Jevtić P et al. (2015), Concentration-dependent Effects of Nuclear Lami...

XB-ART-51369

J Biol Chem 2015 Nov 13;29046:27557-71. doi: 10.1074/jbc.M115.673798.

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Concentration-dependent Effects of Nuclear Lamins on Nuclear Size in Xenopus and Mammalian Cells.

Jevtić P , Edens LJ , Li X , Nguyen T , Chen P , Levy DL .

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A fundamental question in cell biology concerns the regulation of organelle size. While nuclear size is exquisitely controlled in different cell types, inappropriate nuclear enlargement is used to diagnose and stage cancer. Clarifying the functional significance of nuclear size necessitates an understanding of the mechanisms and proteins that control nuclear size. One structural component implicated in the regulation of nuclear morphology is the nuclear lamina, a meshwork of intermediate lamin filaments that lines the inner nuclear membrane. However, there has not been a systematic investigation of how the level and type of lamin expression influences nuclear size, in part due to difficulties in precisely controlling lamin expression levels in vivo. In this study, we circumvent this limitation by studying nuclei in Xenopus laevis egg and embryo extracts, open biochemical systems that allow for precise manipulation of lamin levels by the addition of recombinant proteins. We find that nuclear growth and size are sensitive to the levels of nuclear lamins, with low and high concentrations increasing and decreasing nuclear size, respectively. Interestingly, each type of lamin that we tested (lamins B1, B2, B3, and A) similarly affected nuclear size whether added alone or in combination, suggesting that total lamin concentration, and not lamin type, is more critical to determining nuclear size. Furthermore, we show that altering lamin levels in vivo, both in Xenopus embryos and mammalian tissue culture cells, also impacts nuclear size. These results have implications for normal development and carcinogenesis where both nuclear size and lamin expression levels change.

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Species referenced: Xenopus laevis
Genes referenced: ckap2 gnl3 h2bc21 igf2bp3 lmnb1 lmnb3 ran

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	FIGURE 1. Lamin B3 concentration affects nuclear size in X. laevis egg extract. A) Nuclei were pre- assembled in X. laevis egg extract for 30-35 minutes. Recombinant GFP-LB3 was added to the indicated final concentrations, and the same total volume was added to each reaction. For the control reaction, an equal volume of lamin storage buffer was added. Nuclear growth was allowed to proceed for an additional 75 minutes at 20°C. Nuclei were fixed, spun onto coverslips, processed for immunofluorescence, and stained with mAb414 to visualize the NPC (red) and Hoechst to visualize the DNA (blue). Representative images are shown. White arrows denote intranuclear lamin puncta that form upon addition of high concentrations of recombinant lamin proteins. B) For the representative images at the top of the panel, immunofluorescence against GFP was performed, demonstrating that low concentrations of GFP-LB3 properly localize to the NE. The bar graph shows nuclear size data from one representative experiment. For each bar, the cross-sectional areas of 150-870 nuclei were measured from NPC-stained nuclei and averaged. The error bars are SD. * p<0.05, p<0.01, * p<0.001, NS Not significant. C) Cumulative data from 20 different X. laevis egg extracts are shown. For each data point, the average cross-sectional nuclear area was measured as in B) and normalized to the buffer addition control (bold horizontal line set at 1.0). The red symbols represent data for LB3 protein without a GFP tag. The error bars are SEM. All normalized nuclear area data points greater than 1.05 and less than 0.94 are statistically different from the buffer control (1.0) by at least p<0.05. Due to variability between egg extracts, the data were normalized to the buffer addition control, and SEM error bars are shown rather than larger SD error bars that obscure visualization and interpretation of the data. D) Similar experiments were performed except that mutant LB3 proteins were added at low (2 nM) or high (34 nM) concentrations. The blue data are for LB3 without a GFP tag. The red data are for GFP-LB3 in which the CAAX box was mutated so it cannot be farnesylated (LB3 amino acids 578 and 579 mutated from cysteines to serines). The green data are for GFP-LB3 with an R385P point mutation within LB3 that renders the protein defective for assembly into the nuclear lamina. For each bar, the cross-sectional areas of 152-944 nuclei were measured from NPC-stained nuclei, averaged, and normalized to the buffer addition control (set at 1.0). The error bars are SEM. * p<0.05, p<0.01, * p<0.001, NS Not significant.
	FIGURE 2. Lamin B1, B2, and A concentrations affect nuclear size in X. laevis egg extract. Similar experiments were performed as in Figure 1, using recombinant GFP-LB1, GFP-LB2, and GFP-LA rather than GFP-LB3 (see the Figure Legend for Figure 1). A) Images of representative nuclei. B) Cumulative data from 22 different X. laevis egg extracts are shown. For each data point, the cross-sectional areas of 65-890 nuclei were measured from NPC-stained nuclei, averaged, and normalized to the buffer addition control (bold horizontal line set at 1.0). The open symbols represent data for lamin proteins without a GFP tag. The error bars are SEM. All normalized nuclear area data points greater than 1.06 and less than 0.95 are statistically different from the buffer control (1.0) by at least p<0.05.
	FIGURE 3. Lamin combinations affect nuclear size similarly to single lamins in X. laevis egg extract. A) Western blots were performed to estimate the endogenous amounts of different lamins present in X. laevis embryos during development. Known amounts of recombinant lamins were loaded to the left side of each gel (GFP-LB1: 0.45, 0.9, 1.8 pmol; GFP-LB2: 0.5, 1.25, 2.5 pmol; GFP-LA: 0.1, 0.15, 0.2 pmol). 10 μl each of different stage X. laevis embryo extracts were loaded to the right side of each gel. Blots were probed with antibodies previously shown to be specific to each lamin type (data not shown), as well as Ran as a loading control. Absolute protein amounts were determined by comparing band intensities, quantified by infrared fluorescence, to the known amounts of recombinant lamins on the same blot. Embryo extracts prepared from three different frogs were quantified, and one representative western blot is shown for each lamin. Values below each lane represent the mean ± SD (n=3). LB3 amounts were similarly measured previously as 0.75 pmol in the egg, 2.6 pmol in stage 8, 0.3 pmol in stage 11, and negligible in later stages (6). The bar graph shows average total lamin concentrations present at each stage of development with SD error bars (n=3). B) Similar experiments were performed as in Figure 1, except that different lamin combinations were added to nuclei pre-assembled in egg extract (see the Figure Legend for Figure 1). Lamin combinations that approximated stage 8 were: 175 nM LB3 and 50 nM LB1 (blue diamonds). Lamin combinations that approximated stage 11 were: 50 nM LB1 and 50 nM LB2 (red squares). Lamin combinations that approximated stage 19 were: 20 nM LB1, 20 nM LB2, and 5 nM LA (green triangles). Cumulative data from 5 different X. laevis egg extracts are shown. For each data point, the cross-sectional areas of 85-840 nuclei were measured from NPC-stained nuclei, averaged, and normalized to the buffer addition control (bold horizontal line set at 1.0). The error bars are SEM. All normalized nuclear area data points are statistically different from the buffer control (1.0) by at least p<0.01.
	FIGURE 4. Lamin concentrations affect nuclear size in X. laevis embryo extracts. A) Embryo extracts containing endogenous embryonic nuclei were prepared from X. laevis embryos at stage 7 to early stage 8. Recombinant lamins were added to the indicated final concentrations, and the same total volume was added to each reaction. For the control reaction, an equal volume of lamin storage buffer was added. Nuclei were incubated for 60 minutes at 20°C. Nuclei were then fixed, spun onto coverslips, processed for immunofluorescence, and stained with mAb414 to visualize the NPC (red) and Hoechst to visualize the DNA (blue). Representative images are shown. White arrows denote intranuclear lamin puncta that form upon addition of high concentrations of recombinant lamin proteins. B) Nuclear size data from one representative experiment are shown. For each bar, the cross-sectional areas of 370-1242 nuclei were measured from NPC-stained nuclei and averaged. The error bars are SD. * p<0.05, p<0.01, * p<0.001, NS Not significant. From left to right, the number of nuclei quantified/p-values are: 674/not applicable, 459/10-23, 734/10-12, 513/0.03, 1242/10-18, 704/10-9, 616/0.12, 456/10-31, 763/10-23, 882/10-10, 569/10-45, 370/10-20, 605/0.009. C) Cumulative data from 2 different embryo extracts are shown. For each data point, the average cross-sectional nuclear area was measured as in B) and normalized to the buffer addition control (bold horizontal line set at 1.0). The error bars are SEM. All normalized nuclear area data points greater than 1.05 are statistically different from the buffer control (1.0) by at least p<0.05.
	FIGURE 5. Lamin concentrations affect nuclear size in vivo in X. laevis embryos. A) Single-cell X. laevis embryos were microinjected with the indicated recombinant lamin proteins or lamin storage buffer as a control. The concentrations on the x-axis represent the final concentration of injected lamins in the embryo assuming an embryo volume of 1 μl. For the lamin combinations, the data point at 36 nM = 15 nM LB1 + 15 nM LB2 + 6 nM LA, and the data point at 55 nM = 30 nM LB1 + 25 nM LB2. Embryos were allowed to develop to early stage 8 and embryo extracts were prepared. Extracts containing endogenous embryonic nuclei were fixed, and nuclei were spun onto coverslips and visualized as described in Figure 1. For each data point, 50-100 embryos were microinjected and the cross-sectional areas of 460-2360 nuclei were measured from NPC-stained nuclei, averaged, and normalized to the buffer injected control (bold horizontal line set at 1.0). The error bars are SEM. * p<0.05, *** p<0.001, NS Not significant. B) Single-cell X. laevis embryos were microinjected with mRNA encoding the indicating lamins or water as a control. Embryos were allowed to develop to stage 11-12 and embryo extracts were prepared. Extracts containing endogenous embryonic nuclei were fixed, and nuclei were spun onto coverslips and visualized as described in Figure 1. Representative images are shown. For the control, nuclei stained for the NPC with mAb414 are shown, while in the other images fluorescently labeled lamins are visualized. C) Nuclear size data from the experimental approach described in B) are plotted as a function of the concentration of ectopically expressed lamins. For each data point, 50-100 embryos were microinjected and the cross-sectional areas of 70-2900 NPC-stained nuclei were measured, averaged, and normalized to the water injected control (bold horizontal line set at 1.0). On the x-axis, the concentrations of ectopically expressed lamin proteins were estimated from infrared fluorescence western blots by comparing the amounts of GFP- or mCherry-tagged lamins in extracts from microinjected embryos to known amounts of recombinant lamin proteins loaded on the same gel (data not shown). The error bars are SEM. All normalized nuclear area data points greater than 1.05 and less than 0.98 are statistically different from the control (1.0) by at least p<0.05.
	FIGURE 6. Lamin levels affect nuclear size in mammalian tissue culture cells. A) MRC-5 cells were transiently transfected with plasmids expressing GFP-NLS, GFP-LA, GFP-LB1, or mCherry-LB2. Two days after transfection, cells were transferred to coverslips. After another day, cells were fixed and stained with Hoechst to visualize the DNA (blue). Representative images are shown. B) Nuclear cross-sectional areas were quantified from the red or green channel, depending on the fluorescent label, for transfected MRC-5 cells. For each data point, the cross-sectional areas of 133-608 nuclei were measured and averaged. Error bars are SD. From left to right, the number of nuclei quantified/p-values are: 608/not applicable, 133/10-31, 149/10-54, 215/10-62, 403/not applicable, 163/0.007, 204/10-9, 224/10-50. C) HeLa cells stably expressing GFP-histone H2B were transiently transfected with plasmids expressing GFP- NLS, GFP-LA, GFP-LB1, or mCherry-LB2. Because of the presence of GFP-H2B, all of these plasmids except for mCherry-LB2 were co-transfected with an mCherry plasmid, in order to clearly identify ransfected cells. One day after transfection, cells were transferred to coverslips. After another day, cells were fixed and stained with Hoechst to visualize the DNA (blue). Representative images are shown. For the GFP-LA and GFP-LB1 images, the lamin signal was much stronger than the histone signal, still a weak GFP-H2B signal is visible for all nuclei. Short white arrowheads denote nuclei with intranuclear lamin puncta and/or abnormal nuclear morphology. D) Nuclear cross-sectional areas were quantified from the red or green channel, depending on the fluorescent label, for transfected HeLa cells. For each data point, the cross-sectional areas of 178-2948 nuclei were measured and averaged. Error bars are SD. From left to right, the number of nuclei quantified/p-values are: 2948/not applicable, 195/0.03, 285/10-36, 291/10-38, 178/not applicable, 192/0.13, 356/10-6, 178/10-4, 1006/not applicable, 219/10-30, 362/10-38, 728/10-65. To quantitatively evaluate the level of lamin overexpression, we performed western blots on lysates of transfected HeLa cells (data not shown). Blots were probed with antibodies against LB1, LB2, or LA, as described under Experimental Procedures. To quantify the relative amount of ectopic lamin expression, intensity of the GFP- or mCherry-tagged lamin band was divided by the intensity of the endogenous lamin band. The amount of ectopic lamin expression for Experiments 1, 2, and 3, respectively, was 56%, 27%, 63% for LB1; 35%, 23%, 78% for LB2; 20%, 10%, 20% for LA. For Experiment 4, HeLa cells were transiently transfected with plasmids expressing LA, LB1, or LB2 and co- transfected with an mCherry plasmid to allow for identification of transfected cells. Nuclear cross- sectional areas were quantified from Hoechst-stained cells. E) HeLa cells were transiently transfected with siRNA against the indicated lamin or a control siRNA with no target in the human genome. Cells were co-transfected with an mCherry plasmid to allow for identification of transfected cells. One day after transfection, cells were transferred to coverslips. After another day, cells were fixed and stained with Hoechst. Representative images of Hoechst-stained nuclei are shown. White arrows denote nuclei in cells that received the indicated siRNA, as determined by co-transfection with the mCherry plasmid and corresponding mCherry fluorescence (not shown). (F) For each data point, the cross-sectional areas of 105-1418 nuclei in transfected cells were measured and averaged. From left to right, the number of nuclei quantified/p-values are: 549/not applicable, 229/10-13, 445/10-15, 445/10-79, 1418/not applicable, 163/10-13, 251/10-10, 269/10-4, 1085/not applicable, 105/10-54, 160/10-50, 111/10-51. Error bars are SD. * p<0.05, p<0.01, * p<0.001, NS Not significant.
	FIGURE 7. Cumulative Xenopus nuclear size data plotted as a function of total lamin concentration. Blue diamonds represent all in vitro data from experiments with egg extracts (Fig. 1C, 2B, 3B), red squares represent all in vitro and in vivo data from experiments with early stage embryos (Fig 4C, 5A), and green triangles represent all in vivo data from stage 11-12 embryos (Fig. 5C). The x- axis is total lamin concentration estimated by summing the ectopic lamin concentration (either added recombinant protein or protein expressed from microinjected mRNA) plus the endogenous lamin concentration for that stage of development.

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