Protein farnesylation inhibitors cause donut-shaped cell nuclei attributable to a centrosome separation defect

Abstract

Despite the success of protein farnesyltransferase inhibitors (FTIs) in the treatment of certain malignancies, their mode of action is incompletely understood. Dissecting the molecular pathways affected by FTIs is important, particularly because this group of drugs is now being tested for the treatment of Hutchinson–Gilford progeria syndrome. In the current study, we show that FTI treatment causes a centrosome separation defect, leading to the formation of donut-shaped nuclei in nontransformed cell lines, tumor cell lines, and tissues of FTI-treated mice. Donut-shaped nuclei arise during chromatin decondensation in late mitosis; subsequently, cells with donut-shaped nuclei exhibit defects in karyokinesis, develop aneuploidy, and are often binucleated. Binucleated cells proliferate slowly. We identified lamin B1 and proteasome-mediated degradation of pericentrin as critical components in FTI-induced “donut formation” and binucleation. Reducing pericentrin expression or ectopic expression of nonfarnesylated lamin B1 was sufficient to elicit donut formation and binucleated cells, whereas blocking proteasomal degradation eliminated FTI-induced donut formation. Our studies have uncovered an important role of FTIs on centrosome separation and define pericentrin as a (indirect) target of FTIs affecting centrosome position and bipolar spindle formation, likely explaining some of the anticancer effects of these drugs.

Protein farnesylation is a posttranslational modification that facilitates the binding of proteins to membrane surfaces. Protein farnesyltransferase catalyzes the addition of a 15-carbon farnesyl lipid to proteins containing a carboxyl-terminal CaaX motif consisting of a cysteine (C) followed by two aliphatic amino acids (aa) and a terminal amino acid residue (X), which is often alanine, serine, methionine, or glutamine (1). Among the most familiar examples of farnesylated proteins are the Ras family of proteins, which require the farnesyl lipid for anchoring the protein to the plasma membrane and for proper protein function. Because mutations in RAS oncogenes are involved in ~30% of all human cancers and are associated with poor prognosis and treatment outcome (2, 3), protein farnesyltransferase was seen as an attractive target for anticancer drug therapy, prompting the development of many protein farnesyltransferase inhibitors (FTIs). Interestingly, FTIs exhibit significant efficacy in tumor cells in animal models whether or not they have RAS mutations, suggesting that the efficacy of FTIs cannot be attributed solely to their effects on Ras processing and raising the possibility that other mechanisms underlie their anticancer properties. Recently, FTIs have found another potential application in Hutchinson–Gilford progeria syndrome (HGPS). HGPS is a pediatric progeroid disorder caused by a mutant form of prelamin A that (unlike mature lamin A) retains its farnesyl lipid anchor. Based on salutary effects of FTIs on disease phenotypes in mouse models of progeria (4–6), FTIs are now being tested in children with HGPS. Over the past few years, investigators interested in using FTIs for progeria have recorded anecdotal accounts of misshapen cell nuclei when cells are treated with FTIs (7–9). In the current studies, we investigated the effects of FTIs on nuclear shape and structure in more detail.

Results

Inhibiting Protein Farnesylation Causes Donut-Shaped Nuclei in Vivo and in Vitro.

Treatment of primary human skin fibroblasts with an FTI resulted in a high frequency of cells with donut-shaped nuclei (i.e., nuclei with a central hole devoid of chromatin and lined by a nuclear lamina) (Fig. 1). 3D reconstruction based on confocal images confirmed that the central void penetrates the entire nucleus and is continuous with the cytoplasm; mitochondria and cytoskeletal structures freely pass through the donut hole (Fig. 1 A and B and Movie S1). FTI treatment induced donut-shaped nuclei in fibroblasts from healthy controls and in human cervical cancer (HeLa) cells, human epithelial larynx cancer (HEp2) cells, mouse embryonic fibroblasts (MEFs), and mouse vascular smooth muscle cells (Fig. 1C), indicating that the effect does not depend on the expression of a mutant form of lamin A. To determine if the formation of donut-shaped nuclei is a compound-related effect linked to a specific FTI or the direct result of inhibiting protein farnesylation, we evaluated nuclear shape after treatment with (i) different FTIs; (ii) drugs, such as statins and aminobisphosphonates, that inhibit protein prenylation by affecting other enzymatic steps (8, 10, 11); (iii) triple-drug combinations of the aforementioned drugs, which are currently being tested in children with HGPS; and (iv) shRNA-mediated knockdown of the catalytic β-subunit of protein farnesyltransferase (FNTB). The migration of HDJ-2 on SDS/polyacrylamide gels was used to assess the level of enzyme inhibition (4, 5). Importantly, even low levels of inhibition of protein farnesylation resulted in donut-shaped nuclei, regardless of the treatment approach (Fig. 1D and Figs. S1–S3). These data indicate that the reduction in protein farnesylation, and not any compound-related effect, accounts for the nuclear shape abnormality. To determine if an FTI could also elicit donut-shaped cell nuclei in vivo, we treated mice with an FTI for 6–12 mo (6). FTI treatment resulted in donut-shaped cell nuclei in the intestines and the skin, confirming that FTI-induced nuclear shape abnormalities also occur in vivo (Fig. 2).

Fig. 1.

FTI treatment causes donut-shaped nuclei. (A) 3D surface rendering from a confocal image stack of a donut-shaped nucleus (blue) in an FTI-treated skin fibroblast cell revealing mitochondria (green) passing through the donut hole (Movie S1). (Scale bar: 25 μm.) (B) Single confocal sections through the nucleus of FTI-treated primary human skin fibroblasts stained for lamin A (B1), β-tubulin (B2), and DNA (B3), revealing a nuclear lamina lining the donut hole. (Scale bar: 25 μm.) (C) Induction of donut-shaped nuclei in a variety of cell types after treatment with 10 μM FTI for 3 d. (D) Inhibition of protein farnesylation by different doses of FTI L744832 and lovastatin for 3 d led to more donut-shaped nuclei. At low doses (≤1 μM), lovastatin did not elicit donut-shaped nuclei. When primary human skin fibroblasts were treated with 1 μM tipifarnib (R115777, indicated as R) or 1 μM lonafarnib (SCH66336, indicated as S), or with the combination of 1 μM pravastatin (Pra), 1 μM zoledronate (Zoledr), and either 1 μM tipifarnib (R) or 1 μM lonafarnib (S), a similar amount of nonfarnesylated HDJ-2 (nf-HDJ2) was observed (Lower). A larger number of donut-shaped nuclei were observed in tipifarnib-treated cells than in lonafarnib-treated cells. Combining pravastatin and zoledronate with tipifarnib or lonafarnib led to an increased number of donut-shaped nuclei. More detail is provided in Fig. S3F. *P < 0.05; ***P < 0.001.

Fig. 2.

FTI treatment causes donut-shaped nuclei in vivo. Donut-shaped nuclei are demonstrated in tissue specimens derived from gut (A, H&E) also containing blood vessels (B, stained for lamin A/C) of WT mice (A) and LMNA G608G mice expressing human progerin (B) treated with 2.25 mg of FTI per day for a minimum of 6 mo. (Inset) Close-up of the donut-shaped nucleus in the dashed rectangle. Arrow indicates donut-hole; arrowhead indicates nuclear lamina surrounding donut-hole. (Scale bar: 10 μm.) (C) Large intestine of FTI-treated WT and LMNA G608G mice had significantly increased numbers of donut-shaped nuclei compared with nontreated littermate mice. **P < 0.005; ***P < 0.001.

Donut-Shaped Nuclei Form upon Mitosis.

To investigate the mechanism of nuclear donut formation, we conducted time-lapse video microscopy of human skin fibroblasts stably expressing GFP–lamin A and HEp2 cells stably expressing GFP–histone-3. We observed donut-shaped cell nuclei as early as 90 min after initiating FTI treatment (Fig. 3 A and B and Movie S2), indicating that short periods of enzyme inhibition are sufficient to induce donut formation. Time-lapse series revealed that donut-shaped nuclei arose during chromatin decondensation in late mitosis (Fig. 3 A and B), remained stable for >12 h, and persisted for several days after removing the FTI (Fig. S3D). Blocking mitosis by serum starvation (Fig. 3C) or nocodazole eliminated FTI-induced formation of donut-shaped nuclei, indicating that this abnormality depends on mitosis. When released from the mitotic block, cells in FTI-containing medium quickly acquired donut-shaped cell nuclei.

Fig. 3.

Donut-shaped nuclei form during mitosis attributable to a centrosome separation defect. Time-lapse video microscopy of FTI-treated human skin fibroblasts stably expressing GFP–lamin A (A; Movie S2) and HEp2 cells stably expressing GFP–histone-3 (B) showing donut-shaped nuclei arising during mitosis. (Scale bar: 10 μm.) Arrows indicate donut holes. The asterisk indicates a micronucleus attributable to incomplete chromatin segregation. (C) Serum starvation prohibited mitosis and abolished FTI-induced donut formation. (D) 3D surface rendering of a confocal image stack through an FTI-treated skin fibroblast labeled for β-tubulin (green), DNA (blue), and pericentrin (red), revealing the presence of a thick bundle of microtubules passing through the donut holes and multiple small spots of pericentrin close to and within the donut hole (Movie S3). (E) Measurements showing defective centrosome separation at metaphase and anaphase in FTI-treated HEp2 cells. (F) About 60% of FTI-treated cells had centrosomes facing the equatorial plane (inside). (G) HEp2 cells stably expressing GFP–histone-3 and labeled for pericentrin (red) in late anaphase showing centrosomes on the far side (outside) of evolving daughter nuclei in vehicle-treated cells and on the inside in FTI-treated cells. Note the centrosome in evolving donut holes in FTI-treated HEp2 (G3) and skin fibroblasts labeled for β-tubulin (green) and pericentrin (red) (G4). (H) Presence of rosette-like chromatin distribution (i.e., metaphase ring) instead of normal chromatin alignment at the equatorial plane in FTI-treated HEp2 cells. (I) Treatment of skin fibroblasts with 100 μM monastrol for 20 h increased donut-shaped nuclei. (J) 3D surface rendering of an FTI-treated skin fibroblast cell line at metaphase, revealing rosette-like chromatin distribution (blue) and a monopolar spindle with microtubules (β-tubulin, green) arising from one centrosome (pericentrin, red, arrow). Skin fibroblasts treated with 10 μM FTI for 3 d (K) or 100 μM monastrol for 20 h (L) labeled for pericentrin (red), β-tubulin (green), and DNA (blue) showing the mitotic spindle arising from the center of the rosette-like chromatin ring. (Scale bar: 10 μm.) ***P < 0.001.

Donut-Shaped Nuclei Form Because of a Defect in Centrosome Separation.

The fact that donut-shaped nuclei form during chromatin decondensation in late mitosis suggests that defects in centrosome separation and organization during mitosis might underlie this morphological abnormality. This idea is further supported by the frequent finding of thick bundles of microtubules running through donut holes and (multiple) small centrosomes positioned in or near the donut hole at the end of mitosis in FTI-treated fibroblasts as well as the finding that donut-shaped nuclei were often located in binucleated cells (Fig. 3D and Movie S3). To test the hypothesis that a defect in centrosome separation could elicit donut formation by forcing chromatin to decondense around a centrally located mitotic spindle (Fig. 3G3), we measured the distance between opposing centrosomes in 2D projections of cells expressing GFP–histone-3 and stained for pericentrin, a centrosomal scaffold protein. FTI-treated cells with chromatin aligned in the equatorial plane at metaphase exhibited reduced distances between centrosomes compared with vehicle-treated control cells (Fig. 3E). In addition, ~60% of FTI-treated cells had centrosomes positioned near the equatorial plane between separating daughter nuclei in late mitosis, whereas this feature was never observed in vehicle-treated cells, which always had centrosomes located on the distal sides of the separating nuclei (Fig. 3 F, G1, and G2). FTI-treated cells often (>30%) had another hallmark of incomplete centrosome separation—a rosette-like chromatin distribution at metaphase (Fig. 3 H, J, and K). 3D reconstruction of representative FTI-treated human skin fibroblasts cells at metaphase revealed monopolar spindles with microtubules arising predominantly from one centrosome and a rosette-like chromatin distribution (Fig. 3J). To determine if defective centrosome separation is sufficient to elicit donut formation, we treated cells with monastrol, which inhibits kinesin Eg5, a motor protein required for spindle bipolarity (12). Monastrol resulted in monopolar spindles and led to significantly more donut-shaped nuclei (Fig. 3 I and L), confirming that defective centrosome separation is important for donut formation.

FTI-Mediated Reduction in Pericentrin Causes Donut Formation and Binucleation.

Skin fibroblasts from patients with loss-of-function mutations in the pericentrin (PCNT) gene have spindle defects and binucleation (13), resembling findings in FTI-treated cells. A similar loss of bipolar spindle formation is observed in cells injected with antipericentrin antibodies (14), suggesting a possible role for pericentrin in FTI-induced donut formation and binucleation. Supporting this idea, we found that FTI-treated human skin fibroblasts had reduced levels of the 378-kDa and 250-kDa pericentrin isoforms (Fig. 4 A–C). Furthermore, FTI-treated skin fibroblasts often showed multiple, small, pericentrin-positive spots in late anaphase, suggesting impaired centrosome stability (Fig. S4). Targeting pericentrin by RNAi showed that reduced levels of pericentrin were sufficient to elicit donut-shaped nuclei and binucleation (Fig. 4 D–F), albeit to a lower extent than with FTI treatment. To investigate whether reduced levels of pericentrin were attributable to changes in expression levels or protein stability/degradation, we investigated pericentrin expression after FTI treatment by real-time PCR. The level of pericentrin mRNA in FTI-treated cells was indistinguishable from that of vehicle-treated control cells, suggesting that increased turnover of pericentrin protein could explain the findings during FTI treatment. Of note, blocking proteasome-mediated degradation prevented the FTI-induced loss of pericentrin and significantly reduced the frequency of donut-shaped cell nuclei (Fig. 4G).

Fig. 4.

FTI-induced loss of pericentrin causes formation of donut-shaped nuclei and binucleation. (A) Reduced expression of the large (378 kDa) and small (250 kDa) pericentrin (PCNT) isoforms in FTI-treated primary human skin fibroblasts. MW, molecular weight. Quantification of 378-kDa (B) and 250-kDa (C) PCNT expression levels normalized to β-tubulin and compared with primary skin fibroblasts treated with vehicle alone. (D) RNA interference directed against PCNT resulted in reduced levels of the 378-kDa PCNT product in HeLa cells. Knockdown of PCNT significantly increased binucleation (E) and donut formation (F). (G) Blocking proteasome-mediated degradation reduced donut formation in FTI-treated primary skin fibroblasts. PI, protease inhibitor. (H) Donut formation in MEFs lacking lamin B1 (Lmnb1^Δ/Δ) or lamin B2 (Lmnb2^−/−) and WT controls. (I) Formation of donut-shaped nuclei in HeLa cells after RNAi directed against lamin B1, indicating that lamin B1 is required for FTI-induced donut formation. (J) Percentage of cells with donut-shaped nuclei after expression of a nonfarnesylated version of lamin B1 (LaB1-SAIM) or WT lamin B1. Expression of LaB1-SAIM and, to a lesser extent, lamin B1 increased donut-shaped nuclei even without FTI treatment. (K) Localization of GFP-labeled WT lamin B1 (Upper) or LaB1-SAIM (Lower) in late mitosis showing that nonfarnesylated lamin B1 is mislocalized to the nucleoplasm (more detail is provided in Fig. S6). (Scale bar: 10 μm.) *P < 0.05; **P < 0.005; ***P < 0.001.

Because pericentrin is not a prenylated protein, and therefore not directly affected by FTIs, we investigated other centrosome-associated proteins that could mediate pericentrin stability. Centromere protein (CENP)-F and CENP-E are the only known farnesylated proteins involved in chromatin alignment and segregation (15, 16). Also, in interphase, CENP-F directly interacts with Hook2 (17), a centrosomal protein responsible for the organization of the microtubule network at the centrosome. Transfecting cells with plasmids encoding nonfarnesylated versions of CENP-E and CENP-F was insufficient to induce donut-shaped nuclei; however, those experiments were hampered by difficulties in expressing such large proteins and by the reduced proliferative capacity of transfected cells (18). Interestingly, MEFs from CENP-F–null mice have lower levels of pericentrin, suggesting a role for CENP-F in regulating intracellular pericentrin levels (Fig. S5 A and B). Because FTI treatment had no effect on CENP-F localization and CENP-F was correctly localized to the kinetochore during mitosis (19) (Fig. S5C), we speculate that CENP-F–mediated modulation of pericentrin levels occurs during interphase and that interactions between farnesylated CENP-F and pericentrin at the centrosome protect pericentrin from proteasomal degradation.

Lamin B1 Contributes to FTI-Mediated Donut Formation.

Because lamin B1, a nuclear envelope protein involved in spindle formation during mitosis (20, 21), is a farnesylated protein, we investigated its role in FTI-induced donut formation. WT lamin B1 localized to the nuclear rim in nuclei with decondensing chromatin during late mitosis, but a nonfarnesylated version of lamin B1 (i.e., a mutant lamin B1 terminating in SAIM) was found exclusively in the nucleoplasm of evolving daughter cells during late mitosis, the same stage at which donut-shaped nuclei emerge (Fig. 4K and Fig. S6). Importantly, transient expression of lamin B1-SAIM was sufficient to induce formation of donut-shaped nuclei in the absence of an FTI (Fig. 4J). Also, lamin B1-deficient MEFs (Lmnb1^Δ/Δ) and cells in which lamin B1 expression was knocked down by RNAi did not develop donut-shaped nuclei in the presence of an FTI (Fig. 4 H and I), providing further support for a role of lamin B1 in the formation of donut-shaped nuclei. In contrast, MEFs lacking lamin B2 (Lmnb2^−/−), MEFs lacking exclusively lamin A [“lamin C-only cells” (22)], or MEFs expressing a nonfarnesylated version of prelamin A (23) formed donut-shaped nuclei when incubated with an FTI (Fig. 4H and Fig. S7). Thus, formation of donut-shaped cell nuclei requires lamin B1 but not the other lamins.

Donut-Shaped Nuclei Are Mostly Found in Binucleated Cells.

FTI treatment increased the number of binucleated cells, with most of them also having donut-shaped nuclei (Fig. 5 A and B). In fact, >70% of donut-shaped nuclei were found in binucleated cells (Fig. 5C). Bayesian analysis revealed that the probability of observing donut-shaped nuclei in binucleated cells was ~3.5 times higher than would be predicted for independent events, indicating that the formation of binucleated cells and donut-shaped nuclei is correlated (Fig. 5 D and E and SI Materials and Methods). To assess the functional consequences of donut-shaped nuclei and increased binuclear cells, we followed the fate of these cells for up to 4 d (Fig. 5 F and G). Binucleated cells had a lower propensity to divide, whereas mononucleated cells with donut-shaped nuclei divided at the same rate as cells with normally shaped nuclei (Fig. 5G).

Fig. 5.

FTI treatment results in donut-shaped nuclei and binucleated cells that divide abnormally. FTI-treated primary human skin fibroblasts showing donut-shaped nuclei in binucleated cells in phase-contrast images (A) and in single confocal sections after staining for lamin A (B1), vimentin (B2), and DNA (B3, Hoechst). (B4) Merged image. (Scale bar: 10 μm.) (C) Distribution of donut-shaped nuclei between mononucleated and binucleated cells. (D) FTI treatment caused increased numbers of binucleated cells in two primary human skin fibroblasts (Fibro) lines and in HEp2 cells. (E) Large fraction of binucleated cells contained donut-shaped nuclei. (F) Photoconversion of Dendra2–histone-4 from green to red to track individual nuclei over time. (G) Follow-up study of cell fate for 4 d in FTI-treated skin fibroblasts after photoconversion of mononucleated and binucleated cells with and without donut-shaped nuclei showing a lower probability of cell division in binucleated cells compared with mononucleated cells, regardless of the presence of donut-shaped nuclei. (H and I) Time-lapse video microscopy studies of HEp2 cells showing that ~50% of cells with donut-shaped nuclei divide abnormally, often yielding new donut-shaped nuclei (I, Upper; 55%) or an abnormal number of daughter nuclei (I, Lower; 21%). Arrows indicate daughter nuclei. (Scale bar: 10 μm.) (J) Single confocal sections showing defective karyokinesis in an FTI-treated skin fibroblast after labeling for lamin A (J1), vimentin (J2), and DNA (J3, Hoechst). (Scale bar: 25 μm.) (K) Inactive X-chromosomes in female FTI-treated human skin fibroblasts labeled for the histone macroH2A that preferentially binds to the inactive X-chromosome (arrows). (Scale bar: 10 μm.) (L) Quantification of primary female human skin fibroblasts containing more than one inactive X-chromosome in response to FTI treatment, revealing higher levels of aneuploidy in cells with donut-shaped nuclei. Costaining of macroH2A with Ki67 was used to identify noncycling cells. *P < 0.05; ***P < 0.001.

Donut-Shaped Nuclei Divide Abnormally and Exhibit Aneuploidy.

Time-lapse video microscopy revealed that ~50% of cells with donut-shaped nuclei divided abnormally (Fig. 5H), with most of the abnormal divisions yielding cells with donut-shaped nuclei or multiple nuclei (Fig. 5I and Fig. S8A). Aside from defects in cytokinesis, FTI treatment also interfered with karyokinesis (Fig. 5J, Fig. S8E, and Movie S4). Consequently, donut-shaped nuclei exhibited more aneuploidy than cells with normal cell nuclei (Fig. 5 K and L). Interestingly, despite the dramatic effects of FTI treatment on nuclear shape, we did not observe increased apoptosis in FTI-treated primary skin fibroblasts. Conversely, the nuclei of FTI-treated HEK293 cells exhibited DNA fragmentation.

Discussion

Despite well-documented antiproliferative and proapoptotic effects of FTIs on cancer cells (24), their mode of antitumor activity has remained elusive, in part because of the large number (>100) of protein substrates for protein farnesyltransferase (25, 26). Induction of apoptosis and loss of bipolar spindle formation with arrest of cells in G2-M phase with a rosette-like chromatin distribution have been reported previously in the setting of FTI treatment (24, 27, 28), without any mechanism. Our studies confirm that FTI treatment causes loss of bipolar spindle formation but show that different cell types, including HeLa and HEp2 cancer cells, go on to develop donut-shaped nuclei and binucleated cells. Donut-shaped nuclei could also be detected in vivo in tissues of FTI-treated mice. Interestingly, the PKCβII-selective inhibitor βIIV5-3, a drug used for treating prostate cancer, acts partly by reducing pericentrin levels (29). Hence, reducing pericentrin expression could constitute one mechanism for the antiproliferative effects of FTIs. Alternatively, spindle defects could arise from FTI-mediated loss of farnesyltransferase binding to tubulin (30). Importantly, cells with donut-shaped nuclei did not exhibit apoptosis or senescence and continued to divide, but with a large fraction dividing abnormally and becoming aneuploid.

We identified lower pericentrin levels and lamin B1 as critical for FTI-induced donut formation and binucleation. The precise mechanism by which FTIs reduce pericentrin levels remains to be identified, opening an attractive avenue for future investigation. An earlier study (31) ascribed proteasomal inhibitory activity to FTIs in a breast cancer cell line; however, here, we report data suggesting that FTIs increase proteasomal degradation of pericentrin, in keeping with earlier reports of increased protein degradation in FTI-treated cells (8, 32). Blocking proteasome-mediated degradation prevented the FTI-induced loss of pericentrin and prevented the formation of donut-shaped cell nuclei (Fig. 4G).

Following an ongoing clinical trial in Europe that uses a statin and an aminobisphosphonate, which target different steps of prenylation (8, 11, 33), a triple-drug regimen of an FTI, a statin, and an aminobisphosphonate is currently being tested in the United States as a treatment for progeria. For that reason, we examined the effects of lonafarnib, pravastatin, and zoledronate on donut formation in human skin fibroblasts. We found that the triple-drug regimen induced more donut-shaped nuclei than the FTI alone. Interestingly, low levels of lovastatin [10 nM, similar to plasma levels achieved in patients being treated for hypercholesterolemia (34)] had no perceptible effect on protein farnesylation and did not yield donut-shaped nuclei. In any case, we believe that physicians treating patients who have progeria with an FTI, or the triple-drug combination regimen, should be cognizant of the effects of FTIs on nuclear morphology and pericentrin levels, particularly because loss of pericentrin has been reported to cause dwarfism (13).

Materials and Methods

Mice and FTI Treatment.

WT mice and mice expressing human progerin from a BAC transgene received a 5-g ball of dough each morning containing 2.25 mg of tipifarnib (R115777; Zarnestra) for up to 12 mo (6). All animal use complied with the Animal Care and Use Committee guidelines under Protocol G-03-05 (National Institutes of Health).

Cell Culture, FTI, Lovastatin, and Monastrol Treatment.

For FTI treatment, cells were treated daily with 10 μM FTI L744832 (Biomol) or an equal volume of vehicle (DMSO; 0.025% final concentration) for at least 48 h in the case of MEFs and for 72 h for other cell lines (35). For lovastatin treatment, cells were treated daily with 10 nM and 1, 4, 10, or 20 μM lovastatin (Calbiochem) or equal volumes of vehicle (DMSO) for 72 h. Cells were pretreated with 10 μg/mL cycloheximide (Sigma) for 3 h and thereafter treated daily with 10 μM FTI and 10 μg/mL cycloheximide for 3 d to determine whether only newly synthesized proteins are affected by FTI treatment. Skin fibroblasts were treated for 20 h with 100 μM monastrol (M8515; Sigma) or vehicle (DMSO), fixed, and evaluated for donut-shaped nuclei. Primary skin fibroblasts were cotreated daily with 10 μM lactacystin (Cayman Chemical), which is a proteasome inhibitor, and 10 μM FTI or vehicle (DMSO) alone for a period of 3 d to evaluate if FTI-mediated protein degradation underlies the formation of donut-shaped nuclei.

Time-Lapse Video Microscopy.

HEp2 cells stably expressing GFP–histone-3 (HEp2-GFP–H3) were transfected with a mCherry–β-tubulin plasmid, and SV40-transformed human skin fibroblasts stably expressing GFP–lamin A were imaged on a temperature-controlled dish holder on a motorized stage of an Olympus IX-70 microscope or confocal Zeiss LSM-710 microscope.

Immunofluorescence Microscopy.

Cells were fixed, processed, and incubated with primary antibodies against lamin A/C, CENP-E, CENP-F, vimentin, pericentrin, prelamin A, β-tubulin, macroH2A (generously provided by P. Adams, University of Edinburgh, Glasgow, United Kingdom), or Mitotracker (Invitrogen). They were then counterstained with Hoechst 33342.

Immunohistochemistry.

Tissue sections of gut and skin derived from FTI-treated and nontreated WT mice and LMNA G608G mice were processed for H&E staining or immunostaining with a lamin A/C antibody.

Western Blot Analyses.

Western blot analyses are discussed in SI Materials and Methods.

RNAi Against FNTB, Pericentrin, and Lamin B1.

HeLa cells and SV40-transformed human skin fibroblasts were transfected with shRNA plasmids to reduce expression of FNTB, pericentrin, and lamin B1. The knockdown of each gene was verified by Western blot analysis and real-time PCR assay.

Constructs specifying nonfarnesylated versions of lamin B1, CENP-E, and CENP-F are discussed in SI Materials and Methods.

Photoconversion of Histone 4–Dendra2.

Primary skin fibroblasts were electroporated with a plasmid for Dendra2-tagged histone-4, plated, treated daily with 10 μM FTI for 3 d, and then photoconverted from green to red as described in SI Materials and Methods. Data were acquired in four independent experiments, and at least 60 cells were evaluated for each category.

Statistical Analysis.

Experiments were performed at least three independent times. Images were blinded to the person performing the analysis and randomized before the evaluation. Statistical analyses were performed with PRISM 3.0 and INSTAT software (GraphPad) by an unpaired Student's t test with Welch's correction to allow for different variances. To compare donut formation (a binomial event) between different conditions, results were analyzed by a χ² distribution and Fisher's exact test. For these results, data were expressed as the sample proportion ± SE. All other data were expressed as mean ± SEM. For all experiments, a two-tailed P value ≤0.05 was considered significant.

Image Acquisition and Manipulation.

Phase-contrast and fluorescence images were acquired with a CoolSNAP HQ digital CCD-camera (Photometrics, Roper Scientific) mounted on an Olympus IX70 inverted microscope and controlled by IPLab 4.0 (BD Biosciences) image acquisition software. Experiments to count the fraction of donuts, as well as time-lapse video microscopy experiments, were imaged with an Olympus LCPlanF 20× phase-contrast objective (0.40 N.A.). Immunofluorescence samples were imaged with an Olympus UApo/340 40× water immersion objective (1.15 N.A.). For analysis of Western blots, X-ray films were digitized on an Epson Perfection 2450 scanner with linear intensity settings. Digital images were processed with Adobe Photoshop (version 6.0; Adobe Systems, Inc.) by adjusting the linear image intensity display range, and fluorescence gray-scale images were colorized in ImageJ (National Institutes of Health), Adobe Photoshop, or IPLab (BD Biosciences) 4.0 by selecting a color plane appropriate for the chromophore.

A more detailed description of experimental protocols can be found in SI Materials and Methods.