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Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells.

Daigle N, Beaudouin J, Hartnell L, Imreh G, Hallberg E, Lippincott-Schwartz J, Ellenberg J - J. Cell Biol. (2001)

Bottom Line: No independent movement of single pore complexes was found within the plane of the NE in interphase.During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase.Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.

ABSTRACT
The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121-green fluorescent protein (GFP) and GFP-Nup153, and GFP-lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

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POM121 is immobilized during nuclear assembly in anaphase. FRAP of a NRK cell transiently expressing POM121-EGFP3. (A) Boxed area was photobleached to background levels in metaphase. Recovery was monitored every 9 s in a single image acquired with open pinhole. Note rapid recovery of fluorescence into the ER as the cell reaches metaphase/anaphase transition (02:15). (B) FRAP of the same cell as in A ∼6 min after anaphase onset. Note the absence of complete recovery during the transition to telophase. Times, hh:mm:ss. (C) Plot of recovery in the bleached areas shown in A and B for metaphase (green)and anaphase (black). Data was normalized to total loss of fluorescence, time 0 corresponds to the midpoint of the bleach. Note the difference in IFs 4 min after the bleach. Bars, 5 μm.
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fig6: POM121 is immobilized during nuclear assembly in anaphase. FRAP of a NRK cell transiently expressing POM121-EGFP3. (A) Boxed area was photobleached to background levels in metaphase. Recovery was monitored every 9 s in a single image acquired with open pinhole. Note rapid recovery of fluorescence into the ER as the cell reaches metaphase/anaphase transition (02:15). (B) FRAP of the same cell as in A ∼6 min after anaphase onset. Note the absence of complete recovery during the transition to telophase. Times, hh:mm:ss. (C) Plot of recovery in the bleached areas shown in A and B for metaphase (green)and anaphase (black). Data was normalized to total loss of fluorescence, time 0 corresponds to the midpoint of the bleach. Note the difference in IFs 4 min after the bleach. Bars, 5 μm.

Mentions: Next, we investigated whether assembling NPCs were tightly bound to the chromatin surface in the absence of B type lamins by FRAP analysis of POM121. In metaphase, POM121-EGFP3 diffused freely in the interconnected membranes of the ER with an apparent diffusion constant of DPOM121 = 0.25 μm2/s and an IF <10% (Fig. 6 C), a value comparable to ER membrane proteins (Nehls et al., 2000). Strikingly, performing FRAP experiments in the same cell ∼6 min later during nuclear assembly we found the chromatin-associated pool of POM121-EGFP3 strongly immobilized. In spite of the flux of new material into the growing pronuclei, 60% of POM121 in these structures was immobile over several minutes as the cell progressed into telophase (Fig. 6 B). This occurred well before lamin B1 became detectable around chromosomes (Fig. 5, C and D).


Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells.

Daigle N, Beaudouin J, Hartnell L, Imreh G, Hallberg E, Lippincott-Schwartz J, Ellenberg J - J. Cell Biol. (2001)

POM121 is immobilized during nuclear assembly in anaphase. FRAP of a NRK cell transiently expressing POM121-EGFP3. (A) Boxed area was photobleached to background levels in metaphase. Recovery was monitored every 9 s in a single image acquired with open pinhole. Note rapid recovery of fluorescence into the ER as the cell reaches metaphase/anaphase transition (02:15). (B) FRAP of the same cell as in A ∼6 min after anaphase onset. Note the absence of complete recovery during the transition to telophase. Times, hh:mm:ss. (C) Plot of recovery in the bleached areas shown in A and B for metaphase (green)and anaphase (black). Data was normalized to total loss of fluorescence, time 0 corresponds to the midpoint of the bleach. Note the difference in IFs 4 min after the bleach. Bars, 5 μm.
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Related In: Results  -  Collection

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fig6: POM121 is immobilized during nuclear assembly in anaphase. FRAP of a NRK cell transiently expressing POM121-EGFP3. (A) Boxed area was photobleached to background levels in metaphase. Recovery was monitored every 9 s in a single image acquired with open pinhole. Note rapid recovery of fluorescence into the ER as the cell reaches metaphase/anaphase transition (02:15). (B) FRAP of the same cell as in A ∼6 min after anaphase onset. Note the absence of complete recovery during the transition to telophase. Times, hh:mm:ss. (C) Plot of recovery in the bleached areas shown in A and B for metaphase (green)and anaphase (black). Data was normalized to total loss of fluorescence, time 0 corresponds to the midpoint of the bleach. Note the difference in IFs 4 min after the bleach. Bars, 5 μm.
Mentions: Next, we investigated whether assembling NPCs were tightly bound to the chromatin surface in the absence of B type lamins by FRAP analysis of POM121. In metaphase, POM121-EGFP3 diffused freely in the interconnected membranes of the ER with an apparent diffusion constant of DPOM121 = 0.25 μm2/s and an IF <10% (Fig. 6 C), a value comparable to ER membrane proteins (Nehls et al., 2000). Strikingly, performing FRAP experiments in the same cell ∼6 min later during nuclear assembly we found the chromatin-associated pool of POM121-EGFP3 strongly immobilized. In spite of the flux of new material into the growing pronuclei, 60% of POM121 in these structures was immobile over several minutes as the cell progressed into telophase (Fig. 6 B). This occurred well before lamin B1 became detectable around chromosomes (Fig. 5, C and D).

Bottom Line: No independent movement of single pore complexes was found within the plane of the NE in interphase.During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase.Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.

ABSTRACT
The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121-green fluorescent protein (GFP) and GFP-Nup153, and GFP-lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

Show MeSH
Related in: MedlinePlus