In Vitro Formation of the Endoplasmic Reticulum Occurs Independently of Microtubules by a Controlled Fusion Reaction

Abstract

We have established an in vitro system for the formation of the endoplasmic reticulum (ER). Starting from small membrane vesicles prepared from Xenopus laevis eggs, an elaborate network of membrane tubules is formed in the presence of cytosol. In the absence of cytosol, the vesicles only fuse to form large spheres. Network formation requires a ubiquitous cytosolic protein and nucleoside triphosphates, is sensitive to N-ethylmaleimide and high cytosolic Ca2+ concentrations, and proceeds via an intermediate stage in which vesicles appear to be clustered. Microtubules are not required for membrane tubule and network formation. Formation of the ER network shares significant similarities with formation of the nuclear envelope. Our results suggest that the ER network forms in a process in which cytosolic factors modify and regulate a basic reaction of membrane vesicle fusion.

In vitro reassembly of nuclear envelopes and organelles in Xenopus egg extracts

“…A chelator with a binding constant in this range had a stronger inhibitory effect than that of chelators with lower or higher binding constants [2]. In other systems, similar observations are explained by the fact that cytosolic Ca 2+ gradients can be most effectively dispersed by chelator with a binding constant in the range of the average Ca 2+ concentration in the gradient [33].…”

“…Specific components of the cytosol may thus be required for the vesicle shape change. Thermodynamically, spheres are probably the most stable result of fusion; therefore, one or more cytosolic factors may modify this default reaction to convert it into a fusion reaction that results in a network [2]. In contrast, a low cytosol:vesicle ratio is unfavorable to the formation of the normal bilayer nuclear envelope, instead of the multilayer membrane or multi-double membrane ( Figure 3C and 3D).…”

“…Membranes of most cellular organelles, including the nuclear envelope, the endoplasmic reticulum and Golgi cisternae, and the mitochondrial cristae, have complicated shapes in which two membranes are closely apposed to one another. How these structures are generated and maintained is largely unknown [2].…”

“…It has been shown that in vitro nuclear assembly spontaneously in Xenopus egg interphase extracts a double nuclear membrane with nuclear pore forms around added chromatin, whether natural animal or plant sperm chromatin or exogenously added procaryotic DNA is used [3][4][5]. Some information concerning reassembly of nuclear membranes, organelles, and individual nucleosomes has arisen from the use of cell-free extracts derived from eggs of Xenopus or sea urchin, and somatic cells and embryos of Drosophila or Nicotiana [2,[6][7][8][9][10][11][12][13][14].…”

“…The endoplasmic reticulum, Golgi apparatus, and mitochondria of fertilized ova are present in small membrane tubules and vesicles that, at some point during early development, must fuse to reform the endoplasmic reticulum network, Golgi apparatus, and mitochondria. The machinery for the formation of these structures must also be stored in the egg because there is no synthesis of new material during the first cell divisions [2]. From a biochemical point of view, fragmentation of the cell structures allows their homogenization, with the added advantage that it occurs in such a way as to permit reassembly of the original organelle.…”