Dynamics of PLCγ and Src Family Kinase 1 Interactions during Nuclear Envelope Formation Revealed by FRET-FLIM

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.

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A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.107.1.57 ◽

1988 ◽

Vol 107 (1) ◽

pp. 57-68 ◽

Cited By ~ 87

Author(s):

K L Wilson ◽

J Newport

Keyword(s):

Nuclear Envelope ◽

Protein Interactions ◽

Membrane Vesicles ◽

Integral Membrane Protein ◽

Electron Microscopic ◽

Vitro Assay ◽

Nuclear Envelope Assembly ◽

Microscopic Studies ◽

Nuclear Envelope Formation

The reformation of functioning organelles at the end of mitosis presents a problem in vesicle targeting. Using extracts made from Xenopus laevis frog eggs, we have studied in vitro the vesicles that reform the nuclear envelope. In the in vitro assay, nuclear envelope growth is linear with time. Furthermore, the final surface area of the nuclear envelopes formed is directly dependent upon the amount of membrane vesicles added to the assay. Egg membrane vesicles could be fractionated into two populations, only one of which was competent for nuclear envelope assembly. We found that vesicles active in nuclear envelope assembly contained markers (BiP and alpha-glucosidase II) characteristic of the endoplasmic reticulum (ER), but that the majority of ER-derived vesicles do not contribute to nuclear envelope size. This functional distinction between nuclear vesicles and ER-derived vesicles implies that nuclear vesicles are unique and possess at least one factor required for envelope assembly that is lacking in other vesicles. Consistent with this, treatment of vesicles with trypsin destroyed their ability to form a nuclear envelope; electron microscopic studies indicate that the trypsin-sensitive proteins is required for vesicles to bind to chromatin. However, the protease-sensitive component(s) is resistant to treatments that disrupt protein-protein interactions, such as high salt, EDTA, or low ionic strength solutions. We propose that an integral membrane protein, or protein tightly associated with the membrane, is critical for nuclear vesicle targeting or function.

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PROPHASING OF INTERPHASE NUCLEI AND INDUCTION OF NUCLEAR ENVELOPES AROUND METAPHASE CHROMOSOMES IN HELA AND CHINESE HAMSTER HOMO- AND HETEROKARYONS

The Journal of Cell Biology ◽

10.1083/jcb.62.1.104 ◽

1974 ◽

Vol 62 (1) ◽

pp. 104-113 ◽

Cited By ~ 22

Author(s):

Yoshitaka Obara ◽

Lee S. Chai ◽

Herbert Weinfeld ◽

Avery A. Sandberg

Keyword(s):

Nuclear Envelope ◽

Hela Cells ◽

Interphase Nucleus ◽

Chinese Hamster ◽

Binucleate Cell ◽

Metaphase Chromosomes ◽

Interphase Nuclei ◽

Multinucleate Cells ◽

Interphase Cells ◽

Nuclear Envelope Formation

Fusing human HeLa metaphase cells with HeLa interphase cells resulted within 30 min in either of two phenomena in the resultant binucleate cell: either prophasing of the interphase nucleus or formation of a normal-appearing nuclear envelope around the metaphase chromosomes. The frequency of either occurrence was strongly dependent on environmental pH. At pH's of 6.6–8.0, prophasing predominated; at pH 8.5 nuclear envelope formation predominated. Additionally, the frequencies of the two events in multinucleate cells depended on the metaphase/interphase ratio. When the ratio was 0.33 nuclear envelope formation predominated; when it was 2.0 prophasing predominated. In their general features, the results with fused HeLa cells resembled those reported earlier with fused Chinese hamster Don cells. However, the results provided an indication that between pH 6.6 and 8.0 the HeLa metaphase cells possessed a much greater capacity than the Don metaphase cells to induce prophasing. Fusion of Don metaphase cells with HeLa interphase cells or of Don interphase cells with HeLa metaphase cells at pH 8.0 resulted in nuclear envelope formation or prophasing in each kind of heterokaryon. As in the homokaryons, the frequencies of the two events in the heterokaryons depended on the metaphase/interphase ratio. The statistics of prophasing and nuclear envelope formation in the homo- and heterokaryon populations were consistent with the notion that disruption or formation of the nuclear envelope depends on the balance attained between disruptive and formative processes.

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

Biochemical Society Transactions ◽

10.1042/bst20140224 ◽

2014 ◽

Vol 42 (5) ◽

pp. 1335-1342 ◽

Cited By ~ 4

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.

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A lamin-independent pathway for nuclear envelope assembly.

The Journal of Cell Biology ◽

10.1083/jcb.111.6.2247 ◽

1990 ◽

Vol 111 (6) ◽

pp. 2247-2259 ◽

Cited By ~ 199

Author(s):

J W Newport ◽

K L Wilson ◽

W G Dunphy

Keyword(s):

Nuclear Envelope ◽

Dna Synthesis ◽

Structural Integrity ◽

Nuclear Lamina ◽

Limited Extent ◽

Nuclear Pores ◽

Nuclear Assembly ◽

Nuclear Envelope Assembly ◽

A Cell ◽

Nuclear Envelope Formation

The nuclear envelope is composed of membranes, nuclear pores, and a nuclear lamina. Using a cell-free nuclear assembly extract derived from Xenopus eggs, we have investigated how these three components interact during nuclear assembly. We find that the Xenopus embryonic lamin protein LIII cannot bind directly to chromatin or membranes when each is present alone, but is readily incorporated into nuclei when both of the components are present together in an assembly extract. We find that depleting lamin LIII from an extract does not prevent formation of an envelope consisting of membranes and nuclear pores. However, these lamin-depleted envelopes are extremely fragile and fail to grow beyond a limited extent. This suggests that lamin assembly is not required during the initial steps of nuclear envelope formation, but is required for later growth and for maintaining the structural integrity of the envelope. We also present results showing that lamins may only be incorporated into nuclei after DNA has been encapsulated within an envelope and nuclear transport has been activated. With respect to nuclear function, our results show that the presence of a nuclear lamina is required for DNA synthesis to occur within assembled nuclei.

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