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. 2013 Aug 12;26(3):315-23.
doi: 10.1016/j.devcel.2013.06.016. Epub 2013 Aug 1.

REEP3/4 ensure endoplasmic reticulum clearance from metaphase chromatin and proper nuclear envelope architecture

Anne-Lore Schlaitz  1 , James Thompson, Catherine C L Wong, John R Yates 3rd, Rebecca Heald
Affiliations

REEP3/4 ensure endoplasmic reticulum clearance from metaphase chromatin and proper nuclear envelope architecture

Anne-Lore Schlaitz et al. Dev Cell. .

Abstract

Dynamic interactions between membrane-bound organelles and the microtubule cytoskeleton are crucial to establish, maintain, and remodel the internal organization of cells throughout the cell cycle. However, the molecular nature of these interactions remains poorly understood. We performed a biochemical screen for microtubule-membrane linkers and identified REEP4, a previously uncharacterized endoplasmic reticulum (ER) protein. Depletion of REEP4 and the closely related REEP3 from HeLa cells causes defects in cell division and a proliferation of intranuclear membranes derived from the nuclear envelope. This phenotype originates in mitosis, when ER membranes accumulate on metaphase chromosomes. Microtubule binding and mitotic ER clearance from chromosomes both depend on a short, positively charged amino acid sequence connecting the two hydrophobic domains of REEP4. Our results show that REEP3/4 function redundantly to clear the ER from metaphase chromatin, thereby ensuring correct progression through mitosis and proper nuclear envelope architecture.

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Figures

Figure 1
Figure 1. Depletion of REEP3 and REEP4 causes interphase nuclear envelope defects
(A) Endogenous REEP4 stained with a specific antibody in HeLa cells stably expressing the ER-marker GFP-Sec61β. REEP4 nuclear envelope and ER staining are apparent in control cells but not in REEP4-depleted cells. Intranuclear background staining of the antibody is present in both. (B) HeLa cells expressing GFP-Sec61β stained for Lamin B1. In control cells, GFP-Sec61β and Lamin B1 label is restricted to the nuclear rim, whereas in REEP3/4 double knockdown cells numerous GFP-Sec61β- and Lamin B1-positive structures are visible within the nucleus. (C) HeLa cells co-transfected with siRNA targeting REEP3/4 and plasmids encoding either mcherry or RNAi-resistant versions of HA-tagged REEP3 or REEP4 and stained for HA and Lamin B1. Normal nuclear morphology is restored in cells expressing either REEP3 or REEP4. Scale bars are 10 µm. See Figure S1 and Movie S1.
Figure 2
Figure 2. Nuclear envelope defects arise in mitosis
(A) HeLa cells expressing GFP-Sec61β and histone H2B-mcherry imaged live at metaphase. GFP-Sec61β is mostly excluded from the chromosome area in control cells but shows extensive association with chromatin in REEP3/4 RNAi cells. (B) Still images from time-lapse acquisitions of HeLa cells expressing GFP-Sec61β in mitosis. In control cells, GFP-Sec61β is excluded from chromatin until the onset of nuclear envelope reformation. In REEP3/4 RNAi cells GFP-Sec61β associates with chromosomes throughout anaphase and during nuclear envelope reformation. It is subsequently gradually and incompletely cleared from the inside of the nucleus after nuclear envelope reformation, with some remnants persisting (arrows). For REEP3/4 RNAi a later final time point (39 minutes instead of 20 minutes for control) is shown to illustrate clearing of ER from the nuclear interior and the persistence of intranuclear membranes. See Movie S2. (C) Still images from a time-lapse acquisition of control and REEP3/4 RNAi HeLa cells expressing GFP-Sec61β and H2B-mcherry. The initial frame is shown with both, GFP-Sec61β and H2B-mcherry signal. Below, the time series is shown with only the GFP-Sec61β signal to make ER accumulation discernable. In control cells, the chromatin area remains free of ER over the course of metaphase but in REEP3/4 RNAi cells, ER accumulates at the metaphase plate. (D) Quantification of ER accumulation at the metaphase plate in REEP3/4 RNAi cells as % change compared to the initial frame taken 12 minutes before anaphase onset. Mean pixel intensities for GFP-Sec61β fluorescence at the metaphase plate were measured. Data are mean ± SEM from 10 cells analyzed. Means for each time point are significantly different from t = −12 min to p<0.05 (t = −8 min and t = −4 min), and to p<0.005 for t = 0 min. p-values were obtained using a two-tailed, unpaired student’s t-test. Scale bars represent 10 µm. See Figure S2.
Figure 3
Figure 3. Depletion of REEP3/4 impairs daughter nuclei separation and leads to an increase in chromosome segregation defects
(A) Top panels: Control and REEP3/4 depleted cells in late stages of cell division. Separation between daughter cell nuclei in REEP3/4 RNAi is reduced compared to controls. Bottom panel: Example for chromosome segregation defect in REEP3/4 RNAi cells. Scale bar is 10 µm. (B) Quantification of daughter nuclei separation. When the cleavage furrow had narrowed to ≤ 1 µm, nuclei were separated by 12.5 µm in control cells but only 8.9 µm in REEP3/4 RNAi cells. Data are mean +/− SEM, from three independent experiments. In each experiment, at least 14 control and 22 REEP3/4 RNAi cells were analyzed. Control and REEP3/4 RNAi were significantly different to p<0.001 in each experiment using a two-tailed, unpaired Welch’s t-test. (C) Quantification of chromosome segregation defects (anaphase bridges and lagging chromosomes). 11.1% of control cells and 16.6% of REEP3/4 RNAi cells show chromosome segregation defects. Data shown are mean +/− SEM from seven experiments. At least 50 cells were analyzed per condition and experiment. Control and REEP3/4 RNAi are significantly different to p<0.03 using a two-tailed, unpaired student’s t-test..
Figure 4
Figure 4. Microtubule binding is important for REEP3/4 function
(A) Schematic representation of the REEP protein family. Shaded regions represent the hydrophobic domains. The sequences for the cytoplasmic stretches connecting the hydrophobic domains are shown. (B) Western blot of samples from microtubule co-pelleting assay performed with HA-tagged REEP4 and the REEP4/5 chimeric protein (REEP4dMT). 85% of wild type REEP4-HA but only 13% of REEP4dMT-HA co-pellet with microtubules. (C) HeLa cells expressing GFP-Sec61β and histone H2B-mcherry were imaged live at metaphase. Expression of RNAi-resistant wild type REEP4 restores ER clearance from chromosomes in REEP3/4 RNAi cells whereas expression of RNAi-resistant REEP4dMT does not rescue. Scale bar is 10 µm. (D) Total and metaphase plate GFP-Sec61β fluorescence were quantified from images as in (C). The fraction of cellular GFP-Sec61β fluorescence at the metaphase plate is shown. Data are mean ± SEM from three independent experiments. At least 10 cells were analyzed per experiment and condition. Means for control and REEP3/4 RNAi (p<0.002) as well as control and REEP3/4 RNAi + REEP4dMT-HA (p<0.002) are significantly different. The means for control versus REEP3/4 RNAi + REEP4-HA WT are not significantly different (p>0.4). The means for REEP3/4 RNAi versus REEP3/4 RNAi + REEP4dMT-HA are not significantly different (p>0.7). pvalues were obtained using a two-tailed, unpaired student’s t-test. (E) Model depicting REEP3/4 function. In wild type cells, REEP3/4 transport ER towards microtubule minus ends, thus clearing chromosomes, opposing ER movement towards chromatin and clustering ER at the spindle poles. Without REEP3/4, ER clearance from chromosomes fails and ER invasion of the chromosome area causes accumulation at the metaphase plate. WT: wild type. See Figure S3.
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Comment in

  • PREEParing for mitosis.
    Burke B. Burke B. Dev Cell. 2013 Aug 12;26(3):221-2. doi: 10.1016/j.devcel.2013.07.018. Dev Cell. 2013. PMID: 23948250

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