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How nuclear size is regulated is a fundamental cell-biological question with relevance to cancers, which often exhibit enlarged nuclei. We previously reported that conventional protein kinase C (cPKC) contributes to nuclear size reductions that occur during early Xenopus development. Here we report that PKC-mediated phosphorylation of lamin B3 (LB3) contributes to this mechanism of nuclear size regulation. By mapping PKC phosphorylation sites on LB3 and testing the effects of phosphomutants in Xenopus laevis embryos, we identify the novel site S267 as being an important determinant of nuclear size. Furthermore, FRAP studies demonstrate that phosphorylation at this site increases lamina dynamics, providing a mechanistic explanation for how PKC activity influences nuclear size. We subsequently map this X. laevis LB3 phosphorylation site to a conserved site in mammalian lamin A (LA), S268. Manipulating PKC activity in cultured mammalian cells alters nuclear size, as does expression of LA-S268 phosphomutants. Taken together, these data demonstrate that PKC-mediated lamin phosphorylation is a conserved mechanism of nuclear size regulation.
Nuclear size varies in different cell types and during cell differentiation and development (Conklin, 1912 ; Wilson, 1925 ; Edens et al., 2013 ; Borsos and Torres-Padilla, 2016 ), and nuclear size is often inappropriately enlarged in cancer (Zink et al., 2004 ; Jevtic and Levy, 2014 ). It is largely unknown whether nuclear size directly affects cell function, motivating our studies on mechanisms of nuclear size regulation. Early Xenopus laevis development is a robust system in which to study nuclear size, as the early embryo undergoes a series of rapid cell divisions accompanied by reductions in both cell and nuclear size. After 12 cleavage cell cycles, the embryo reaches the midblastula transition (MBT), or stage 8, at which time zygotic transcription is up-regulated and cell divisions slow and become asynchronous (Nieuwkoop and Faber, 1967 ; Newport and Kirschner, 1982 ). From fertilization to the MBT, average nuclear volume decreases approximately fivefold, driven at least in part by reduced nuclear import kinetics and levels of cytoplasmic importin α. Nuclear size continues to scale smaller in post-MBT embryos, with a greater-than-threefold reduction in nuclear volume between stages 8 and 12. Furthermore, whereas pre-MBT nuclei continuously expand during brief 15- to 20-min interphase periods, nuclei in post-MBT embryos reach a steady-state size (Gerhart, 1980 ; Levy and Heald, 2010 ; Jevtic and Levy, 2015 ).
We previously reported an in vitro nuclear resizing assay in which large nuclei assembled de novo in X. laevis egg extract become smaller when incubated in cytoplasm isolated from stage 12 post-MBT X. laevis embryos (i.e., late-embryo extract). We showed that this nuclear shrinking depends on conventional protein kinase C (cPKC), a kinase family that includes PKC α and β and depends on diacyglycerol and calcium for activity (Newton, 2003 ). Furthermore, we showed that developmental reductions in nuclear size correlate with increased cPKC nuclear localization and activity, and manipulating cPKC activity in live X. laevis embryos led to concomitant changes in nuclear size within interphase. Taken together, these results implicated cPKC as a key regulator of nuclear size in post-MBT embryos (Edens and Levy, 2014a , 2016 ). An important question resulting from this earlier work was the identity of the cPKC substrates that control nuclear size.
Nuclear lamins—intermediate filament proteins that form a meshwork on the nucleoplasmic face of the nuclear envelope (NE)—are known PKC substrates (Simon and Wilson, 2013 ). During open mitosis, NE disassembly requires dissolution of the nuclear lamina, mediated by lamin phosphorylation by PKC and cyclin-dependent kinases (Heald and McKeon, 1990 ; Mall et al., 2012 ). Compared to global NE PKC activity at mitosis, localized PKC-mediated phosphorylation of the nuclear lamina is required for nuclear egress of certain viruses and large messenger ribonucleoprotein (mRNP) particles (Muranyi et al., 2002 ; Reynolds et al., 2004 ; Park and Baines, 2006 ; Speese et al., 2012 ; Hatch and Hetzer, 2014 ). In our nuclear shrinking assay, we observed PKC-dependent NE removal of lamin B3 (LB3), the primary lamin isoform found in X. laevis eggs and early embryos (Stick and Hausen, 1985 ), prompting us to test the hypothesis that nuclear size is regulated by direct cPKC-mediated phosphorylation of nuclear lamins.
In this study, we identify a novel cPKC phosphorylation site in LB3 that influences both nuclear lamina dynamics and nuclear size in X. laevis embryos and extracts. Furthermore, we show that cPKC activity affects nuclear size in several cultured mammalian cell lines. Finally, we demonstrate that the phosphorylation site identified in Xenopus LB3 is conserved in human lamin A (LA) and that phosphorylation at this site influences the association of LA with the NE and nuclear size. We propose that cPKC-mediated phosphorylation of nuclear lamins represents a conserved mechanism of nuclear size regulation by which lamin association with the NE is decreased by interphase phosphorylation, resulting in concomitant reductions in nuclear size.
We previously demonstrated that nuclei assembled in X. laevis egg extract become smaller when incubated in cytoplasm isolated from stage 12 embryos (Figure 1A). This PKC-dependent nuclear shrinking is accompanied by an approximately fivefold increase in cPKC nuclear staining (Edens and Levy, 2014a ). To determine whether PKC is acting within the nucleus to affect nuclear size, we performed the nuclear shrinking assay in the presence of wheat germ agglutinin (WGA), which binds glycosylated FG-nucleoporins at the nuclear pore complex (NPC), thereby blocking nucleocytoplasmic transport. In the presence of WGA, nuclei failed to shrink and nuclear cPKC staining was significantly reduced (Supplemental Figure S1), indicating that the target of cPKC is intranuclear. Having previously demonstrated that nuclear shrinking is accompanied by PKC-dependent removal of nuclear lamins (Edens and Levy, 2014a ), we next tested whether PKC directly phosphorylates nuclear lamins to effect changes in nuclear size.
We incubated recombinant green fluorescent protein (GFP)-LB3 with purified cPKC protein or late-embryo extract and resolved the reisolated GFP-LB3 on a gel that maximizes phosphorylation-induced band shifts. A fraction of both the late embryo extract–treated and cPKC-treated GFP–LB3 protein exhibited reduced gel mobility (Figure 1B and Supplemental Figure S2A). To identify sites directly phosphorylated by cPKC, we subjected the LB3 samples treated with recombinant cPKC protein to phosphorylation-site mapping by mass spectrometry. Although several putative phosphorylation sites were identified (Figure 1C and Supplemental Figure S2B), two sites had the highest Ascores and could be unequivocally assigned (Beausoleil et al., 2006 ), representing likely authentic PKC phosphorylation sites. We were particularly interested in these two sites because they appeared to be novel, having not been previously identified in studies of mitotic lamin PKC phosphorylation (Simon and Wilson, 2013 ), with Thr-223 found in the second linker region and Ser-267 found in the 2B coil-coiled region (Figure 1D).
To test whether these phosphorylation sites influence nuclear size, we generated LB3 phospho-null alanine mutants. Single-cell X. laevis embryos were comicroinjected with morpholinos to reduce endogenous LB3 levels (Supplemental Figure S3, A–D) and mRNA encoding GFP-LB3 wild-type (wt), LB3-S267A, or LB3-T223A and allowed to develop to stage 12 (Figure 1E). We previously demonstrated that the GFP tag does not interfere with the nuclear localization of LB3 or its concentration-dependent effect on nuclear size (Jevtic et al., 2015 ). Embryos expressing the LB3-S267A mutant exhibited significantly larger nuclei than the LB3-wt– or LB3-T223A–microinjected embryos (Figure 1F), and no developmental defects were observed. Measurements of nuclear GFP-LB3 intensity showed that LB3-wt, LB3-S267A, and LB3-T223A were present at similar levels within nuclei (Supplemental Figure S3E), demonstrating that the nuclear import efficiency of the phospho-null mutants was not affected. We next treated the LB3-wt– and LB3-S267A–microinjected embryos with phorbol 12-myristate 13-acetate (PMA), a PKC activator. PMA treatment reduced nuclear size in LB3-wt–microinjected embryos to a greater extent than in embryos expressing LB3-S267A (Figure 1G), supporting the idea that S267 is normally phosphorylated by PKC. The fact that PMA still reduced nuclear size, albeit to a lesser extent, in LB3-S267A–expressing embryos might be explained by incomplete morpholino knockdown of endogenous LB3 and/or the presence of additional LB3 PKC phosphorylation sites or PKC substrates relevant to nuclear size control. Consistent with the phosphorylation status of S267 influencing nuclear size, microinjection of the phosphomimetic mutant GFP-LB3-S267E resulted in smaller nuclei (Figure 1H). Taken together, these data indicate that PKC-mediated phosphorylation of LB3 at S267 contributes to the regulation of nuclear size during early X. laevis development.
Having demonstrated that phosphorylation of LB3 by cPKC influences nuclear size, we next asked whether the mechanism of action was by altering the dynamic association of LB3 with the nuclear lamina. We assembled nuclei in X. laevis egg extract supplemented with GFP-LB3 and resuspended isolated nuclei in late-embryo extract. To quantify GFP-LB3 dynamics, we performed confocal fluorescence recovery after photobleaching (FRAP) on the surface of nuclei and quantified the recovery of GFP-LB3 signal into the bleached spot (Figure 2, A and B, and Supplemental Videos S1–S3). We chose to monitor LB3 dynamics in Xenopus extract rather than in embryos because time-lapse imaging in stage 12 embryos is complicated by rapid cell and nuclear movements and small nuclear size (Edens and Levy, 2014a ). Consistent with previous nuclear lamin FRAP studies (Moir et al., 2000 ; Zaremba-Czogalla et al., 2012 ; Buxboim et al., 2014 ; Kochin et al., 2014 ), we observed that the association of LB3 with the NE was not very dynamic, exhibiting a mobile fraction of 16%. Nonetheless, inhibiting PKC activity with the small molecule chelerythrine significantly reduced the LB3 mobile fraction to <10%, comparable to that observed when nuclei were treated with heat-inactivated late-embryo extract (Figure 2C). It was also evident that spreading of photobleached GFP-LB3 throughout the NE was greatest for nuclei treated with late-embryo extract, consistent with LB3 being more dynamic in the presence of active PKC (Figure 2A).
We also examined the dynamics of the LB3-S267 phosphomutant proteins using the same FRAP assay. We found that LB3-S267A exhibited reduced mobility relative to wt (Figure 2D), consistent with phosphorylation at this site being important for increased LB3 mobility. The LB3-S267E mutant exhibited a similar mobile fraction to wt, suggesting that a significant fraction of LB3-wt molecules are normally phosphorylated at this site. Whereas PKC inhibition with chelerythrine reduced LB3-wt mobility to a nominal level, LB3-S267E mobility was unaffected by PKC inhibition (Figure 2D), strongly indicating that S267 phosphorylation is a primary determinant of PKC-dependent LB3 mobility. These data support a model by which PKC-dependent LB3-S267 phosphorylation modulates nuclear lamina dynamics, thereby affecting nuclear size.
To determine whether cPKC-dependent nuclear size regulation is conserved outside of Xenopus, we investigated three cultured mammalian cell lines: HeLa (human cervical cancer cells), Ptk2 (rat-kangaroo epithelial kidney cells), and MRC-5 (human fetal lung fibroblasts). Nearly confluent cell monolayers were treated for 90 min with a PKC activator (PMA) or a cPKC inhibitor (Gö 6976). Consistent with our Xenopus findings, PMA treatment resulted in significantly smaller nuclei in all three cell lines. However, PKC inhibition increased nuclear size only in HeLa cells, perhaps because these cells are cancer derived and more amenable to nuclear size alterations (Figure 3A). The fact that we observed nuclear size changes after brief treatments with PMA or Gö 6976 indicates that these changes occurred within interphase and were not caused by postmitotic effects on nuclear size.
To explore further the effect of specific cPKC isoforms on nuclear size, we performed transient transfections with plasmids expressing constitutively active forms of PKC α and β (Supplemental Figure S4A). Expression of constitutively active PKC βII significantly reduced cross-sectional nuclear area in HeLa, Ptk2, and MRC-5 cells by up to 25% (Figure 3B). Conversely, knocking down PKC β levels by transient small interfering RNA (siRNA) transfection (Supplemental Figure S4B) increased cross-sectional nuclear area by up to 22% (Figure 3C). These results demonstrate that PKC β levels and activity influence nuclear size in mammalian cells to a greater extent than PKC α, consistent with Xenopus studies (Edens and Levy, 2014a ). Taken together, these data suggest that cPKC represents a conserved mechanism of nuclear size regulation.
Having demonstrated that cPKC influences nuclear size in mammalian cells, we next sought to determine whether the S267 phosphorylation site identified in Xenopus LB3 was conserved in mammalian lamins. Sequence alignments indicated that, although not found in B-type lamins, a conserved serine residue is present at S268 in lamin A (LA; Figure 4A). In addition, recombinant LA treated with cPKC or late-embryo extract exhibited a gel shift similar to that observed with LB3, indicating that LA is a substrate for PKC phosphorylation (Figure 4B). Note that A-type lamins are generally phosphorylated by PKC to mediate nuclear egress of viruses and large mRNP particles (Muranyi et al., 2002 ; Reynolds et al., 2004 ; Speese et al., 2012 ).
To test the functional significance of the conserved serine residue in human LA, we generated LA phosphomutants. HeLa cells were transiently transfected with plasmids expressing GFP-LA-wt, LA-S268A, or LA-S268E (Supplemental Figure S4, C and D). The LA mutants exhibited striking differences in their nuclear distribution patterns (Figure 4C) regardless of whether endogenous LA levels were knocked down by siRNA (unpublished data). We observed enhanced NE localization of LA-S268A and the accumulation of LA-S268A plaques lining the NE. GFP-LA intensity line scans through representative nuclei (Figure 4D) revealed a nearly twofold increase in the GFP-LA NE peak widths for LA-S268A compared with wt (Figure 4E), suggesting a greater accumulation of the phospho-null LA mutant at the NE. In contrast, LA-S268E exhibited primarily nucleoplasmic puncta, with very little localization to the NE rim (Figure 4, C and D).
We previously reported that ectopic GFP-LA expression increases nuclear size in both HeLa and MRC-5 cells and that LA lacking the GFP tag has a similar effect (Jevtic et al., 2015 ). In HeLa cells transiently cotransfected with siRNA against endogenous LA and plasmids expressing GFP-LA, we again noted a positive correlation between nuclear size and the level of LA expression, as quantified by nuclear GFP-LA intensity (Figure 5, A and B). Linear regression analysis suggested that LA-S268A expression increased nuclear size relative to LA-wt (Figure 5B), consistent with our Xenopus LB3-S267A findings (Figure 1F). To evaluate these data further and normalize for LA expression levels, we divided nuclei into bins based on their GFP-LA intensity. Across all but the highest GFP-LA–intensity bins, LA-S268A expression resulted in larger nuclei compared with LA-wt. The effect of LA-S268E on nuclear size varied, generally exhibiting nuclear sizes intermediate between those of LA-wt and LA-S268A (Figure 5C). When nuclear sizes were averaged across multiple experiments irrespective of LA expression levels, both LA-S268A and LA-S268E expression resulted in nuclear cross-sectional areas ~20% larger than wt (Figure 5D). Of importance, the magnitude of nuclear size changes reported here is potentially physiologically relevant, falling within the clinical range of nuclear size increases in cancer cells (Wang et al., 1992 ; Mossbacher et al., 1996 ; Abdalla et al., 2009 ; Vukovic et al., 2016 ), as well as being sufficient to alter developmental timing in Xenopus embryos (Jevtic and Levy, 2015 ). Whereas PMA treatment reduced nuclear size in cells expressing LA-wt, nuclear size was refractory to PMA in cells expressing LA-S268A or LA-S268E (Supplemental Figure S4E), supporting the idea that S268 is a key PKC target in nuclear size regulation.
Although we have not demonstrated that S268 of LA is directly phosphorylated by PKC, this supposition is supported by 1) all of our experiments with the conserved serine residue in Xenopus lamin B3 (Figures 1 and and2),2), 2) observed effects of LA-S268 mutations on LA localization and nuclear size (Figures 4 and and5),5), and 3) our finding that activating PKC with PMA fails to reduce nuclear size in cells expressing LA-S268 mutants (Supplemental Figure S4E). Whereas our human LA and Xenopus LB3 results generally agreed with each other, one discrepancy concerned the phosphomimetic mutant—namely, LB3-S267E decreased nuclear size in Xenopus embryos, whereas LA-S268E increased HeLa nuclear size. This difference could be due to 1) the punctate nuclear localization of LA-S268E in HeLa cells potentially affecting mechanical properties of the nucleus (Lammerding et al., 2006 ; Denais and Lammerding, 2014 ; Gruenbaum and Foisner, 2015 ; Zwerger et al., 2015 ), 2) functional and structural differences between LB3 and LA, and/or 3) confounding effects of different B-type lamins being present in HeLa cells, whereas only one primary lamin protein is found in early Xenopus embryos (i.e., LB3). Note that at the highest LA expression levels, LA-S268E nuclei were smaller than wt (Figure 5C). Nonetheless, the novel lamin point mutants we describe here are, to the best of our knowledge, among the most precise and specific ways to manipulate nuclear size in cells and embryos. In the future, we anticipate that these mutants will be important tools in dissecting the functional significance of nuclear size with respect to chromatin organization and gene expression.
We show that PKC-mediated lamin phosphorylation is a conserved mechanism of nuclear size regulation. We propose that steady-state nuclear size is determined by a balance between nuclear import–mediated nuclear growth (Levy and Heald, 2010 ) and modulation of nuclear lamina dynamics by PKC activity and localization. The new results presented here have potentially important disease implications. An Emery–Dreifuss muscular dystrophy patient was identified with a LA-S268P mutation (Scharner et al., 2011 ), and mutations in the adjacent amino acid, LA-Y267, are known to be pathogenic (Vytopil et al., 2003 ; Carboni et al., 2008 ). Cancer cells are often identified by their increased nuclear size (Zink et al., 2004 ; Jevtic and Levy, 2014 ), and carcinogenesis is frequently associated with prognostic changes in lamin expression levels (Foster et al., 2010 ; Chow et al., 2012 ) and PKC signaling (Lonne et al., 2010 ; Mochly-Rosen et al., 2012 ; Urtreger et al., 2012 ). In addition, altered PKC signaling and nuclear size have both been proposed as priming events in carcinogenesis (Prevostel et al., 1997 ; Gokmen-Polar et al., 2001 ; Chai and Brown, 2009 ). It is intriguing to speculate that the two might be causally related and that modulating the levels or activity of PKC and lamins might restore a normal nuclear size in some cancers, potentially abrogating features of the disease.
Chelerythrine chloride (C2932; Sigma-Aldrich), Gö 6976 (G1171; Sigma-Aldrich), and WGA (L9640; Sigma-Aldrich) were reconstituted in water. PMA (P8139; Sigma-Aldrich) was reconstituted in dimethyl sulfoxide (DMSO). For in vitro Xenopus experiments, chelerythrine was used at 0.4 mM. For Xenopus embryo and mammalian cell culture experiments, PMA was used at 6 nM and Gö 6976 at 2 µM.
We used mammalian expression vectors (pHACE) expressing constitutively active human PKC α (21233; Addgene) and mouse PKC βII (16383; Addgene). In each case, the N-terminal 29 amino acids encompassing the pseudosubstrate domain had been deleted (dNPS), giving rise to constitutive activity (Soh and Weinstein, 2003 ). Recombinant GFP-LB3 protein was expressed from GFP-LB3 pDEST17 (pDL5; Levy and Heald, 2010 ). Site-directed mutagenesis of pDL5 was performed to generate the S267A mutant (pDL58), T223A mutant (pDL60), and S267E mutant (pDL85). GFP-LB3 mRNA was synthesized from plasmid pCS2+ GFP-LB3 (pDL19; Levy and Heald, 2010 ). GFP-LB3-S267A was cloned from pDL58 into pCS2+ at BamHI/XhoI (pDL71). GFP-LB3-T223A was cloned from pDL60 into pCS2+ at BamHI/XhoI (pDL73). GFP-LB3-S267E was cloned from pDL85 into pCS2+ at BamHI/XhoI (pDL86). Transfection of cultured mammalian cells was performed with GFP-lamin A pcDNA3.1 (pDL74; Jevtic et al., 2015 ). Site-directed mutagenesis of pDL74 was performed to generate the S268A mutant (pDL83) and S268E mutant (pDL84). The H2B-mCherry mammalian expression vector was obtained from Anne Schlaitz (Zentrum für Molekulare Biologie der Universität Heidelberg).
To knock down expression of PKC, the following siRNAs were used: PKC ζ (SASI_Hs01_00116520; Sigma-Aldrich), PKC α (SASI_Hs01_00018815; Sigma-Aldrich), PKC β (SASI_Hs01_00071183; Sigma-Aldrich), and universal negative control (SIC001; Sigma-Aldrich). For LA knockdown, we used a DsiRNA (IDT): sense, 5′-GACUCCGAGCAGUCUCUGUCCUUCGAC-3′, and antisense, 5′-GUCGAAGGACAGAGACUGCUCGGAGUC-3′. This DsiRNA sequence is based on a previously described shRNA that targets the 5′ untranslated region (UTR) of the LA transcript (Lund et al., 2013 ) and therefore does not target GFP-LA expressed from plasmids pDL74, pDL83, and pDL84.
All Xenopus procedures and studies were conducted in compliance with the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals. Protocols were approved by the University of Wyoming Institutional Animal Care and Use Committee (Assurance #A-3216-01). The following procedures have been previously described (Edens and Levy, 2014a , 2016 ). X. laevis egg extracts and sperm nuclei were used to assemble nuclei in vitro. Reactions were supplemented with ~28 nM recombinant wt or mutant GFP-LB3 protein 45 min after initiating nuclear assembly. Egg extract nuclei were isolated and resuspended in late-embryo extract to induce nuclear shrinking. For immunofluorescence, nuclei in egg or embryo extract were fixed, spun onto coverslips, and processed for immunofluorescence. A primary rabbit antibody that recognizes phospho–PKC α/β (bs-3333R; Bioss USA) was used at a dilution of 1:100. For LB3 staining, we used a previously described rabbit LB3 antibody at 1:500 (Levy and Heald, 2010 ). For NPC staining, we used mAb414 (Covance) at 1:1000. Secondary antibodies were Alexa Fluor 488 or 568 anti-mouse or anti-rabbit immunoglobulin G (Molecular Probes) used at 1:1000.
In vitro phosphorylation of GFP-LB3 and GFP-LA was performed with the Promega PepTag Non-Radioactive PKC Assay kit (V5330). Reactions included 0.2 µM recombinant GFP-LB3 or GFP-LA, 10 ng of recombinant PKC α/β, phosphatidylserine micelles, reaction buffer, and protease inhibitors in a final volume of 25 µl. In some cases where indicated, recombinant GFP-LB3 or GFP-LA was treated with 25 µl of late-embryo extract rather than recombinant PKC. Reactions were incubated at 30°C for 30 min before isolating GFP-LB3 or GFP-LA using Ni–nitrilotriacetic acid (NTA) resin (Qiagen) under denaturing conditions. Protein samples were supplemented with SDS–PAGE sample buffer, boiled for 5 min, and separated on a 10% SuperSep Phos-tag gel (Wako). For mapping of phosphorylation sites, the cPKC reaction was scaled up fourfold to 100 µl, gels were stained with Coomassie, and the lower-mobility GFP-LB3 band resulting from phosphorylation was excised. Western blots were performed as previously described (Edens and Levy, 2014a ; Jevtic et al., 2015 ) with the following antibodies: rabbit anti-GFP antibody (A-6455; Invitrogen) used at 1:2000, mouse anti-actin antibody (AM1965b; Abgent) used at 1:200, rabbit pan-PKC antibody (sc-10800; Santa Cruz Biotechnology) used at 1:1000, and mouse Ran antibody used as previously described (Levy and Heald, 2010 ). Xenopus embryo or HeLa cell extracts were probed by Western blot using a rabbit antibody that specifically recognizes LB3 (R12-2960; Assay Biotech) at 1:500 or a mouse antibody against lamin A (MCA-4C4; EnCor Biotechnology) at 1:10,000, respectively. We verified that these lamin antibodies were unique for a given lamin type by testing them against recombinant lamin proteins (Jevtic et al., 2015 ). Secondary antibodies included a 1:15,000 dilution of goat anti-rabbit horseradish peroxidase (HRP; ImmunoPure Antibody 31460; Thermo Scientific) and 1:20,000 dilution of IRDye 800CW anti-rabbit (926-32211; Li-Cor) or IRDye 680RD anti-mouse (925-68070; Li-Cor). Detection was performed as previously described for HRP (Edens and Levy, 2014a ) or infrared fluorescence (Jevtic et al., 2015 ). Band quantification was performed with ImageStudio.
Phosphorylation-site mapping was performed by the Taplin Mass Spectrometry Facility (Cell Biology Department, Harvard Medical School, Boston, MA). Excised gel bands were cut into ~1 mm3 pieces. The samples were reduced with 1 mM dithiothreitol for 30 min at 60°C and then alkylated with 5 mM iodoacetamide for 15 min in the dark at room temperature. Gel pieces were then subjected to a modified in-gel trypsin digestion procedure (Shevchenko et al., 1996 ). Gel pieces were washed and dehydrated with acetonitrile for 10 min, followed by removal of acetonitrile. Pieces were then completely dried in a SpeedVac. Rehydration of the gel pieces was with 50 mM ammonium bicarbonate solution containing 12.5 ng/µl modified sequencing-grade trypsin (Promega, Madison, WI) at 4°C. Samples were then placed in a 37°C room overnight. Peptides were later extracted by removing the ammonium bicarbonate solution, followed by one wash with a solution containing 50% acetonitrile and 1% formic acid. The extracts were then dried in a SpeedVac (~1 h) and stored at 4°C until analysis.
On the day of analysis, the samples were reconstituted in 5–10 µl of HPLC solvent A (2.5% acetonitrile, 0.1% formic acid). A nanoscale reverse-phase HPLC capillary column was created by packing 2.6-µm C18 spherical silica beads into a fused silica capillary (100-µm inner diameter by ~30-cm length) with a flame-drawn tip (Peng and Gygi, 2001 ). After equilibrating the column, each sample was loaded via a Famos autosampler (LC Packings, San Francisco, CA) onto the column. A gradient was formed, and peptides were eluted with increasing concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid). As each peptide was eluted, it was subjected to electrospray ionization and then entered into an LTQ Orbitrap Velos Pro ion-trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Eluting peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein or translated nucleotide databases with the acquired fragmentation pattern by the software program Sequest (ThermoFinnigan, San Jose, CA; Eng et al., 1994 ). The modification of 79.9663 mass units to serine, threonine, and tyrosine was included in the database searches to determine phosphopeptides. Phosphorylation assignments were determined by the Ascore algorithm (Beausoleil et al., 2006 ). All databases include a reversed version of all of the sequences, and the data were filtered to between a 1 and 2% peptide false discovery rate.
X. laevis embryos were generated, dejellied, and microinjected as previously described (Jevtic and Levy, 2015 ). One-cell embryos were comicroinjected with a 10-nl mixture of 1 mM LB3 morpholino oligonucleotides (Gene Tools) and 50 ng/µl GFP-LB3 mRNA using a Picospritzer III and allowed to develop to stage 12. A mixture of two morpholinos was used to target both LB3 isoforms: 5′-GGCCATAGTTTCCCTGTGAGTCCA-3′ and 5′-CGTAGCCATATTGTCTCTGAGTCCC-3′. These oligos target the 5′ UTR of endogenous LB3 transcripts and not the microinjected GFP-LB3 mRNA. In some experiments, a standard negative control morpholino oligo was used: 5′-CCTCTTACCTCAGTTACAATTTATA-3′. To generate mRNA for microinjections, plasmids pDL19, pDL71, pDL73, and pDL86 were linearized with NotI, and mRNA was expressed from the SP6 promoter using the mMessage mMachine kit (Ambion). Embryo extract was prepared from 15–50 embryos, and nuclei were fixed, spun onto coverslips, and stained with Hoechst as previously described (Edens and Levy, 2014a ). GFP-LB3 fluorescence was used to quantify nuclear size.
Wide-field microscopy was performed with an Olympus BX51 fluorescence microscope using an Olympus UPlanFl 20×/0.50 Ph1 objective. Images were acquired with a QIClick Digital CCD Camera, Mono, 12-bit (model QIClick-F-M-12), at room temperature using Olympus cellSens software. Images for measuring fluorescence intensity were acquired using the same exposure times. Total fluorescence intensity and cross-sectional nuclear area were measured from the original thresholded images using MetaMorph software (Molecular Devices). For publication, images were cropped using ImageJ but were otherwise unaltered. We previously demonstrated that cross-sectional nuclear area serves as an accurate proxy for total nuclear surface area and volume (Edens and Levy, 2014a ; Jevtic and Levy, 2015 ).
Where indicated, confocal imaging was performed on a spinning-disk confocal microscope based on an Olympus IX71 microscope stand equipped with a five-line LMM5 laser launch (Spectral Applied Research) and switchable two-fiber output to facilitate imaging through either a Yokogawa CSU-X1 spinning-disk head or total internal reflection fluorescence illuminator. Confocal images were acquired with an electron-multiplying charge-coupled device camera (ImagEM; Hamamatsu) using an Olympus APO N 60× O/1.49 numerical aperture objective. Z-axis focus was controlled using a Piezo Pi-Foc (Physik Instrumente), and multiposition imaging was achieved using a motorized Ludl stage. Image acquisition and all system components were controlled using MetaMorph software, and images for measuring fluorescence intensity were acquired using the same exposure times.
Nuclei were assembled in X. laevis egg extract at room temperature. At 30–40 min after initiating nuclear assembly, reactions were supplemented with ~28 nM GFP-LB3. After an additional 60-min incubation, nuclei were isolated and resuspended in late-embryo extract as depicted in Figure 1A. Where indicated, we used heat-inactivated late-embryo extract treated at 56°C for 30 min or late-embryo extract supplemented with ~0.4 mM chelerythrine to inhibit PKC activity. Specific details of this nuclear shrinking assay are given elsewhere (Edens and Levy, 2014b , 2016 ). Reactions were supplemented with an oxygen-scavenging mix to minimize photodamage (Aitken et al., 2008 ) and sealed between a slide and coverslip using a Pap pen. Imaging of GFP-LB3 incorporated into nuclei was accomplished by spinning-disk confocal microscopy using an Olympus UPlanApo 60×/1.20W objective. The surface of the NE juxtaposed to the coverslip was brought into focus, and a 3.6-µm-radius circular spot on the surface of the nucleus was photobleached using an iLas2 system (BioVision Technologies) with a 54-ms pulse of a 405-nm laser set at 30% power. After photobleaching, GFP-LB3 FRAP was detected by imaging every 5 s for a total of 120 s. For each FRAP time-lapse experiment, ImageJ was used to measure mean GFP-LB3 fluorescence intensity of the photobleached region, entire nucleus, and a background region at each time point. These data were analyzed using the easyFRAP application (Rapsomaniki et al., 2012 ), and subjected to double normalization. Prebleach intensities were normalized to 100%.
Cell lines used were MRC-5 normal human lung fibroblasts, HeLa cells with or without stable H2B-GFP expression, and Ptk2 rat-kangaroo epithelial kidney cells. All cell lines were cultured at 37°C with 5% CO2 using DMEM for MRC-5 and HeLa-H2B-GFP cell lines, Eagle’s minimum essential medium for the unlabeled HeLa cell line, and RPMI for Ptk2 cells. All media were supplemented with 10% fetal bovine serum and 50 IU/ml penicillin and streptomycin. For transient transfections, cells were grown in 24-well plates to 70–90% confluency. Transient plasmid transfections were performed with Lipofectamine 3000 (Invitrogen). Briefly, in one tube, 1.5 µg of DNA was added to 25 µl of Opti-MEM medium plus 3 µl of P3000 reagent. In a separate tube, 1.5 µl of Lipofectamine 3000 reagent was added to 25 µl of Opti-MEM media. The two were mixed and incubated at room temperature for 5 min. Medium was removed from the 24-well plates and replaced with 500 µl/well of fresh culture medium and 50 µl of DNA–Lipofectamine complex. At 6 h (HeLa), 12 h (Ptk2), or 48 h (MRC-5) after transfection, cells were seeded onto acid-washed 18-mm square coverslips in 35-mm2 dishes with 2 ml of fresh culture medium. At 24 h later, coverslips were washed with phosphate-buffered saline (without Mg2+ and Ca2+), fixed with 4% paraformaldehyde for 20 min, washed, stained with 5 μg/ml Hoechst for 5 min, washed, mounted in Vectashield (Vector Laboratories), and sealed with nail polish. Transient siRNA transfections were performed with Lipofectamine RNAiMAX (Invitrogen). Briefly, 3 µl of 10 µM siRNA was diluted in 50 µl of Opti-MEM medium, 3 µl of Lipofectamine RNAiMAX reagent was diluted in 50 µl of Opti-MEM, and the two were mixed and incubated at room temperature for 5 min. The 50-µl transfection mixture was added to 500 µl of culture medium and incubated for 1 d (HeLa) or 3 d (MRC-5). Where indicated and to identify transfected cells, cotransfections were performed with 1.5 µg of H2B-mCherry plasmid. Cells were then seeded onto coverslips, cultured an additional 24 h, and fixed and processed as described for plasmid transfections. For small-molecule treatment, cells were seeded onto acid-washed 18-mm-square coverslips in 35-mm2 dishes with 2 ml of fresh culture medium. After 24 h, the medium was supplemented with PMA (6 nM) or Gö 6976 (2 μM). After a 90-min treatment, cells were fixed and processed as described for plasmid transfections. Images were acquired and nuclear sizes quantified as described under Microscopy and image quantification.
Unless otherwise noted, nuclear cross-sectional areas were measured from thresholded images in MetaMorph (Molecular Devices). For each coverslip, at least 105, and usually >310, nuclei were quantified, and areas were averaged. Unless otherwise noted, nuclear area measurements were normalized to controls. Averaging and statistical analysis were performed for independently repeated experiments. Two-tailed Student’s t tests assuming equal variances were performed in Excel (Microsoft) to evaluate statistical significance. The p values, number of independent experiments, and error bars are given in the figure legends.
We thank members of the Levy lab, as well as our colleagues in the Department of Molecular Biology, for helpful advice and discussions; Jay Gatlin, Jason Gigley, and Anya Lyuksyutova for cultured mammalian cell lines; and Karen White for constructive comments on the manuscript. This work was supported by the National Institutes of Health/National Institute of General Medical Sciences (R01GM113028) and the American Cancer Society (RSG-15-035-01-DDC).
This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E16-11-0786) on March 29, 2017.