Dynamics of lamin-A processing following precursor accumulation.
ABSTRACT Lamin A (LaA) is a component of the nuclear lamina, an intermediate filament meshwork that underlies the inner nuclear membrane (INM) of the nuclear envelope (NE). Newly synthesized prelamin A (PreA) undergoes extensive processing involving C-terminal farnesylation followed by proteolysis yielding non-farnesylated mature lamin A. Different inhibitors of these processing events are currently used therapeutically. Hutchinson-Gilford Progeria Syndrome (HGPS) is most commonly caused by mutations leading to an accumulation of a farnesylated LaA isoform, prompting a clinical trial using farnesyltransferase inhibitors (FTI) to reduce this modification. At therapeutic levels, HIV protease inhibitors (PI) can unexpectedly inhibit the final processing step in PreA maturation. We have examined the dynamics of LaA processing and associated cellular effects during PI or FTI treatment and following inhibitor washout. While PI reversibility was rapid, with respect to both LaA maturation and associated cellular phenotype, recovery from FTI treatment was more gradual. FTI reversibility is influenced by both cell type and rate of proliferation. These results suggest a less static lamin network than has previously been observed.
- SourceAvailable from: Brian Burke[show abstract] [hide abstract]
ABSTRACT: A group of human diseases, known as 'laminopathies', are associated with defects in proteins of the nuclear envelope. Most laminopathy mutations have been mapped to the A-type lamin gene, which is expressed in most adult cell types. So, why should different mutations in a near-ubiquitously expressed gene be associated with various discrete tissue-restricted diseases? Attempts to resolve this paradox are uncovering new molecular interactions #151; both inside the nucleus and at its periphery -- which indicate that the nuclear envelope has functions that go beyond mere housekeeping.Nature Reviews Molecular Cell Biology 09/2002; 3(8):575-85. · 37.16 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: We have determined the structural organization of the human gene that encodes nuclear lamins A and C, intermediate filament proteins of the nuclear lamina. Sequencing and restriction mapping show that the coding region spans approximately 24 kilobases. The 5'-proximal promoter region contains several GC-rich stretches, a CCAAT box, and a TATA-like element of sequence TATTA. The lamin A/C gene contains 12 exons. Alternative splicing within exon 10 gives rise to two different mRNAs that code for pre-lamin A and lamin C. Consequently, two proteins are generated, only one of which, pre-lamin A, can be modified by isoprenylation. The intron positions in the human lamin A/C gene are generally conserved in the previously characterized genes for Xenopus lamin LIII and mouse lamin B2, but different from those in a Drosophila lamin gene. In the regions coding for the central rod domains, the intron positions are also conserved when compared with the intron positions in the genes for most cytoplasmic intermediate filament proteins except those for nestin and neurofilaments. Analysis of the intron positions in these genes supports the hypothesis that the nuclear lamins and other intermediate filament proteins arose from a common ancestor.Journal of Biological Chemistry 09/1993; 268(22):16321-6. · 4.65 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The nuclear lamina is a protein meshwork associated with the nucleoplasmic surface of the inner nuclear membrane, that is suggested to be important for organizing nuclear envelope and interphase chromosome architecture. To investigate the structural organization of the lamina, we have analysed rat liver nuclear envelopes by various chemical extraction procedures. From these studies, we have defined conditions that yield a nuclear envelope subfraction that is both highly enriched in the lamina and devoid of pore complexes. This fraction contains mostly lamins A, B and C, the three major lamina polypeptides that are apparently arranged in a polymeric assembly. Our chemical extraction studies also indicate that lamin B has a stronger interaction with nuclear membranes than the other two lamins, and support the possibility that lamin B is important for attaching the lamina to the inner nuclear membrane. We have examined the synthesis and assembly of the lamins during interphase in tissue-culture cells to investigate lamina structure by a second approach. We found that all three lamins are synthesized at similar rates throughout the cell cycle in synchronized Chinese hamster ovary cells, and that their biosynthesis is not temporally coupled to DNA replication. Our studies indicate that newly synthesized lamins are rapidly assembled into an insoluble lamina structure but that the apparent half-time for lamina insertion differs for individual lamins. We have also observed that lamin A is synthesized as an apparent precursor molecule that is converted to mature lamin A only after integration into the lamina structure. The lamina is reversibly depolymerized during cell division, a process that may be mediated by enzymic phosphorylation of the lamins. To investigate this possibility further, we have analysed charge-altering modifications of the lamins on two-dimensional gels, and have found that phosphorylation is the only detectable modification of these proteins that occurs specifically during mitosis. Furthermore, we have determined that when the lamins are disassembled during metaphase, each lamin has approximately 2 moles of associated phosphate/mole lamin, a value that is four to sevenfold higher than the average interphase level. Considering this information, we discuss a model by which depolymerization and reassembly of the lamina can regulate the reversible disassembly of the nuclear envelope during mitosis.Journal of cell science. Supplement 02/1984; 1:137-60.
Dynamics of Lamin-A Processing Following Precursor
Qian Liu1,2, Dae In Kim2, Janet Syme2, Phyllis LuValle2, Brian Burke2,3, Kyle J. Roux2*
1Department of Histology and Embryology, Shandong University School of Medicine, Jinan, Shandong, China, 2Department of Anatomy and Cell Biology, University of
Florida, Gainesville, Florida, United States of America, 3Institute of Medical Biology, Immunos, Singapore, Singapore
Lamin A (LaA) is a component of the nuclear lamina, an intermediate filament meshwork that underlies the inner nuclear
membrane (INM) of the nuclear envelope (NE). Newly synthesized prelamin A (PreA) undergoes extensive processing
involving C-terminal farnesylation followed by proteolysis yielding non-farnesylated mature lamin A. Different inhibitors of
these processing events are currently used therapeutically. Hutchinson-Gilford Progeria Syndrome (HGPS) is most
commonly caused by mutations leading to an accumulation of a farnesylated LaA isoform, prompting a clinical trial using
farnesyltransferase inhibitors (FTI) to reduce this modification. At therapeutic levels, HIV protease inhibitors (PI) can
unexpectedly inhibit the final processing step in PreA maturation. We have examined the dynamics of LaA processing and
associated cellular effects during PI or FTI treatment and following inhibitor washout. While PI reversibility was rapid, with
respect to both LaA maturation and associated cellular phenotype, recovery from FTI treatment was more gradual. FTI
reversibility is influenced by both cell type and rate of proliferation. These results suggest a less static lamin network than
has previously been observed.
Citation: Liu Q, Kim DI, Syme J, LuValle P, Burke B, et al. (2010) Dynamics of Lamin-A Processing Following Precursor Accumulation. PLoS ONE 5(5): e10874.
Editor: Peter Sommer, Institut Pasteur Korea, Republic of Korea
Received February 2, 2010; Accepted May 6, 2010; Published May 28, 2010
Copyright: ? 2010 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Bankhead-Coley New Investigator Research (NIR) 08BN-05 to K.J.R. (http://www.floridabiomed.com/funding_bc.html) and
National Institutes of Health (NIH) RO1 5R01GM084085-02 to B.B. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
The nuclear lamina is an intermediate filament meshwork
composed of A- and B-type lamins. In mammalian somatic cells
the A-type lamins are represented by lamins A and C (LaA/C),
which arise through alternative splicing of the LMNA gene. Several
diseases are associated with mutations in LMNA, including
progeria, lipodystrophy, muscular dystrophy and peripheral
neuropathy . LaA and LaC differ only by virtue of unique C-
terminal extensions, 96 residues for LaA and six for LaC .
Unlike LaC, LaA undergoes multistep posttranslational processing
at its C-terminal CaaX motif involving 3–4 enzymes. Initially a
farnesyl moiety is added to the cysteine by farnesyltransferase,
followed by cleavage of the last three amino acids (-aaXing) by
either Rce1 or ZmpSte24. This C-terminal cysteine is carbox-
(ICMT) to generate farnesylated/carboxymethylated-PreA. With-
in approximately 90 min of synthesis , ZmpSte24 cleaves PreA
[4,5,6] 14 amino acids upstream of the C-terminus to generate
mature LaA [7,8]. PreA is the only known substrate of ZmpSte24
Defective LaA processing has been implicated in both familial
and acquired forms of lipodystrophy. Mutations in LMNA that
alter certain charged residues on the surface of the Ig-fold region
common to both LaA and LaC are associated with Dunnigan-type
familial partial lipodystrophy (FPLD) [10,11]. FPLD patients
exhibit peri-pubertal onset of subcutaneous fat loss from the
extremities and trunk, hypercholesterolemia and type-II diabetes
[12,13]. There are reports of PreA accumulation in FPLD-patient
fibroblasts through an unknown mechanism [14,15]. PreA
accumulation is also observed in cells from HIV-infected patients
with acquired lipodystrophy [15,16]. This is likely related to
certain HIV PIs used in highly active antiretroviral therapy
(HAART) that inhibit the activity of ZmpSte24 [17,18,19].
Conclusive evidence for an involvement of PreA in HAART-
associated lipodystrophy has yet to be presented.
Incomplete LaA processing is also associated with the rare
premature aging disorder, HGPS, in which patients begin to
exhibit a phenocopy of premature ageing around 1–2 years and
die of cardiovascular-related illness by around 13 years of age
[20,21,22]. The most common HGPS mutation (LaA G608G)
generates a cryptic splice site within exon 11 of LMNA resulting in
deletion of 50 amino acid residues within the LaA C-terminus.
This truncated LaA, termed progerin, lacks the second cleavage
site for ZmpSte24, resulting in retention of the farnesylated and
carboxymethylated C-terminal cysteine [23,24,25]. In support of
farnesylated-PreA or -progerin toxicity [9,26] is restrictive
dermopathy (RD), a perinatal lethal disease with progeroid
features in which farnesylated PreA accumulates due to mutations
in ZMPSTE24 or LMNA [26,27]. Although the mechanism of
progerin or farnesylated-PreA toxicity remains unclear, these
observations led to studies that tested the efficacy of FTI in
ameliorating HGPS symptoms by inhibiting farnesylation of LaA
along with the other CaaX-motif proteins (there are ,100
predicted in the human genome). In cell culture and mouse
progeria models, FTIs yielded promising results with improved
PLoS ONE | www.plosone.org1May 2010 | Volume 5 | Issue 5 | e10874
nuclear morphology in vitro [28,29,30,31,32], and weight gain with
increased viability in vivo [33,34,35]. Thus, a clinical trial for
HGPS patients with the FTI, lonafarnib, was rapidly established,
the outcome of which remains unreported .
Despite the clinical use of the LaA processing inhibitors, FTIs
and HIV PIs, there are fundamental deficits in our understanding
of the consequences. After prolonged treatment with either form of
inhibitor, the nuclear lamina will contain considerable levels of
PreA, either farnesylated or non-farnesylated. Would rapid
processing of this PreA occur following inhibitor release, or would
lamina disassembly during cell division be required? This question
has implications in terms of the reversibility of both FTIs and PIs,
which may in turn impact their clinical usage. Such reversibility,
or lack thereof, would also shed new light on lamina dynamics and
interactions within the nuclear lamina. In this study, we have
explored the recovery rates for LaA processing following
accumulation of both farnesylated and non-farnesylated forms of
PreA within the nuclear lamina.
Reagents and Treatment
HIV protease inhibitors were obtained from the National
Institutes of Health AIDS Research and Reference Reagent
Program. Nelfinavir, lopinavir and atazanavir sulfate were
prepared at a stock solution at 20mM in DMSO, indinavir sulfate
at 20mM in water, tipranavir at 20mM in ethyl acetate. FTI-277
(Sigma) was prepared as a stock at 10mM. Lov (Sigma, St. Louis,
MO) was used at 10mM and GGTI-2147 (Calbiochem) at 20mM.
BMS-214662 was a gift from M. Gelb, University of Washington.
Unless otherwise noted, Saos-2 cells were treated for 48 hr at
20mM for HIV PIs, Lov and GGTI-2147, and at 10mM for FTI-
277. Cycloheximide was used at 10mg/ml. Mitomycin C was used
at 10 mg/ml (2 hrs at 37uC). Control cells were incubated with the
vehicle DMSO or ethyl acetate. HGPS fibroblasts were treated
with FTI-277 at a dose of 10mM for 4–9 days.
Saos-2 cells were maintained in 6% CO2 at 37uC in DMEM
(GIBCO BRL) with 10% FBS (Hyclone), and 10% penicillin/
streptomycin (GIBCO BRL). Human G608G HGPS fibroblasts
were obtained from the Coriell Cell Repository (repository
nos. AG01972) and maintained in 6% CO2 at 37uC in DMEM
with 15% FBS, 10% penicillin/streptomycin and 26 concentra-
tion of essential and non-essential amino acids. To washout the
inhibitors, cells were washed with PBS times and culture media
three times. Cells were transfected as described previously .
The following antibodies were used in this study: mouse anti-
LaA/C , anti-Nup153 , anti-HDJ-2 (MS-225, Thermo
Scientific); rabbit anti-Sun-2 , anti-nesprin-3 (gift from A.
Sonnenberg, Netherlands Cancer Institute, Amsterdam, NL), anti-
emerin (gift from G. Morris, Robert Jones and Agnes Hunt
Orthopaedic Hospital, Oswestry, UK), anti-LaA/C (Cell Signal-
ing), anti-LaB1 (ab16048, Abcam), anti-LaA (SC20680) and goat
anti-LaA (SC6214), anti-LaA/C (SC621) (Santa Cruz Biotechnol-
ogy). Secondary antibodies conjugated with AlexaFluor dyes
(Invitrogen) or peroxidase (Biosource International) were used as
previously described .
For immunofluorescence microscopy, cells were grown on glass
coverslips and fixed, permeabilized, immunolabeled and observed
as described previously . Image quantification was performed
using IPLab software and NIH ImageJ Circularity software.
Unpaired t-tests were performed to evaluate the significance of the
Immunoblotting-pulse chase immunoprecipitation
Subconfluent 35-mm dishes of Saos-2 cells, either untreated or
treated with DMSO, Lop, Lov, or FTI-277 for 48 hrs, were
incubated in 90% DMEM with 10% FBS, and 10% L-cysteine
and L-methionine-free media (MP Biolabs) with 25 mCi
Translabel (MP Biolabs). After 18 hrs, Saos-2 cells were washed
once with PBS and refed with DMEM plus 10% FBS with or
without inhibitors. One hr later, Saos-2 cells were washed twice
with PBS and either incubated in DMEM with 10% FBS for an
additional 1–24 hr or lysed immediately. For cell lysis, cells were
washed three times in PBS and incubated in 800ml lysis buffer
(50 mM Tris-HCl, pH 9.0; 500 mM NaCl, 0.4% SDS, 2% TX-
100, 1mM dithiothreitol 10mg/ml in DMSO each of chymostatin,
leupeptin, antipain, and pepstatin) on ice for 5 min. The cells were
then scraped off and sheared 86through a 26-gauge needle. After
centrifugation for 10 min at 10,0006G, the supernatant was
incubated overnight at 4uC with rabbit anti-LaA/C and protein
A-sepharose beads. Beads were washed three times in lysis buffer
and once in wash buffer (50 mM Tris-HCl, pH 7.4; 50 mM
NaCl). Samples were processed for SDS-PAGE and fluorography
. All Densitometry was performed using NIH ImageJ.
To explore PreA processing following treatment of human cells
with HIV protease inhibitors, we first examined their efficacy in
promoting farnesylated PreA accumulation. Saos-2 cells were
treated for 48h with various HIV-PIs and the relative levels of
PreA determined by immunoblot analysis. Lopinavir (Lop) and
nelfinavir were most effective in blocking the PreA maturation,
followed by atazanavir; whereas indinavir and tipranavir had little
effect on PreA accumulation (Figure 1A). As Lop provided the best
balance between cellular toxicity and PreA accumulation, we used
it exclusively for the remainder of our studies.
In order to determine the susceptibility of PreA accumulated at
the NE to proteolytic maturation, Saos-2 cells were treated for
48 hrs with Lop, prior to washout of the drug. Before drug
withdrawal, more than half of the total cellular LaA was detected
as PreA (Figure 1B, upper panel). However, by 3 hrs after Lop
washout ,50% of PreA was processed to maturity and by 5–7 hrs
the levels approached that of the control. Parallel immunofluo-
rescence experiments reveal the accumulation of PreA at the NE
(Figure 1C). Loss of PreA following Lop washout follows a time-
course similar to that observed by western blot. Complementary
pulse-chase experiments were performed on Saos-2 cells labeled
overnight with35S Cys/Met in the presence of Lop (Figure 1B,
lower panel). After labeling, the cells were cells were ‘‘chased’’ for
1h in medium containing both Lop and excess non-radioactive
Cys/Met to ensure newly synthesized labeled lamins were
incorporated into the nuclear lamina. The chase was continued
for 7h in the absence of Lop. Roughly 50% of the accumulated
PreA is processed to the mature form by 3h, and by 7h it is fully
processed (Figure 1B, lower panel). To confirm that our results do
not reflect turnover of accumulated PreA in combination with de
novo synthesis, protein synthesis was inhibited with cyclohexamide
1 hr prior to and during the Lop washout (Figure S1). The rate of
PreA processing appears enhanced in the presence of cyclohex-
imide, most likely due to the elimination of any newly synthesized
PreA as a competitive substrate for ZmpSte24. In normal tissue
Dynamics of Lamin-A Processing
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culture cells, the half-time for newly synthesized PreA processing is
,1.5 hrs . It is remarkable that the recovery half-time from
extended Lop treatment is only ,3h, especially as the bulk of the
total PreA in the Lop-treated cells appears integrated into the
Following extended treatment with PIs, and concomitant with
PreA accumulation, cells acquire irregular nuclear profiles with
quantifiably decreased circularity (Figure 2A–C). Normal nuclear
morphology recovers independent of new protein synthesis over a
period of 7–15h following Lop washout (Figure 2C). To determine
if the effect of Lop on nuclear morphology is LaA-dependent Saos-
2 cells were depleted of LaA and LaC by RNAi in conjunction
with Lop treatment (Figure 2D–E). Cells depleted of LaA and LaC
retained normal nuclear morphology and circularity over the 48h
period of the experiment suggesting that PreA is the mediator of
Lop-induced nuclear dysmorphology. Another consequence of
HIV PI-treatment is the aberrant accumulation of LaA and LaC
in cytoplasmic aggregates in both mitotic and early G1 cells
(Figure 2F–K). In the former, aggregates tend to be adjacent to the
spindle poles. Similar aggregates have been described in cells
expressing progerin [42,43]. We also observed a range of other NE
proteins, including emerin, LaB1 (Figure 2G, J), sun-2 and
nesprin-3 (Figure S2) that were also retained within both the
mitotic and G1 aggregates. After Lop washout the frequency of
these aggregates diminished to control levels over a period of 7–
15h, paralleling the restoration of normal nuclear morphology
(Figure 2H, K).
Considering the half-time of PreA maturation is ,3hrs after
Lop washout, what is the cause of this delay in morphological
recovery? One explanation is that nuclear and/or nuclear
envelope remodeling must take place during the lag period.
Another possibility is that the level of farnesylated-PreA at the time
of washout is far in excess of that required to induce these nuclear
abnormalities. To attempt to resolve this issue, we first determined
the lowest concentration of Lop that after 48h would produce an
accumulation of farnesylated-PreA equivalent to that observed 5–
7h after washout of our normal 20mm Lop (Figure 3). The
concentration of Lop chosen was 5mm. Subsequently we compared
nuclear circularity and appearance of LaA/C aggregates during
metaphase and early G1 from cells treated with 5mM or 20mM
Lop (Figure 3B). 5mM Lop was unable to induce a significant
change in nuclear morphology or to induce LaA/C aggregation.
These data imply that nuclear or nuclear envelope remodeling
likely accounts for the lag period, although a threshold for PreA
accumulation causing nuclear dysmorphology cannot be entirely
discounted. We next determined whether such remodeling might
require cell division. Cells were treated with mitomycin C prior to
Lop washout to prevent cell division. In treated cells, we observed
no difference in the maturation rate of farnesylated PreA
(Figure 3C). However, we did observe a significant delay in
recovery of nuclear circularity (Figure 3D). The suggestion is that
postmitotic nuclear reassembly partially contributes to the
restoration of normal nuclear morphology. However, we consider
it likely that ongoing synthesis of NE components and their
incorporation into interphase nuclei may also contribute to
recovery from Lop treatment.
Our next question was whether an accumulation of lamina-
associated non-farnesylated PreA, such as following long-term FTI
treatment, would be processed to maturity with similar efficiency
to that observed for farnesylated-PreA. We first examined the
efficacy of both lovastatin (Lov) and the farnesyl transferase
inhibitor FTI-277 in accumulating non-farnesylated PreA in Saos-
2 cells. Lov is an inhibitor of the enzyme HMG-CoA reductase
which catalyses the production of mevalonic acid, a key precursor
of isoprenoid synthesis. Treatment with Lov will consequently
block prenylation involving both farnesyl (C15) and geranylgeranyl
Figure 1. HIV PIs block LaA maturation, which is reversed rapidly following PI washout. (A) Blotted with PreA specific and LaA/C
antibodies, extracts from Saos-2 cells treated with vehicle (DMSO), Tip, Nel, Ata, Ind and Lop reveal different levels of PreA accumulation. Percentage
of mature LaA is listed under each lane. (B) Immunoblot of extracts from Saos-2 cells treated with Lop followed by washout indicates that half time of
LaA maturation is approximately 3 hrs. The processing rate of the overnight35S Cys/Met labeled LaA following Lop washout is similar. Percentage of
mature LaA is listed under each lane. (C) Immunofluorescence microscopy of Saos-2 cells double labeled with the antibodies again PreA and Nup153.
PreA is accumulated on the NE after Lop treatment and disappears following Lop washout. All images are taken at the same exposure time. In the
merged images, DNA, revealed by staining with Hoechst dye, is shown in blue. Bar, 15mm.
Dynamics of Lamin-A Processing
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Figure 2. Recovery from aberrant cellular phenotypes is delayed following Lop washout. (A) Immunofluorescence microscopy of Saos-2
cells. Altered interphase nuclear morphology and abnormal accumulation of LaA/C and emerin in the cytoplasm are evident after Lop treatment and
recover within 15 hrs following Lop washout. (B) The aberrant cytoplasmic aggregates after Lop treatment contain LaB1, predominantly colocalized
with LaA/C. (C) Nuclear circularity following Lop washout is significantly altered by PI treatment and following washout either without (grey bars) or
with cycloheximide (white bars) (error bars=SEM; N=.96). (D) Double labeled with antibodies against LaA/C and LaB1, Saos-2 cells exhibit normal
nuclear morphology after Lop treatment following LaA/C RNAi. (E) Nuclear circularity is higher in LaA/C RNAi treated cells (error bars=SEM; N=.65).
(F) Nuclear components such as LaA/C, emerin and LaB1 are diffuse in control cells during metaphase. (G) Lop treatment leads to aberrant
aggregation of LaA/C adjacent to metaphase chromosomes. Emerin and LaB1 are also retained in these aggregates (white arrows). (H) Measurements
of percentage of cells with metaphase aggregates indicate that mitotic abnormality recovers by ,15 hrs after Lop washout (error bars=SEM; N=3,
.75 cells counted each experiment). (I) LaA/C, emerin and LaB1 localize on the NE in early G1. (J) Lop treatment leads to aberrant aggregation of LaA/
C in the cytoplasm in early G1. These aggregates also contain emerin and LaB1 (white arrows). (K) Measurements of percentage of cells with early G1
aggregates indicate that recovery of cytoplasmic abnormality is ,15 hrs after Lop washout (error bars=SEM; N=3, .75 cells counted each
experiment). Bars, 5mm.
Dynamics of Lamin-A Processing
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(C20) modifications. Both inhibitors induced accumulation of non-
farnesylated PreA at the NE of Saos-2 cells treated for 48 hrs
(Figure 4A). Following Lov washout the half-time of mature LaA
recovery was ,7h as determined by both western blot and pulse-
chase analysis (Figure 4B). Similar studies with FTI-277,
employing both western blot and pulse-chase analyses, revealed
a considerably slower restoration of non-farnesylated PreA
processing with a recovery half-time of ,12h (Figure 4C). With
either treatment, inhibition of new protein synthesis with
cyclohexamide added at drug washout accelerated PreA matura-
tion, likely due to reduced competition from newly synthesized
substrates. Analysis of the processing of HDJ-2, another
farnesylated protein, reveals that washout of lovastatin is rapid
whereas the processing of LaA is delayed (Figure 4B). However, in
the case of FTI washout, recovery of HDJ-2 farnesylation is
slowed, but not as markedly so as the maturation of non-
farnesylated pre-LaA (Figure 4C). We repeated the same
experiments with other FTIs such as L-744, 832 and BMS-
214662 and observed the same effect (Figure S3).
Both Lov and FTIs led to significant accumulation of the slower
migrating non-farnesylated-PreA, however lovastatin was consid-
erably more effective than any of the FTIs. Recently it was shown
that in the absence of farnesylation, PreA could be a substrate for
geranylgeranylation . To explore this possibility, Saos-2 cells
were treated with geranylgeranyl transferase inhibitor (GGTI)-
2147 alone or in combination with FTI (Figure 4D). While GGTI-
alone had no effect on LaA maturation, and the FTI alone had
only a partial effect, in combination the GGTI and FTI largely
blocked LaA processing, yielding results similar to those obtained
with lovastatin. These results support the concept that farnesyl-
transferase inhibition induces geranylgeranlyation and maturation
of a substantial fraction of LaA. If the cytotoxic affects of Lop,
including nuclear dysmorphology and the appearance of mitotic
and G1 LaA/C-containing aggregates, can be ascribed to the
accumulation of farnesylated PreA, then inhibition of farnesyl-
transferase should abrogate these effects. To test this idea, Saos-2
cells were treated with Lop, both with and without FTI, for
periods of up to 72h (Figure 5A–B). Lop alone, as expected, caused
Figure 3. Recovery of nuclear morphology following Lop washout is time dependent and enhanced in cycling cells. (A) Probed with
LaA/C antibody, immunoblot of Saos-2 cells treated with increased concentrations of Lop from 0 to 40 mM reveals increased PreA accumulation in
parallel. Percentage of mature LaA is listed under each lane. (B) Measurements of interphase nuclear circularity (error bars=SEM; N=.80),
percentage of cells with metaphase and early G1 aggregates (error bars=SEM; N=3, .75 cells counted each experiment) reveal that the effects of
5mM Lop differs significantly from 20mM Lop, with similar level to vehicle (DMSO) control. (C) Accumulated PreA in Lop treated cells complete its
maturation by 7 hrs following drug washout. Mitomycin C treatment prior to Lop removal does not affect PreA processing rate. Percentage of mature
LaA is listed under each lane. (D) Measurement of nuclear circularity indicates that mitomycin delays recovery of aberrant nuclear shape at 15 hrs
following Lop washout (error bars=SEM; N=.92).
Dynamics of Lamin-A Processing
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nuclear deformation (measured as loss of circularity) and mitotic
and G1 lamin-positive aggregates. Inclusion of FTI partially
suppressed these effects; whereas FTI and GGTI together almost
eliminated Lop-induced nuclear dismorphology and cytoplasmic
lamin aggregates. Subsequent washout of both FTI and GGTI
together or of FTI alone (leaving GGTI in the medium) resulted in
the re-appearance of Lop cytotoxicity over a period of about 24h.
Reappearance of dysmorphology was more rapid when both FTI
and GGTI were washed out together. The implication is that non-
prenylated PreA that has been accumulated over an extended
period can be utilized as a substrate by geranylgeranyl transferase
even in the presence of active farnesyl transferase.
That FTI treatment permits geranylgeranylation of LaA is of
particular significance given that FTI treatment of HGPS patients
is currently the subject of a clinical trial. The effectiveness of this
treatment strategy could be compromised by alternative lamin
prenylation. To begin to address this issue we first established that
FTI treatment indeed restored normal nuclear morphology, as
previously reported [28,29,30,31,32], and eliminated cytoplasmic
lamin-positive aggregates in HGPS dermal fibroblasts (Figure 6A).
Upon washout of the FTI, it took on the order of 48–72h to
reacquire the aberrant HGPS morphology. Western blot analysis
of cells after FTI treatment reveals a partial shift to slower
migrating forms for both progerin and LaA. While progerin does
not undergo ZmpSte24-mediated maturation, it does display a
mobility shift linked to prenylation and -aaXing (Figure 6B, S4A).
FTI washout revealed slow processing of both non-farnesylated
PreA and non-farnesylated progerin over a period of about 30h,
significantly longer than that observed in Saos-2 cells. Similarly,
HDJ-2 required a longer period to mature in HGPS cells (Figure
S4B). Combined treatment of HGPS fibroblasts with both FTI
and GGTI resulted in a more substantial shift of LaA to PreA
(Figure 6B). Albeit less dramatic, an increase in the accumulation
of non-farnesylated progerin was also observed. Thus our data
support the findings that in the absence of farnesylation, both full-
length PreA and, to a lesser extent, progerin can be geranylger-
anylated in HGPS dermal fibroblasts . However, washout of
both FTI and GGTI did not appreciably enhance the rate of
return to the mature forms.
In this study we have evaluated the processability of PreA
localized to the NE following the use of HIV PIs or FTIs.
Significant levels of farnesylated PreA accumulated by ZmpSte24
inhibition are rapidly processed upon enzyme activation, yet
abnormal nuclear phenotypes resulting from this accumulated
PreA are slower to resolve. Recovery occurs more rapidly in
proliferative cells, yet too quickly to require nuclear envelope
breakdown, suggesting a structural reorganization of the lamina
may constantly occur in cycling cells. We observed a much slower
recovery of endogenous LaA maturation following washout of FTI
or Lov than for Lop. This may not be surprising as our readout of
LaA maturation requires only ZmpSte24 cleavage for Lop
inhibition, whereas farnesylation, aaXing, carboxylmethylation
and then ZmpSte24 cleavage all must occur following FTI or Lov
Figure 4. Nonfarnesylated-PreA accumulated at the NE by prenylation inhibitors gradually matures following drug washout. (A)
Immunofluorescence microscopy of Saos-2 cells reveals significant accumulation of nonfarnesylated-PreA on the NE after a treatment of Lov or FTI-
277. DNA, revealed by staining with Hoechst dye, is shown at bottom column. Bar, 5mm (B) Immunoblot of extracts from Saos-2 cells treated with Lov
followed by washout indicates that half-time of PreA maturation is ,7 hrs. Cyclohexamide chase upon release from Lov block enhances maturation.
Accumulations of slower migrating non-farnesylated HDJ-2 regain farnesylation by 2 hrs following Lov washout. Processing rate of overnight35S Cys/
Met labeled LaA following Lov removal is similar to unlabeled total population. (C) In similar experiments with FTI-277, the half-time of PreA
maturation following FTI-277 washout is ,15 hrs in total lysates, ,7 hrs with cyclohexamide chase and ,12 hrs in an overnight35S Cys/Met labeled
population. HDJ-2 is farnesylated at a faster rate than LaA is processed upon FTI-277 washout. (D) Immunoblot of extracts of Saos-2 cells probed with
LaA antibody. In contrast to the incomplete PreA accumulation by FTI-277 treatment, a combinatorial treatment of FTI-277 and GGTI-2147 or Lov-
alone lead to more complete accumulation of non-prenylated PreA. Percentage of mature LaA or HDJ-2 is listed under each lane.
Dynamics of Lamin-A Processing
PLoS ONE | www.plosone.org6 May 2010 | Volume 5 | Issue 5 | e10874
treatment. Furthermore, in the case of ZmpSte24 there are no
known substrates other than LaA, but there are ,30–100 other
substrates to compete for limited farnesylation, aaXing and
These observations suggest that components of the nuclear
lamina are readily accessible to processing enzymes such as the
soluble farnesyltransferase and the integral membrane ZmpSte24,
Rce1 and ICMT. Thus, the lamina may in fact be less static than
is generally envisaged. Previous studies utilizing FRAP analyses
have described GFP-LaA as essentially immobile [45,46].
However, in addition to relying on exogenous lamins with large
N-terminal fusion proteins, this technique only accounts for rather
large-scale movement of lamins within the nucleus. If lamins do
form structures similar to other intermediate filaments, then it is
difficult to imagine how an entire population of PreA assembled
into a static lamina could be readily accessible to the integral
membrane processing enzymes such as ZmpSte24. If all of the C-
terminal ‘tails’ were exposed on the surface of a lamin filament
that was capable of dynamic ‘rolling’ and situated adjacent to the
surface of the INM then this might permit sufficient association
with the processing enzymes. Or there could be focal assembly and
disassembly of lamin dimers that form the filaments, a model that
has been proposed for cytoplasmic intermediate filaments .
However, this possibility would be hard to reconcile with the A-
type lamin photobleaching data. An alternative explanation for the
rapid processing of lamina-associated PreA by ZmpSte14 would
be the lack of a filamentous lamina, at least in the conventional
sense.. In the absence of direct observations on the organization of
the somatic cell lamina, this latter suggestion cannot be entirely
The mechanism by which farnesylated PreA leads to HGPS or
RD remains unknown. However, there is considerable evidence
that a shift in the ratio of farnesylated A-type lamins is seriously
detrimental [9,26]. Previously, aberrant mitotic and post-mitotic
aggregates of progerin have been described with a suggested role
in the etiology of HGPS [42,43]. We have observed identical
results in ZmpSte24-null cells. Although the purpose of this study
does not include investigating the mechanisms of PreA or progerin
toxicity, our findings have added to the long list of associated
phenotypes. We have found not only A-type lamins, emerin and
LaB1 in these aggregates (Dechat et al., 2007), but also Sun2 and
Nesprin 3, both LINC complex components. The displacement of
these NE constituents from the newly forming post-mitotic NE is a
possible mechanism by which aberrantly farnesylated lamins may
Figure 5. Aberrant cellular phenotypes recover rapidly in PI treated cells following washout of FTI and GGTI. (A) Labeled with LaA/C
antibody, immunofluorescence microscopy of Saos-2 cells reveals that the combination of FTI-277 and GGTI-2147, but not FTI-alone, rectify aberrant
nuclear morphology and abnormal nucleoplasmic aggregation caused by Lop. Release of FTI and GGTI enhance this phenotypic rebound. Bar 10mm.
(B) Measurements of nuclear circularity and percentage of cells with metaphase and early G1 aggregates reveal that corresponding cellular
phenotypes are reversed by 15 hrs following FTI-277 and GGTI-2147 washout and by 24 hrs following FTI-277 washout (error bars=SEM; N=3, .75
cells counted each experiment).
Dynamics of Lamin-A Processing
PLoS ONE | www.plosone.org7 May 2010 | Volume 5 | Issue 5 | e10874
be toxic. This could perturb the organization of nuclear structures
such as chromatin and NPCs as well as the way in which the
nucleus interacts with the cytoskeleton via the envelope spanning
A role for farnesylated PreA in acquired lipodystrophy in HIV
patients receiving HAART treatment has not been conclusively
proven. However, there is considerable circumstantial evidence
that inhibition of ZmpSte24 by certain PIs may indeed contribute
to this lipodystophy [15,16]. And although it is unclear how PreA
may induce lipodystrophy, expression of progerin has been
reported to inhibit adipogenesis in human mesenchymal stem
In the case of nonfarnesylated-PreA and HDJ-2, maturation
following FTI washout in Saos-2 cells is considerably slower than is
found for Lov washout. This discrepancy may result from both
reduced clearance of the FTI and the geranylgeranylation of LaA
. In support of PreA geranylgeranylation, we have observed
that GGTI must be utilized with the FTI to quantitatively inhibit
maturation of LaA. When the rapidly diving Saos-2 cells were
treated combinatorially with Lop, FTI and GGTI, the prenylation
inhibitors blocked the aberrant cellular phenotype associated with
Lop treatment-alone. However, upon washout of the FTI and
GGTI the cells rapidly acquired the nuclear abnormalities and
cytoplasmic aggregates that are normally observed following Lop
treatment. We attribute this to the shift of accumulated
nonprenylated PreA within the nuclear lamina to a toxic
prenylated state that rapidly induces an aberrant cellular
phenotype. Recently Lee et al. have reported that in cells lacking
the b-subunit of farnesyl transferase there is a significant
accumulation of PreA which is described as nonfarnesylated
. However, the apparent presence of mature LaA in these cells
also suggests geranylgeranylation or some alternative mechanism
is promoting lamin A processing. Another recent study addressing
the subcellular localization of LaA processing described the
relatively rapid recovery of mature LaA following FTI washout
in the presence of cyclohexamide . The discrepancy with our
rather slower recovery (1.5h versus 4–5h with cyclohexamide) may
be a reflection of the fact that these cells were expressing
exogenous GFP-tagged lamins that appear localized in nucleo-
plasmic aggregates during FTI treatment. It is conceivable that
this altered localization might enhance the exposure of PreA to the
various lamin processing enzymes.
Although HGPS cells required 3 days to reacquire their
aberrant nuclear profiles and cytoplasmic lamin aggregates after
FTI washout, we suspect that this delayed recovery likely results
from the extremely limited proliferative potential of these cells.
Additionally, we cannot rule out that there may be different levels
or activities of processing enzymes in these cells. Thus, it may be
useful to determine the reversibility of lonafarnib, the FTI
currently used in a clinical trial for HGPS, in order to evaluate
any potential risks associated with skipped doses or rapid
termination of the treatment in HGPS patients. Furthermore,
the rate of PreA and progerin geranylgeranylation during
lonafarnib treatment should be examined as this modification
can delay proteolytic maturation of wild type PreA  which in
turn could cause unintended accumulation of toxic PreA and
progerin. We did observed that at doses sufficient to block PreA
processing both Lov and the combinatorial FTI/GGTI treatment
led to an obvious inhibition of cell growth over 48 hrs in Saos-2
cells. Thus, effective inhibition of progerin prenylation may
require inhibitor concentrations too toxic for clinical use.
processing following PI washout. Saos-2 cells were treated with
cyclohexamide at the time of Lop washout. PreA levels
dramatically disappear by 3 hrs following washout. Percent of
mature LaA listed below.
Found at: doi:10.1371/journal.pone.0010874.s001 (0.08 MB TIF)
Inhibition of protein synthesis does not impair PreA
cytoplasmic aggregates during mitosis and early G1 during Lop
treatment. A 48hr treatment of Saos-2 cells with Lop led to the
accumulation of Sun 2 and Nesprin-3 at LaA/C immunoreactive
Sun-2 and Nesprin-3 are present in abnormal
Figure 6. LaA and progerin are slow to mature in HGPS cells following FTI washout. (A) FTI-277 treatment of HGPS cells for 96 hrs inhibits
abnormal nuclear morphology as observed by LaA/C imunofluorescence (error bars=SEM; N=.80). Bar, 10mm. (B) After a 9 day FTI-277 treatment,
,50% of the LaA is PreA and nonfarnesylated-progerin (non-farD50). In contrast FTI-277 and GGTI-2147 lead to a more complete accumulation of
PreA and non-farD50. Following FTI or FTI and GGTI washout, mature LaA (matA) and farnesylated progerin (farD50) slowly recover, but at relatively
similar rates. Percentage of mature LaA or LaAD50 is listed above or below each lane, respectively.
Dynamics of Lamin-A Processing
PLoS ONE | www.plosone.org8 May 2010 | Volume 5 | Issue 5 | e10874
cytoplasmic aggregates in metaphase (upper panels) and early G1
(lower panels). DNA is labeled by Hoechst dye in blue. Bar, 10mm.
Found at: doi:10.1371/journal.pone.0010874.s002 (0.46 MB TIF)
PreA following washout. As detected by anti-LaA immunoblots of
Saos-2 cell lysates, PreA was refractory to processing following
washout of 10mm L-744, 832 or 1mm BMS-214662. Percent of
mature LaA listed below.
Found at: doi:10.1371/journal.pone.0010874.s003 (0.79 MB TIF)
Multiple FTIs failed to permit rapid processivity of
following FTI washout. (A) An anti-HA immunoblot of extracts
from WT human fibroblasts expressing exogenous HA-progerin
In HGPS cells, HDJ-2 exhibits prolonged maturation
were either treated with DMSO (control) or FTI-277 for 48 hrs
immediately following transfection. The FTI-277 treated progerin
migrates more slowly. (B) In HGPS cells treated with FTI-277 for
96hrs, HDJ-2 is incompletely processed by 24hrs following FTI-
277. Percent of mature HDJ-2 listed above.
Found at: doi:10.1371/journal.pone.0010874.s004 (0.10 MB TIF)
Conceived and designed the experiments: BB KJR. Performed the
experiments: QL DIK JS KJR. Analyzed the data: QL PL BB KJR.
Wrote the paper: BB KJR.
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