth inhibition was alleviated by the addition of Vps4 and ATP. High-speed atomic force microscopy further revealed highly dynamic arrays of growing and shrinking ESCRT-III spirals in the presence of Vps4. Continuous ESCRT-III remodelling by subunit turnover might facilitate shape adaptions to variable membrane geometries, with broad implications for diverse cellular processes.

Dynamic remodeling of the dynamin helix during membrane constriction

Significance The GTPase dynamin catalyzes membrane fission and is essential in endocytosis and other events such as organelle division. Dynamin is a unique molecular motor with torsional and contractile abilities. Because these abilities involve a conformational change at the whole-polymer level, standard structural biology tools have not been able to fully unravel the mechanism by which it constricts and twists. Here we used high-speed atomic force microscopy to image the constriction and fission of dynamin-coated tubules with subnanometer and subsecond resolution. Our results provide important findings to establish the contribution of the various constriction mechanisms. Dynamin is a dimeric GTPase that assembles into a helix around the neck of endocytic buds. Upon GTP hydrolysis, dynamin breaks these necks, a reaction called membrane fission. Fission requires dynamin to first constrict the membrane. It is unclear, however, how dynamin helix constriction works. Here we undertake a direct high-speed atomic force microscopy imaging analysis to visualize the constriction of single dynamin-coated membrane tubules. We show GTP-induced dynamic rearrangements of the dynamin helix turns: the average distances between turns reduce with GTP hydrolysis. These distances vary, however, over time because helical turns were observed to transiently pair and dissociate. At fission sites, these cycles of association and dissociation were correlated with relative lateral displacement of the turns and constriction. Our findings show relative longitudinal and lateral displacements of helical turns related to constriction. Our work highlights the potential of high-speed atomic force microscopy for the observation of mechanochemical proteins onto membranes during action at almost molecular resolution.

Dynamic and elastic shape transitions in curved ESCRT-III filaments

The ESCRT-III complex is an evolutionary ancient and conserved complex that catalyzes fission of lipid membranes from the lumen of the neck in many, if not all processes requiring this specific fission reaction. The ESCRT-III membrane remodeling complex is unique as its molecular and polymeric structures do not intuitively suggests how it could deform and break lipid membranes. Here we review the common structural features of the ESCRT-III subunits, and the shape diversity of the various filamentous forms. We propose a simple geometry and elasticity framework that could help to isolate which features of the ESCRT-III filaments are common to all filamentous forms as well as to explain their diversity. We speculate on how these features could provide mechanistic insights into the many functions of the ESCRT-III complex.

Decrease in plasma membrane tension triggers PtdIns(4,5)P 2 phase separation to inactivate TORC2

The target of rapamycin complex 2 (TORC2) plays a key role in maintaining the homeostasis of plasma membrane (PM) tension. TORC2 activation following increased PM tension involves redistribution of the Slm1 and 2 paralogues from PM invaginations known as eisosomes into membrane compartments containing TORC2. How Slm1/2 relocalization is triggered, and if/how this plays a role in TORC2 inactivation with decreased PM tension, is unknown. Using osmotic shocks and palmitoylcarnitine as orthogonal tools to manipulate PM tension, we demonstrate that decreased PM tension triggers spontaneous, energy-independent reorganization of pre-existing phosphatidylinositol-4,5-bisphosphate into discrete invaginated membrane domains, which cluster and inactivate TORC2. These results demonstrate that increased and decreased membrane tension are sensed through different mechanisms, highlighting a role for membrane lipid phase separation in mechanotransduction.Using a small-molecule modulator of TORC2 signalling and a mechanosensitive probe, Riggi et al. reveal that decreased plasma membrane tension induces distinct PIP2-enriched domains that sequester and inactivate TORC2.

Lysophospholipids Facilitate COPII Vesicle Formation

Summary Coat protein complex II (COPII) proteins form vesicles from the endoplasmic reticulum to export cargo molecules to the Golgi apparatus. Among the many proteins involved in this process, Sec12 is a key regulator, functioning as the guanosine diphosphate (GDP) exchange factor for Sar1p, the small guanosine triphosphatase (GTPase) that initiates COPII assembly. Here we show that overexpression of phospholipase B3 in the thermosensitive sec12-4 mutant partially restores growth and protein transport at non-permissive temperatures. Lipidomics analyses of these cells show a higher content of lysophosphatidylinositol (lysoPI), consistent with the lipid specificity of PLB3. Furthermore, we show that lysoPI is specifically enriched in COPII vesicles isolated from in vitro budding assays. As these results suggested that lysophospholipids could facilitate budding under conditions of defective COPII coat dynamics, we reconstituted COPII binding onto giant liposomes with purified proteins and showed that lysoPI decreases membrane rigidity and enhances COPII recruitment to liposomes. Our results support a mechanical facilitation of COPII budding by lysophospholipids.

A Novel Method to Couple Electrophysiological Measurements and Fluorescence Imaging of Suspended Lipid Membranes: The Example of T5 Bacteriophage DNA Ejection

We present an innovative method to couple electrophysiological measurements with fluorescence imaging of functionalized suspended bilayers. Our method combines several advantages: it is well suited to study transmembrane proteins that are difficult to incorporate in suspended bilayers, it allows single molecule resolution both in terms of electrophysiological measurements and fluorescence imaging, and it enables mechanical stimulations of the membrane. The approach comprises of two steps: first the reconstitution of membrane proteins in giant unilamellar vesicles; then the formation of a suspended bilayer spanning a 5 to 15 micron-wide aperture that can be visualized by high NA microscope objectives. We exemplified how the technique can be used to detect in real time the translocation of T5 DNA across the bilayer during its ejection from the bacteriophage capsid.

LimeSeg: A coarsed-grained lipid membrane simulation for 3D image segmentation

Bioimage analysis is an important preliminary step required for data representation and quantitative studies. To carry out these tasks, we developed LimeSeg, an easy-to-use, efficient and modular 3D image segmentation method. Based on the idea of SURFace ELements, LimeSeg resembles a highly coarse-grained simulation of a lipid membrane in which a set of particles, analogous to lipid molecules, are attracted to local image maxima. The particles are self-generating and self-destructing thus providing the ability for the membrane to evolve towards the contour of the object of interest. We characterize the emergent mechanical properties of this system and show how it can be used to segment many 3D objects from numerous types of image of biological samples (brain MRI, cell epithelium, cellular organelles). LimeSeg is available as a Fiji plugin that includes simple commands, a 3D visualizer, and customization options via ImageJ scripting.

Optical control of cytoplasmic flows

Cytoplasmic flows are essential for various cellular processes. However, tools to manipulate these flows within cells are still lacking. Now research shows that an optical tool allows for control of cytoplasmic flows and can be used as a subcellular rheometer.