QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online January 24, 2007
doi: 10.1242/10.1242/jcs.03355

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ma, Y. Articles by Zhang, C. Search for Related Content PubMed PubMed Citation Articles by Ma, Y. Articles by Zhang, C. Social Bookmarking                         What's this?

Lamin B receptor plays a role in stimulating nuclear envelope production and targeting membrane vesicles to chromatin during nuclear envelope assembly through direct interaction with importin β

The Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education, The National Key Laboratory of Bio-membrane and Membrane Biotechnology, the College of Life Sciences, Peking University, Beijing 100871, China

View larger version (76K): [in this window] [in a new window]   Fig. 1. GFP-xLBR overexpression leads to nuclear membrane overproduction. (A) Location of GFP-xLBR expression during the cell cycle. HeLa cells were transfected with GFP-xLBR expression vector and visualized under a fluorescence microscope to judge the expression levels of the fusion protein. HeLa cells expressing low levels of full-length GFP-xLBR of various stages of cell cycle were fixed and visualized for GFP or stained with DAPI for DNA. Note that the fusion protein was located to the NE and ER in interphase and had normal cell cycle distribution dynamics. (B) High level expression of GFP-xLBR caused nuclear membrane overproduction. Note that with increasing expression of GFP-xLBR, the excess NE either folded into the nucleoplasm (arrows) and/or formed vesicular aggregates (arrowheads) in the cytoplasm. DNA was stained blue with DAPI. Bars, 10 µm.

View larger version (78K): [in this window] [in a new window]   Fig. 2. Perinuclear aggregates of GFP-xLBR bud off from the nuclear membrane and form vesicles with membrane stacks that do not contain lamin B or nucleoporins. (A) HeLa cells overexpressing GFP-xLBR were viewed by time-lapse microscopy. At the beginning, the vesicle was very small and was connected through its edges with the nuclear membrane (NE, arrows). Within several hours, the vesicle gradually and progressively pinched off from nuclear membrane. Also, the small vesicle aggregates could fuse to form a large one (arrowheads). The area indicated by the arrow in the top panels was enlarged and shown in the bottom panels. Bar, 10 µm. (B) HeLa cells overexpressing GFP-xLBR were visualized by fluorescence microscopy and then processed for TEM. The nuclear membrane in the transfected cells appeared normal (B1-B4; the two transfected and two nontransfected cells look similar). TEM examination of aggregates showed that vesicles had numerous bilayered stacks of NE-like membranes (indicated by arrows). In B4 the boxed areas a and b are shown at higher magnification in Ba and Bb. The insets in a and b are higher magnification of the boxed areas. N, nucleus. Bars, 10 µm in the upper panels and 1 µm in the lower panels. (C,D) HeLa cells overexpressing GFP-xLBR were fixed and stained with anti-nucleoporin monoclonal antibody mAb414 (C) or anti-lamin B (D). Neither the nuclear pore complex component nor Lamin B was observed on the membrane stacks. (E,F) Immunofluorescence of HeLa cells overexpressing GFP-xLBR using the antibody against a Golgi marker (E) or the ER marker (F). Bar, 10 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 3. xLBR domain requirement for NE localization and aggregation production in both HeLa and XTC cells. (A) GFP-fused LBR or its deletion mutants were overexpressed in HeLa or XTC cells. The cells were examined under a fluorescence microscope. Note that GFP-xLBR1-210 was concentrated in nucleoplasm, whereas the GFP-LBR90-210 was distributed throughout the whole cell, indicating that a nuclear localization signal is present within amino acids 1-90. GFP-LBR211-621 was located on the nuclear rim and forms aggregates although less than those of full length xLBR, whereas GFP-LBR309-621 was located in the whole cell body, suggesting that the first transmembrane segment within amino acids 211 to 309 is necessary for locating the protein to the NE and formation of the aggregates. Bar, 10 µm. (B) A schematic diagram of the domain structure of LBR.

View larger version (43K): [in this window] [in a new window]   Fig. 4. LBR binds to importin β in vivo through the N-terminal 1-210 domain. (A) Interphase and mitotic HeLa cells were analyzed by indirect immunofluorescence microscopy using anti-importin β and LBR antibodies, and shows colocalization of these endogenous substances. (B) The N-terminal domain of xLBR binds to importin β in mitotic HeLa extract. CNBr-activated Sepharose 4B-coated with GFP-xLBR1-210 was incubated with mitotic HeLa extract. The proteins bound to the beads were isolated and subjected to western blot analysis for the presence of importin β. (C) A fluorescence protein binding assay shows direct interaction of LBR with importin β. Purified GFP-xLBR1-210 bound to importin β but not GFP beads. (D) The purified N-terminal domain of xLBR binds to importin β in the presence of 100 mM or and 300 mM NaCl but not in the presence of 500 mM NaCl. Proteins associated with the importin β beads were subjected to western blot with anti-His antibody. (E) In Xenopus egg extract, GFP-xLBR1-210 but not GFP beads could specifically pull-down the endogenous importin β (arrowhead). Proteins associated with the beads were separated on a SDS gel followed by silver staining. The molecular mass markers are shown on the left. (F) Western blot analysis of the precipitated protein in E and egg extract for the presence of importin β.

View larger version (52K): [in this window] [in a new window]   Fig. 5. LBR binds to importin β via amino acids 45 to 90. (A) Schematic diagram of N-terminal deletion constructs of xLBR1-210. The cDNAs for the different truncated N-terminal domains of xLBR were subcloned into pET28a to express the devised polypeptides. (B) Western analysis of the in vitro binding assay using the truncated LBR fragments and GST-importin β. Note that deletion past aa 45 reduced the binding, and past aa 90 abolished binding. (C) Quantification of the in vitro binding assayed in B by densitometry. 10% of the input for each fragment was set to 100%. The data is shown as the mean percentage bound plus the standard deviation. (D) Schematic diagram of the C-terminal deletion mutants of xLBR1-210. The cDNAs for the different domains of xLBR were subcloned into pET28a-GFP to express the polypeptides fused with GFP. (E) In vitro binding assay using the C-terminally truncated LBR proteins and importin β. Note that GFP-xLBR1-53 did not bind to importin β and GFP-xLBR1-81 had weak binding. (F) Quantification of the in vitro binding assayed in E, which was identical to the method used in C. (G) Analysis of the binding of GFP-xLBR45-90 to importin β. GFP-xLBR45-90 bound to importin β as efficiently as GFP-xLBR1-210. (H) 1. GFP-xLBR45-90 pulled down importin β from mitotic HeLa extract. 2. GFP-xLBR45-90 pulled down importin β from Xenopus egg extract.

View larger version (23K): [in this window] [in a new window]   Fig. 6. LBR binding to importin β is regulated by the nucleotide state of Ran and is independent of importin . (A) An in vitro assay of for the release of GST-importin β-bound His-xLBR1-210 in the absence (no Ran, lanes 1 and 2) and presence of His-RanQ69L-GTP (RanQ69L, lanes 3 and 4) or His-RanT24N-GDP (RanT24N, lanes 5 and 6). The soluble (S) and pelleted (P) (bound to GST-importin β) proteins were analyzed for His-xLBR1-210 with anti-His antibody. (B) An in vitro assay for the release of GST-importin β45-876-bound His-xLBR1-210 in the absence (no Ran, lanes 1 and 2) and presence of His-RanQ69L-GTP (RanQ69L, lanes 3 and 4) or His-RanT24N-GDP (RanT24N, lanes 5 and 6) as in A. (C,D) Pull-down assays of the endogenous importin β of mitotic HeLa cell extract (C) or Xenopus egg extract (D) by CNBr-activated Sepharose 4B-coated with His-xLBR1-210 in the presence of buffer alone, His-RanQ69L-GTP or His-RanT24N-GDP. The LBR-bound importin β was probed with the anti-importin β antibody on western blots. (E) An in vitro binding assay of purified GST-importin β with His-xLBR1-210 in the absence or presence of importin . The LBR was probed with the anti-His antibody. (F) His-xLBR1-210 binding assay with GST-importin β or GST-importin β1-462 in vitro. His-xLBR1-210 was probed with the anti-His antibody.

View larger version (70K): [in this window] [in a new window]   Fig. 7. LBR-containing NE precursor vesicles are recruited to participate in NE assembly through importin β. (Aa). Importin β-coated Sepharose beads were blocked in advance with BSA or xLBR1-210 and used in the NE assembly assay in mitotic HeLa extract prepared from constitutive GFP-xLBR-expressing HeLa cells. Note that the incorporation of GFP-xLBR into NE could be blocked by xLBR1-210 (indicated by arrow) but not by BSA (indicated by arrowhead). (Ab 1 and 2) Importin β-coated Dyna beads were blocked in advance with BSA (Ab 1) or xLBR1-210 (Ab 2) and used in the NE assembly assay, followed by analysis with TEM. The double-layered NE (arrows) could be clearly seen. Note that xLBR1-210 blocked the NE precursor vesicle recruitment and the NE assembly around the importin β-coated beads (Ab 2). Scale bar, 500 nm. (Ab 3) Negative staining of the NE with typical NPCs assembled on the surface of importin β-coated TEM grids. NPCs are indicated by asterisks. (Ba) RanGDP-coated beads induced NE assembly in mitotic HeLa extract prepared from constitutive GFP-xLBR-expressing HeLa cells in the presence of His-xLBR90-210 or His-xLBR1-210. The result showed that His-xLBR1-210 (arrow) but not His-xLBR90-210 (arrowhead) could specifically prevent recruitment of GFP-xLBR-bound NE precursor vesicles onto the RanGDP-beads to assemble the NE. (Bb) Statistical analysis of the NE assembly in the presence of His-xLBR90-210 or His-xLBR1-210. The data is shown as the mean percentage decorated beads plus the standard deviation. (C) NE assembly assay in the extract prepared from constitutive GFP-xLBR-expressing HeLa cells and depleted of importin β with RanQ69L. (Ca) Western blot analysis showed that more than 90% of the endogenous importin β in the extract was depleted by RanQ69L-GTP. The untreated extract (1), extract mock-depleted with control beads (2), extract depleted with Q69L beads (3), proteins bound to control (4) or Q69L (5) beads were subjected to Western blot analysis for importin β. (Cb) When NE assembly was induced with RanGDP-beads in the importin β-depleted HeLa extract, the NE assembly was efficiently blocked (indicated by arrow) compared with that in the mock-depleted extract (indicated by arrowhead). If importin β-coated beads were added to the importin β-depleted extract, the NE assembly could occur efficiently (indicated by arrowhead,). The addition of herparin to a final concentration of 0.5% did not influence the NE assembly around importin β-coated beads. (Da) When His-xLBR90-210 or His-xLBR1-210 was added to the importin β-depleted extract, the NE assembly around the added importin β-beads could only occur in the extract containing added His-xLBR90-210 (indicated by arrow) but not His-xLBR1-210 (indicated by arrowhead), indicating His-xLBR1-210 prevented the access of the NE precursor vesicles to the importin β on the beads. (Db) Statistical analysis of the NE assembly in the presence of His-xLBR90-210 or His-xLBR1-210. The data is shown as the mean percentage (plus the standard deviation) of the beads decorated with the NE.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter