Thomas J. Pucadyil

Geometric Catalysis of Membrane Fission Driven by Flexible Dynamin Rings

Making the Cut Dynamin is the prototypical member of a large family of structurally related guanosine triphosphatases involved in membrane fission and fusion. A variety of models have been suggested to explain how dynamin works. Shnyrova et al. (p. 1433; see the Perspective by Holz) reconstituted dynamin-mediated membrane scission on lipid nanotubes and suggest a molecular model for dynamin activity that takes into consideration all known aspects of dynamin function. Guanosine triphosphate hydrolysis limits polymerization of the membrane protein dynamin on lipid nanotubes into short, metastable collars. [Also see Perspective by Holz] Biological membrane fission requires protein-driven stress. The guanosine triphosphatase (GTPase) dynamin builds up membrane stress by polymerizing into a helical collar that constricts the neck of budding vesicles. How this curvature stress mediates nonleaky membrane remodeling is actively debated. Using lipid nanotubes as substrates to directly measure geometric intermediates of the fission pathway, we found that GTP hydrolysis limits dynamin polymerization into short, metastable collars that are optimal for fission. Collars as short as two rungs translated radial constriction to reversible hemifission via membrane wedging of the pleckstrin homology domains (PHDs) of dynamin. Modeling revealed that tilting of the PHDs to conform with membrane deformations creates the low-energy pathway for hemifission. This local coordination of dynamin and lipids suggests how membranes can be remodeled in cells.

Spatial Control of Epsin-induced Clathrin Assembly by Membrane Curvature

Background: The clathrin adaptor epsin is indispensable for clathrin-mediated endocytosis, but the mechanism by which it regulates clathrin assembly remains unclear. Results: Epsin shows a preference to localize to regions of high membrane curvature. Conclusion: Epsins membrane curvature sensing directs clathrin assembly to highly curved membranes. Significance: Membrane curvature could determine the hierarchy of molecular interactions during clathrin-mediated membrane budding. 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.

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.

The pleckstrin-homology domain of dynamin is dispensable for membrane constriction and fission

Classical dynamins engage in rapid vesicle release during synaptic vesicle recycling and contain a lipid-binding domain called the pleckstrin-homology domain (PHD). An analysis of a reengineered dynamin construct lacking the PHD shows that the PHD acts as a catalyst to enhance the rates of dynamin-induced membrane fission.

Comparative analysis of adaptor-mediated clathrin assembly reveals general principles for adaptor clustering

Clathrin-mediated endocytosis sorts the bulk of membrane proteins and is a process that starts with adaptor-induced clathrin assembly. Real-time fluorescence analysis shows that adaptor sorting is determined not by the extent of clathrin recruited or the degree of clathrin clustered but instead by the rate of clathrin assembly.

Dynamic Remodeling of Membranes Catalyzed by Dynamin

Publisher Summary This chapter discusses the dynamic remodeling of membranes catalyzed by dynamin. Members of the dynamin family of large GTPases are unique among proteins involved in remodeling cell membranes. They readily polymerize on favorable membrane templates and display reversible modes of membrane association that is coupled to their GTPase cycle. Recent studies with dynamin-1—a prototypical member of the dynamin superfamily—have highlighted their capacity to catalyze membrane fission under conditions of constant GTP turnover. Eukaryotic cells are characterized by an elaborate endomembrane system encapsulated by the plasma membrane. Comprised of the endoplasmic reticulum, Golgi apparatus, endosomes and lysosomes, the endomembrane system, and the plasma membrane constitute a dynamic membrane network. The striking observation that dynamin can spontaneously form highly ordered helical spirals to a large extent contributed to defining mechanochemical models for dynamin function. Preassembled helical spirals of dynamin are an obvious starting point as nucleotide-based conformational changes have traditionally compared the apo- and nucleotide-bound states.

The 2018 biomembrane curvature and remodeling roadmap

The importance of curvature as a structural feature of biological membranes has been recognized for many years and has fascinated scientists from a wide range of different backgrounds. On the one hand, changes in membrane morphology are involved in a plethora of phenomena involving the plasma membrane of eukaryotic cells, including endo- and exocytosis, phagocytosis and filopodia formation. On the other hand, a multitude of intracellular processes at the level of organelles rely on generation, modulation, and maintenance of membrane curvature to maintain the organelle shape and functionality. The contribution of biophysicists and biologists is essential for shedding light on the mechanistic understanding and quantification of these processes. Given the vast complexity of phenomena and mechanisms involved in the coupling between membrane shape and function, it is not always clear in what direction to advance to eventually arrive at an exhaustive understanding of this important research area. The 2018 Biomembrane Curvature and Remodeling Roadmap of Journal of Physics D: Applied Physics addresses this need for clarity and is intended to provide guidance both for students who have just entered the field as well as established scientists who would like to improve their orientation within this fascinating area.

Excess area dependent scaling behavior of nano-sized membrane tethers

Thermal fluctuations in cell membranes manifest as an excess area ([Formula: see text]) which governs a multitude of physical process at the sub-micron scale. We present a theoretical framework, based on an in silico tether pulling method, which may be used to reliably estimate [Formula: see text] in live cells. We perform our simulations in two different thermodynamic ensembles: (i) the constant projected area and (ii) the constant frame tension ensembles and show the equivalence of our results in the two. The tether forces estimated from our simulations compare well with our experimental measurements for tethers extracted from ruptured GUVs and HeLa cells. We demonstrate the significance and validity of our method by showing that all our calculations performed in the initial tether formation regime (i.e. when the length of the tether is comparable to its radius) along with experiments of tether extraction in 15 different cell types collapse onto two unified scaling relationships mapping tether force, tether radius, bending stiffness κ, and membrane tension σ. We show that [Formula: see text] is an important determinant of the radius of the extracted tether, which is equal to the characteristic length [Formula: see text] for [Formula: see text], and is equal to [Formula: see text] for [Formula: see text]. We also find that the estimated excess area follows a linear scaling behavior that only depends on the true value of [Formula: see text] for the membrane, based on which we propose a self-consistent technique to estimate the range of excess membrane areas in a cell.

SMrT Assay for Real-Time Visualization and Analysis of Clathrin Assembly Reactions

Clathrin-mediated endocytosis manages the vesicular transport of the bulk of membrane proteins from the plasma membrane and the trans-Golgi network. During this process, discrete sets of adaptor proteins recognize specific classes of membrane proteins, which recruit and assemble clathrin lattices on the membrane. An important determinant to the success of this vesicular transport reaction is the intrinsic ability of adaptors to polymerize clathrin on a membrane surface. Adaptor-induced clathrin assembly has traditionally been analyzed using static electron microscopy-based approaches. Here, we describe a methodology to follow adaptor-induced clathrin assembly in real-time using fluorescence microscopy on a facile model membrane assay system of supported membrane tubes (SMrT). Results from such assays can be conveniently run through routine image analysis procedures to extract kinetic parameters of the clathrin assembly reaction.

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.