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In this report, the authors present evidence on the restitution of nuclear …

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- Homeostatic restitution of cell membranes. Nuclear membrane lipid biogenesis and transport of protein from cytosol to intranuclear spaces

4. Discussion

In response to myriad of stimuli, the receptors expressed by the cell regulate cellular signaling pathways impacting cell metabolism, secretory properties, electrical activity, shape, motility, and virtually all functions within the realm of genome [1,30,31,35]. Several operative mechanisms to accomplish this goal have been delineated, and the concept that transmission of extracellular signals to the interior of the cell is one of the most fundamental processes of cell membrane receptors is used without reproach. Yet, within this global concept, there are still numerous factors to investigate and provide clear depiction of the cellular elements that contribute to appearance of the final cell and its function. One of them is the cell membrane lipid network, the membrane lipid biogenesis, and the fidelity of the processes charged with membrane restitution. It is not explored how the receptor proteins are delivered to cell membrane and incessantly provided with the same elemental environment, how the transmembrane architecture of the receptors is ceaselessly reproduced, or how the movement of the cytosolic proteins to nucleus is achieved [6,7,19,22,32,36,37]. Today, almost entire emphasis in cellular signaling is placed on the functional roles of RNAs in cytosol [10,28,38,39], and mutation-induced conformational changes in protein structure and function, simulated in most rudimentary lipid environment provided by the arbitrary cocktails of extraneous lipid mixtures or localized with aid of colored labels.

In our investigations we concentrated on the role of specific lipids in cellular membrane biogenesis that accompany the signaling processes, materialized in transcription, translation, posttranslational modification and homeostatic restitution of the cell [13-18]. Initially, we established that lipid biogenesis in ER is the elemental process initiating the intracellular membranes and vesicular transport from ER to Golgi [13-15]. The ER vesicles, consisting of newly synthesized lipids and proteins, transfer the synthesized cargo of proteins and newly assembled membranes from ER to Golgi. Further maturation processes of the delivered membrane, and the subcellular specificity of the membrane occurs in Golgi. The Cer of the membrane is used to synthesize sphingomyelin and glycosphingolipids, whereas PI is phosphorylated to PI3P and PI4P [14-18]. The transport vesicles with secretory proteins and the membrane containing PI3P and glycosphingolipids, displayed affinity for and fused with apical membranes, while the vesicles containing PI4P in their membrane were delivered to endosomes [16,18].

The specific biomembrane-protein assembly, and the lipid synthesized in ER and specifically modified in Golgi were in the entirety introduced to cell membrane or intracellular organelle [13-18]. Moreover, the vesicles capable of fusing with apical membrane would not fuse with ER or endosomes [13,14,18]. Further, the transport vesicles that remained integral part of endosomes would not integrate into the apical cell membrane, and in the experiments with apical membranes were found as portion of unattached vesicles remaining in the cytosol [16-18]. In both events, as determined by lipid composition of the products, the transport vesicles by fusing with membranes, delivered en bloc lipids and membrane proteins. The analysis of lipid composition of the products confirmed the specificity and irreversibility of the processes that allow ER membranes be free of SM and GLL. In our opinion, these data generate a solid support for our concept that transport is highly lipid membrane-specific, irreversible and that the retrograde transport is not returning empty transport vesicles to ER [26], since neither apical membrane specific lipids nor NIP-labeled apical membrane and caveolea [29,40] were identified in cell organelles.

If in the apical vesicular transport, the vesicles were recovered by the way of retrograde transport, fused with ER and resumed another round of transport, the composition of intracellular membranes would be uniform throughout, including cell and ER membrane. This is certainly not the case, since each organelle displays its specific membrane make-up [13-18]. It may be argued that the intracellular organelle modification is associated with delivery of lipids from the cytosol [5,33]. However, as we have shown in our reports, the lipids suspended in the cytosol do not intercalate into specific membranes, but some in a fashion similar to transport vesicles [16-18] may merely associate with membrane proteins. For instance, the addition of Cer to cytosol does not stimulate production of transport vesicles, yields more vesicles or vesicles with higher concentration of Cer [16,18]. With the use of some artificial lipid mixtures that encase protein receptors, some retention of the complex on the membrane may occur, but certainly not the genuine insertion or multiple intercalations into the membrane is taking place.

The process of membrane renewal is coordinated with protein and biomembrane synthesis in ER [18]. Only if both processes coincide, the specific intercalation of the protein is feasible. In keeping with our hypothesis that specific lipids and proteins are synthesized at designated sites of ER are the findings on cotranslational assembly of a protein complexes encoded by a family of colocalized mRNAs [9,10]. Local recruitment of mRNAs to coordinate synthesis of the complexes contributing to overall cell motility by supplying new protein (new actin monomers precisely at the sites where they are needed) is an example of the location-specific translation [39]. Although data on cotranslational assembly of a protein complexes encoded by a family of colocalized mRNAs are derived from completely unrelated to membrane biogenesis experimental paradigm [10], it firmly attests to the significance of the assembly of protein complexes at the specific sites of ER.

While the vesicular transport is subject of many reviews and highly imaginative interpretations are offered to explain secretory transport processes, the issue of the transport to nucleus remains ambiguous [1,20,23,27-31]. The question still is how the cytosol protein can be transported onto the inner nuclear membrane surface [1-5,11,12, 28,35,38]. In recent studies on nuclear receptors, the structural analyses revealed phosphatidylinositides as ligands for the NR5 orphan receptors and showed that PIP phospholipids are involved in the receptor transport [6,21,38]. While elegant structural paradigms present the activation of lipid ligands-regulated receptors, the conformation of the interacting molecules is deduced from studies with artificial liposomes [2].

Our study presented in this report addresses similar issue of protein delivery from cytosol to the nucleus, and show that ER membrane directly appose to nuclear membrane is involved. The results demonstrate that nuclear membrane synthesized by ER-initiated synthesis of new membrane lipids and the enlargement of the outer layer of the nuclear membrane must generate lateral movement of the membrane between nuclear pores. Such movement not only allows restitution of the nuclear inner membrane but also imports the lipid ligand-associated proteins from the cytosol to nucleus, and perhaps transfers the nuclear components to the cytosol. The fact that nuclear envelope consists of two layers of uninterrupted membrane connected with ER, provides most sensible explanation that lateral movement of the membrane is the most feasible process that connects nuclear, ER, and cytosolic events [11].

Interestingly, when the ER membranes, other than outer nuclear membrane were involved in the synthesis of lipids and generation of transport vesicles, the incubation of the ONM/ER with cytosol generated PC, PI, PIPs and PA but transport vesicles were not formed. The lipids were synthesized when the intact nuclei were incubated with the cytosol but not in the presence of the inner nuclear contents. On the other hand, the experiments performed with INM and ONM derived from intact nuclei but with cytosol afforded synthesis of new lipids in both membranes. The intriguing finding that INM is enriched with new lipids when incubated with the cytosol is in agreement with histochemical studies and the in vitro data suggesting the functional relationship between phospholipids and gene expression/transcription [19]. Yet, the existence of intranuclear synthesis of phospholipids is contradicted by the results of our experiments demonstrating that nuclear contents from purified nuclei is not capable to generate new phospholipids. Only, if the nuclear membrane movement is considered, our findings and those described above [19] can be reconciled by the interpretation that the appearance of new phospholipids in the INM is the consequence of ONM lateral movement-induced transfer. Such movement of the membrane would provide continuous transport of the lipid ligand-associated cytosolic protein into the nucleus, restitution of the nuclear INM phosphatidylinositides, and steady growth of ER membrane.

In keeping with the hypothesis is the fact that lipid ligand such as PIP2 interacts with cytosolic protein, but upon delivery to the nucleus the complex is dissociated. The NIP-30 initially in complex with PIP2 appears as free of lipid nuclear protein, whereas the PIP2 vanishes. At this stage we cannot provide evidence whether the PIPs degradation is taking place during release of the protein, or later before the membrane reenters cytosolic environment. The possibility exists that PIPs of INM are degraded by nuclear PLC and serve as a source of inositol phosphate in the nucleus. Recent study on RNA editing provide evidence that nuclear inositol phosphates are implicated in the formation of an active site of adenosine deaminase, and are important in the in vivo and in vitro deamination of adenosine [38].

The evidence that outer nuclear membrane/endoplasmic reticulum is involved in the synthesis of phospholipids and restitution of nuclear membranes, whereas the endoplasmic reticulum engaged in synthesis of transport vesicles containing ceramides is used for restitution of Golgi, endosomes and apical membranes, provides initial view of two highly dependent pathways responsible for reproduction of cell components and prevention of the cell demise. The dynamics of the pathways appear to control homeostatic restitution of the cell and its organelles, and all is linked by the cell endogenous cytosol. Hence, the reassembly of the specific units of the cell is determined by (1) the type of cell, (2) the protein induced, (3) the site of the protein function, and (4) the metabolic status of the cytosol. Therefore, the replacement of native cytosol with the one derived from different cells, may indeed impact cellular appearance, physiological functions, and the processes associated with transcription, mRNA translation and small RNAs functions [33,38,39].

As the transcriptome represents the transcripts of genes expressed in the cell and informs which genes are induced or repressed by the metabolic status and physical environment of such cell, the path between the signal translation into the gene transcription and the translation of the transcripts into cellular components was not spatially discerned heretofore. Our studies show that the intracellular and cellular membranes' biogenesis links the signaling events into a path that uniformly restores the cell structure and function. The signals from cell membrane through the cytosol link engage simultaneously the nuclear and organellar events [13-18]. Together, and in perfect synchrony, the ER-initiated processes contribute to the restitution of cell components, retention of the precisely controlled cell structure, and the designated function.

Conflict of interests

Declared none.

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