By choosing the cells which are extremely active in the synthesis and transport of the secretory proteins we were able to determine the specificity and lipid composition of the biomembrane that delivers the ER products to Golgi, determine the fine adjustments in the biomembrane composition for the restoration of the apical epithelial cell membrane, and the membrane which was provided for the restitution of endosomes . Our previous studies have demonstrated that intracellular vesicular transport is dependent on the finely tuned synthesis of the cell-specific protein and lipids that assemble a precise vesicular biomembrane in ER [13-18]. Precise and explicit intercalation of the protein into the membrane can only take place when the translation of mRNA and synthesis of biomembrane proceed concomitantly within the ER space capable to generate specific lipids and translate specific mRNA [10,18]. Moreover, the composition of lipids and the membrane protein of a new membrane and the vesicular cargo determine the generation and directional transport of ER vesicles to Golgi. Thus, we found out that the synthesis of ceramides determines the quantity of transport vesicles that deliver protein to Golgi . Concurrently, the substrates in the in situ medium (the cell cytosol) defined the process of transport vesicles synthesis and delivery to the next organelle, since depletion of mRNA from the cytosol reduced the overall transport to Golgi, and was rescued with reinstatement of the initial cytosolic mRNA . Together, these results allowed us to hypothesize that synthesis of the intracellular biomembrane and the protein in ER must be initiated in a explicit site of the organelle, the site that is capable to assemble constitutive protein, specifically formulated biomembrane, and deliver it en bloc to the destination point.
In this report, our studies on nuclei and the inner and outer nuclear/ER membrane reveal feasible path associated with nuclear membrane restitution and transport of the cytosolic protein into the nucleus. While the previous studies [13-18] determined the requirements for the transport and restitution of biomembrane that constitute the outer apical portion of cell membrane and the membranes of Golgi and endosomes, the restitution of ER and the cell nucleus membranes were not apparent or consequential of the same pathway. Also, it was evident that ER is not renewed by the retrograde vesicular transport, since the lipids of ER and nucleus are not identical with the lipids of the apical cell membrane [25,26,29-32]. In addition, we could not substantiate a popular explanation of the subcellular membrane modification through the transferring of the individual lipids from cytosol into ER , or nuclear membrane [29,34], or bi-directional transport between ER and Golgi . As shown in our previous investigation  and here in Fig. 1 (panel A, lanes 1,2,3), the incubation of ER, IN, ONM with cytosol enriched with sphingolipid extracts from Golgi vesicles, cell membrane rafts and caveolea, and apical cell membrane was not facilitating lipid integration into the membranes, and the lipids remained in cytosol (lanes 4,5). On the other hand, addition of lipid precursors ([3H]inositol, [3H]palmitate or [3H]choline) to CC produced membranes with newly synthesized integrated radiolabeled phospholipids and ceramides (lanes 7,8,9).
In our opinion, the separated membranes from IN subjected to prolonged incubation with CC represent the ER scaffold that reentered cytosol and is used for ceramides synthesis and mRNA translation.
Based on the obtained results, we formed hypothesis that the region of ER representing ONM is involved in the synthesis of membrane that generates outer and inner nuclear membrane. The enlargement of the outer area of the nuclear membrane, causes lateral movement of the nuclear membrane in-between nuclear pores and introduces the membrane with newly synthesized lipids to INM, which include PIPs (Fig. 2B) that import cytosol-derived protein into the nucleus. At the same time, continuation of the lateral movement out of the nucleus contributes to the membrane re-emergence from nucleus, transport of nuclear products to cytosol and, restitution of PI, PC, and PA-enriched ER membrane (Fig. 5D). Such movement would explain the presence of radiolabeled PIP2 lipids in the INM obtained from incubation of the IN with CC (Fig. 2A,B lack of lipid synthesis on INM facing nuclear contents, and appearance of the membranes that separate from purified and incubated IN that contain PI, PC and PA, the lipids which perhaps contribute to the restitution of ER membranes distant from ER/nuclear envelope.
Based on the lack of incorporation of NIP-labeled apical membranes and NIP-labeled caveolea into IN and ER (not shown since the NIP-labeled apical membrane proteins could not be detected in ER and nuclear membranes), we conclude that retrograde transport is neither contributing to the restitution of the ER/nuclear membrane nor to the transport of the phosphatidylinositides and cell cytosol protein to the nucleus. The incubation (up to 120 min) of the surface labeled hepatocytes with NIP has not introduced any NIP-labeled protein to either IN, ER or Golgi membrane. If any, the retrograde transport of biomembrane was not occurring in the detectable quantity, or the quantity that could restore the lipids and membranes used for constitutive secretory processes. The phosphatidylinositides detected in the nuclear envelope membranes were not derived from the pool of cell membrane, but were synthesized directly in the outer membrane of the nucleus. Their composition in INM phosphatidylinositides apparently is changing with PIPs hapten-associated protein, as the appearance of free NIP-30 protein in nuclear contents coincided with diminishing and changing PIPs profile in the INM (Fig.2C, Fig.4 C, D, Fig. 5).
Together, our studies suggest divergent and synchronous processes that restore cellular membranes and cell organelles. The CC impacts in synchrony the vesicular transport-assisted cellular and intracellular membranes synthesis restoring Golgi, endosomes and cell membrane, and the nuclear transport connected to nuclear membrane restitution, and ER membrane replacement. The divergent processes initiated in ER are concurrently impacted by the signaling clues deposited in the cell cytosol. And thus, the constitutive delivery of protein to the nucleus mediated through the synthesis of ER/ONM provides the link between genomic inherited design set in motion to restore cell components in homeostatic mode, as well as it may respond to signal-induced pathological cell differentiation in disease.