Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation

Nanotube-Mediated Path to Protocell Formation

Cellular compartments are membrane-enclosed, spatially distinct microenvironments that confine and protect biochemical reactions in the biological cell. On the early Earth, the autonomous formation of compartments is thought to have led to the encapsulation of nucleotides, thereby satisfying a starting condition for the emergence of life. Recently, surfaces have come into focus as potential platforms for the self-assembly of prebiotic compartments, as significantly enhanced vesicle formation was reported in the presence of solid interfaces. The detailed mechanism of such formation at the mesoscale is still under discussion. We report here on the spontaneous transformation of solid-surface-adhered lipid deposits to unilamellar membrane compartments through a straightforward sequence of topological changes, proceeding via a network of interconnected lipid nanotubes. We show that this transformation is entirely driven by surface-free energy minimization and does not require hydrolysis of organic molecules or external stimuli such as electrical currents or mechanical agitation. The vesicular structures take up and encapsulate their external environment during formation and can subsequently separate and migrate upon exposure to hydrodynamic flow. This may link the self-directed transition from weakly organized bioamphiphile assemblies on solid surfaces to protocells with secluded internal contents

Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases

Mycobacterium tuberculosis adenylyl cyclase (AC) Rv1625c/Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular signalling is well established, the function of their transmembrane (TM) regions remains unknown. Here, we describe the cryo- EM structure of Cya bound to a stabilizing nanobody at 3.6 & Aring; resolution. The TM helices 1-5 form a structurally conserved domain that facilitates the assembly of the helical and catalytic domains. The TM region contains discrete pockets accessible from the extracellular and cytosolic side of the membrane. Neutralization of the negatively charged extracellular pocket Ex1 destabilizes the cytosolic helical domain and reduces the catalytic activity of the enzyme. The TM domain acts as a functional component of Cya, guiding the assembly of the catalytic domain and providing the means for direct regulation of catalytic activity in response to extracellular ligands.ISSN:2050-084

Styrene maleic acid copolymer induces pores in biomembranes

We investigated the interactions between styrene–maleic acid (SMA) copolymers and phospholipid bilayers, using confocal microscopy and surface acoustic wave resonance (SAR) sensing. For the first time we experimentally observed and followed pore formation by SMA copolymers in intact supported bilayers and unilamellar vesicles, showing that fluorescein, a water-soluble organic compound with a mean diameter of 6.9 Å, can traverse the membrane. Our findings are in agreement with recent theoretical predictions, which suggested that SMA copolymers may insert along the plane of the bilayer to form stable toroidal pores