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

Abstract

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 Al 2 O 3 substrate. The adhesion is mediated by Ca 2+ in the ambient buffer. Subsequent depletion of Ca 2+ 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 Al 2 O 3 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. Video Link The video component of this article can be found at https://www.jove.com/video/58923/ 4. In this method, an ER model is fabricated with a bottom-up approach using only a few elements, in which the molecules of interest can be integrated to add complexity. The network represents ER structure and dynamics. Furthermore, reversible transformation between the planar membrane and the tubes, vesicle formation from the tubes, tube fusion, sliding and retraction can all be observed. In addition to serving as a bottom-up model for the incompletely understood cellular ER, the lipid route to nanotube networks described in this protocol can be applicable for researchers studying self-assembly, nanofluidics, single-molecule and colloid

Subcompartmentalization and Pseudo‐Division of Model Protocells

“…To prepare the lipid suspensions, the dehydration and rehydration method [ 14,65 ] was used. Various types of lipids were utilized, namely l ‐α‐phosphatidylcholine (Soy), 18: dioleoylphosphoethanolamine, l ‐α‐phosphoethanolamine ( Escherichia coli ), l ‐α –phosphatidylglycerol ( E.coli ), Cardiolipin ( E.coli ) and E.coli polar lipid extract.…”

Colony-like Protocell Superstructures

“…Preparation of Lipid Vesicles. The dehydration and rehydration method 79,80 was used to prepare the lipid suspensions. Briefly, lipids (99 wt %) and lipid-conjugated fluorophores (1 wt %) were dissolved in chloroform to a final concentration of 10 mg/mL (cf.…”

Colony-like Protocell Superstructures

“…The dehydration and rehydration method 61, 62 was used to prepare the lipid suspensions. Briefly, lipids (99 wt %) and lipid-conjugated fluorophores (1 wt %) were dissolved in chloroform to a final concentration of 10 mg/ml ( cf.…”