Man1, an inner nuclear membrane protein, regulates vascular remodeling by modulating transforming growth factor beta signaling

A growing number of integral inner nuclear membrane (INM) proteins have been implicated in diverse cellular functions. Man1, an INM protein, has recently been shown to regulate transforming growth factor (Tgf) beta superfamily signaling by interacting with receptor-associated Smads. However, the in vivo roles of Man1 have not been fully characterized. Here, we show that Man1 regulates vascular remodeling by analyzing Man1-deficient embryos lacking the Smad interacting domain. Man1-deficient embryos die at midgestation because of defects in embryonic vasculature; the primary capillary plexus forms, but subsequent remodeling is perturbed. It has been proposed that the angiogenesis process is divided into two balanced phases, the activation and resolution/maturation phases, both of which are regulated by Tgfbeta1. We have demonstrated, in Man1-deficient embryos, the expression of Tgfb1 is upregulated and Smad2/3 signaling is abnormally activated, resulting in increased extracellular matrix deposition, a hallmark of the resolution phase of angiogenesis. We have also showed that the recruitment of mural cells to the vascular wall is severely disturbed in mutants, which may lead to disruption of intercellular communication between endothelial and mural cells required for proper vascular remodeling. These results have revealed a novel role for Man1 in angiogenesis and provide the first evidence that vascular remodeling can be regulated at the INM through the interaction between Man1 and Smads.     

Integral membrane proteins specific to the inner nuclear membrane and associated with the nuclear lamina

We obtained a monoclonal antibody (RL13) that identifies three integral membrane proteins specific to the nuclear envelope of rat liver, a major 75-kD polypeptide and two more minor components of 68 and 55 kD. Immunogold labeling of isolated nuclear envelopes demonstrates that these antigens are localized specifically to the inner nuclear membrane, and that the RL13 epitope occurs on the inner membrane's nucleoplasmic surface where the nuclear lamina is found. When nuclear envelopes are extracted with solutions containing nonionic detergent and high salt to solubilize nuclear membranes and pore complexes, most of these integral proteins remain associated with the insoluble lamina. Since the polypeptides recognized by RL13 are relatively abundant, they may function as lamina attachment sites in the inner nuclear membrane. Major cross-reacting antigens are found by immunoblotting and immunofluorescence microscopy in all rat cells examined. Therefore, these integral proteins are biochemical markers for the inner nuclear membrane and will be useful models for studying nuclear membrane biogenesis.     

Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury

Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N(2)A cells (EC(50) in nm range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca(2+) in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress.     

Lipopolysaccharide transport to the cell surface: biosynthesis and extraction from the inner membrane

The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane. The accompanying paper (May JM, Sherman DJ, Simpson BW, Ruiz N, Kahne D. 2015 Phil. Trans. R. Soc. B 370, 20150027. (doi:10.1098/rstb.2015.0027)) describes the subsequent steps as LPS travels through the periplasm and the outer membrane to its final destination at the cell surface.     

Epidermal growth factor induces the accumulation of calpactin II on the cell surface during membrane ruffling

Confluent and proliferatively quiescent T51B rat liver epithelial cells provide a cellular model for the study of epidermal growth factor (EGF) effects in non-neoplastic cells. Immunoreactive calpactin II, a well-known substrate for EGF-receptor kinase, was found predominantly in the cytosol, although a second immunoreactive pool was found in a Triton X-100-extractable membrane fraction. Stimulation with EGF resulted in a rapid and transient (2-5 min) formation of ruffles at the cell surface and at the cell-cell contacts. Both calpactin II and filamentous actin were found co-localized at the membrane ruffles. Immunoprecipitations of membrane-bound calpactin II from 32P-labeled cells indicate a transient EGF-dependent phosphorylation of calpactin II correlating with membrane ruffling. These results suggest a temporal (2-5 min) function for calpactin II at the plasma membrane during the EGF-induced mitogenesis of T51B cells.     

Highway to the inner nuclear membrane: rules for the road

To enter the nucleus a protein must be chaperoned by a transport factor through the nuclear pore complex or it must be small enough to pass through by diffusion. Although these principles have long described the nuclear import of soluble proteins, recent evidence indicates that they also apply to the import of integral inner nuclear membrane proteins. Here we develop a set of rules that might govern the transport of proteins to the inner nuclear membrane.     

Free diffusion to and from the inner nuclear membrane of newly synthesized plasma membrane glycoproteins

Sindbis virus-infected baby hamster kidney (BHK) cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies was used in conjunction with colloidal gold-conjugated protein A. As we previously reported (Torrisi, M. R., and S. Bonatti, 1985, J. Cell Biol., 101:1300-1306), Sindbis transmembrane glycoproteins are present in the inner nuclear membrane as well as in the outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes. Viral glycoproteins located on the inner nuclear membrane resemble those present on the outer membrane in terms of amount, distribution, and preferential partition after fracture. We show in this paper that Sindbis glycoproteins after treatment with cycloheximide are removed from the inner nuclear membrane with the same kinetics as their counterparts present on the outer membrane. This finding strongly suggests that newly synthesized transmembrane glycoproteins may freely diffuse to and from the inner nuclear membrane before entering into the intracellular transport pathway to the plasma membrane.     

Signals and structural features involved in integral membrane protein targeting to the inner nuclear membrane

We have examined transfected cells by immunofluorescence microscopy to determine the signals and structural features required for the targeting of integral membrane proteins to the inner nuclear membrane. Lamin B receptor (LBR) is a resident protein of the nuclear envelope inner membrane that has a nucleoplasmic, amino-terminal domain and a carboxyl-terminal domain with eight putative transmembrane segments. The amino-terminal domain of LBR can target both a cytosolic protein to the nucleus and a type II integral protein to the inner nuclear membrane. Neither a nuclear localization signal (NLS) of a soluble protein, nor full-length histone H1, can target an integral protein to the inner nuclear membrane although they can target cytosolic proteins to the nucleus. The addition of an NLS to a protein normally located in the inner nuclear membrane, however, does not inhibit its targeting. When the amino-terminal domain of LBR is increased in size from approximately 22.5 to approximately 70 kD, the chimeric protein cannot reach the inner nuclear membrane. The carboxyl-terminal domain of LBR, separated from the amino-terminal domain, also concentrates in the inner nuclear membrane, demonstrating two nonoverlapping targeting signals in this protein. Signals and structural features required for the inner nuclear membrane targeting of proteins are distinct from those involved in targeting soluble polypeptides to the nucleoplasm. The structure of the nucleocytoplasmic domain of an inner nuclear membrane protein also influences targeting, possibly because of size constraints dictated by the lateral channels of the nuclear pore complexes.     

Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore

Resident integral proteins of the inner nuclear membrane (INM) are synthesized as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and are transported to the INM throughout interphase using an unknown trafficking mechanism. To study this transport, we developed a live cell assay that measures the movement of transmembrane reporters from the ER to the INM by rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM. However, increasing the size of either domain by 47 kD strongly inhibited movement. Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect. Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex.     

Hepatoma-derived growth factor stimulates cell growth after translocation to the nucleus by nuclear localization signals.

Hepatoma-derived growth factor (HDGF) is the original member of the HDGF family of proteins, which contains a well-conserved N-terminal amino acid sequence (homologous to the amino terminus of HDGF; hath) and nuclear localization signals (NLSs) in gene-specific regions other than the hath region. In addition to a bipartite NLS in a gene-specific region, an NLS-like sequence is also found in the hath region. In cells expressing green fluorescence protein (GFP)-HDGF, green fluorescence was observed in the nucleus, whereas it was detected in the cytoplasm of cells expressing GFP-HDGF with both NLSs mutated or deleted. GFP-hath protein (GFP-HATH) was distributed mainly in the nucleus, although some was present in the cytoplasm, whereas GFP-HDGF with a deleted hath region (HDGFnonHATH) was found only in the nucleus. Exogenously supplied GFP-HDGF was internalized and translocated to the nucleus. GFP-HATH was internalized, whereas GFP-HDGFnonHATH was not. Overexpression of HDGF stimulated DNA synthesis and cellular proliferation, although HDGF with both NLSs deleted did not. Overexpression of HDGFnonHATH caused a significant stimulation of DNA synthesis, whereas that of hath protein did not. HDGF containing the NLS sequence of p53 instead of the bipartite NLS did not stimulate DNA synthesis, and truncated forms without the C- or N-terminal side of NLS2 did not. These findings suggest that the gene-specific region, at least the bipartite NLS sequence and the N- and C-terminal neighboring portions, is essential for the mitogenic activity of HDGF after nuclear translocation.     

Functional K(v)10.1 channels localize to the inner nuclear membrane

Ectopically expressed human K(V)10.1 channels are relevant players in tumor biology. However, their function as ion channels at the plasma membrane does not totally explain their crucial role in tumors. Both in native and heterologous systems, it has been observed that a majority of K(V)10.1 channels remain at intracellular locations. In this study we investigated the localization and possible roles of perinuclear K(V)10.1. We show that K(V)10.1 is expressed at the inner nuclear membrane in both human and rat models; it co-purifies with established inner nuclear membrane markers, shows resistance to detergent extraction and restricted mobility, all of them typical features of proteins at the inner nuclear membrane. K(V)10.1 channels at the inner nuclear membrane are not all transported directly from the ER but rather have been exposed to the extracellular milieu. Patch clamp experiments on nuclei devoid of external nuclear membrane reveal the existence of channel activity compatible with K(V)10.1. We hypothesize that K(V)10.1 channels at the nuclear envelope might participate in the homeostasis of nuclear K(+), or indirectly interact with heterochromatin, both factors known to affect gene expression.     

Multiple mechanisms actively target the SUN protein UNC-84 to the inner nuclear membrane

Approximately 100 proteins are targeted to the inner nuclear membrane (INM), where they regulate chromatin and nuclear dynamics. The mechanisms underlying trafficking to the INM are poorly understood. The Caenorhabditis elegans SUN protein UNC-84 is an excellent model to investigate such mechanisms. UNC-84 recruits KASH proteins to the outer nuclear membrane to bridge the nuclear envelope (NE), mediating nuclear positioning. UNC-84 has four targeting sequences: two classical nuclear localization signals, an INM sorting motif, and a signal conserved in mammalian Sun1, the SUN--nuclear envelope localization signal. Mutations in some signals disrupt the timing of UNC-84 nuclear envelope localization, showing that diffusion is not sufficient to move all UNC-84 to the NE. Thus targeting UNC-84 requires an initial step that actively transports UNC-84 from the peripheral endoplasmic reticulum to the NE. Only when all four signals are simultaneously disrupted does UNC-84 completely fail to localize and to function in nuclear migration, meaning that at least three signals function, in part, redundantly to ensure proper targeting of UNC-84. Multiple mechanisms might also be used to target other proteins to the INM, thereby ensuring their proper and timely localization for essential cellular and developmental functions.     

Synthesis, transport and incorporation into the nuclear envelope of A-type lamins and inner nuclear membrane proteins

The mammalian NE (nuclear envelope), which separates the nucleus from the cytoplasm, is a complex structure composed of nuclear pore complexes, the outer and inner nuclear membranes, the perinuclear space and the nuclear lamina (A- and B-type lamins). The NE is completely disassembled and reassembled at each cell division. In the present paper, we review recent advances in the understanding of the mechanisms implicated in the transport of inner nuclear membrane and nuclear lamina proteins from the endoplasmic reticulum to the nucleus in interphase cells and mitosis, with special attention to A-type lamins.     

Regulation of transferrin receptor expression at the cell surface by insulin-like growth factors, epidermal growth factor and platelet-derived growth factor.

Addition of platelet-derived growth factor (PDGF), recombinant insulin-like growth factor I (rIGF-I) or epidermal growth factor (EGF) to BALB/c 3T3 fibroblasts causes a marked increase in the binding of [125I]diferric transferrin to cell surface receptors. This effect is very rapid and is complete within 5 min. The effect of EGF is transient, with [125I]diferric transferrin binding returning to control values within 25 min. In contrast, PDGF and rIGF-I cause a prolonged stimulation of [125I]diferric transferrin binding that could be observed for up to 2 h. The increase in the binding of [125I]diferric transferrin caused by growth factors was investigated by analysis of the binding isotherm. Epidermal growth factor, PDGF and rIGF-I were found to increase the cell surface expression of transferrin receptors rather than to alter the affinity of the transferrin receptors. This result was confirmed in human fibroblasts by the demonstration that EGF, PDGF and rIGF-I could stimulate the binding of a monoclonal antibody directed against the transferrin receptor (OKT9) to the cell surface. Furthermore, PDGF and rIGF-I stimulated the sustained uptake of [59Fe]diferric transferrin by BALB/c 3T3 fibroblasts, while EGF transiently increased uptake. Thus the effect of these growth factors to increase the cell surface expression of the transferrin receptor appears to have an important physiological consequence.     

Blocking translocation of cell surface molecules from the ER to the cell surface by intracellular antibodies targeted to the ER

Intracellular antibodies (intrabodies) constitute a potent tool to neutralize the function of target proteins inside specific cell compartments (cytosol, nucleus, mitochondria and ER). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals and complements or replaces knockdown techniques such as antisense-RNA, RNAi and RNA aptamers. This article focuses on intrabodies targeted to the ER. Intracellular anti-bodies expressed and retained inside the ER (ER intrabodies) are shown to be highly efficient in blocking the translocation of secreted and cell surface molecules from the ER to the cell surface.The advantage of ER intrabodies over cytoplasmic intrabodies is that they are correctly folded and easier to select. A particular advantage of the intrabody technology over existing ones is the possibility of inhibiting selectively post-translational modifications of proteins.The main applications of ER intrabodies so far have been (i) inactivation of oncogenic receptors and (ii) functional inhibition of virus envelope proteins and virus-receptor molecules on the surface of host cells.In cancer research, the number of in vivo mouse models for evaluation of the therapeutic potential of intrabodies is increasing.In the future, endosomal localized receptors involved in bacterial and viral infections, intracellular oncogenic receptors and enzymes involved in glycosylation of tumour antigens might be new targets for ER intrabodies.