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<div><p>Nuclear lamins are important structural and functional proteins in mammalian cells, but little is known about the mechanisms and cofactors that regulate their traffic into the nucleus. Here, we demonstrate that trafficking of lamin A, but not lamin B1, and its assembly into the nuclear envelope are regulated by sorting nexin 6 (SNX6), a major component of the retromer that targets proteins and other molecules to specific subcellular locations. SNX6 interacts with lamin A <i>in vitro</i> and <i>in vivo</i> and links it to the outer surface of the endoplasmic reticulum in human and mouse cells. SNX6 transports its lamin A cargo to the nuclear envelope in a process that takes several hours. Lamin A protein levels in the nucleus augment or decrease, respectively, upon gain or loss of SNX6 function. We further show that SNX6-dependent lamin A nuclear import occurs across the nuclear pore complex via a RAN-GTP-dependent mechanism. These results identify SNX6 as a key regulator of lamin A synthesis and incorporation into the nuclear envelope.</p></div

SNX6 overexpression alters lamin A/C subcellular distribution.

<p>U2OS cells were transiently transfected as indicated and analyzed by confocal microscopy. (<b>A</b>) Cells cotransfected with FLAG-lamin A and YFP localized the mature lamin A protein in the perinucleus (top images, YFP in yellow, FLAG-LMNA in red). Cotransfection of FLAG-LMNA with YFP-SNX6 (bottom images) revealed perinuclear expression of SNX6 together with its high accumulation in external vesicles around the nucleus (left panel, bottom). This co-expression within the cell coincided with the partial re-distribution of FLAG-LMNA into distinctive extra-perinuclear vesicles and the partial loss of the smoothened perinuclear shape. (<b>B</b>) Similar results were obtained in cells cotransfected with HA-LMNA and YFP-SNX6. (<b>C</b>) Quantification of cells with an aberrant (extranuclear) distribution of HA-lamin A after cotransfection with YFP or YFP-SNX6 (n = 3 independent transfections). (<b>D</b>) Cells transfected with YFP alone (top images) or YFP-SNX6 (bottom images) also exhibited an altered distribution of endogenous lamin A/C upon SNX6 overexpression. (<b>E</b>) Quantification of cells with an extranuclear expression pattern of endogenous lamin A/C upon transfection with YFP alone or YFP-SNX6 (n = 3 independent transfections). (<b>F</b>) Quantification of cells with an extranuclear expression pattern of CFP-lamin A two days after silencing of endogenous SNX6 with specific siRNA (siRNA-SNX6). In controls, cells were transfected with siRNA-CTRL (n = 3 independent transfections). (<b>G</b>) Cells were cotransfected with CFP-lamin A, YFP-lamin B1 and either HA alone (top) or HA-SNX6 (bottom). The arrow marks one perinuclear region with significant content of lamin A but no significant changes in the distribution lamin B1. When cotransfected with SNX6, CFP-LMNA, but not YFP-LMNB1, also displays a more diffuse pattern and accumulates in small vesicles.</p

SNX6-dependent lamin A incorporation into the nucleus occurs via a RAN-dependent mechanism and is independent of ER tubule-forming proteins.

<p>(<b>A</b>) Flow cytometry analysis of nuclei isolated from U2OS cells cotransfected with CFP-lamin A and either HA alone or HA-SNX6. When indicated, cells were also cotransfected with reticulon 3. (<b>B</b>) Confocal microscopy analysis of U2OS cells cotransfected as indicated. The intensity of the CFP signal across the cell nucleus and cytoplasm (arrows) is shown for each cell in the graphs below. The arrows across the cells correspond to the sections along which CFP-Lamin A signals were quantified. (<b>C</b>) Nuclei from cells transfected as in (B) were isolated and analyzed by flow cytometry.</p

In vivo shuttling of lamin A to the nucleus.

<p>Time-lapse analysis of U2OS cells cotransfected with GFP-lamin A and HA-SNX6 to enhance GFP-lamin A extranuclear accumulation. Over a period of 8 hours, the extranuclear GFP-Lamin A progressively incorporated into the nucleus of the transfected cell. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115571#pone.0115571.s006" target="_blank">S2 Video</a>.</p

SNX6 interacts with lamin A/C <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A</b>) Cell extracts from U2OS cells overexpressing HA-lamin A (left) or mouse smooth muscle cells (SMCs) expressing endogenous lamin A (right) were subjected to pull-down with GST-SNX6 or GST alone. Pelleted material was probed by immunoblot with the indicated antibodies. Input lane corresponds to an aliquot of the total protein mixture before each pull down experiment. (<b>B</b>) <i>In vivo</i> interaction between SNX6 and lamin A was quantified by fluorescence resonance energy transfer (FRET) using the acceptor photobleaching method. Data in the graph represent the mean±SE of three independent experiments. The images show a representative example of cells cotransfected with YFP-SNX6 and CFP-LMNA before and after YFP photobleaching. (<b>C</b>) U2OS cells were transiently transfected with YFP-SNX6 together with either HA-lamin A or HA alone as indicated. Cell lysates were immunoprecipitated (IP) with anti-GFP antibodies or control immunoglobulins and immunocomplexes were further analyzed by immunoblotting with anti-HA (top blot) to visualize specific interactions or with anti-GFP (bottom blot) to validate the experimental procedure. Ctrl- indicates the use of unrelated antibodies for immunoprecipitation. (<b>D</b>) Interaction between endogenous lamin A and SNX6. Cell extracts from mouse embryonic fibroblasts (MEFs) and U2OS cells (right) were immunoprecipitated with antibodies against lamin A (LMNA) or against unrelated proteins (SP1 and UCP2). Samples were analyzed by Western blot with the indicated antibodies.</p

SNX6 and lamin A do not colocalize in early endosomes, and SNX6 overexpression increases the amount of lamin A protein without affecting its proteasomal degradation or mRNA expression.

<p>(<b>A</b>) Double immunofluorescence confocal microscopy images showing a high degree of colocalization between endogenous SNX6 and EEA1. The graph shows the intensity for each fluorochrome along the arrowed line in the merge image. (<b>B</b>) U2OS cells were transfected with CFP-lamin A and two days later were left untreated (top images, un-extracted) or subjected to <i>in situ</i> extraction with cytoskeleton buffer (CSK) prior to fixing (lower images, in situ-extracted). Cells were then incubated with FITC-coupled anti-EEA1 antibodies and examined by confocal microscopy. Images reveal the efficient extraction of EEA1 from early endosomes upon in situ extraction with CSK, in contrast to lamin A, which remained in the NE. (<b>C</b>) U2OS cells were cotransfected with YFP and CFP-lamin A and treated as in B. Images show the <i>in situ</i> extraction of ubiquitously expressed YFP but not of lamin A. (<b>D</b>) U2OS cells were cotransfected with YFP-SNX6 and CFP-lamin A and treated as in B. Treatment with CSK did not extract either lamin A or SNX6. (<b>E</b>) Western blot analysis of U2OS cells transfected with control siRNA (siRNA-CTRL) or with siRNA targeting SNX6 (siRNA-SNX6). After two days, cultures were treated for 16 h with either cycloheximide (CHX, 10 µg/ml, Sigma), the proteasome inhibitor MG132 (25 µM, Sigma) or vehicle (DMSO). Efficient SNX6 knockdown in cells transfected with siRNA-SNX6 was verified with anti-SNX6 antibody. ERK2 levels were analyzed as a loading control. (<b>F</b>) Representative confocal microscopy image showing the correlation between high overexpression of YFP-SNX6 and CFP-lamin A. Pictures were taken of U2OS cells two days after transfection with both plasmids. (<b>G</b>) Flow cytometry analysis of cells cotransfected with CFP-lamin A and either YFP or YFP-SNX6, corroborating higher CFP-lamin A expression upon cotransfection of YFP-SNX6. (<b>H</b>) Western blot analysis of U2OS cells transfected with YFP or YFP-SNX6 using primary antibodies against lamin A (top), ERK2 (middle) or GFP (bottom). (<b>I</b>) RT-qPCR analysis of total RNA isolated from U2OS cells two days post-transfection with either YFP or YFP-SNX6. Relative lamin A/C mRNA levels were determined using three sets of primers (primers1, primers2 and primers3) and calculated relative to values obtained in cells transfected with YFP alone (n = 3 experiments).</p

Colocalization of lamin A and SNX6 at the outer surface of the endoplasmic reticulum.

<p>(<b>A</b>) <i>In vivo</i> time-lapse confocal microscopy analysis of U2OS cells cotransfected with GFP-lamin A, HA-SNX6 (to promote extranuclear lamin A accumulation) and RFP-SEC61 (ER label). The top left image shows a representative transfected cell with labeled ER (red) and GFP-lamin A (green) and colocalization of both (yellow). The bottom left image shows an Imaris 3D reconstruction of the same cell. The images on the right show details of 3D reconstructions of the same cell imaged at ten minute intervals. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115571#pone.0115571.s005" target="_blank">S1 Video</a>. (<b>B</b>) U2OS cells were transfected with plasmids encoding GFP-Lamin A and HA-SNX6 and processed two days later for immunofluorescence analysis. Cells were non-permeabilized or permeabilized with either Triton X-100 (to permeabilize all membranes) or digitonin (to permeabilize only the plasma membrane). Cells were incubated with anti-GFP antibodies (left) or anti-lamin A/C antibodies (right). Ectopic lamin A was directly visualized by its GFP fluorescence (green) and indirectly from the signals of anti-GFP or anti-lamin A/C antibodies (red). (<b>C</b>) Subcellular fractions were prepared from lysates of subconfluent cultures of U2OS cells and the indicated fractions were analyzed by western blot with antibodies against lamin A, SNX6 and the ER marker GRP94.</p