The four main types of biomolecules are nucleic acids, proteins, carbohydrates and lipids. The knowledge about their respective interactions is as important as the individual understanding of each of them. However, while, for example, the interaction of proteins with the other three groups is extensively studied, that of nucleic acids and lipids is, in comparison, very poorly explored. An iconic paradigm of physical (and likely functional) proximity between DNA and lipids is the case of the genomic DNA in eukaryotes: enclosed within the nucleus by two concentric lipid bilayers, the wealth of implications of this interaction, for example in genome stability, remains underassessed. Nuclear lipid-related phenotypes have been observed for 50 years, yet in most cases kept as mere anecdotical descriptions. In this review, we will bring together the evidence connecting lipids with both the nuclear envelope and the nucleoplasm, and will make critical analyses of these descriptions. Our exploration establishes a scenario in which lipids irrefutably play a role in nuclear homeostasis.

Calcium (Ca2+) is a highly versatile intracellular messenger that regulates several cellular processes. Although it is unclear how a single-second messenger coordinates various effects within a cell, there is growing evidence that spatial patterns of Ca2+ signals play an essential role in determining their specificity. Ca2+ signaling patterns can differ in various cell regions, and Ca2+ signals in the nuclear and cytoplasmic compartments have been observed to occur independently. The initiation and function of Ca2+ signaling within the nucleus are not yet fully understood. Receptor tyrosine kinases (RTKs) induce Ca2+ signaling resulting from phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and inositol 1,4,5-trisphosphate (InsP3) formation within the nucleus. This signaling mechanism may be responsible for the effects of specific growth factors on cell proliferation and gene transcription. This review highlights the recent advances in RTK trafficking to the nucleus and explains how these receptors initiate nuclear calcium signaling.

Metastases are responsible for the vast majority of cancer deaths, yet most therapeutic efforts have focused on targeting and interrupting tumor growth rather than impairing the metastatic process. Traditionally, cancer metastasis is attributed to the dissemination of neoplastic cells from the primary tumor to distant organs through blood and lymphatic circulation. A thorough understanding of the metastatic process is essential to develop new therapeutic strategies that improve cancer survival. Since Paget\'s original description of the \"Seed and Soil\" hypothesis over a hundred years ago, alternative theories and new players have been proposed. In particular, the role of extracellular vesicles (EVs) released by cancer cells and their uptake by neighboring cells or at distinct anatomical sites has been explored. Here, we will outline and discuss these alternative theories and emphasize the horizontal transfer of EV-associated biomolecules as a possibly major event leading to cell transformation and the induction of metastases. We will also highlight the recently discovered intracellular pathway used by EVs to deliver their cargoes into the nucleus of recipient cells, which is a potential target for novel anti-metastatic strategies.

Nucleus is at the center stage of cellular drama orchestrated in the life of a cell and the nucleoplasm is surrounded by a double membranous compartment constituting the Nuclear membrane/envelope (NE) that separates it from the cytoplasm in nucleated cells. The initial understanding of the NE was that of a border security entity between the nucleus and the cytoplasm, separating gene regulation and transcription in the nucleus from translation in the cytoplasm. However, the discovery of a wide array of inherited diseases caused by mutations in genes encoding proteins that reside or interact with NE diverted the interest into deciphering the lipid-protein-rich environment of the NE. Today, the NE is considered a dynamic organelle which forms a functional linkage between the nucleus and the rest of the cell. The exposure of NE to constant mechanical constraints by its connectivity to the large polymer network of the lamina and chromatin on one side, and to the cytoskeleton on the other side results, in a variety of shape changes. We discuss two such deformation, the formation of nuclear blebs and nucleoplasmic reticulum (NER). Although the protein and the lipid composition of NE comprises a small fraction of the total lipid-protein load of the cell, the ability to define the lipid-protein composition of Inner nuclear membrane (INM) and Outer nuclear membrane (ONM) with precision is crucial for obtaining a deeper mechanistic understanding of their lipid-protein interaction and the various signaling pathways that are triggered by them. In addition, this allows us to further understand the direct and indirect roles of NE machinery in the chromosomal organization and gene regulation.

Invaginations of the nuclear membrane occur in different shapes, sizes, and compositions. Part of these pleiomorphic invaginations make up the nucleoplasmic reticulum (NR), while others are merely nuclear folds. We define the NR as tubular invaginations consisting of either both the inner and outer nuclear membrane, or only the inner nuclear membrane. Specifically, invaginations of both the inner and outer nuclear membrane are also called type II NR, while those of only the inner nuclear membrane are defined as type I NR. The formation and structure of the NR is determined by proteins associated to the nuclear membrane, which induce a high membrane curvature leading to tubular invaginations. Here we review and discuss the current knowledge of nuclear invaginations and the NR in particular. An increase in tubular invaginations of the nuclear envelope is associated with several pathologies, such as laminopathies, cancer, (reversible) heart failure, and Alzheimer\'s disease. Furthermore, viruses can induce both type I and II NR. In laminopathies, the amount of A-type lamins throughout the nucleus is generally decreased or the organization of lamins or lamin-associated proteins is disturbed. Also, lamin overexpression or modulation of lamin farnesylation status impacts NR formation, confirming the importance of lamin processing in NR formation. Virus infections reorganize the nuclear lamina via (de)phosphorylation of lamins, leading to an uneven thickness of the nuclear lamina and in turn lobulation of the nuclear membrane and the formation of invaginations of the inner nuclear membrane. Since most studies on the NR have been performed with cell cultures, we present additional proof for the existence of these structures in vivo, focusing on a variety of differentiated cardiovascular and hematopoietic cells. Furthermore, we substantiate the knowledge of the lamin composition of the NR by super-resolution images of the lamin A/C and B1 organization. Finally, we further highlight the essential role of lamins in NR formation by demonstrating that (over)expression of lamins can induce aberrant NR structures.

Tapetal cells comprise an anther tissue fundamental to pollen grain development. They are associated with endoreduplication events, which culminate in polyploid and multinucleated cells, high metabolic activity, and different organelle arrangements to support all the development of the pollen grains. Passiflora species present a secretory tapetum, with diversity in the number and size of nuclei. Tapetal cells undergo numerous changes in a short period of development when compared to the plant\'s life span. To improve our knowledge of tapetum development, tests assessing ploidy levels, anatomy, cytochemistry, transmission electron microscopy, flow cytometry, as well as conventional and molecular cytogenetics were used in Passiflora actinia and P. elegans. The current data show striking differences in nuclear organisation during tapetal cell development, including mono to quadrinucleate cells, and ploidy levels from 2n to 32n. One of the most peculiar features was the atypical behaviour of the endoplasmic reticulum (ER), which accumulated in the cell border, similar to a \'cER\', as well as large dictyosomes. This endomembrane configuration may be related to the tapetum nutritional network and secretion of compounds at the end of meiosis. Another atypical feature of the ER was the formation of an invagination to establish \'binucleated\' polyploid cells. This membrane projection appears when the nuclei form two lobes, as well as when it organises a nucleoplasmic reticulum. These data demonstrate that there are important ultrastructural changes in tapetal cells, including organelle arrangements, ploidy levels, and nuclear activity, common to P. actinia and P. elegans, but different from the plant model A. thaliana.

Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pathological conditions, including the induction of pro-metastatic traits, but it is not yet known how and where functional cargoes of EVs are delivered to their targets in host cell compartments. We have described that after endocytosis, EVs reach Rab7+ late endosomes and a fraction of these enter the nucleoplasmic reticulum and transport EV biomaterials to the host cell nucleoplasm. Their entry therein and docking to outer nuclear membrane occur through a tripartite complex formed by the proteins VAP-A, ORP3 and Rab7 (VOR complex). Here, we report that the antifungal compound itraconazole (ICZ), but not its main metabolite hydroxy-ICZ or ketoconazole, disrupts the binding of Rab7 to ORP3-VAP-A complexes, leading to inhibition of EV-mediated pro-metastatic morphological changes including cell migration behaviour of colon cancer cells. With novel, smaller chemical drugs, inhibition of the VOR complex was maintained, although the ICZ moieties responsible for antifungal activity and interference with intracellular cholesterol distribution were removed. Knowing that cancer cells hijack their microenvironment and that EVs derived from them determine the pre-metastatic niche, small-sized inhibitors of nuclear transfer of EV cargo into host cells could find cancer therapeutic applications, particularly in combination with direct targeting of cancer cells.

The shape of plant nuclei varies among different species, tissues, and cell types. In Arabidopsis thaliana seedlings, nuclei in meristems and guard cells are nearly spherical, whereas those of epidermal cells in differentiated tissues are elongated spindle-shaped. The vegetative nuclei in pollen grains are irregularly shaped in angiosperms. In the past few decades, it has been revealed that several nuclear envelope (NE) proteins play the main role in the regulation of the nuclear shape in plants. Some plant NE proteins that regulate nuclear shape are also involved in nuclear or cellular functions, such as nuclear migration, maintenance of chromatin structure, gene expression, calcium and reactive oxygen species signaling, plant growth, reproduction, and plant immunity. The shape of the nucleus has been assessed both by labeling internal components (for instance chromatin) and by labeling membranes, including the NE or endoplasmic reticulum in interphase cells and viral-infected cells of plants. Changes in NE are correlated with the formation of invaginations of the NE, collectively called the nucleoplasmic reticulum. In this review, what is known and what is unknown about nuclear shape determination are presented, and the physiological significance of the control of the nuclear shape in plants is discussed.

Expansion microscopy (ExM) is a magnification method that allows achieving super-resolved images using a conventional light microscope. In ExM, biomolecules, fluorescent proteins, and dyes are functionalized with specific handles to link a dense polyelectrolyte hydrogel, which can achieve an isotropic expansion of 4.5-fold in water. The use of ExM coupled with STED nanoscopy allows examining macromolecular machinery in life science, like the nuclear pore complex (NPC). In particular, in this chapter, we show a general protocol for labeling one of its subunit, i.e. the Nup153. Such method shows the nanoscale isotropy of the expansion process and enables precise measurement of the expansion factor. Finally, we used ExM for the visualization of a peculiar nuclear invagination in normal and aged cells.

Extracellular membrane vesicles (EVs) are emerging as new vehicles in intercellular communication, but how the biological information contained in EVs is shared between cells remains elusive. Several mechanisms have been described to explain their release from donor cells and the initial step of their uptake by recipient cells, which triggers a cellular response. Yet, the intracellular routes and subcellular fate of EV content upon internalization remain poorly characterized. This is particularly true for EV-associated proteins and nucleic acids that shuttle to the nucleus of host cells. In this review, we will describe and discuss the release of EVs from donor cells, their uptake by recipient cells, and the fate of their cargoes, focusing on a novel intracellular route wherein small GTPase Rab7+ late endosomes containing endocytosed EVs enter into nuclear envelope invaginations and deliver their cargo components to the nucleoplasm of recipient cells. A tripartite protein complex composed of (VAMP)-associated protein A (VAP-A), oxysterol-binding protein (OSBP)-related protein-3 (ORP3), and Rab7 is essential for the transfer of EV-derived components to the nuclear compartment by orchestrating the particular localization of late endosomes in the nucleoplasmic reticulum.