Study of the interaction of the matrix protein of the newcastle disease virus with lipid bilayers: implications for the mechanism of viral budding

La regulación dinámica de la forma de la membrana celular mediante interacciones proteolipídicas es importante para procesos intra- e inter-celulares. Se sabe que la gemación de muchos virus con envoltura se basa en la acción excusiva de una única proteína: la proteína matriz ó M. En el presente trabajo la gemación viral ha sido reconstituida usando la proteína M del virus de la enfermedad de Newcastle (NDV) y diferentes sistemas lipídicos modelo (membranas planas, vesículas unilamelares gigantes y grandes, y monocapas lipídicas). Así, la gemación por parte de la proteína M de NDV ha sido caracterizada mediante diferentes técnicas experimentales, tales como medidas de admitancia eléctrica, microscopía de fluorescencia, técnicas espectroscópicas, microscopía electrónica y medidas de presión superficial. Los datos experimentales obtenidos señalan que la interacción de la proteína matriz del NDV con los sistemas lipídicos modelo lleva a la gemación de membrana y producción de vesículas cuya distribución de tamaños es similar a la del NDV. De ésta forma, la proteína matriz del NDV es capaz de inducir la gemación de la membrana lipídica en ausencia de otros componentes de la maquinaria celular o viral. La adsorción y gemación por parte de la proteína matriz del NDV depende de la composición lipídica de la membrana, viendose ámbas favorecidas por la presencia de moleculas como el colesterol. Además, se observa que la proteína M del NDV organiza dominios fuidos sobre la membrana lipídica antes de que ocurra la gemación de la membrana. La unidireccionalidad de la gemación producida por la proteína M indica que el autoensamblaje de la proteína sobre la superficie de la membrana lipídica induce una curvatura negativa en la membrana. La formación de dominios proteolipídicos líquidos sobre la superfície de la membrana se propone como el mecanismo para la gemación de membrana celular por parte de la proteína M del NDV.Dynamic regulation of membrane shape by lipid-protein interactions is imperative for many intra and intercellular processes. Matrix proteins of many enveloped viruses have been suggested as the main responsible of the shape transformation of the cellular membrane into the viral vesicle. In order to investigate the molecular mechanism behind the matrix protein driven budding this process has been reconstituted with M protein purified from the NDV and different model lipid systems (planar lipid membranes, large and giant unilamellar vesicles and lipid monolayer). M protein budding activity has been characterized by different experimental approaches, including electric admittance measurements, fluorescence microscopy, spectroscopic approaches, electron microscopy and surface pressure measurements. The experimental data had shown that the interaction of the matrix protein from NDV with model lipid system results in membrane budding and production of membrane vesicles with a size distribution similar to that of the NDV. Thus, NDV matrix protein does not need the presence of any cellular component in order to activate its budding activity. NDV matrix protein binding and budding activities were dependent on the membrane lipid composition, being enhanced in the presence of molecules such as cholesterol in the membrane. Moreover, it was observed that the M protein of NDV organizes fluid-like domains on the lipid membrane prior to the membrane budding. The unidirectionality of budding produced by M protein indicated that the self-assembly of the protein on the membrane surface induced creation of a negative curvature. Fluid-like proteolipid domain creation had been proposed as the mechanism behind the cellular membrane budding induced by the matrix protein of the Newcastle Disease Virus

Reply to Roy and Pucadyil: A gain of function by a GTPase-impaired Drp1

A.V.S. was supported by Spain Ministry of Science and Innovation/National Research Agency/European Regional Development Fund grant PGC2018-099971-B-I00. I.P.J. acknowledges a predoctoral fellowship from the University of the Basque Country. P.M.M. and R.R. were supported by NIH R01 grant GM121583

Vesicle formation by self-assembly of membrane-bound matrix proteins into a fluidlike budding domain

The shape of enveloped viruses depends critically on an internal protein matrix, yet it remains unclear how the matrix proteins control the geometry of the envelope membrane. We found that matrix proteins purified from Newcastle disease virus adsorb on a phospholipid bilayer and condense into fluidlike domains that cause membrane deformation and budding of spherical vesicles, as seen by fluorescent and electron microscopy. Measurements of the electrical admittance of the membrane resolved the gradual growth and rapid closure of a bud followed by its separation to form a free vesicle. The vesicle size distribution, confined by intrinsic curvature of budding domains, but broadened by their merger, matched the virus size distribution. Thus, matrix proteins implement domain-driven mechanism of budding, which suffices to control the shape of these proteolipid vesicles

Microfluidic chip with pillar arrays for controlled production and observation of lipid membrane nanotubes

Lipid membrane nanotubes (NTs) are a widespread template forin vitrostudies of cellular processes happening at high membrane curvature. Traditionally NTs are manufactured one by one, using sophisticated membrane micromanipulations, while simplified methods for controlled batch production of NTs are in growing demand. Here we propose a lab-on-a-chip (LOC) approach to the simultaneous formation of multiple NTs with length and radius controlled by the chip design. The NTs form upon rolling silica microbeads covered by lipid lamellas over the pillars of a polymer micropillar array. The array's design and surface chemistry set the geometry of the resulting free-standing NTs. The integration of the array inside a microfluidic chamber further enables fast and turbulence-free addition of components, such as proteins, to multiple preformed NTs. This LOC approach to NT production is compatible with the use of high power objectives of a fluorescence microscope, making real-time quantification of the different modes of the protein activity in a single experiment possible.This work was partially supported by the Spanish Ministry of Science, Innovation, and Universities grants PGC2018-099971-B-I00, EUR2019-103830, RYC-2014-01419 and BIO2016-80417-P and the Basque Government grants IT1270-19 and IT1271-19. JMMG and MGH acknowledge the predoctoral fellowships from the University of the Basque Country (UPV/EHU). The authors are grateful for the technical support provided by SGIker (UPV/EHU and ERDF, EU) for the SEM experiments

Nonlinear material and ionic transport through membrane nanotubes

Membrane nanotubes (NTs) and their networks play an important role in intracellular membrane transport and intercellular communications. The transport characteristics of the NT lumen resemble those of conventional solid-state nanopores. However, unlike the rigid pores, the soft membrane wall of the NT can be deformed by forces driving the transport through the NT lumen. This intrinsic coupling between the NT geometry and transport properties remains poorly explored. Using synchronized fluorescence microscopy and conductance measurements, we revealed that the NT shape was changed by both electric and hydrostatic forces driving the ionic and solute fluxes through the NT lumen. Far from the shape instability, the strength of the force effect is determined by the lateral membrane tension and is scaled with membrane elasticity so that the NT can be operated as a linear elastic sensor. Near shape instabilities, the transport forces triggered large-scale shape transformations, both stochastic and periodic. The periodic oscillations were coupled to a vesicle passage along the NT axis, resembling peristaltic transport. The oscillations were parametrically controlled by the electric field, making NT a highly nonlinear nanofluidic circuitry element with biological and technological implications.This work was partially supported by NIGMS of the National Institutes of Health under award R01GM121725, RYC-2014-01419 to A.V.S.; Spanish Ministry of Science, Innovation and Universities grants PGC2018-099971-B-I00 and EUR2019-103830 to A.V.S.; Basque Government grant IT1270-19; and the Ministry of Science and Higher Education of the Russian Federation to P.I.K. and G.T.R

Human ATG3 binding to lipid bilayers: role of lipid geometry, and electric charge

Specific protein-lipid interactions lead to a gradual recruitment of AuTophaGy-related (ATG) proteins to the nascent membrane during autophagosome (AP) formation. ATG3, a key protein in the movement of LC3 towards the isolation membrane, has been proposed to facilitate LC3/GABARAP lipidation in highly curved membranes. In this work we have performed a biophysical study of human ATG3 interaction with membranes containing phosphatidylethanolamine, phosphatidylcholine and anionic phospholipids. We have found that ATG3 interacts more strongly with negatively-charged phospholipid vesicles or nanotubes than with electrically neutral model membranes, cone-shaped anionic phospholipids (cardiolipin and phosphatidic acid) being particularly active in promoting binding. Moreover, an increase in membrane curvature facilitates ATG3 recruitment to membranes although addition of anionic lipid molecules makes the curvature factor relatively less important. The predicted N-terminus amphipathic a-helix of ATG3 would be responsible for membrane curvature detection, the positive residues Lys 9 and 11 being essential in the recognition of phospholipid negative moieties. We have also observed membrane aggregation induced by ATG3 in vitro, which could point to a more complex function of this protein in AP biogenesis. Moreover, in vitro GABARAP lipidation assays suggest that ATG3-membrane interaction could facilitate the lipidation of ATG8 homologues.This article is part of COST (European Cooperation in Science and Technology) Actions (PROTEOSTASIS, BM1307, TRANSAUTOPHAGY, CA15138). The authors thank Dr. Isei Tanida (National Institute of Infectious Diseases, Tokyo, Japan) for providing human ATG3 and GABARAP plasmids, and to Dr. Martin B. Ulmschneider (Johns Hopkins University, Baltimore, MD) for Fig. 1B. They are also indebted to Ms Araceli Marcos for technical support. This work was supported in part by grants from the Spanish Ministry of Economy and FEDER (BFU 2011-28566, BFU 2015-66306-P, AGL2011-24758), and from the Basque Government (IT838-13, IT84913). A.S. acknowledges support from RyC Program of the Spanish Ministry of Economy. J.H.H and Z.A. were predoctoral students supported by the University of the Basque Country. Editoria

NMR identification of a conserved Drp1 cardiolipin-binding motif essential for stress-induced mitochondrial fission

Mitochondria form tubular networks that undergo coordinated cycles of fission and fusion. Emerging evidence suggests that a direct yet unresolved interaction of the mechanoenzymatic GTPase dynamin-related protein 1 (Drp1) with mitochondrial outer membrane-localized cardiolipin (CL), externalized under stress conditions including mitophagy, catalyzes essential mitochondrial hyperfragmentation. Here, using a comprehensive set of structural, biophysical, and cell biological tools, we have uncovered a CL-binding motif (CBM) conserved between the Drp1 variable domain (VD) and the unrelated ADP/ATP carrier (AAC/ANT) that intercalates into the membrane core to effect specific CL interactions. CBM mutations that weaken VD-CL interactions manifestly impair Drp1-dependent fission under stress conditions and induce "donut" mitochondria formation. Importantly, VD membrane insertion and GTP-dependent conformational rearrangements mediate only transient CL nonbilayer topological forays and high local membrane constriction, indicating that Drp1-CL interactions alone are insufficient for fission. Our studies establish the structural and mechanistic bases of Drp1-CL interactions in stress-induced mitochondrial fission

Dynamic constriction and fission of endoplasmic reticulum membranes by reticulon

The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. Here we find that the ER has an embedded fragmentation mechanism based upon the ability of reticulon to produce fission of elongating network branches. In Drosophila, Rtnl1-facilitated fission is counterbalanced by atlastin-driven fusion, with the prevalence of Rtnl1 leading to ER fragmentation. Ectopic expression of Drosophila reticulon in COS-7 cells reveals individual fission events in dynamic ER tubules. Consistently, in vitro analyses show that reticulon produces velocity-dependent constriction of lipid nanotubes leading to stochastic fission via a hemifission mechanism. Fission occurs at elongation rates and pulling force ranges intrinsic to the ER, thus suggesting a principle whereby the dynamic balance between fusion and fission controlling organelle morphology depends on membrane motility

Dynamic constriction andfission of endoplasmicreticulum membranes by reticulon

The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. Here we find that the ER has an embedded fragmentation mechanism based upon the ability of reticulon to produce fission of elongating network branches. In Drosophila, Rtnl1-facilitated fission is counterbalanced by atlastin-driven fusion, with the prevalence of Rtnl1 leading to ER fragmentation. Ectopic expression of Drosophila reticulon in COS-7 cells reveals individual fission events in dynamic ER tubules. Consistently, in vitro analyses show that reticulon produces velocity-dependent constriction of lipid nanotubes leading to stochastic fission via a hemifission mechanism. Fission occurs at elongation rates and pulling force ranges intrinsic to the ER, thus suggesting a principle whereby the dynamic balance between fusion and fission controlling organelle morphology depends on membrane motility.This work was partially supported by NIH R01GM121725 to V.A.F., a 5x1000 grant from the Italian Ministry of Health and Telethon GGP11189 to A.D., Spanish Ministry of Science, Innovation and Universities grants BFU2015-70552-P to V.A.F. and A.V.S., and BFU2015-63714-R and PGC2018-099341-B-I00 to B.I., Basque Government grant IT1196-19, Russian Science Foundation Grant No. 17-75-30064 and Ministry of Science and Higher Education of the Russian Federation

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