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Disassembly and reassembly of the nuclear pore complex
Södertörn University, School of Life Sciences. Karolinska Institutet.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The nuclear pore complexes (NPCs) are multiprotein communicative channels spanning the nuclear envelope. In higher eukaryotes NPCs reversibly disassemble during mitosis into distinct nucleoporin subcomplexes. Some cell types (e.g. oocytes and early embryonic cells) also contain mimics of NPCs of unknown function, which are located in cytoplasmic membranes. They are termed annulate lamellae pore complexes (ALPC). This study was aimed at understanding the process of mitotic disassembly and reassembly of the NPC and at elucidating the function of ALPCs. Using syncytial Drosophila embryos as a model we have tested the proposed function of ALPCs as a storage compartment for nucleoporins fueling assembly of new NPCs in rapidly proliferating cells. Surprisingly, we found that ALPCs are not depleted during assembly of new NPCs and that they represent only a minor fraction of the total embryonic nucleoporins while the major fraction is persistently soluble. We conclude that in Drosophila, ALPCs play only a minor role as a storage compartment for nucleoporins. We developed a novel in vivo model system based on syncytial Drosophila embryos to study mitotic disassembly/reassembly of the NPC. We found that the major mitotic kinase Cdk1 is the key regulator of both NPC and ALPC disassembly/reassembly in vivo and that Cdk1 activity is able to phosphorylate and solubilize nucleoporins in vitro. We also found that phosphatase activity, sensitive to okadaic acid (OA), is required for reassembly of both NPCs and ALPCs in vivo. Additionally, we showed that the Ran GTPase system, that drives active nucleocytoplasmic transport during intephase, is selectively required for post-mitotic reassembly of NPCs but not ALPCs in vivo. Our findings suggest that in live cells NPC assembly is regulated by a dynamic equilibrium between kinase (Cdk1) and phospahatase (sensitive to OA) activity and that it is spatially coordinated by the Ran GTPase system. Finally. using the nucleoporin gp210 as a model. we have tested a role of mitotic phosphorylation of nucleoporins in disassembly of the NPC. We present evidence that a single mitotic phosphorylation of gp210 weakens its binding to the NPC and interferes with its postmitotic recruitment to the newly formed NE. These findings represent the first direct evidence that mitotic nucleoporin phosphorylation functions in disassembly of the NPC.

Place, publisher, year, edition, pages
Stockholm: Karolinska instiutet , 2006. , 65 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:sh:diva-31961ISBN: 91-7140-929-7 (print)OAI: oai:DiVA.org:sh-31961DiVA: diva2:1071786
Public defence
2006-10-13, MA648, Alfred Nobels allé 7, Huddinge, 09:00 (English)
Opponent
Supervisors
Available from: 2017-02-06 Created: 2017-02-06 Last updated: 2017-02-06Bibliographically approved
List of papers
1. Annulate lamellae play only a minor role in the storage of excess nucleoporins in Drosophila embryos
Open this publication in new window or tab >>Annulate lamellae play only a minor role in the storage of excess nucleoporins in Drosophila embryos
Show others...
2004 (English)In: Traffic: the International Journal of Intracellular Transport, ISSN 1398-9219, E-ISSN 1600-0854, Vol. 5, no 3, 152-164 p.Article in journal (Refereed) Published
Abstract [en]

The nuclear pore complexes (NPCs), multiprotein assemblies embedded in the nuclear envelope, conduct nucleo-cytoplasmic traffic of macromolecules. Mimics of NPCs, called annulate lamellae pore complexes (ALPCs), are usually found in cytoplasmic membranous stacks in oocytes and early embryonic cells. They are believed to constitute storage compartments for excess premade nucleoporins. To evaluate the extent to which ALPCs store nucleoporins in early embryonic cells we took advantage of syncytial Drosophila embryos, containing both AL and rapidly proliferating nuclei in the common cytoplasm. Electron microscopic morphometric analysis showed that the number of ALPCs did not decrease to compensate for the growing number of NPCs during syncytial development. We performed Western blot analysis to quantify seven different nucleoporins and analyzed their intraembryonal distribution by confocal microscopy and subcellular fractionation. Syncytial embryos contained a large maternally contributed stockpile of nucleoporins. However, even during interphases, only a small fraction of the excess nucleoporins was assembled into ALPCs, whereas the major fraction was soluble and contained at least one phosphorylated nucleoporin. We conclude that in Drosophila embryos ALPCs play only a minor role in storing the excess maternally contributed nucleoporins. Factors that may prevent nucleoporins from assembly into ALPCs are discussed.

National Category
Cell Biology
Identifiers
urn:nbn:se:sh:diva-15486 (URN)10.1111/j.1600-0854.2004.0166.x (DOI)000189142900004 ()15086791 (PubMedID)2-s2.0-1642376626 (ScopusID)
Available from: 2012-02-21 Created: 2012-02-20 Last updated: 2017-02-06Bibliographically approved
2. Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos
Open this publication in new window or tab >>Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos
2005 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 16, no 11, 5152-5162 p.Article in journal (Refereed) Published
Abstract [en]

Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In Agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC.

National Category
Cell Biology
Identifiers
urn:nbn:se:sh:diva-14439 (URN)10.1091/mbc.E05-07-0642 (DOI)000233025000011 ()16120647 (PubMedID)2-s2.0-27644495950 (ScopusID)
Available from: 2012-01-31 Created: 2011-12-23 Last updated: 2017-02-06Bibliographically approved
3. Phosphomimetic mutation of the mitotically phosphorylated serine 1880 compromises the interaction of the transmembrane nucleoporin gp210 with the nuclear pore complex
Open this publication in new window or tab >>Phosphomimetic mutation of the mitotically phosphorylated serine 1880 compromises the interaction of the transmembrane nucleoporin gp210 with the nuclear pore complex
2007 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 313, no 12, 2744-2751 p.Article in journal (Refereed) Published
Abstract [en]

The nuclear pore complexes (NPCs) reversibly disassemble and reassemble during mitosis. Disassembly of the NPC is accompanied by phosphorylation of many nucleoporins although the function of this is not clear. It was previously shown that in the transmembrane nucleoporin gp210 a single serine residue at position 1880 is specifically phosphorylated during mitosis. Using amino acid substitution combined with live cell imaging, time-lapse microscopy and FRAP, we investigated the role of serine 1880 in binding of gp210 to the NPC in vivo An alanine subtitutions mutant (S1880A) was significantly more dynamic at the NPC compared to the wild-type protein, suggesting that serine 1880 is important for binding of gp210 to the NPC. Moreover a glutamate substitution (S1880E) closely mimicking phosphorylated serine specifically interfered with incorporation of gp210 into the NPC and compromised its post-mitotic recruitment to the nuclear envelope of daughter nuclei. our findings are consistent with the idea that mitotic phosphorylation acts to dissociate gp210 from the structural elements of the NPC.

National Category
Cancer and Oncology Cell Biology
Identifiers
urn:nbn:se:sh:diva-14217 (URN)10.1016/j.yexcr.2007.05.011 (DOI)000248112300019 ()17559836 (PubMedID)2-s2.0-34250885134 (ScopusID)
Note

Som manuskript i avhandling. As manuscript in dissertation with title:

Mitotic phosphorylation regulates binding of gp210 to the nuclear pore complex

Available from: 2011-12-20 Created: 2011-12-19 Last updated: 2017-02-06Bibliographically approved
4. Differential requirement of RanGTP production for assembly of pore complexes in the nuclear envelope and annulate lamellae
Open this publication in new window or tab >>Differential requirement of RanGTP production for assembly of pore complexes in the nuclear envelope and annulate lamellae
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-31960 (URN)
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2017-02-06 Created: 2017-02-06 Last updated: 2017-02-06Bibliographically approved

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