Principle of duality in phospholipids: regulators of membrane morphology and dynamics

2014 ◽  
Vol 42 (5) ◽  
pp. 1335-1342 ◽  
Author(s):  
Banafshé Larijani ◽  
Fadi Hamati ◽  
Aupola Kundu ◽  
Gary C. Chung ◽  
Marie-Charlotte Domart ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Physical Properties ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Second Messengers ◽  
Physical Structure ◽  
Membrane Dynamics ◽  
Direct Role ◽  
Membrane Morphology ◽  
Nuclear Envelope Formation

To suggest and develop intelligent strategies to comprehend the regulation of organelle formation, a deeper mechanistic interpretation requires more than just the involvement of proteins. Our approaches link the formation of endomembranes with both signalling and membrane physical properties. Hitherto, membrane morphology, local physical structure and signalling have not been well integrated. Our studies derive from a cross-disciplinary approach undertaken to determine the molecular mechanisms of nuclear envelope assembly in echinoderm and mammalian cells. Our findings have led to the demonstration of a direct role for phosphoinositides and their derivatives in nuclear membrane formation. We have shown that phosphoinositides and their derivatives, as well as acting as second messengers, are modulators of membrane morphology, and their modifying enzymes regulate nuclear envelope formation. In addition, we have shown that echinoderm eggs can be exploited as a milieu to directly study the roles of phospholipids in maintaining organelle shape. The use of the echinoderm egg is a significant step forward in obtaining direct information about membrane physical properties in situ rather than using simpler models which do not provide a complete mechanistic insight into the role of phospholipids in membrane dynamics.

scholarly journals Reshaping of the endoplasmic reticulum limits the rate for nuclear envelope formation

2008 ◽  
Vol 182 (5) ◽  
pp. 911-924 ◽  
Author(s):  
Daniel J. Anderson ◽  
Martin W. Hetzer
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Mammalian Cells ◽  
Time Lapse ◽  
Principal Mechanism ◽  
Nuclear Assembly ◽  
Nuclear Envelope Formation ◽  
Rate Limiting

During mitosis in metazoans, segregated chromosomes become enclosed by the nuclear envelope (NE), a double membrane that is continuous with the endoplasmic reticulum (ER). Recent in vitro data suggest that NE formation occurs by chromatin-mediated reorganization of the tubular ER; however, the basic principles of such a membrane-reshaping process remain uncharacterized. Here, we present a quantitative analysis of nuclear membrane assembly in mammalian cells using time-lapse microscopy. From the initial recruitment of ER tubules to chromatin, the formation of a membrane-enclosed, transport-competent nucleus occurs within ∼12 min. Overexpression of the ER tubule-forming proteins reticulon 3, reticulon 4, and DP1 inhibits NE formation and nuclear expansion, whereas their knockdown accelerates nuclear assembly. This suggests that the transition from membrane tubules to sheets is rate-limiting for nuclear assembly. Our results provide evidence that ER-shaping proteins are directly involved in the reconstruction of the nuclear compartment and that morphological restructuring of the ER is the principal mechanism of NE formation in vivo.

KASH-domain proteins and the cytoskeletal landscapes of the nuclear envelope

2008 ◽  
Vol 36 (6) ◽  
pp. 1368-1372 ◽  
Author(s):  
Maria Schneider ◽  
Angelika A. Noegel ◽  
Iakowos Karakesisoglou
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Nuclear Architecture ◽  
Membrane Interface ◽  
Novel Proteins ◽  
Evolutionarily Conserved ◽  
Membrane Tethering ◽  
Novel Model

Over the last few years, several novel proteins have been identified that facilitate the physical integration of the nucleus with the cytoplasmic compartment. The majority belong to the evolutionarily conserved KASH [klarsicht/ANC-1 (anchorage 1)/SYNE (synaptic nuclear envelope protein) homology]-domain family, which function primarily as exclusive outer nuclear membrane scaffolds that associate with the cytoskeleton, the centrosome and the motor protein apparatus. In the present paper, we propose a novel model, which may explain why these proteins also determine nuclear architecture. Moreover, we discuss further nuclear membrane-tethering devices, which indicate collectively the presence of specific molecular mechanisms that organize the cytoplasmic–nuclear membrane interface in mammalian cells.

Initiation of DNA synthesis by nuclei from scrape-ruptured quiescent mammalian cells in high-speed supernatants of Xenopus egg extracts

1994 ◽  
Vol 107 (11) ◽  
pp. 3045-3053 ◽  
Author(s):  
M. Hola ◽  
S. Castleden ◽  
M. Howard ◽  
R.F. Brooks
Keyword(s):  
Nuclear Envelope ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
High Speed ◽  
Nuclear Assembly ◽  
Naked Dna ◽  
Egg Extracts ◽  
Eukaryotic Dna ◽  
Nuclear Envelope Formation ◽  
Xenopus Egg Extracts

Demembranated sperm heads, detergent-isolated somatic nuclei and even naked DNA are efficiently replicated in cytoplasmic extracts of activated amphibian eggs, but only after nuclear assembly and the formation of an intact nuclear envelope. DNA synthesis has not previously been shown to be initiated in high-speed (200,000 g) supernatants of egg cytoplasm because they are depleted of the vesicular material required to support nuclear envelope formation. Here we show that mammalian nuclei prepared by scrape-rupture are able to initiate DNA replication in such high-speed supernatants. These nuclei begin DNA synthesis asynchronously. This asynchrony cannot be attributed to differences in the time taken for nuclear assembly. Instead, we suggest that the asynchrony reflects intrinsic differences between nuclei and that these differences are a major cause of cell cycle variability. Our demonstration of initiation in high-speed supernatants now enables the initiation of eukaryotic DNA synthesis to be studied independently of nuclear assembly.

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

A Possible Modulation Mechanism of Intramolecular and Intermolecular Interactions for NCAM Polysialylation and Cell Migration

2019 ◽  
Vol 19 (25) ◽  
pp. 2271-2282 ◽  
Author(s):  
Bo Lu ◽  
Xue-Hui Liu ◽  
Si-Ming Liao ◽  
Zhi-Long Lu ◽  
Dong Chen ◽  
...  
Keyword(s):  
Cell Migration ◽  
Intermolecular Interactions ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Intramolecular Interaction ◽  
Polysialic Acid ◽  
Tumor Cell Migration ◽  
Neural Cell Adhesion ◽  
Polybasic Domains ◽  
Novel Model

Polysialic acid (polySia) is a novel glycan that posttranslationally modifies neural cell adhesion molecules (NCAMs) in mammalian cells. Up-regulation of polySia-NCAM expression or NCAM polysialylation is associated with tumor cell migration and progression in many metastatic cancers and neurocognition. It has been known that two highly homologous mammalian polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST), can catalyze polysialylation of NCAM, and two polybasic domains, polybasic region (PBR) and polysialyltransferase domain (PSTD) in polySTs play key roles in affecting polyST activity or NCAM polysialylation. However, the molecular mechanisms of NCAM polysialylation and cell migration are still not entirely clear. In this minireview, the recent research results about the intermolecular interactions between the PBR and NCAM, the PSTD and cytidine monophosphate-sialic acid (CMP-Sia), the PSTD and polySia, and as well as the intramolecular interaction between the PBR and the PSTD within the polyST, are summarized. Based on these cooperative interactions, we have built a novel model of NCAM polysialylation and cell migration mechanisms, which may be helpful to design and develop new polysialyltransferase inhibitors.

scholarly journals The Saccharomyces cerevisiae RAD6 Group Is Composed of an Error-Prone and Two Error-Free Postreplication Repair Pathways

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1633-1641 ◽  
Author(s):  
Wei Xiao ◽  
Barbara L Chow ◽  
Stacey Broomfield ◽  
Michelle Hanna
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Signal Transducer ◽  
Repair Pathway ◽  
Polyubiquitin Chain ◽  
Postreplication Repair ◽  
Translesion Dna Synthesis ◽  
Radiation Repair ◽  
Repair Pathways ◽  
High Degree

Abstract The RAD6 postreplication repair and mutagenesis pathway is the only major radiation repair pathway yet to be extensively characterized. It has been previously speculated that the RAD6 pathway consists of two parallel subpathways, one error free and another error prone (mutagenic). Here we show that the RAD6 group genes can be exclusively divided into three rather than two independent subpathways represented by the RAD5, POL30, and REV3 genes; the REV3 pathway is largely mutagenic, whereas the RAD5 and the POL30 pathways are deemed error free. Mutants carrying characteristic mutations in each of the three subpathways are phenotypically indistinguishable from a single mutant such as rad18, which is defective in the entire RAD6 postreplication repair/tolerance pathway. Furthermore, the rad18 mutation is epistatic to all single or combined mutations in any of the above three subpathways. Our data also suggest that MMS2 and UBC13 play a key role in coordinating the response of the error-free subpathways; Mms2 and Ubc13 form a complex required for a novel polyubiquitin chain assembly, which probably serves as a signal transducer to promote both RAD5 and POL30 error-free postreplication repair pathways. The model established by this study will facilitate further research into the molecular mechanisms of postreplication repair and translesion DNA synthesis. In view of the high degree of sequence conservation of the RAD6 pathway genes among all eukaryotes, the model presented in this study may also apply to mammalian cells and predicts links to human diseases.

scholarly journals Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

1998 ◽  
Vol 18 (9) ◽  
pp. 5208-5218 ◽  
Author(s):  
Michael Gale ◽  
Collin M. Blakely ◽  
Bart Kwieciszewski ◽  
Seng-Lai Tan ◽  
Michelle Dossett ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Functional Assay ◽  
Binding Region ◽  
Dimerization Domain ◽  
Antiviral Effects

ABSTRACT The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2α phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

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