Cisternal organization of the endoplasmic reticulum during mitosis - PubMed (original) (raw)

Cisternal organization of the endoplasmic reticulum during mitosis

Lei Lu et al. Mol Biol Cell. 2009 Aug.

Abstract

The endoplasmic reticulum (ER) of animal cells is a single, dynamic, and continuous membrane network of interconnected cisternae and tubules spread out throughout the cytosol in direct contact with the nuclear envelope. During mitosis, the nuclear envelope undergoes a major rearrangement, as it rapidly partitions its membrane-bound contents into the ER. It is therefore of great interest to determine whether any major transformation in the architecture of the ER also occurs during cell division. We present structural evidence, from rapid, live-cell, three-dimensional imaging with confirmation from high-resolution electron microscopy tomography of samples preserved by high-pressure freezing and freeze substitution, unambiguously showing that from prometaphase to telophase of mammalian cells, most of the ER is organized as extended cisternae, with a very small fraction remaining organized as tubules. In contrast, during interphase, the ER displays the familiar reticular network of convolved cisternae linked to tubules.

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Figures

Figure 1.

Figure 1.

Changes in appearance of the ER during cell division. Spinning disk confocal fluorescence images of live HeLa cells at different stages during the cell cycle; the ER and chromatin are marked by expression of GFP-Sec61β (green) and H2B-mRFP (red), respectively. The upper two rows correspond to equatorial middle sections; the bottom row is from a section adjacent to the glass coverslip. (a and a′) HeLa cell during interphase. (b1–b5 and b2′–b5′) Sequential images from a second HeLa cell starting with late prophase and ending with metaphase. (c1–c4) Sequential images from a third HeLa cell starting with anaphase and ending, after abscission, with early interphase; the images in c4 and c4′ correspond to one of the daughter cells depicted in c3. NEBD, nuclear envelope breakdown. Bar, 10 μm.

Figure 2.

Figure 2.

Comparison of ER organization in interphase and metaphase. Sequential spinning disk confocal sections along the _z_-axis and 3D renderings of live-cell images acquired from HeLa cells expressing GFP-Sec61β. (A) HeLa cell imaged during interphase. Top left, planes a–e selected for display in the subsequent panels, with plane e closest to the coverslip. Sequences f and g are from the regions boxed in e; they display every third plane from the sequential acquisition along the _z_-axis; their absolute position is indicated in micrometers. (f′ and g′) are parts of the full z-stack in 3D rendering; they correspond to regions f and g, respectively; their orientations are presented with respect to the coordinate frame in a (the same scheme is followed throughout this article). Bar, 10 μm (e) and 1 μm (f and g). N, nucleus. (B) HeLa cell imaged during metaphase. Top left, planes a–e selected for display. Note that the cell has rounded up and is therefore much thicker than the cell in A. Sequences f and g are from the boxed region in d and c, respectively; they display every third plane from the sequential acquisition along the _z_-axis; their absolute position is indicated in micrometers. The intensity was rescaled in g to help highlight the ER tubules. f′ and g′ are parts of the full z-stack in 3D rendering; they correspond to regions f and g, respectively. Renderings (h and i) show the full z-stack in the middle of the cell, between planes c and d, and between planes d and e, respectively; see Supplemental Movies 11 and 12 for the interactive QTVR movies. Bar, 10 μm (e) and 1 μm (f and g).

Figure 3.

Figure 3.

Partial alignment of ER tubules with the spindle in mitotic cells. Sequential spinning disk confocal, live-cell images of a HeLa cell expressing GFP-Sec61β (green) and cherry-tubulin (red) to illustrate the partial association of mitotic ER tubules with microtubules of the spindle (see arrowheads in the enlarged views). Most of the ER tubules are in the central zone of the cell; they seem to radiate from the spindle poles positioned in different sections of the z-stack. A fraction of the ER cisternae converge onto the spindle pole (dotted circle). The intensity in the enlarged views of GFP-Sec61β was rescaled to help highlight the ER tubules. Bars, 10 μm (left column) or 1 μm (enlarged views).

Figure 4.

Figure 4.

Uniform redistribution of inner nuclear membrane proteins throughout the ER cisternae in mitosis. Optical sections from a 3D z-stack of HeLa cells expressing either (A) LBR-GFP or (B) emerin-GFP together with cherry-Sec61β and imaged during interphase or metaphase. The Pearson's r (R) was used to estimate the extent of colocalization between LBR-GFP or emerin-GFP and cherry-Sec61β and was calculated using the complete 3D image z-stack, which encompassed the entire volume of the cells examined. Bar, 10 μm.

Figure 5.

Figure 5.

Redistribution of Rtn4HD to the extended ER cisternae. (A) Bottom plane adjacent to the glass coverslip from an interphase HeLa cell expressing GFP-Rtn4HD (green) and cherry-Sec61β (red) obtained using live-cell spinning disk confocal microscopy. The serial sections in the enlarged views are from the boxed region and correspond to every third consecutive plane from the z-stack with their absolute positions along the _z_-axis shown in micrometers. Bar, 1 μm. The section with z = −0.27 highlights the preferential location of Rtn4HD to ER regions of high curvature composed of tubules (arrow) and edges of cisternae (empty arrowhead) and its exclusion from flatter regions of the cisternae (solid arrowhead); the nuclear envelope (NE) is devoid of reticulon. Bar, 10 μm. The scatter plot in the right panel shows the extent of colocalization between GFP-Rtn4HD and cherry-Sec61β; the Pearson's r (R) for this cell is 0.81 and 0.83 ± 0.02 (mean ± SD) for three cells. These values were calculated using the complete 3D image z-stack, which covered the entire cell volume and the nuclear envelope was not included in these calculations because Rtn4HD is excluded from this region. Bar, 10 μm. (B) Equatorial plane from a metaphase HeLa cell expressing GFP-Rtn4HD (green) and cherry-Sec61β (red) obtained using live-cell spinning disk confocal microscopy; bar, 10 μm. The serial sections in the enlarged views are from the boxed region and correspond to every third consecutive plane from the z-stack with their absolute positions along the _z_-axis shown in micrometers. Bar, 1 μm. The scatter plot in the right panel shows the significantly higher extent of colocalization (than in A) of GFP-Rtn4HD and cherry-Sec61β; the Pearson's r (R) for this cell is 0.96 and 0.94 ± 0.02 (mean ± SD) for three cells. (C) Equatorial plane from an interphase HeLa cell expressing GFP-Rtn4HD and cherry-Sec61β treated with 33 μM nocodazole for 2 h. The scatter plot in the right panel shows the significant increase, over the cell panel A, in colocalization of GFP-Rtn4HD and cherry-Sec61β; the Pearson's r (R) for this cell is 0.91. The Pearson's r was calculated using the full z-stack; the nuclear envelope was not included in the calculation. N, nucleus. Bar, 10 μm.

Figure 6.

Figure 6.

EM tomographic reconstructions from the ER during metaphase. EM tomographic reconstruction from metaphase BSC1 cells prepared by high-pressure freezing and freeze substitution. (a and c) Low-magnification views of different metaphase BSC1 cells identified by their condensed chromosomes (chrom.); examples of curvilinear membrane ER profiles (arrows) are shown in a. Bar, 10 μm. (b) Example, at higher magnification, of a tomographic 1.1-nm slice showing examples of curvilinear membrane profiles, identified as ER by their close association with ribosomes (see Supplemental Movie 3 for the complete set of serial slices spanning ∼200 nm that illustrates presence of hollow cisternae and absence of tubules). Bar, 200 nm. (d and e) The reconstruction is composed of four serial 200-nm sections, representing ∼800 nm of cellular thickness from the boxed region in c; see Supplemental Movie 4 for the complete set of serial slices. Representative tomographic slice of 1.1 nm in thickness without (d) and with (e) superimposition of the model contour tracing of the ER membrane. Identity of the ER membrane (green) was established by the presence of ribosomes (magenta) adhering to its cytosolic surface. Bar, 200 nm. Panels f–h show three views of the rendered 3D model also depicted in Supplemental Movie 7. The ER in the sample conforms primarily to a cisternal structure; the hollow cisterna is evident in f–h. Bar, 200 nm.

Figure 7.

Figure 7.

Microtubule depolymerization by nocodazole treatment results in loss of ER tubules and gain of extended ER cisternae. (A) A BSC1 cell expressing GFP-Sec61β (top row) and cherry-tubulin (bottom row) was exposed during interphase to 33 μM nocodazole; the images are from a single-plane, time-lapse series acquired using the spinning disk confocal microscope and correspond to an optical section close to the glass coverslip (see Supplemental Movie 8). The arrows highlight a peripheral region containing tubular ER joined by three-way junctions and its replacement with extended cisternae after microtubule depolymerization. Bar, 10 μm. (B) Spinning disk confocal z-sections and 3D rendering of a BSC1 cell stably expressing GFP-Sec61β imaged during interphase and showing the effects of nocodazole treatment. Left, plane a selected for display. Sequences b and c display enlarged views from the boxed regions in a and correspond to every third plane along the _z_-axis; their absolute position along the _z_-axis is indicated in micrometers. The intensity was rescaled in b and c to help highlight the ER cisternae. Views (b′) and (c′) are 3D renderings of regions b and c, respectively, from the full 3D z-stack. Bar, 10 μm (a) and 1 μm (c). N, nucleus. (C) EM tomographic reconstruction of the ER in an NRK cell treated with nocodazole during interphase. The reconstruction was made from a 200-nm section. Representative tomographic slice of 1.1 nm in thickness without (a) and with (b) superimposition of the contour of a model that traces the ER membrane. Identity of the ER membrane (green) was established by its close association with small particles corresponding to ribosomes (magenta), which cover the cytosolic face of ER cisternae. c–e show three rendered views of the 3D model also depicted in Supplemental Movie 9. Note that a budding domain is present on the right side of the reconstruction. Bars, 200 nm.

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