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doi: 10.1111/j.1600-0854.2008.00721.xTraffic 2008; 9: 708–724Blackwell Munksgaard

Dictyostelium Sun-1 Connects the Centrosome toChromatin and Ensures Genome Stability

Huajiang Xiong1,2,3, Francisco Rivero1,2,3,4,

Ursula Euteneuer5, Subhanjan Mondal1,2,3,

Sebastian Mana-Capelli6, Denis Larochelle6,

Annette Vogel5, Berthold Gassen1,2,3 and

Angelika A. Noegel1,2,3,*

1Center for Biochemistry I, Medical Faculty, University ofCologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne,Germany2Center for Molecular Medicine Cologne (CMMC),University of Cologne, Joseph-Stelzmann-Strasse 52,50931 Cologne, Germany3Cologne Excellence Cluster on Cellular StressResponses in Aging-Associated Diseases (CECAD),University of Cologne, Joseph-Stelzmann-Strasse 52,50931 Cologne, Germany4Current address: The Hull York Medical School,University of Hull, HU6 7RX Hull, UK5Institute for Cell Biology and Munich Center forIntegrated Protein Science (CIPSM), University ofMunich, Schillerstrasse 42, 80336 Munich, Germany6Department of Biology, Clark University, 950 MainStreet, Worcester, MA 01610, USA*Corresponding author: Angelika A. Noegel,[email protected]

The centrosome-nucleus attachment is a prerequisite

for faithful chromosome segregation during mitosis. We

addressed the function of the nuclear envelope (NE)

protein Sun-1 in centrosome-nucleus connection and

the maintenance of genome stability in Dictyostelium

discoideum. We provide evidence that Sun-1 requires

direct chromatin binding for its inner nuclear membrane

targeting. Truncation of the cryptic N-terminal chromatin-

binding domain of Sun-1 induces dramatic separation of

the inner from the outer nuclear membrane and deforma-

tions in nuclear morphology, which are also observed

using a Sun-1 RNAi construct. Thus, chromatin binding

of Sun-1 defines the integrity of the nuclear architecture.

In addition to its role as a NE scaffold, we find that

abrogation of the chromatin binding of Sun-1 dissociates

the centrosome-nucleus connection, demonstrating that

Sun-1 provides an essential link between the chromatin

and the centrosome. Moreover, loss of the centrosome-

nucleus connection causes severe centrosomehyperampli-

fication and defective spindle formation, which enhances

aneuploidy and cell death significantly. We highlight an

important new aspect for Sun-1 in coupling the centro-

some and nuclear division duringmitosis to ensure faithful

chromosome segregation.

Key words: aneuploidy, centrosome hyperamplification,

nuclear envelope architecture, spindle formation defects,

Unc-84

Received 23 October 2007, revised and accepted for

publication 7 February 2008, uncorrected manuscript

published online 11 February 2008, published online 12

March 2008

The nuclear envelope (NE) separates the nuclear compart-

ment from the cytoplasm. It is composed of two mem-

branes: the outer nuclear membrane (ONM) and the inner

nuclear membrane (INM). The lumen between the two

membranes is the perinuclear space (PNS). The ONM is

continuous with the endoplasmic reticulum (ER), whereas

the INM harbors a unique set of proteins. INM and ONM

proteins can interact within the PNS. Underneath the INM,

the nuclear lamina is located, which is formed by interme-

diate filament (IF) proteins and associated proteins. The

lamina forms the nucleoskeleton and associates with the

INM, chromatin and nuclear pore complexes. Proteins of

the NE have important roles. They are involved in nuclear

migration and positioning and are essential for many pro-

cesses such as mitosis, meiosis, differentiation and cell

migration. Furthermore, several of the NE proteins have

been associated with inherited diseases (1,2).

Research in mammalian cells and in Caenorhabditis elegans

has identified conserved components of the NE that link the

nucleoskeleton to the cytoskeleton. In C. elegans, two

putative INM proteins, matefin/SUN-1 and UNC-84, bind

to the nuclear lamina and extend their C-terminus into the

PNSwhere they interactwith the C-termini of KASH domain

proteins (Klarsicht/Anc-1/Syne homology, designated KASH

domain). Matefin/SUN-1 and UNC-84 belong to the SUN

family of proteins based on the presence of the conserved

SUN (Sad1/UNC-84 homology) domain at their C-terminus.

KASH domain proteins are type II transmembrane proteins

of the NE and have been identified as molecular linkers

connecting the nucleus to actin filaments [filamentous actin

(F-actin)], IFs andmicrotubules (MTs) (3).C. elegans harbors

three KASH domain proteins, Anc-1, a huge ONM protein

with an F-actin-binding domain (ABD) of the a-actinin type

at its N-terminus with which it can link to the actin cyto-

skeleton, UNC-83 that associates with MTs, and ZYG-12,

which provides a link to the centrosome. Anc-1 and UNC-

83 interact with UNC-84, ZYG-12 with matefin/SUN-1 (1).

In mammalian cells, Nesprin-1/Enaptin and Nesprin-2/

NUANCE are homologues of Anc-1, and their largest iso-

forms also have ABDs for associationwith F-actin; Nesprin-3

binds to the ABD of plectin, which itself can bind to the IF

cytoskeleton. SUN-1 and SUN-2 are the best characterized

SUN/UNC-84 homologues in mammalian cells and, like the

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C. elegans proteins, interact with the C-terminus of the

Nesprins in the PNS (3). Taken together, by combination of

the SUN domain proteins with diverse KASH domain

proteins, the nucleus can be linked simultaneously to

different cytoskeletal elements and to the centrosome.

In yeasts, recent studies revealed that SUN domain proteins

organize the chromatin during homologous chromosome

pairing in meiosis. In Saccharomyces cerevisiae, the SUN

domain protein Mps3 that is involved in karyogamy and

sister chromatid cohesion interacts with the meiotic telo-

mere protein Ndj1 to cluster the telomeres to the NE, which

is known as the bouquet formation (4–6). In Schizosacchar-

omyces pombe, the bouquet formation is mediated by the

complex containing the SUN domain protein Sad1 and the

linker proteins Bqt1 and Bqt2. Sad1 is associated with the

spindle pole body that is the centrosome equivalent in

fission yeast. The movement of the chromosomes toward

their appropriate homologues occurs through the interaction

of Sad1 with the KASH domain protein Kms1 that engages

a cytoplasmic dynein motor complex (7). Similarly, in C.

elegans, the complex containing matefin/Sun-1 and Zyg-12

tethers and moves the chromosomal pairing centers along

the NE to allow homologue recognition and clustering.

Accordingly, mammalian Sun-1 attaches the telomeres to

the NE to promote homologous chromosome pairing and

synapsis formation in meiotic prophase I (8–10).

To date, little is known about the NE and NE proteins in

Dictyostelium discoideum. Like yeast, D. discoideum does

not have lamins and undergoes a closed mitosis. The first

NE protein described so far, interaptin (11), may function

as a KASH domain protein in analogy to the findings in

higher eukaryotes. The prediction of SUN domain proteins

in the D. discoideum genome prompted us to investigate

the function of Sun-1 and its interplay with interaptin. Here,

we report that Sun-1 is retained in the INM through binding

to chromatin and defines the spacing of the NE lumen as

well as the centrosome-nucleus juxtaposition. Abrogation

of the Sun-1 binding to chromatin induces dramatic sep-

aration of the two nuclear membranes and loss of the

centrosome-nucleus connection that ultimately results in

centrosome hyperamplification causing defective mitotic

spindle formation that induces chromosome missegrega-

tion and genome instability.

Results

Sun-1 is an INM protein and binds to chromatin

The 105 kDa D. discoideum Sun-1 (DDB0219949) homo-

logue is composed of an N-terminal coiled-coil domain

(amino acids 170–221), a single transmembrane domain

(aa 291–313), a pair of coiled-coil domains (aa 412–457 and

507–571) and a C-terminal SUN domain (aa 712–859) that

terminates in a putative ER retention signal SDEL that is

unique for D. discoideum Sun-1 (Figure 1A). Sun-1 has

features found in different SUN domain protein homo-

logues: (i) it shares the single transmembrane domain with

the C. elegans UNC-84 and the S. pombe Sad1. (ii) Like the

mammalian SUN domain proteins, Sun-1 possesses

coiled-coil domains that are absent in Ce UNC-84 and Sp

Sad1 (12,13). The SUN domain of Dd Sun-1 shows highest

homology to the one of vertebrate Sun proteins (Figure

1A). Sun-2 (DDB0186751) represents the second SUN

domain protein in D. discoideum. It has a different domain

structure with a centrally located SUN domain and belongs

to the SUN-like proteins, which are also present in the

proteome of yeast and flies (14).

As the SUN domain proteins that are involved in nuclear

positioning andmigration possess a C-terminal SUNdomain,

we focused on the analysis of Sun-1 in D. discoideum that

may represent an orthologue of the classical SUN domain

proteins.

In wild-type AX2 cells, Sun-1 was present at the NE, where

it colocalized with green fluorescent protein (GFP)-tagged

nuclear pore protein Nup43 and to some extent also in the

cytoplasm (Figure 1B,B9 and B0 and Figure S1) using

monoclonal antibody (mAb) K55-460-1 generated against

a polypeptide encompassing the two central coiled-coil

domains (aa 344–653; Figure 1A). Upon fractionation of

total cell lysates on discontinuous sucrose gradients, Sun-

1 was exclusively present in the fractions of highest

density, which correspond to the nuclear fractions and

ER membranes (15) as we find the ER marker PDI in these

fractions as well (Figure 1C). The Dictyostelium KASH

domain protein interaptin (11) showed a similar distribution

but was also present in lighter fractions resembling the

distribution of PDI (Figure 1C). Coimmunofluorescence

studies using calreticulin–GFP and calnexin–GFP express-

ing cells as well as PDI antibodies supported this localiza-

tion (Figure S1). Sun-1 was partially extracted from nuclei

by addition of Triton-X-100, urea or a combination of both

reagents, indicating that Sun-1 is hydrophobic and strongly

associated with the NE (Figure 1D). Similarly, mammalian

Sun-2 is resistant to Triton-X-100 and urea extraction,

characteristics that are thought to be because of its

immobilization on lamins (16,17). Distinct from higher

eukaryotes, D. discoideum lacks lamins; thus, Sun-1 may

bind to other nuclear components.

To investigate the targeting and retention of Sun-1, we first

addressed its membrane topology in proteinase K pro-

tection assays using intact nuclei. A 70 kDa Sun-1 frag-

ment representing the complete C-terminus including the

transmembrane domain (aa 291–905) was protected from

proteolysis (Figure 1E). As the low molecular mass of

proteinase K enables it to diffuse into the nucleoplasm

and degrade epitopes on the INM, we were unable to

determine the precise localization of Sun-1 at the INM and/

or ONM from these assays. In conclusion, Sun-1 is a type II

transmembrane protein, which exposes its N-terminus to

either the nucleoplasm and/or the cytoplasm and its

C-terminus to the PNS. A location of the N-terminus to

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Sun-1 Roles at the Nuclear Envelope

the nucleoplasm is, however, favored based on the find-

ings that all SUN domain proteins studied so far interact

directly or indirectly with lamins, although their INM

targeting is lamin independent (18–21).

AsDictyostelium lacks lamins, we hypothesized that Sun-1

may be retained in the INM through interaction with

chromatin. We did not expect DNA sequence specificity

for Sun-1, as the chromatin binding is supposed to anchor

Figure 1: Legend on next page.

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Xiong et al.

Sun-1. Therefore, we analyzed the protein–DNA interac-

tion by polymerase chain reaction (PCR) amplification of the

housekeeping gene actin-8. In chromatin immunoprecipi-

tation (ChIP) experiments, chromatin was coprecipitated

with the endogenous Sun-1 (Figure 2A), indicating that

chromatin binding may represent a lamin-independent

INM retention mechanism and that the N-terminus of

Sun-1 is most likely involved in chromatin binding. Next,

we subjected purified recombinant Sun-1 N-terminus

[glutathione S-transferase (GST)-Sun1N400] to electromo-

bility shift assays (EMSA) to validate whether the Sun-1

N-terminus interacts directly with DNA. GST-Sun1N400

interacted directly with radiolabeled DNA, whereas GST

did not (Figure 2B). Furthermore, we analyzed the DNA

binding using non-induced and induced bacterial total

lysates containingGST-Sun1N400orGST-Sun1N800, a poly-

peptide encompassing the Sun1N400 sequences, using the

Southwestern technique. Both GST-Sun1N400 and GST-

Sun1N800 interact directly with radiolabeled DNA, whereas

non-induced bacterial lysates or a GST-fusion protein con-

taining the coiled-coil domains of Sun-1 (Sun1CT) did not

showDNA interaction (Figure 2C and Figure S2). In addition,

both GST-Sun1N400 and GST-Sun1N800 interact not only

with the DNA sequence of D. discoideum hp1 but also with

that of D. discoideum actin-8 and mammalian DNA, sug-

gesting that the Sun-1 N-terminus does not recognize

specific DNA sequences (Figure S2). Collectively, these

data obtained from ChIP and EMSA demonstrate that the

Sun-1 N-terminus binds to chromatin and DNA. Distinct

frommammalian Sun-1 that possesses a zinc-finger domain

at the N-terminus, the N-terminus of D. discoideum Sun-1

does not contain known DNA-binding motifs, but it is likely

that Sun-1 has a cryptic DNA-binding domain with strong

DNA-binding affinity that allows the INM retention of Sun-1.

By extension, this interaction may be responsible for the

INM targeting and retention of SUN domain proteins in

other organisms.

Sun-1 forms homodimers and higher oligomers

Many SUN domain proteins contain predicted coiled-coil

domains through which homodimerization might occur

(17). Analysis of the Dictyostelium Sun-1 sequence re-

vealed that the central pair of coiled-coil domains harbors

hot spots of conserved amino acid residues such as R,

Y, F, H and M, which have the potential to promote strong

protein–protein interactions, as well as L and I that

contribute moderate protein–protein interfaces (Figure 2D)

(22,23). We tested the capability of Sun-1 to oligomerize

using recombinant SunCT1 (residues 344–653) that encom-

passes the pair of coiled-coil domains. First, we used circular

dichroism (CD) spectrum analysis to confirm that the

bacterially expressed polypeptide had folded. The CD spec-

trum showed twominima near 210 and 220 nm typical of an

a-helical protein and indicated that the protein had folded

(Figure S3). In the presence of the cross-linking reagent

glutaraldehyde, the polypeptide formed dimers and higher

oligomers (Figure 2E). Notably, the native non-cross-linked

SunCT1 sample contained some amount of dimers (approx-

imately 80 kDa) and trimers (approximately 100 kDa) that

were not disassembled into monomers (40 kDa) under

denaturing conditions (Figure 2E). To further strengthen

a self-interaction of SunCT1, the native and cross-linked

protein was analyzed by gel filtration chromatography using

a Sephadex G-75 column. In both cases, Sun1CT eluted

primarily in fraction 10 corresponding to the dimer and

trimer. The monomer eluted in fraction 11 (Figure 2F).

N-terminal truncation of Sun-1 abolishes

INM targeting

To investigate whether the INM targeting and retention of

Sun-1 requires chromatin binding, the construct GFP-

DNSun-1 in which the Sun-1 N-terminus was replaced by

a GFP tag was stably expressed in the wild-type AX2

genetic background that did not affect the viability of the

cells. GFP-DNSun-1 is localized to the NE like the endo-

genous Sun-1, suggesting that the N-terminus is dispens-

able for the NE targeting of Sun-1 (Figure 3A,E). As GFP-

DNSun-1 contains the complete C-terminus, it is likely that

that the C-terminus of Sun-1 determines its NE localization.

Within the mutant strain, we compared the distribution of

GFP-DNSun-1 in the nuclear membranes with that of the

endogenous Sun-1 using sequential digitonin and Triton-

X-100 permeabilization. First, the plasma membrane was

selectively permeabilized with digitonin to address cyto-

plasmic epitopes on the ONM. The polyclonal rabbit

Figure 1: Sun-1 is a type II NE protein inD. discoideum. A) Comparison ofD. discoideum Sun-1 (Dd Sun-1) with C. elegansUNC-84 (Ce

UNC-84), C. elegans matefin/Sun-1 (Ce mtf/Sun-1), S. pombe Sad1 (Sp Sad1), human Sun-1 and Sun-2. The putative domains are

highlighted: coiled-coil domains (blue, CC), SUN domain (pink box) and transmembrane domain (gray box, T). Dd Sun-1 possesses an ER

retention signal at its C-terminus (green box). The sequence identity of the SUN domains is indicated. The Sun-1 monoclonal antibodies

(Sun-1 mAb) were generated against a polypeptide encompassing the two central coiled-coil domains (aa 344–653, bar). (B and B0) Sun-1

was localized to the NE and to some cytoplasmic compartments using the mAb K55-460-1 (B, gray scale; B0 red), the NE is delineated by

GFP-tagged Nup43 (B9, gray scale; B0, green), DNA was labeled with DAPI (B0, blue). C) The subcellular localization of Sun-1 was

determined by fractionation of a total cell lysate of strain AX2 on a discontinuous sucrose gradient. Sun-1 was detected by mAb K55-432-2,

the ER marker PDI by mAb 221-135-1 and the KASH domain protein interaptin, which exists as two isoforms, by mAb 260-60-10. D)

Solubilization of Sun-1. AX2 total cell lysate (L) was separated into cytoplasmic (C) and nuclear (N) fraction, and the nuclear fraction further

treatedwith PBS and subjected to extractions by addition of 1% Triton-X-100 (TX-100), 8 M urea or a combination of both reagents (TX/urea)

resulting in supernatant (NS) and pellet (NP). Western blots (WB) were probed with mAb K55-432-2. E) The C-terminus of Sun-1 projects into

the PNS. Proteinase K protection assays of intact nuclei were carried out. Proteinase K generated a 70-kDa fragment representing the Sun-1

C-terminus. Complete degradation of Sun-1 was observed in the presence of the membrane solubilizing detergent Triton-X-100.

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Figure 2: Sun-1 binds to chromatin and DNA and forms oligomers. A) In ChIP assays, chromatin was coprecipitated with the Sun-1

mAb K55-460-1 (Sun-1). PCR amplification of the housekeeping gene actin-8 (act8) was used to detect the precipitated chromatin. The AX2

lane shows the amplification from purified genomic DNA. In control experiments using an a-tubulin-specific mAb (alphaTub) (611B1 (46);

a kind gift from Michael Koonce) or protein A–Sepharose (beads), act8 was not amplified by PCR. Immunoprecipitated Sun-1 is shown

(Western blot: Sun-1). The antibody heavy chain (Ab HC) is displayed for control. B) The Sun-1 N-terminus (GST-Sun1N400) binds directly to

hp1 DNA in EMSA but not GST. C) Sun-1 N-terminal polypeptides (GST-Sun1N400 and GST-Sun1N800) bind radiolabeled hp1 DNA in

Southwestern blots. Western blots of non-induced (�) and isopropyl-b-D-thiogalactopyranosid (IPTG)-induced (þ) bacterial lysates were

incubated with radiolabeled DNA. D) The amino acid sequence encompassing the central coiled-coil domains (blue) contains hot spots of

conserved residues providing protein interfaces of strong (F, H, M, Y, R; underlined) and weak affinity (L and I; underlined). E) Five

micrograms of the recombinant coiled-coil domains (SunCT1) was incubated with 0.001% of the cross-linker glutaraldehyde. The native

SunCT1 occurs as a monomer (40 kDa, Mo), low amounts of dimers (80 kDa, Di) and trimers (approximately 100 kDa, Tri) are also present.

The amount of dimers, trimers, tetramers (160 kDa, Tet) and pentamers (200 kDa, Pen) was increased by adding glutaraldehyde. Samples

were taken 5, 10 and 20 min after addition of glutaraldehyde. F) Analysis of native cross-linked SunCT1 by gel filtration analysis followed by

SDS–PAGE and Western blot. The proteins eluted in fractions corresponding to the dimer (fraction 10) and the monomer (fraction 11) in

case of the native material.

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GFP antibodies (pAb GFP) detected the N-terminus of

GFP-DNSun-1 at the NE facing the cytoplasm (Figure 3B).

Under this condition, the mAb K55-460-1 failed to stain

GFP-DNSun-1 and the endogenous Sun-1, confirming that

both proteins locate their C-terminus in the PNS (Figure 3F).

The inaccessibility of the coiled-coil domains to the mAb

K55-460-1 in the PNS also proved the NE integrity upon

digitonin treatment. In the second step, all cellular mem-

branes were permeabilized with Triton-X-100 application

that enables the mAb K55-460-1 to access the coiled-coil

domains of both GFP-DNSun-1 and the endogenous Sun-1.

In the analysis, we frequently observed separation of the

INM from the ONM at several sites of the GFP-DNSun-1-positive nuclei (Figure 3C,D).

Intriguingly, at sites of the separated nuclear membranes,

the endogenous Sun-1 was targeted to the INM and was

also observed in the nucleoplasm (Figure 3C,D, arrow),

whereas GFP-DNSun-1 accumulated in the ONM (Figure

3D, arrowhead), indicating that the chromatin binding of

Sun-1 is essential for INM retention presumably through

its N-terminus. As a control, pAb GFP staining after Triton-

X-100 permeabilization did not reveal separation between

the INM and the ONM, and we assume that GFP-DNSun-1was restricted to the ONM (Figure 3G,H). In immunoe-

lectron microcopy studies using GFP antibodies, the

ONM accumulation of GFP-DNSun-1 was confirmed

(Figure 3I,I9, arrowheads) as well as the separation of

the INM and ONM (Figure 3I,I9, asterisks) implying that

Sun-1 may regulate the spacing of the NE lumen, prob-

ably by interaction with an ONM protein. According to this

hypothesis, GFP-DNSun-1 in the ONM may disturb this

interaction by binding with its C-terminus to the yet

unknown ONM partner leading to the separation of the

INM and the ONM. To some extent, GFP-DNSun-1 was

also detected in the INM that might be because of its

heterodimerisation with the endogenous Sun-1 allowing

the translocation of the truncated protein to the INM

(Figure 3I,I9, arrows).

Moreover, we analyzed the nuclear and cell size as well as

the integrity of the NE in GFP-DNSun-1 as well as in Sun-1-

depleted cells. In comparison with AX2 cells that exhibit

a diameter of 10–15 mm in 98% of cells, 30% of the GFP-

DNSun-1 cells and 10% of the Sun-1 RNAi cells displayed

an increase in nuclear and cellular diameter (Figure 3J). To

examine the integrity of the NE, the 49-6-diamidino-2-

phenylindole (DAPI) staining was superimposed with the

GFP-DNSun-1 image (Figure 3K), whereas the NE of Sun-1

RNAi cells was visualized by immunofluorescence of

interaptin that localized properly to the NE (Figure 3L).

The nuclear and cellular expansion upon GFP-DNSun-1expression and Sun-1 RNAi was accompanied by severe

nuclear deformations, such as formation of NE blebs that

did not contain DNA (Figure 3K,L, arrowheads). We

observed NE deformations in 95% of the huge GFP-

DNSun-1 cells nuclei and in 65% of the huge Sun-1 RNAi

cells (Figure 3J). The extent of abnormal nuclear morphol-

ogy correlated with nuclear and cellular volume expansion,

which suggests that GFP-DNSun-1 and Sun-1 RNAi cells

suffer from inefficient co-ordination of nuclear and cell

division. The relatively milder effect on nuclear and cellular

expansion and NE deformation in Sun-1 RNAi cells com-

pared with GFP-DNSun-1 cells may be because of the

limited efficiency of the Sun-1 depletion that was achieved

(approximately 40% of AX2 levels) (Figure 3J). However,

we were not able either to improve the efficiency of Sun-1

downregulation or to generate a sun-1 knockout strain, as

both events are most likely lethal for D. discoideum,

indicating that Sun-1 may play an essential role.

GFP-DNSun-1 disconnects the centrosome

from the nucleus

The nuclear and cellular enlargement suggests defects in

nuclear and cell division; thus, we investigated the fidelity

of chromosome segregation in GFP-DNSun-1 cells by

evaluation of the karyotype. In AX2 cells, the genome is

distributed on six chromosomes (Figure 4A). In marked

contrast, GFP-DNSun-1 cells displayed a significant

increase of aneuploid nuclei containing different numbers

of chromosomes ranging from three to five (Figure 4A)

confirming a loss of chromosomes during segregation. In

concordance with the numerical chromosome aberrations,

we observed reduced proliferation capacity and a fivefold

increase in cell death upon GFP-DNSun-1 expression as

well as Sun-1 RNAi (Figure 4B).

Figure 3: The N-terminus of Sun-1 is required for its INM retention. Truncation of the Sun-1 N-terminus (GFP-DNSun-1) did not affect

its NE targeting but caused an altered nuclear morphology (A, E, deltaN, GFP fluorescence). After selective digitonin permeabilization of the

plasmamembrane, the N-terminal GFP tag of GFP-DNSun-1was recognized by GFP-specific polyclonal rabbit antibodies (pAb GFP) (B). The

coiled-coil domains of GFP-DNSun-1 and the endogenous Sun-1 were inaccessible for the mAb K55-460-1 (F). After permeabilization of the

NE by Triton-X-100, the mAb K55-460-1 detected both GFP-DNSun-1 and the endogenous Sun-1 (C and G) as well as the staining of GFP-

DNSun-1 by pAb GFP (H). In C and D, a separation of the INM from the ONM at several sites is observed. Endogenous Sun-1 is targeted to

the INM (D, arrow), whereas GFP-DNSun-1 accumulates in the ONM (D, arrowhead). (I and I9) Immunoelectron microcopy of the nuclei

fromGFP-DNSun1 expressing cells after labelingwith gold-conjugated GFP pAb showsGFP-DNSun1 in the ONM (I and I9, arrowheads) and

reduced amounts at the INM (I and I9, arrows). Asterisks indicate separations of the INM and ONM. J) Cell size and NE deformations were

determined for AX2 (transparent), GFP-DNSun-1 (green) and Sun-1 RNAi (blue) cells (n > 400). In the enlarged cells, the percentage of

deformed nuclei was determined. The efficiency of RNAi was probed by Western blotting, actin served as control. (K and L) Nuclear

deformations in GFP-DNSun-1 and Sun-1 RNAi cells (arrowheads). The arrows point to normal sized cells with normal nuclei. Nuclear

deformation in Sun-1 RNAi cells was determined after staining of the NE using interaptin antibody mAb 260-60-10 (Int) (L). DNA was

stained by DAPI.

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As faithful chromosome segregation depends on the

centrosome stability, we studied the centrosome-nucleus

connection in GFP-DNSun-1 and Sun-1 RNAi cells. In AX2

cells, each nucleus is intimately connected to one centro-

some during interphase (Figure 4C). In comparison, in

90% of the GFP-DNSun-1 cells the centrosomes are

located far away from the nuclei (Figure 4D,E). In regular-

sized cells, the NE formed protrusions extending toward

the centrosome, which bridged a distance up to one third

of the cell diameter (Figure 4D). Notably, GFP-DNSun-1decorated NE protrusions that were attached to the

periphery of the centrosomes, indicating that GFP-

DNSun-1 may participate in a putative protein complex,

which forms the centrosome-nucleus connection (Figure

4D, boxes; Figure 4G). Although the centrosome-nucleus

vicinity was abolished in the regular-sized GFP-DNSun-1cells, the centrosome number correlated strictly with that

of the nuclei as in AX2 cells (Figure 4C,D). On the contrary,

NE protrusions were absent in the huge GFP-DNSun-1cells. Instead, the nuclei in almost all huge cells were

completely disconnected from the centrosome. Moreover,

we observed a dramatic centrosome hyperamplification

and cells with two nuclei had up to five centrosomes

(Figure 4E). Although centrosome hyperamplification was

also found in Sun-1 RNAi cells, fewer nuclei have lost the

vicinity to the centrosomes (Figure 4F). Probably, the

remaining amount of Sun-1 can maintain the centrosome-

nucleus connection to some extent.

The quantification of centrosome and nuclear number

revealed that the centrosome-nucleus number strictly

correlated within AX2 cells, whereas GFP-DNSun-1 cells

showed diverse combinations in centrosome-nucleus

number. Although the nuclear number was not dramati-

cally increased in GFP-DNSun-1 and Sun-1 siRNA cells,

GFP-DNSun-1 exhibited a severe centrosome hyperampli-

fication. Sun-1 siRNA cells were remarkable in that they

mainly had a single nucleus that in the majority of cases

was associated with one centrosome. Few cells had

supernumerary centrosomes (Figure 4H). As the down-

regulation of Sun-1 did not enhance centrosome hyper-

amplification to the severe extent that was observed in

GFP-DNSun-1 expressing cells, it appears likely that the

remaining amount of Sun-1 in Sun-1 RNAi cells is sufficient

to tether the chromatin to the centrosome and thereby to

maintain the centrosome-nucleus connection and mitotic

spindle stability.

We also carried out rescue experiments with Sun-1 RNAi

cells by expressing a full-length GFP-tagged Sun-1 in order

to prove that the observed effects were specific. Such

cells had normal nuclei, and centrosome amplification was

no longer observed, whereas centrosomes were often

detached from the nuclei (Figure S4A). The latter pheno-

type appears to be caused by GFP-Sun-1 overexpression

as detachment of centrosomes was also observed in AX2

cells overexpressing GFP-Sun-1. In fact, this strain ex-

hibited the highest number of cells with detached centro-

somes (Figure S4A,B). Furthermore, in sequential

permeabilization experiments, GFP-Sun1 was also de-

tected at the ONM in AX2 cells (Figure S4C).

The observed aneuploidy in GFP-DNSun-1 cells prompted

us to analyze the distribution of DNA during mitosis. In

contrast to AX2 cells in which the chromosomes are

partitioned and connected to the centrosomes during

mitosis (Figure 4I,I0), we found that chromosomes were

detached from the centrosomes and lost along the spindle

in GFP-DNSun-1 cells (Figure 4J,J0), indicating that Sun-1

may facilitate chromosome attachment to the centrosome

during mitosis. In addition, centrosome-nucleus detach-

ment and centrosome hyperamplification in GFP-DNSun-1cells yielded the formation of monopolar and multipolar

mitotic spindles that represent various forms of defective

spindles, whereas some bipolar spindles performed asym-

metric nuclear division (Figure S5).

Sun-1 competes with interaptin for NE localization

SUN and KASH domain proteins interact through their

C-termini in the PNS to provide a physical connection

of the nucleoskeleton with the cytoskeleton (16–19). In

D. discoideum, interaptin exhibits the conserved domain

architecture of the KASH domain proteins that includes

an N-terminal actin-binding domain, a central stretch of

coiled-coil repeats and a C-terminal transmembrane

domain with a short tail harboring a KASH motif (11). In

AX2 cells, both interaptin and Sun-1 were localized in the

NE (Figure 5A,A9 and B,B9). Surprisingly, while in an

interaptin-deficient strain Sun-1 was targeted to the NE

(Figure 5C,C9 and D,D9), Sun-1 was displaced from the NE

when interaptin was overexpressed (Figure 5E,E9 and

F,F9), which implies a competitive localization of Sun-1

and interaptin in the NE ofD. discoideum, that is in contrast

to the vertebrate model (3).

Figure 4: GFP-DNSun-1 induces centrosome hyperamplification and aneuploidy. A) The Dictyostelium genome is distributed on six

chromosomes in wild-type cells (n > 400, white). In GFP-DNSun-1 cells, the karyotype varied from three to six chromosomes (n > 400),

indicating a tendency toward aneuploidy (gray). DNAwas stained using DAPI. B) Quantification of cell death in AX2, GFP-DNSun-1 and Sun-

1 RNAi cells was carried out after propidium iodide staining (n > 400). C) In AX2 cells, each nucleus is localized in the vicinity of one

centrosome labeled by mAb K29-359-31 (Cent, red). D) Regular-sized cells expressing GFP-DNSun-1 (deltaN, green) have NE protrusions

toward the centrosome (K29-359-31, red) (boxes). (E and F) The nuclei of the huge cells are disconnected even from closely located

centrosomes (magnified box). G) Immunoelectron microscopy of a GFP-DNSun-1-positive nucleus attached to a centrosome (arrowhead).

GFP-DNSun-1 is detectedwith the GFP pAb (arrows). H) Centrosome hyperamplification in GFP-DNSun-1 (deltaN) and Sun-1 RNAi (siSun1)

cells (n > 400). (I, I0 and J, J0). Partitioning of chromosomes during mitosis in AX2 (I and I0) and GFP-DNSun-1 cells (J and J0). The mitotic

spindle that was visualized using mouse a-tubulin (alphaTub) antibody (red).

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Comparison of the interaptin C-terminal tail with those

from C. elegans Anc-1, Drosophila melanogasterMsp-300/

Nesprin and mammalian Nesprin-1 and -2 pointed to the

terminal amino acids PT as the relevant KASH motif for NE

targeting. Previously, we have shown that the truncated

interaptin GFP-IntCT, which encompasses the C-terminal

transmembrane domain and the KASHmotif, displaces the

endogenous interaptin from the NE (11) (Figure 5G,G9).

Now, we show that the terminal amino acids PT are

essential for NE targeting of interaptin, as proteins carrying

amutation of proline to alanine (GFP-IntP/A, Figure 5I,J9) or

a truncation of PT (GFP-IntDPT, Figure 5K,L9) failed to

localize to the NE and did not compete with endogenous

interaptin for NE localization (Figure 5I,K). As interaptin

was absent from the NE protrusions and interaptin mu-

tants did not suffer from centrosome-nucleus disconnec-

tion or centrosome hyperamplification (data not shown), it

is apparently not involved in the centrosome-nucleus

complex but may have other functions particularly during

development.

NE localization of Sun-1 was abolished upon overexpres-

sion of GFP-IntCT (Figure 5H,H9) but was not affected by

GFP-IntCT-P/A or GFP-IntCT-DPT (Figure 5J,J9 and L,L9),

which demonstrates that the interaptin C-terminus exerts

a dominant-negative effect on the NE localization of Sun-1

and that the KASH motif is involved in a competition with

Sun-1. Conversely, overexpression of GFP-DNSun-1reduced the NE localization of interaptin (Figure 5M,M9),

indicating that Sun-1 and interaptin may compete for

a common binding partner in the NE. Similarly, C. elegans

UNC-84 may interact indirectly with the KASH domain

protein Anc-1 (24), implying that their interaction may be

mediated by an additional partner. In D. discoideum, Sun-1

may interact with a centrosome-attached KASH domain

protein to establish centrosome-nucleus connection,

whereas nuclear positioning may require KASH domain

proteins other than interaptin.

Furthermore, we observed in live cell imaging analysis

that nuclei of GFP-DNSun-1 cells experienced severe

deformations but were able to move, although they

were disconnected from the centrosomes. In com-

parison, wild-type nuclei in which the NE was labeled

by GFP-IntCT maintained a nearly perfectly round shape

(Figure 6).

Discussion

Sun-1 is a chromatin-binding protein of the INM

In this study, we have shown that Sun-1 is an INM protein

in D. discoideum, which extends its N-terminus into the

nucleoplasm and the C-terminus into the PNS. The local-

ization and the topology of Sun-1 are conserved with that

of SUN domain proteins in other species (3,17). We have

provided evidence that the central coiled-coil domains

promote dimerization and oligomerization of Sun-1 in vitro.

Based on this, it is possible that Sun-1 forms dimeric and/

or oligomeric complexes in the INM that serve as a plat-

form for ONM proteins.

In general, SUN domain proteins do not contain nuclear

localization signal sequences; therefore, their specific

targeting mechanism to the INM was unexplained. Some

SUN domain proteins from higher eukaryotes, such as

C. elegansmatefin/Sun-1 and mammalian Sun-1 and Sun-2

interact with lamins but can localize to the INM after

depletion of lamins, indicating that targeting of the SUN

domain proteins may involve other nuclear components

(18,20). Here, we provide evidence that Sun-1 is targeted

and retained in the INM through binding to chromatin

through its N-terminus in D. discoideum that is a lamin-free

model system. Notably, the N-terminus of Sun-1 is dis-

pensable for NE localization per se but determines its

targeting to the INM as GFP-DNSun-1 mostly failed to

localize to the INM and accumulated in the ONM, demon-

strating that chromatin binding of the Sun-1 N-terminus

represents an INM retention mechanism. Mislocalization

of GFP-DNSun-1 causes separation of the INM and ONM,

indicating that Sun-1 regulates the spacing of the NE

through interaction with an ONM protein that may be

bypassed by GFP-DNSun-1 in the ONM. Dilation of the

PNS, nuclear deformation and the increased nuclear

volume both in GFP-DNSun-1 and in Sun-1 RNAi cells

suggest that Sun-1 may define the spacing of the NE

lumen and the integrity of NE morphology.

Sun-1 establishes centrosome-nucleus connection

We observed loss of centrosome-nucleus juxtaposition

because of NE protrusions toward the centrosome in

regular-sized cells, whereas the nuclei in huge GFP-DNSun-1cells were completely disconnected from the centrosomes

Figure 5: Sun-1 and interaptin localize to the NE in a competitive manner. In AX2 cells, interaptin was predominantly present at the

NE (A and A9) as was Sun-1 (B and B9). In the interaptin-deficient strain abpD� (C and C9), the NE localization of Sun-1 was unaffected (D and

D9). When interaptin was overexpressed (abpDþ) (E and E9), Sun-1 was displaced from the NE and accumulated in the cytoplasm (F and F9).

GFP-IntCT encoding the KASH domain of interaptin (green) is capable to displace the endogenous interaptin (G and G9, red) and Sun-1 (H

and H9, red). Mutation of proline 1736 to alanine within the KASH domain abolished the NE targeting of GFP-IntP/A (I9 and J9, green) but

allowed that of the endogenous interaptin (I and I9, red) and Sun-1 (J and J9, red). GFP-IntDPT (K9 and L9, green) containing a KASH domain

lacking the terminal PT failed to localize to the NE, whereas the endogenous interaptin (K and K9, red) and Sun-1 (L and L9, red) were found

at the NE. Conversely, the NE localization of the endogenous interaptin (M, red) was reduced upon overexpression of GFP-DNSun-1 (M9,

green). DNA was stained with DAPI (blue).

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Xiong et al.

and dramatic centrosome hyperamplification occurred that

caused chromosome missegregation, aneuploidy and

a fivefold increase in cell death. Our findings agreed well

with the neoplastic characteristics of malignant tumors in

which centrosome hyperamplification was always associ-

ated with aneuploidy (25–29).

To date, the mechanisms for centrosome amplification are

not completely understood and so far most efforts were

focused on regulation of centrosome duplication. Interest-

ingly, a centrosome-nucleus cross talk seems to be

mediated by shuttling centrosomal and nuclear proteins

between these organelles. In particular, during mitosis,

the nuclear proteins p53, Orc2, Orc6 and RAD51 are

required at the centrosome to control its duplication only

once during the cell cycle (30–33). Conversely, the Ran/

importin-dependent shuttle of the centrosomal proteins

centrin-1 and pericentrin/kendrin to the nucleus is pro-

posed to control MT dynamics and nucleation at the

centrosome (34), whereas centrin-2 functions in a complex

with Xeroderma pigmentosum group C protein during

nucleotide excision repair (35). According to this shuttling

model, the centrosome-nucleus juxtaposition may facili-

tate the rapid signaling of regulatory proteins between

these organelles to co-ordinate their accurate duplication

and division. Our data suggest that the physical attach-

ment of centrosome and chromatin co-ordinate their

duplication in a concerted fashion and that Sun-1 parti-

cipates in a complex with further centrosome-specific

proteins of the ONM to establish centrosome-nucleus

juxtaposition in D. discoideum. Thus, loss of centrosome-

nucleus vicinity (coupling) disables the cross talk of these

organelles, thereby causing centrosome and chromosome

instability.

Recent studies revealed that in S. cerevisiae, the SUN

domain protein Mps3 is required as a positive regulator for

the duplication of the spindle pole body (14). In contrast,

C. elegans matefin/Sun-1 has been reported to act as

a suppressor for the centrosome duplication promoting

kinase Zyg-1 (36). However, our findings agree rather with

the negative role of C. elegans matefin/Sun-1 as over-

expression of GFP-DNSun-1 or Sun-1 RNAi induces centro-

some hyperamplification. Furthermore, D. discoideum

Sun-1 may also involve cell cycle regulators to couple the

centrosome and nuclear duplication.

Figure 6: Sun-1 is responsible for maintaining the nuclear shape and the centrosome-nucleus connection. Live cell imaging of

wild-type cells with nuclei visualized by GFP-IntCT and of GFP-DNSun-1 cells. Arrowheads point to highly deformed nuclei and arrows point

to protrusions of the nuclear membrane. Live cell images were acquired every 10 seconds for a total of 15 min 50 seconds.

Representative images with a 50-second time lapse are displayed.

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In addition to their mechanical role in nuclear positioning,

SUN domain proteins are also involved in more diverse

cellular processes, such as organization of the telomeres,

influence on the fat metabolism and germ line maturation

(7,20,37,38). In meiotic cells, however, S. cerevisiae

Mps3, S. pombe Sad1, C. elegans matefin/Sun-1 as well

as mouse Sun-1 may facilitate chromosome pairing (8,10).

This aspect cannot be investigated in D. discoideum as the

laboratory strain AX2 occurs as a haploid organism and

under laboratory conditions does not undergo meiosis.

The fivefold increased cell death in both GFP-DNSun-1 and

Sun-1 RNAi cells as well as the unavailability of a sun-1

knockout strain demonstrate that Sun-1 may play an

essential role for cell viability. The mild extent of centro-

some hyperamplification in Sun-1 RNAi cells may be

because of the residual amount of Sun-1 that may be

sufficient to propagate the centrosome-nucleus connec-

tion and proper spindle formation (data not shown) but

failed to maintain the nuclear and cellular morphology. Our

data demonstrate that Sun-1 may have two functions:

during interphase, it defines the integrity of the NE and

establishes centrosome-nucleus connection by interaction

with chromatin, whereas during mitosis, Sun-1 ensures

the accuracy of chromosome segregation and genome

stability by tethering chromosomes to the centrosome.

Similarly, depletion of C. elegans matefin/Sun-1 was

observed with abnormally condensed chromatin and

enhanced apoptosis (20,39). In contrast to the vertebrate

model, the KASH domain protein interaptin does not

bind to Sun-1 to position the nucleus on F-actin or the

centrosome, although it is likely that interaptin may have

other functions in the NE during the development of

D. discoideum. Yet, the identification of the centrosome

linker of that complex is a future challenge that will provide

more insight into the function of the SUN domain proteins.

Taken together, we propose that Sun-1 forms dimers or

higher oligomers in vivo that are retained in the INM by

binding to chromatin with their N-termini. The Sun-1

C-terminus interacts with an ONM protein and a centro-

some linker in the PNS to define the spacing of the NE

lumen and to ensure the centrosome-nucleus vicinity.

Alternatively, Sun-1 may bind indirectly to interaptin or

other KASH domain proteins to position the nucleus on

the actin cytoskeleton (Figure 7). Truncation of the Sun-1

N-terminus abrogates chromatin binding as well as the

INM localization of GFP-DNSun-1. Low amounts of GFP-

DNSun-1 can be escorted in a complex with the endoge-

nous Sun-1 to the INM, whereas the majority of GFP-

DNSun-1 accumulate in the ONM and counteract the

connection of the centrosome linker with Sun-1, resulting

in the formation of NE protrusions (Figure 7). These NE

protrusions were likely formed because of the mechanical

force emerging between the cytoskeleton and the nucleus

leading to disconnection of the centrosomes from the

nucleus. Subsequently, increase in centrosome-nucleus

distance leads to loss of the centrosome connection

Figure 7: Model of Sun-1 function in the NE of D. discoideum. Sun-1 may form dimers or higher oligomers and interacts with

chromatin to be retained in the INM. The SUN domain of Sun-1 may form a complex with centrosome linker proteins of the ONM (X) in the

PNS to establish the centrosome-nucleus juxtaposition. Sun-1 may connect the nucleus to F-actin by indirect interaction with the interaptin

KASHmotif (PT) through a linker (Y). Sun-1may also bind to further ONMproteins to define the spacing of the PNS. Truncation of the Sun-1

N-terminus abrogates the INM retention of GFP-DNSun-1 that accumulates in the ONM. GFP-DNSun-1 can bypass the connection

between the endogenous Sun-1 and the centrosome linker (X) leading to the formation of NE protrusions. The increase of centrosome-

nucleus distance may ultimately facilitate their disconnection that results in centrosome hyperamplification, chromosome instability and

aneuploidy.

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Xiong et al.

promoting the centrosome hyperamplification and genome

instability such as aneuploidy. Based on this, we propose

that SUN domain proteins will also play an important role

during carcinogenesis in mammalian cells.

Material and Methods

Generation of Sun-1 constructs and D. discoideum

strains usedFor GST-SunCT1, a PCR fragment extending from cDNA position 1032–

1959 (encompassing the central two coiled-coil domains) was generated as

a 59-XmaI and a 39-XhoI fragment that was cloned into the expression

vector pGEX4T2. The GST-SunCT1 polypeptide was expressed in the

Escherichia coli strain XL1-Blue. GFP-DNSun-1 containing the nucleotide

region 853–2718 as a ClaI fragment encoding the aa 283–905 was

generated using the vector pDEX-79 (40). Full-length Sun-1 cDNA was also

cloned into pDEX-79 to generate a GFP fusion for rescue experiments. To

generate a NE marker, a full-length cDNA encoding D. discoideum

nucleoporin Nup43 was cloned into pDEX-79. GFP-IntDPT was generated

by excluding the final six base pairs during PCR amplification using GFP-

IntCT as template that contains the C-terminal transmembrane domain and

part of the tail sequence of interaptin (11). GFP-IntP/A was generated by

site-directed mutagenesis using GFP-IntCT. Selection of D. discoideum

strain AX2 transformants was with G418 (4 mg/mL).

The Sun1 RNAi construct contains the sequence 59-AAGAGCTTAAAC-

TAGTTAAACTT-39 that targets the Sun-1 cDNA at the position 1187–

1209 bp. The target sequence is flanked by complementary sequences that

form a hairpin. Selection of transformants was by blasticidin (3.5 mg/mL).

AX2 was used as host strain for all transformations. Cells expressing

GFP-tagged calreticulin and calnexin as ER markers were obtained from

Dr G. Gerisch (41). The interaptin-deficient strain abpD� was described

previously (11).

Preparation of total cell lysates and intact nucleiCellswerewashed twice with Soerensen phosphate buffer (2 mMNa2HPO4,

14.6 mM KH2PO4, pH 6.0) before resuspending in TMS buffer [50 mM

Tris/HCl, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 250 mM sucrose, 1 mM

ethylenediaminetetraacetic acid (EDTA), 1 mM EGTA, 1 mM DTT, 1 mM

benzamidine, 1 mM phenylmethylsulphonyl fluoride (PMSF)]. Total cell

lysates were obtained by passage through Nuclepore membrane (5 mm

diameter, Whatman) and used in further experiments. Intact nuclei were

collected after lysis through Nuclepore membrane by spinning for 5 min at

4000 g.

Proteinase K protection assayIntact nuclei were isolated from AX2 cells and incubated in proteinase K

protection assay buffer (10 mM Tris/HCl, pH 7.4, 250 mM sucrose, 1 mg/mL

proteinase K) with or without addition of 0.5% Triton-X-100 (v/v) on ice.

Samples were collected from both reactions after 5, 10, 30, 45 min, and the

proteinase K was immediately inactivated by adding PMSF to a final

concentration of 1 mM and heating (958C, 5 min) in sodium dodecyl sulfate

(SDS) sample buffer. The samples were further analyzed by SDS–PAGE

and Western blot.

Chromatin immunoprecipitationAbout 5 � 107 cells per ChIP reaction were harvested and lysed in TMS

buffer (see above). To reduce the viscosity of genomic DNA and disrupt the

NE, total cell lysates were sonified twice with 10 pulses. The appropriate

antibodies were coupled to protein A–Sepharose beads and then incubated

with sonified total cell lysate (2 h on a vertical rotator, 48C). Unspecific

protein and DNA binding were removed by five times washing with PBS

and once with TE buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA) and divided

into two aliquots. One aliquot of each ChIP reaction was used for elution of

DNA by adding 100 mL of TE buffer containing 1% SDS. After phenol/

chloroform extraction and ethanol precipitation, DNA was resuspended in

50 mL water; 5 mL of the DNA was subsequently used for PCR analysis

using actin-8 gene-specific primers. The second aliquot was subjected to

SDS–PAGE and Western blot analysis.

Electromobility shift assay and Southwestern blottingGST-Sun1N400 (aa 1–135) was cloned using EcoRI/NsiI fragment and

GST-Sun1N800 (aa 1–255) using EcoRI/NcoI fragment into pGEX-4T3

(Amersham Pharmacia). Protein purification from E. coli XL1-Blue using

GST-Sepharose beads and elution from the beads was carried out following

user’s manual (Amersham Pharmacia). About 20 or 40 ng of each protein

and GST for control were mixed with 2000 cpm radiolabeled DNA probe

(720 bp D. discoideum hp1-fragment produced by EcoRI digestion of

pGEM-Teasy-Ddhp1) in the EMSA buffer containing 50 mM Tris/HCl, pH

8.0, 0.5 mM EDTA, 50 mM NaCl, 0.1% Triton-X-100. The protein–DNA

binding was carried out for 3 h at room temperature. The samples were

separated on 5% polyacrylamide gels containing 12.5 mM Tris/Cl, pH 8.4,

95 mM glycine and 0.5 mM EDTA. DNA was visualized by autoradiography

of dried gels.

Southwestern analysis of GST-Sun1N400 and GST-Sun1N800 were per-

formed as described (42). Radiolabeled DNA probes (D. discoideum hp1

and actin-8 or human CAP2 gene sequences) were incubated overnight

with the protein membrane at room temperature. After three times

washing with the renaturation buffer, the Southwestern blot was exposed

to an X-ray film.

Metaphase arrest of cell division (karyotyping)Cells were allowed to attach to coverslips for 2 h before adding nocodazole

to a final concentration of 33 mM to the medium. The incubation with

nocodazole was maintained for 4 h to increase the number of cells arrested

in metaphase. The solution was replaced by cold water for 10 min at 48C.Chromosomes were then fixed on the coverslips in ethanol/acetic acid 3/1

(v/v) for 1 h on ice, followed by a change of fresh fixative for an additional

10 min on ice, before proceeding with DAPI staining.

In vitro cross-linking and gel filtration

chromatographyTo address the multimerisation behavior, GST was removed from SunCT1

by thrombin cleavage. Ten micrograms of SunCT1 was incubated at room

temperature in the phosphate potassium buffer (pH 7.4) containing 0.001%

(v/v) glutaraldehyde. The reaction was stopped by addition of glycine to

a final concentration of 0.1 M. After 5, 10 and 20 min, samples were

analyzed by SDS–PAGE (10% acrylamide) andWestern blotting. Native and

chemically cross-linked SunCT1 were also analyzed by gel filtration chro-

matography using a Sephadex G-75 column. About 100-ml fractions were

collected and analyzed by SDS–PAGE followed by Western blotting (43).

Analytical gel filtration employed the SMART system (GE Healthcare). For

calibration, BSA (66 kDa), ovalbumin (43 kDa) and chymotrypsin (25 kDa)

were used.

Indirect immunofluorescence microscopyApproximately 1 � 106 cells were transferred onto a coverslip (10 mm

diameter) and allowed to adhere for 20 min at room temperature. If not

mentioned otherwise, standard immunofluorescence stainings were car-

ried out using ice-cold methanol as fixative (5 min, �208C). Cells were

treated twice for 15 min (room temperature) with blocking solution (1 �PBS containing 0.5% (w/v) BSA and 0.1% (v/v) fish gelatin). The appropriate

antibodies were diluted in the blocking solution and applied for 1 h at room

temperature; the excess of antibodies was removed by washing with the

blocking solution prior to the 1 h of incubation with the according secondary

antibodies.

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For sequential digitonin/Triton-X-100 permeabilization experiments, cells

adhered to the coverslips were fixed by application of 4% (w/v) para-

formaldehyde for 20 min. After three times washing with PBS, cells

were first permeabilized by short incubation with prechilled digitonin

(10 mg/mL in PBS) (5 min, on ice) and washed five times with PBS.

Blocking reaction and incubation with the primary antibodies was

performed as described above. After removal of excess of the primary

antibodies (six washes with PBS), cells were permeabilized by applica-

tion of 0.2% Triton-X-100 in PBS (10 min, room temperature). The

permeabilized sample was blocked before probing with the second

epitope-specific antibodies. The two specific primary antibodies were

finally visualized by simultaneous incubation with the according second-

ary antibodies. Confocal images were acquired with an inverted Leica

TCS-SP laser scanning microscope using �40 and �100 Neofluar oil

immersion objectives.

Primary antibodies used in this study were: mouse monoclonal anti-GFP

antibody (44), rabbit polyclonal anti-GFP antibody (gift from M. Schleicher),

rabbit polyclonal anti-GST antibody (unpublished), mouse monoclonal

anti-interaptin antibody (260-60-10) (11), mouse monoclonal anti-PDI-1

antibody (221-135-1) (45), mouse monoclonal anti-Sun-1 antibody (K55-

432-2, Western blot analysis, this study), mouse monoclonal anti-Sun-1

antibody (K55-460-1, immunofluorescence, this study), mouse mono-

clonal antitubulin antibody (K29-359-31) that was generated against

a tubulin fraction from the green alga Spermatozopsis similis and which

recognizes the D. discoideum centrosome (gift from Dr K. Herkner and

Dr M. Melkonian), mouse monoclonal anti-a-tubulin antibody (46), rat

monoclonal anti-a-tubulin antibody (YL1/2) (47). The appropriate second-

ary antibodies were: Cy3-conjugated goat anti-mouse immunoglobulin G

(IgG) (Sigma); Cy5-conjugated goat anti-mouse IgG (Sigma), Alexa 568-

conjugated goat anti-mouse IgG (Molecular Probes), Alexa 568-conjugated

goat anti-rat IgG (Molecular Probes). DNA was stained with DAPI or ToPro-3

(Invitrogen).

Electron microscopyNuclei of GFP-DNSun-1 cells were isolated using Nuclepore membranes

(5 mm; Corning). The cells were washed twice in cold PBS and then

resuspended in Dicty-PHEM (48) containing a protease inhibitor cocktail and

PMSF. They were pressed through the filter assembly three to five times.

The resulting mixture was layered onto a 30% sucrose cushion and

centrifuged at 2200 � g for 10 min at 48C. The pellet containing mainly

nuclei was resuspended in Dicty-PHEM and centrifuged at 2200 � g for

5 min onto 12-mm coverslips.

The nuclei were fixed with 2% formaldehyde in Dicty-PHEM for 15 min.

They were washed and treated with rabbit anti-GFP antibody (P. A. Silver,

Harvard) for 45 min and with 5 nm PAG-Gold (Department of Cell Biology,

Utrecht University) overnight. Alternatively, mouse monoclonal anti-GFP

antibody (J. Wehland, Braunschweig) was detected using 6-nm goat anti-

mouse antibody (Aurion, Biotrend). Dehydration and embedment were

carried out according to standard procedures.

The nuclei were sectioned using a Reichert Ultracut E and viewed in a JEOL

1200C equipped with a TEM camera Keenview-10/12 and iTEM imaging

system (Soft Imaging System).

Acknowledgments

We thank Rolf Muller for help throughout the course of this work. We

thank Drs M. Melkonian and K. Herkner for providing a centrosome-

specific antibody, V. Peche for CAP2 cDNA, Dr G. Gerisch for cells

expressing GFP-tagged calreticulin and calnexin and Sabrina Rosenbaum

for help with CD spectroscopy. H. X. was a member of the International

Graduate School in Genetics and Functional Genomics of the University

of Cologne.

Supplementary Materials

Figure S1: Colocalization of ER markers and a nuclear pore complex

protein with Sun-1 at the NE. Cells expressing GFP-tagged calnexin,

calreticulin and Nup43 were stained with mAb K55-460-1 for Sun-1. Cells

expressing GFP-Sun-1 were stained with PDI-specific antibodies. Nuclei

were stained with DAPI. Bar ¼ 10 mm.

Figure S2: Sun-1 N-terminus interacts directly with DNA. Non-induced

(�) and induced (þ) bacterial lysates containing the GST-Sun1N400, GST-

Sun1N800 and GST-Sun1CT were subjected to Southwestern blotting. The

blots were probed with radiolabeled human CAP2 cDNA or D. discoideum

actin-8 DNA fragment. The corresponding Coomassie Blue stained gel is

shown. The location of the GST-fusion proteins is indicated (box).

Figure S3: Circular dichroism (CD) spectrum of bacterially expressed

SUNCT1. Recording was performed at 208C in 20 mM Tris/HCl (pH 8.0) and

50 mM NaCl using a Jobin Yvon Dichrograph Mark IV (Instruments S. A.).

Figure S4: Expression of GFP-tagged full-length Sun-1 in Sun-1 RNAi

and AX2 cells. A) Analysis of centrosome detachment in various strains. B)

Upper panel, GFP-SUN-1 colocalizes with the endogenous protein at the NE

in AX2 transformants. Lower panel, GFP-Sun-1 induces detachment of the

centrosome in AX2 cells. K55-460-1 recognizes Sun-1, K29-356-31 the

centrosome. C) Sequential permeabilization experiments show that GFP-

Sun-1 is detectable at the ONM in AX2.

Figure S5: GFP-DNSun-1 cells exhibit various defective mitotic spin-

dles. (A9 and A0) GFP-DNSun-1 cells formmonopolar spindles, although two

centrosomes were present in one cell. (B9 and B0) Multipolar spindles may

represent a consequence of centrosome hyperamplification that cause the

formation of a random network of spindle MTs instead of a bipolar spindle.

(C9 and C0) Bipolar spindles were found to carry out an asymmetric nuclear

division in which two nuclei were formed but were attached to the same

centrosome. Mitotic spindles were stained using mouse a-tubulin antibody

(red), DNA using DAPI.

Supplemental materials are available as part of the online article at http://

www.blackwell-synergy.com

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