Manipulating integrin signaling for anti-thrombotic benefits

A hallmark of neurogenesis in vertebrate is the apical-basal fluctuation of radial glia nuclei. Such a phenomenon, called INM, has been known for decades and is closely associated with mitosis but still puzzles scientists. An impressive step in the molecular understanding of INM has recently been achieved by Tsai and coworkers. Using RNA interference associated with time-lapse imaging, these authors demonstrated a dual motor system that can push/pull the nuclei accordingly with the cell cycle stages.     

Mechanism and a Peptide Motif for Targeting Peripheral Proteins to the Yeast Inner Nuclear Membrane

Trm1 is a tRNA specific m₂²G methyltransferase shared by nuclei and mitochondria in Saccharomyces cerevisiae . In nuclei, Trm1 is peripherally associated with the inner nuclear membrane (INM). We investigated the mechanism delivering/tethering Trm1 to the INM. Analyses of mutations of the Ran pathway and nuclear pore components showed that Trm1 accesses the nucleoplasm via the classical nuclear import pathway. We identified a Trm1 cis-acting sequence sufficient to target passenger proteins to the INM. Detailed mutagenesis of this region uncovered specific amino acids necessary for authentic Trm1 to locate at the INM. The INM information is contained within a sequence of less than 20 amino acids, defining the first motif for addressing a peripheral protein to this important subnuclear location. The combined studies provide a multi-step process to direct Trm1 to the INM: (i) translation in the cytoplasm; (ii) Ran-dependent import into the nucleoplasm; and (iii) redistribution from the nucleoplasm to the INM via the INM motif. Furthermore, we demonstrate that the Trm1 mitochondrial targeting and nuclear localization signals are in competition with each other, as Trm1 becomes mitochondrial if prevented from entering the nucleus.     

Interkinetic nuclear migration: a mysterious process in search of a function

During interkinetic nuclear migration (INM), the nuclei in many epithelial cells migrate between the apical and basal surfaces, coordinating with the cell cycle, and undergoing cytokinesis at the apical surface. INM is observed in a wide variety of tissues and species. Recent advances in time-lapse microscopy have provided clues about the mechanisms and functions of INM. Whether actin or microtubules are responsible for nuclear migration is controversial. How mitosis is initiated during INM is poorly understood, as is the relationship between the cell cycle and nuclear movement. It is possible that the disagreements stem from differences in the tissues being studied, since epithelia undergoing INM vary greatly in terms of cell height and cell fates. In this review we examine the reports addressing the mode and mechanisms that regulate INM and suggest possible functions for this dramatic event.     

Regulation of interkinetic nuclear migration by cell cycle-coupled active and passive mechanisms in the developing brain

A hallmark of neurogenesis in the vertebrate brain is the apical-basal nuclear oscillation in polarized neural progenitor cells. Known as interkinetic nuclear migration (INM), these movements are synchronized with the cell cycle such that nuclei move basally during G1-phase and apically during G2-phase. However, it is unknown how the direction of movement and the cell cycle are tightly coupled. Here, we show that INM proceeds through the cell cycle-dependent linkage of cell-autonomous and non-autonomous mechanisms. During S to G2 progression, the microtubule-associated protein Tpx2 redistributes from the nucleus to the apical process, and promotes nuclear migration during G2-phase by altering microtubule organization. Thus, Tpx2 links cell-cycle progression and autonomous apical nuclear migration. In contrast, in vivo observations of implanted microbeads, acute S-phase arrest of surrounding cells and computational modelling suggest that the basal migration of G1-phase nuclei depends on a displacement effect by G2-phase nuclei migrating apically. Our model for INM explains how the dynamics of neural progenitors harmonize their extensive proliferation with the epithelial architecture in the developing brain.     

Making and breaking the inner nuclear membrane proteome

The nuclear envelope (NE) is the defining feature of eukaryotic cells, separating the nucleus from the cytoplasm. It has a complex architecture consisting of two lipid bilayers that, despite being continuous between them and with the endoplasmic reticulum, have different protein compositions consistent with their distinct functions. In particular, the unique composition of the inner nuclear membrane (INM), facing the nucleoplasm and its underlying nuclear lamina, is critical for the organisation and function of nuclear processes, from cell fate to gene regulation and DNA repair. Mutations in INM proteins affecting this organisation are associated with muscular dystrophies and premature ageing syndromes highlighting the role of INM architecture in cell homeostasis. Here, we discuss recent progress in understanding how specific proteins concentrate at the INM, as well as the quality control mechanisms involved in remodelling and maintaining INM protein homeostasis.     

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

Our studies on homeostatic restitution of cellular and subcellular membranes showed that vesicular intracellular transport is engaged in systematic and coordinated replacement of lipids and proteins in the membranes of the secretory, non-dividing epithelial cells (Slomiany et al., J. Physiol. Pharmacol. 2004; 55: 837-860). In this report, we present evidence on the homeostatic restitution of lipids in the biomembranes that constitute nuclear envelopes. We investigated nuclear membranes lipid synthesis by employing purified intact nuclei (IN), the outer nuclear membrane (ONM), the inner nuclear membrane (INM) and the cell cytosol (CC). In contrast to Endoplasmic Reticulum (ER) which in the presence of CC generates new biomembrane that forms ER vesicles transporting ER products to Golgi, the IN, ONM and INM are not producing transport vesicles. Instead, the newly synthesized lipids remain in the nuclear membranes. The membranes (INM, ONM) of IN incubated with CC become enriched with newly synthesized phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA). The incubation of separated ONM and INM with CC also enriched the membranes with IN specific lipids identified above. Moreover, the incubation of IN or its membranes with CC afforded retention of numerous CC proteins on the nuclear membrane. Here, we concentrated on 30kDa CC protein that displayed affinity to nuclear membrane PIP2. The 30kDa CC protein bound to PIP2 of IN, INM, and ONM. With IN, initially the PIP2-30kDa CC protein complex was detected on ONM, after 30-120 min of incubation, was found on INM and in nuclear contents. At the same time when the 30 kDa protein was released from INM and found in nuclear contents, the PIP2 of INM and ONM became undetectable, while the lipid extract from the membrane displaced from IN contained labeled PI only. Since ONM is an uninterrupted continuum of ER and INM, we speculate that the synthesis of the lipids in the ER, in the region adjacent to nucleus, is defining nuclear outer and inner biomembrane composition, is responsible for transport of the cytosolic protein into the nucleus and, replenishment of ER membrane used for vesicular transport.     

Two wrongs make a right: deficits in reversal learning after orbitofrontal damage are improved by amygdala ablation

The nuclei of dividing neural progenitors undergo a cell-cycle-dependent change in position along the apico-basal axis known as interkinetic nuclear migration (INM). The functional relationship between INM and the mode of division of neural progenitors remains elusive, in part because its regulation at the molecular level is poorly understood. In this issue of Neuron, Xie et al. identify two centrosomal proteins (Cep120 and TACCs) regulating the INM of cortical neural progenitors.     

Phosphorylation-dependent mitotic SUMOylation drives nuclear envelope–chromatin interactions

In eukaryotes, chromatin binding to the inner nuclear membrane (INM) and nuclear pore complexes (NPCs) contributes to spatial organization of the genome and epigenetic programs important for gene expression. In mitosis, chromatin–nuclear envelope (NE) interactions are lost and then formed again as sister chromosomes segregate to postmitotic nuclei. Investigating these processes in S. cerevisiae, we identified temporally and spatially controlled phosphorylation-dependent SUMOylation events that positively regulate postmetaphase chromatin association with the NE. Our work establishes a phosphorylation-mediated targeting mechanism of the SUMO ligase Siz2 to the INM during mitosis, where Siz2 binds to and SUMOylates the VAP protein Scs2. The recruitment of Siz2 through Scs2 is further responsible for a wave of SUMOylation along the INM that supports the assembly and anchorage of subtelomeric chromatin at the INM and localization of an active gene (INO1) to NPCs during the later stages of mitosis and into G1-phase.     

The inner nuclear membrane protein Lem2 is critical for normal nuclear envelope morphology

The inner nuclear membrane (INM) of eukaryotic cells is characterized by a unique set of transmembrane proteins which interact with chromatin and/or the nuclear lamina. The number of identified INM proteins is steadily increasing, mainly as a result of proteomic and computational approaches. However, despite a link between mutation of several of these proteins and disease, the function of most transmembrane proteins of the INM remains unknown and depletion of many of these proteins from a variety of systems did not produce an obvious phenotype in the affected cells. Here, we report that depletion of the conserved INM protein Lem2 from human cell lines leads to abnormally shaped nuclei and severely reduces cell survival. We suggest that interactions of Lem2 with lamins or chromatin are critical for maintaining the integrity of the nuclear envelope.     

The microtubule cytoskeleton of radial glial progenitor cells

A high number of genetic mutations associated with cortical malformations are found in genes coding for microtubule-related factors. This has stimulated research to understand how the various microtubule-based processes are regulated to build a functional cerebral cortex. Here, we focus our review on the radial glial progenitor cells, the stem cells of the developing neocortex, summarizing research mostly performed in rodents and humans. We highlight how the centrosomal and acentrosomal microtubule networks are organized during interphase to support polarized transport and proper attachment of the apical and basal processes. We describe the molecular mechanism for interkinetic nuclear migration (INM), a microtubule-dependent oscillation of the nucleus. Finally, we describe how the mitotic spindle is built to ensure proper chromosome segregation, with a strong focus on factors mutated in microcephaly.     

Regulation of neurogenesis by interkinetic nuclear migration through an apical-basal notch gradient

The different cell types in the central nervous system develop from a common pool of progenitor cells. The nuclei of progenitors move between the apical and basal surfaces of the neuroepithelium in phase with their cell cycle, a process termed interkinetic nuclear migration (INM). In the retina of zebrafish mikre oko (mok) mutants, in which the motor protein Dynactin-1 is disrupted, interkinetic nuclei migrate more rapidly and deeply to the basal side and more slowly to the apical side. We found that Notch signaling is predominantly activated on the apical side in both mutants and wild-type. Mutant progenitors are, thus, less exposed to Notch and exit the cell cycle prematurely. This leads to an overproduction of early-born retinal ganglion cells (RGCs) at the expense of later-born interneurons and glia. Our data indicate that the function of INM is to balance the exposure of progenitor nuclei to neurogenic versus proliferative signals.     

Live Imaging of Whole Mouse Embryos during Gastrulation: Migration Analyses of Epiblast and Mesodermal Cells

During gastrulation in the mouse embryo, dynamic cell movements including epiblast invagination and mesodermal layer expansion lead to the establishment of the three-layered body plan. The precise details of these movements, however, are sometimes elusive, because of the limitations in live imaging. To overcome this problem, we developed techniques to enable observation of living mouse embryos with digital scanned light sheet microscope (DSLM). The achieved deep and high time-resolution images of GFP-expressing nuclei and following 3D tracking analysis revealed the following findings: (i) Interkinetic nuclear migration (INM) occurs in the epiblast at embryonic day (E)6 and 6.5. (ii) INM-like migration occurs in the E5.5 embryo, when the epiblast is a monolayer and not yet pseudostratified. (iii) Primary driving force for INM at E6.5 is not pressure from neighboring nuclei. (iv) Mesodermal cells migrate not as a sheet but as individual cells without coordination.     

A role for CK2 upon interkinetic nuclear migration in the cell cycle of retinal progenitor cells

In developing retina, the nucleus of the elongated neuroepithelial cells undergoes interkinetic nuclear migration (INM), that is it migrates back and forth across the proliferative layer during the cell cycle. S-phase occurs at the basal side, while M-phase occurs at the apical margin of the retinal progenitors. G1 and G2-phases occur along the nuclear migration pathway. We tested whether this feature of the retinal cell cycle is controlled by CK2, which, among its many substrates, phosphorylates both molecular motors and cytoskeletal components. Double immunolabeling showed that CK2 is contained in BrdU-labeled retinal progenitors. INM was examined after pulse labeling the retina of newborn rats with BrdU, by plotting nuclear movement from basal to apical sides of the retinal progenitors during G2. The CK2 specific inhibitor 4,5,6,7-tetrabromobenzotriazole inhibited the activity of rat retinal CK2, and blocked nuclear movement proper in a dose-dependent way. No apoptosis was detected, and total numbers of BrdU-labeled nuclei remained constant following treatment. Immunohistochemistry showed that, following inhibition of CK2, the tubulin cytoskeleton is disorganized, with reduced acetylated and increased tyrosinated tubulin. This indicates a reduction in stable microtubules, with accumulation of free tubulin dimers. The results show that CK2 activity is required for INM in retinal progenitor cells.     

Expression of yeast INM1 encoding inositol monophosphatase is regulated by inositol, carbon source and growth stage and is decreased by lithium and valproate

Inositol monophosphatase plays a vital role in the de novo biosynthesis of inositol and in the phosphoinositide second messenger signalling pathway. We cloned the Saccharomyces cerevisiae open reading frame (ORF) YHR046c (termed INM1), which encodes inositol monophosphatase, characterized the protein Inm1p and analysed expression of the INM1 gene. INM1 was expressed in bacteria under the control of the lacZ promoter. The purified protein has inositol monophosphatase activity that is inhibited by the antibipolar drug lithium, but not valproate. In the inm1Delta:URA3 null mutant, inositol monophosphatase activity was reduced but not eliminated. The disruption had little effect on growth in the presence of lithium or valproate and no effect on growth in the absence of inositol. To characterize the regulation of INM1, we examined the effects of inositol, carbon source, growth phase, and the antibipolar drugs lithium and valproate on INM1 expression using an INM1-lacZ reporter gene. Unlike all other phospholipid biosynthetic enzyme-encoding genes studied, which contain the UASINO regulatory element, INM1 expression is increased in the presence of inositol. In addition, INM1 expression was repressed during growth in glycerol and derepressed as glucose-grown cells entered stationary. Both lithium and valproate, which cause a decrease in intracellular inositol, effect a decrease in INM1 expression. A model is presented to account for regulation of INM1 expression.     

Nuclear localization of NCX: Role in Ca2+ handling and pathophysiological implications

Numerous lines of evidence indicate that nuclear calcium concentration ([Ca²⁺]_n) may be controlled independently from cytosolic events by a local machinery. In particular, the perinuclear space between the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) of the nuclear envelope (NE) likely serves as an intracellular store for Ca²⁺ ions. Since ONM is contiguous with the endoplasmic reticulum (ER), the perinuclear space is adjacent to the lumen of ER thus allowing a direct exchange of ions and factors between the two organelles. Moreover, INM and ONM are fused at the nuclear pore complex (NPC), which provides the only direct passageway between the nucleoplasm and cytoplasm. However, due to the presence of ion channels, exchangers and transporters, it has been generally accepted that nuclear ion fluxes may occur across ONM and INM. Within the INM, the Na⁺/Ca²⁺ exchanger (NCX) isoform 1 seems to play an important role in handling Ca²⁺ through the different nuclear compartments. Particularly, nuclear NCX preferentially allows local Ca²⁺ flowing from nucleoplasm into NE lumen thanks to the Na⁺ gradient created by the juxtaposed Na⁺/K⁺-ATPase. Such transfer reduces abnormal elevation of [Ca²⁺]_n within the nucleoplasm thus modulating specific transductional pathways and providing a protective mechanism against cell death. Despite very few studies on this issue, here we discuss those making major contribution to the field, also addressing the pathophysiological implication of nuclear NCX malfunction.     

Membrane-bound trafficking regulates nuclear transport of integral epidermal growth factor receptor (EGFR) and ErbB-2

Nuclear localization of multiple receptor-tyrosine kinases (RTKs), such as EGF receptor (EGFR), ErbB-2, FGF receptor (FGFR), and many others, has been reported by several groups. We previously showed that cell surface EGFR is trafficked to the nucleus through a retrograde pathway from the Golgi to the endoplasmic reticulum (ER) and that EGFR is then translocated to the inner nuclear membrane (INM) through the INTERNET (integral trafficking from the ER to the nuclear envelope transport) pathway. However, the nuclear trafficking mechanisms of other membrane RTKs, apart from EGFR, remain unclear. The purpose of this study was to compare the nuclear transport of EGFR family proteins with that of FGFR-1. Interestingly, we found that digitonin permeabilization, which selectively releases soluble nuclear transporters from the cytoplasm and has been shown to inhibit nuclear transport of FGFR-1, had no effects on EGFR nuclear transport, raising the possibility that EGFR and FGFR-1 use different pathways to be translocated into the nucleus. Using the subnuclear fractionation assay, we further demonstrated that biotinylated cell surface ErbB-2, but not FGFR-1, is targeted to the INM, associating with Sec61β in the INM, similar to the nuclear trafficking of EGFR. Thus, ErbB-2, but not FGFR-1, shows a similar trafficking pathway to EGFR for translocation to the nucleus, indicating that at least two different pathways of nuclear transport exist for cell surface receptors. This finding provides a new direction for investigating the trafficking mechanisms of various nuclear RTKs.     

Cep120 and TACCs control interkinetic nuclear migration and the neural progenitor pool

Centrosome- and microtubule-associated proteins have been shown to be important for maintaining the neural progenitor pool during neocortical development by regulating the mitotic spindle. It remains unclear whether these proteins may control neurogenesis by regulating other microtubule-dependent processes such as nuclear migration. Here, we identify Cep120, a centrosomal protein preferentially expressed in neural progenitors during neocortical development. We demonstrate that silencing Cep120 in the developing neocortex impairs both interkinetic nuclear migration (INM), a characteristic pattern of nuclear movement in neural progenitors, and neural progenitor self-renewal. Furthermore, we show that Cep120 interacts with transforming acidic coiled-coil proteins (TACCs) and that silencing TACCs also causes defects in INM and neural progenitor self-renewal. Our data suggest a critical role for Cep120 and TACCs in both INM and neurogenesis. We propose that sustaining INM may be a mechanism by which microtubule-regulating proteins maintain the neural progenitor pool during neocortical development.     

A perinuclear α-helix with amphipathic features in Brl1 promotes NPC assembly

How nuclear pore complexes (NPCs) assemble in the intact nuclear envelope (NE) is only rudimentarily understood. Nucleoporins (Nups) accumulate at the inner nuclear membrane (INM) and deform this membrane toward the outer nuclear membrane (ONM), and eventually INM and ONM fuse by an unclear mechanism. In budding yeast, the integral membrane protein Brl1 that transiently associates with NPC assembly intermediates is involved in INM/ONM fusion during NPC assembly but leaving the molecular mechanism open. AlphaFold predictions indicate that Brl1-like proteins carry as common motifs an α-helix with amphipathic features (AαH) and a disulfide-stabilized, anti-parallel helix bundle (DAH) in the perinuclear space. Mutants with defective AαH (brl1^F391E, brl1^F391P, brl1^L402E) impair the essential function of BRL1. Overexpression of brl1^F391E promotes the formation of INM and ONM enclosed petal-like structures that carry Nups at their base, suggesting that they are derived from an NPC assembly attempt with failed INM/ONM fusion. Accordingly, brl1^F391E expression triggers mislocalization of Nup159 and Nup42 and to a lesser extent Nsp1, which localize on the cytoplasmic face of the NPC. The DAH also contributes to the function of Brl1, and AαH has functions independent of DAH. We propose that AαH and DAH in Brl1 promote INM/ONM fusion during NPC assembly.