Metabolic and immune-sensitive contacts between lipid droplets and endoplasmic reticulum reconstituted in vitro.

Coordinated cell function requires a variety of subcellular organelles to exchange proteins and lipids across physical contacts that are also referred to as membrane contact sites. Such organelle-to-organelle contacts also evoke interest because they can appear in response to metabolic changes, immune activation, and possibly other stimuli. The microscopic size and complex, crowded geometry of these contacts, however, makes them difficult to visualize, manipulate, and understand inside cells. To address this shortcoming, we deposited endoplasmic reticulum (ER)-enriched microsomes purified from rat liver or from cultured cells on a coverslip in the form of a proteinaceous planar membrane. We visualized real-time lipid and protein exchange across contacts that form between this ER-mimicking membrane and lipid droplets (LDs) purified from the liver of rat. The high-throughput imaging possible in this geometry reveals that in vitro LD-ER contacts increase dramatically when the metabolic state is changed by feeding the animal and also when the immune system is activated. Contact formation in both cases requires Rab18 GTPase and phosphatidic acid, thus revealing common molecular targets operative in two very different biological pathways. An optical trap is used to demonstrate physical tethering of individual LDs to the ER-mimicking membrane and to estimate the strength of this tether. These methodologies can potentially be adapted to understand and target abnormal contact formation between different cellular organelles in the context of neurological and metabolic disorders or pathogen infection.

Validity of point-mass model in off-resonance dynamic atomic force microscopy.

The quantitative measurement of viscoelasticity of nano-scale entities is an important goal of nanotechnology research and there is considerable progress with advent of dynamic atomic force microscopy. The hydrodynamics of cantilever, the force sensor in AFM measurements, plays a pivotal role in quantitative estimates of nano-scale viscoelasticity. The point-mass (PM) model, wherein the AFM cantilever is approximated as a point-mass with mass-less spring is widely used in dynamic AFM analysis and its validity, particularly in liquid environments, is debated. It is suggested that the cantilever must be treated as a continuous rectangular beam to obtain accurate estimates of nano-scale viscoelasticity of materials it is probing. Here, we derived equations, which relate stiffness and damping coefficient of the material under investigation to measured parameters, by approximating cantilever as a point-mass and also considering the full geometric details. These equations are derived for both tip-excited as well as base-excited cantilevers. We have performed off-resonance dynamic atomic force spectroscopy on a single protein molecule to investigate the validity of widely used PM model. We performed measurements with AFMs equipped with different cantilever excitation methods as well as detection schemes to measure cantilever response. The data was analyzed using both, continuous beam model and the PM model. We found that both models yield same results when the experiments are performed in truly off-resonance regime with small amplitudes and the cantilever stiffness is much higher than the interaction stiffness. Our findings suggest that a simple PM approximation based model is adequate to describe the dynamics, provided care is taken while performing experiments so that the approximations used in these models are valid.

A Screen for Membrane Fission Catalysts Identifies the ATPase EHD1.

Membrane fission manifests during cell division, synaptic transmission, vesicular transport, and organelle biogenesis, yet identifying proteins that catalyze fission remains a challenge. Using a facile and robust assay system of supported membrane tubes in a microscopic screen that directly monitors membrane tube scission, we detect robust GTP- and ATP-dependent as well as nucleotide-independent fission activity in the brain cytosol. Using previously established interacting partner proteins as bait for pulldowns, we attribute the GTP-dependent fission activity to dynamin. Biochemical fractionation followed by mass spectrometric analyses identifies the Eps15-homology domain-containing protein1 (EHD1) as a novel ATP-dependent membrane fission catalyst. Together, our approach establishes an experimental workflow for the discovery of novel membrane fission catalysts.

ATP-dependent membrane remodeling links EHD1 functions to endocytic recycling.

Endocytic and recycling pathways generate cargo-laden transport carriers by membrane fission. Classical dynamins, which generate transport carriers during endocytosis, constrict and cause fission of membrane tubes in response to GTP hydrolysis. Relatively, less is known about the ATP-binding Eps15-homology domain-containing protein1 (EHD1), a dynamin family member that functions at the endocytic-recycling compartment. Here, we show using cross complementation assays in C. elegans that EHD1's membrane binding and ATP hydrolysis activities are necessary for endocytic recycling. Further, we show that ATP-bound EHD1 forms membrane-active scaffolds that bulge tubular model membranes. ATP hydrolysis promotes scaffold self-assembly, causing the bulge to extend and thin down intermediate regions on the tube. On tubes below 25 nm in radius, such thinning leads to scission. Molecular dynamics simulations corroborate this scission pathway. Deletion of N-terminal residues causes defects in stable scaffolding, scission and endocytic recycling. Thus, ATP hydrolysis-dependent membrane remodeling links EHD1 functions to endocytic recycling.

Dynamin-related protein 1 has membrane constricting and severing abilities sufficient for mitochondrial and peroxisomal fission.

Dynamin-related protein 1 (Drp1) is essential for mitochondrial and peroxisomal fission. Recent studies propose that Drp1 does not sever but rather constricts mitochondrial membranes allowing dynamin 2 (Dnm2) to execute final scission. Here, we report that unlike Drp1, Dnm2 is dispensable for peroxisomal and mitochondrial fission, as these events occurred in Dnm2 knockout cells. Fission events were also observed in mouse embryonic fibroblasts lacking Dnm1, 2 and 3. Using reconstitution experiments on preformed membrane tubes, we show that Drp1 alone both constricts and severs membrane tubes. Scission required the membrane binding, self-assembling and GTPase activities of Drp1 and occurred on tubes up to 250 nm in radius. In contrast, Dnm2 exhibited severely restricted fission capacity with occasional severing of tubes below 50 nm in radius. We conclude that Drp1 has both membrane constricting and severing abilities and is the dominant dynamin performing mitochondrial and peroxisomal fission.

Salmonella SipA mimics a cognate SNARE for host Syntaxin8 to promote fusion with early endosomes.

SipA is a major effector of Salmonella, which causes gastroenteritis and enteric fever. Caspase-3 cleaves SipA into two domains: the C-terminal domain regulates actin polymerization, whereas the function of the N terminus is unknown. We show that the cleaved SipA N terminus binds and recruits host Syntaxin8 (Syn8) to Salmonella-containing vacuoles (SCVs). The SipA N terminus contains a SNARE motif with a conserved arginine residue like mammalian R-SNAREs. SipAR204Q and SipA1-435R204Q do not bind Syn8, demonstrating that SipA mimics a cognate R-SNARE for Syn8. Consequently, Salmonella lacking SipA or that express the SipA1-435R204Q SNARE mutant are unable to recruit Syn8 to SCVs. Finally, we show that SipA mimicking an R-SNARE recruits Syn8, Syn13, and Syn7 to the SCV and promotes its fusion with early endosomes to potentially arrest its maturation. Our results reveal that SipA functionally substitutes endogenous SNAREs in order to hijack the host trafficking pathway and promote Salmonella survival.

Use of the supported membrane tube assay system for real-time analysis of membrane fission reactions.

The process of membrane fission is fundamental to diverse cellular processes such as nutrient uptake, synaptic transmission and organelle biogenesis, and it involves the localized application of curvature stress to a tubular membrane intermediate, forcing it to undergo scission. Alternative techniques for creating such substrates necessitate the use of micromanipulators or sophisticated optical traps and require a high level of technical expertise. We present a facile method to generate an array of membrane tubes supported on a passivated glass coverslip, which we refer to as supported membrane tubes (SMrTs). SMrT templates are formed upon hydration of a dry lipid mix in physiological buffer and subsequent flow-induced extrusion of the lipid reservoir into long membrane tubes with variable dimensions. Following surface passivation of coverslips, these templates can be formed from a variety of lipids, with as little as 1-2 nmol of lipid in a matter of 2 h, and can be used in membrane-curvature-sensitive fission assays.

A high-throughput platform for real-time analysis of membrane fission reactions reveals dynamin function.

Dynamin, the paradigmatic membrane fission catalyst, assembles as helical scaffolds that hydrolyse GTP to sever the tubular necks of clathrin-coated pits. Using a facile assay system of supported membrane tubes (SMrT) engineered to mimic the dimensions of necks of clathrin-coated pits, we monitor the dynamics of a dynamin-catalysed tube-severing reaction in real time using fluorescence microscopy. We find that GTP hydrolysis by an intact helical scaffold causes progressive constriction of the underlying membrane tube. On reaching a critical dimension of 7.3 nm in radius, the tube undergoes scission and concomitant splitting of the scaffold. In a constant GTP turnover scenario, scaffold assembly and GTP hydrolysis-induced tube constriction are kinetically inseparable events leading to tube-severing reactions occurring at timescales similar to the characteristic fission times seen in vivo. We anticipate SMrT templates to allow dynamic fluorescence-based detection of conformational changes occurring in self-assembling proteins that remodel membranes.

Spatial Control of Epsin-induced Clathrin Assembly by Membrane Curvature.

Epsins belong to the family of highly conserved clathrin-associated sorting proteins that are indispensable for clathrin-mediated endocytosis, but their precise functions remain unclear. We have developed an assay system of budded supported membrane tubes displaying planar and highly curved membrane surfaces to analyze intrinsic membrane curvature preference shown by clathrin-associated sorting proteins. Using real-time fluorescence microscopy, we find that epsin preferentially partitions to and assembles clathrin on highly curved membrane surfaces. Sorting of epsin to regions of high curvature strictly depends on binding to phosphatidylinositol 4,5-bisphosphate. Fluorescently labeled clathrins rapidly assemble as foci, which in turn cluster epsin, while maintaining tube integrity. Clathrin foci grow in intensity with a typical time constant of ∼75 s, similar to the time scales for coated pit formation seen in cells. Epsin therefore effectively senses membrane curvature to spatially control clathrin assembly. Our results highlight the potential role of membrane curvature in orchestrating the myriad molecular interactions necessary for the success of clathrin-mediated membrane budding.