The Nuclear Envelope During Mitosis

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Breaking down the wall: the nuclear envelope during mitosis

1 1,2

Christine J Smoyer and Sue L Jaspersen

A deﬁning feature of eukaryotic cells is the nucleus, which assemble an intranuclear spindle. As we discuss below,

houses the genome inside the nuclear envelope (NE): a double eukaryotic cells utilize both strategies, as well as vari-

lipid bilayer that separates the nuclear and cytoplasmic ations in between (Figure 1), offering glimpses into how

materials. Although the NE is commonly viewed as a barrier that the nucleus evolved as an organelle to organize and

is overcome only by embedded nuclear pore complexes protect the genome and why mutations in NE proteins

(NPCs) that facilitate nuclear-cytoplasmic trafﬁcking, recent result in a broad spectrum of human diseases, including

work in a wide range of eukaryotes reveals that the NE is a cancer [1,2].

dynamic organelle that is modiﬁed each time the cell divides to

ultimately establish two functional daughter nuclei. Here, we The nuclear envelope

review how studies of divergent mitotic strategies have helped Although multiple theories exist on how the eukaryotic

elucidate common properties of NE biology that allow it to nucleus arose during evolution, most share the idea that

function throughout the cell cycle. the NE and its associated proteins have ancient roots in

Addresses division of the genome [1,3]. The nucleus likely co-

Stowers Institute for Medical Research, Kansas City, MO 64110, United evolved with other parts of the endomembrane system.

States

2 However, unlike other cellular membranes, the NE is a

Department of Molecular and Integrative Physiology, University of

double lipid bilayer composed of an outer (ONM) and

Kansas Medical Center, Kansas City, KS 66160, United States

inner membrane (INM) separated by a perinuclear space.

Corresponding author: Jaspersen, Sue L ([email protected]) The ONM is contiguous with the ER and is thought to

contain many of the same proteins and lipids. In contrast,

the INM is distinct—proteomic analyses suggest that it

Current Opinion in Cell Biology 2014, 26:1–9

contains at least 100 unique components and may contain

This review comes from a themed issue on Cell architecture

over 1000 proteins, many of which interact with chromo-

Edited by Sue Biggins and Matthew D Welch somes and/or the nuclear lamina [4,5,6].

For a complete overview see the Issue and the Editorial

The NE is typically regarded as a hydrophobic barrier:

Available online 4th September 2013

transport of macromolecules into and out of the nucleus

0955-0674/$ – see front matter, # 2013 The Authors. Published by

occurs through nuclear pore complexes (NPCs) that are

Elsevier Ltd. This is an open access article under the CC BY-NC-SA

embedded in the NE at sites where the ONM and INM

license (http://creativecommons.org/licenses/by-nc-sa/3.0/.

are contiguous (reviewed in [7,8]). The NPC has often

http://dx.doi.org/10.1016/j.ceb.2013.08.002

been considered to be the sole route for crossing the NE;

however, a recent study found that large ribonucleopro-

tein particles involved in Wnt signaling at the neuromus-

Introduction cular junction of Drosophila larvae cross the NE by a

During cell division, genetic material and cytoplasmic vesicle budding and fusion pathway that is NPC-inde-

components are partitioned into two daughter cells. In pendent [9 ]. This pathway, in which a vesicle buds from

eukaryotic cells, the DNA is segregated away from the the INM and then docks and fuses with the ONM, is

cytoplasm by the nuclear envelope (NE). After DNA is reminiscent of the nuclear egress mechanism used by

duplicated, it must be physically separated, which is certain virus particles to escape the NE (reviewed in

facilitated by a microtubule-based structure known as [10,11]). It is unclear if vesicle-mediated transport can

the mitotic spindle. Key regulators of microtubule nuclea- be used for nuclear entry as well as for nuclear export or if

tion, including tubulin, microtubule-associated proteins it is a widely used mechanism of nuclear-cytoplasmic

and the microtubule-organizing center (MTOC), are pre- trafﬁcking, but it is tantalizing to speculate that there

dominately localized in the cytoplasm of interphase cells. are multiple routes across the NE border.

In order to allow microtubules access to chromosomes,

the NE must either become permeable, or tubulin and

The budding and ﬁssion yeast closed mitosis

microtubule-associated proteins need to be imported into Although the NE barrier is advantageous in terms of

the nucleus and the MTOC inserted into the NE to

regulation of transcription, translation and DNA repli-

cation, it poses a unique challenge during mitosis: how

§ to distribute chromosomes inside the nucleus into the

This is an open-access article distributed under the terms of the

two daughter cells using the cytoplasmic microtubule

Creative Commons Attribution-NonCommercial-ShareAlike License,

which permits non-commercial use, distribution, and reproduction in cytoskeleton. Some organisms, such as the budding

any medium, provided the original author and source are credited. yeast Saccharomyces cerevisiae and the ﬁssion yeast

www.sciencedirect.com Current Opinion in Cell Biology 2014, 26:1–9

2 Cell architecture

Figure 1

interphase mitosis

open

semi-open

closed

closed-rupture

Current Opinion in Cell Biology

Mitotic strategies used in eukaryotes. Shown are interphase (left) and mitotic (right) cells undergoing open, semi-open and closed mitosis as well as the

closed mitosis of S. japonicus in which the NE ruptures during anaphase. MTOCs are depicted in purple, chromosomes in blue, microtubules in black

and membranes in white.

Schizzosaccharomyces pombe, undergo a closed mitosis Cytological and molecular analysis of the SPB in

where the NE does not break down (Figure 1). In budding yeast suggests that its assembly is similar to

order to facilitate the organization of chromosomes de novo assembly of NPCs in all eukaryotes. Both

on the mitotic spindle, the MTOC, known as the SPBs and NPCs are assembled as subcomplexes in

spindle pole body (SPB), is anchored in the NE during the cytoplasm and nucleoplasm; NE remodeling and

all or part of the cell cycle [12–14]. As we discuss below, stabilization are required to allow these complexes to

despite structural differences between the fungal SPB associate in the NE to create an intact and

and the metazoan centrosome, analysis of SPB dupli- functional SPB or NPC (Figure 2) (reviewed in

cation and tethering in the NE in both budding and [7,15]). Because SPB insertion into the NE occurs at

ﬁssion yeast has led to important insights into NE a speciﬁc time (late G1 phase) and place (adjacent to

biology. the existing SPB), it is a useful model for analysis of

Current Opinion in Cell Biology 2014, 26:1–9 www.sciencedirect.com

Nuclear envelope during mitosis Smoyer and Jaspersen 3

Figure 2 Figure 3

ONM reticulons/Yop1 Poms

INM ONM

approximation

INM

hemifusion

fusion pore membrane ALPS proteins

insertion

Current Opinion in Cell Biology

Molecular mechanism of pore membrane formation. It is thought that the

INM and ONM approximation is achieved by lumenal interactions or

pore membrane proteins (Poms), which may also recruit other

NPC SPB

membrane remodeling enzymes such as the reticulons/Yop1 that insert

Current Opinion in Cell Biology

into the outer leaflet to induce membrane curvature. SUN proteins on the

INM have been proposed to result in localized changes in membrane

composition to facilitate membrane curvature, which is then stabilized at

The pore membrane. Depicted are the double lipid bilayers of the ONM

least in part by ALPS proteins, such as Nbp1.

and INM. Formation of the pore membrane is thought to begin by

approximation of the inner membrane leaflets, which fuse to form a

hemifused membrane structure. The outer leaflets then fuse, and the

pore membrane is created. Further expansion of the pore allows for

assembly of a NPC or SPB into the NE. Both complexes are anchored in the NPC assembly defects seen may be due to titration of

the pore membrane by integral membrane proteins, which appear as

proteins away from the NPC to the SPB [23].

hooks in the electron microscope.

Converting a lipid bilayer to a curved structure requires

specialized proteins that speciﬁcally act on the membrane

events at the NE required for complex insertion to induce curvature. Reticulons and the related Yop1/

(reviewed in [15,16]). DP1 are ER membrane bending proteins that are thought

to induce membrane curvature by insertion into the outer

Examination of NE structure by electron microscopy and leaﬂet of the membrane (Figure 3) [7,24]. Previous work

tomography shows that SPBs, like NPCs, are located in in yeast and Xenopus illustrated a role for the reticulons in

pores: regions of the NE where the ONM and INM are NPC assembly, most likely through the creation of a NE

contiguous [12,17,18]. Localized changes in the NE are pore, although other models have also been proposed

thought to allow transition from a lipid bilayer to a curved [25,26]. Further analysis of S. cerevisiae cells lacking both

pore membrane for insertion of SPBs and NPCs. The RTN1 (reticulon) and YOP1 revealed a defect in SPB

connection between membrane remodeling events integrity, including an inability to tether the SPB in the

required for SPB insertion and NPC assembly was origin- NE [27 ]. Rtn1 and Yop1 physically interact with Ndc1,

ally proposed by Chial and colleagues following charac- pointing to a model in which Ndc1 recruits membrane-

terization of Ndc1, an evolutionarily conserved integral remodeling factors to the site of SPB and/or NPC assem-

membrane protein that is a component of both complexes bly [27 ].

[19]. Mutations in NDC1 result primarily in SPB dupli-

cation defects, although some alleles, as well as NDC1 Ndc1 may recruit additional factors to the SPB to form the

depletion, display defects in NPC assembly [19–22]. It is pore membrane. In addition to binding to reticulons, it

unclear if Ndc1 function is essential for NPC assembly or also associates with Nbp1, which contains an ALPS motif

is partially redundant with other pore membrane proteins; (for ArfGAP1 lipid packing sensor) [28 ]. This domain,

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4 Cell architecture

Figure 4

which is also present in several NPC components, forms a

hydrophobic patch that allows for association with lipid

tails, which are unavailable in a planar membrane but ONM

partially exposed in a highly curved membrane [29–31]. INM

Binding of ALPS domain-containing proteins is thought

to form a coat-like structure, stabilizing the membrane

and serving as a docking site for additional proteins ONM

(Figure 3) [8,29,30]. Consistent with this idea, depletion INM

of NBP1 resulted in SPB insertion and assembly defects.

Curiously, Nbp1 shows no speciﬁcity for planar or curved

membranes in vitro. However, Nbp1’s structure may ONM

impose a level of regulation; adjacent to the Nbp1 ALPS INM

motif is a nuclear localization sequence that may be

obscured until it has been transported to the nucleus ONM

and the bound karyopherin removed [28 ]. This interest- INM

ing twist to regulation of NE remodeling may explain how

the INM as well as the ONM are remodeled during SPB

assembly.

Although ﬁssion yeast also undergo a closed mitosis, their

SPB bears little structural similarity to that of budding

yeast. Moreover, the S. pombe SPB is predominately

Current Opinion in Cell Biology

cytoplasmic during most of the cell cycle, residing on

the surface of the NE until mitosis when it is enveloped

Insertion of the S. pombe SPB into the NE. The SPB is associated with

by the NE (Figures 1 and 4) [14,32]. After completion of

the NE during interphase with a specialized region of the INM that likely

anaphase, the SPBs are extruded from the NE and the

contains Sad1. Late in G2, the SPB duplicates to form two SPBs

pore membrane is sealed. A genome-wide comparison of connected by a bridge. When the cell enters mitosis, electron dense

genetic interactions in budding and ﬁssion yeast contain- material in a NE fenestra appears before SPB insertion into the NE by an

unknown mechanism. The two SPBs separate to form the poles of the

ing mutations or deletions of SPB components revealed

spindle. At the end of mitosis, the fenestra close, pushing SPBs back

differences in their biology as well. In ﬁssion yeast,

into the cytoplasm.

deletion of the ESCRT (endosomal sorting complex

required for transport) components Vps4 or Vps32 led

to synthetic lethality with SPB mutants; ESCRT mutants

also contain multiple foci of the SPB component Cut12- homology) family of INM proteins [39,40]. SUN proteins

CFP, which is consistent with defects in SPB duplication span the INM, interacting with chromatin and/or nuclear

or stability [33 ]. Importantly, these ﬁndings are consist- lamina inside the nucleus while binding to ONM proteins

ent with data from HeLa cells showing that human in the perinuclear space, forming a bridge across the NE

ESCRTIII and Vps4 are required for centrosome main- (reviewed in [41,42]). Often, the ONM binding partner

tenance [34 ]. In this way, the closed mitosis of S. pombe contains a poorly conserved motif known as the KASH

exhibits some similarity to the open mitosis of higher domain (for Klarsich-ANC-1-Syne-1 homology).

eukaryotes and offers another area for researching the

ESCRT complex and its role in centrosome-dependent A yeast two-hybrid screen using Sad1 as bait revealed

NE remodeling. interactions not only with KASH proteins, but also other

NE proteins, including factors involved in lipid remodel-

It is also unclear if microtubule nucleation from the S. ing (Figure 2) [43]. Evidence from budding yeast is also

pombe SPB is required for mitosis. Analysis of ﬁssion yeast consistent with the idea that SUN proteins may play a role

grown in the presence of the microtubule depolymerizing in lipid homeostasis: speciﬁc alleles of MPS3, which

drug carbendazim revealed that the NE itself is sufﬁcient encodes the sole SUN protein in budding yeast, show

to drive chromosome segregation in the absence of micro- changes in lipid composition and/or overproliferation of

tubules [35]. This mitosis resembles bacterial chromo- the INM that result in defects in SPB insertion

some segregation and ﬁssion, requiring insertion of the [44 ,45,46 ]. Several mps3 mutants also exacerbate the

SPB into the NE and the conserved INM protein Ima1/ growth defect of spo7D and/or nem1D mutants, which

Net5 [36–38]. It is possible that SPB insertion may simply form a NE ‘ﬂare’ (a region of the membrane devoid of

tether the genome to the NE, and thus reﬂect the ancient underlying chromatin that protrudes in the cytoplasm and

origins of NE proteins in partitioning of the genome. likely impedes nuclear division) due to mis-regulation of

Tethering of the SPB to the NE requires Sad1, the Pah1/lipin, the phosphatide (PA) phosphatase that con-

founding member of the SUN (for Sad1-UNC-84 trols the conversion of PA to diacylglycerol [47–52]. Mps3

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Nuclear envelope during mitosis Smoyer and Jaspersen 5

is not the only SUN protein implicated in membrane precursor cells the KASH protein UNC-83 and the SUN

homeostasis; mutations in C. elegans UNC-84 are reported protein UNC-84 are important for microtubule tethering

to decrease fat levels [53]. Although considerable work is in a centrosome-independent pathway [56,71]. In cul-

needed to validate this model, this novel function for tured mammalian neurons, glial cells or kerotinocytes,

SUN proteins could explain their requirement for de novo Sun1/2 and the KASH proteins Syne/Nesprin-1/2 make

assembly of NPCs in metazoans [54]. A study of inter- the connection between the centrosome and the nucleus,

phase NPC assembly showed that the pore membrane while centrosome tethering in human aortic endothelial

protein Pom121 and Sun1 collaborate in the early steps of cells is dependent on nesprin-3 and intermediate ﬁla-

NPC assembly by driving juxtaposition of the INM and ments [58,69,72–74]. However, studies in HeLa cells

ONM [55]. It might also be linked to NE changes before reveal a dependence on nucleoporins for centrosome

mitosis that occur in the vicinity of centrosomes, which tethering at the NE while reports from human and murine

are tethered at the NE via SUN/KASH proteins in many ﬁbroblast investigations have shown the INM protein

cell types [56–58,59 ]. emerin mediates the centrosome-NE attachment through

an interaction with tubulin [69 ,75,76]. Importantly, the

Another set of proteins functions in NE remodeling at the Nup133-mediated centrosome tethering occurs by a

ﬁssion yeast SPB; the integral membrane proteins Brr6 dynein and RanBP2-Bicaudal D2 independent mechan-

and Apq12 are required for insertion and extrusion of the ism, suggesting that the NPC may directly associate with

SPB [60 ]. In budding yeast, Brr6 and its paralog Brl1, as certain spindle and/or centrosomal proteins [69 ].

well as Apq12 are important for NPC assembly possibly

through regulation of membrane composition [61–63]. Tethering of the centrosome to the NE may serve

While it is possible that Brr6 and Apq12 perform different multiple purposes. First, the NE provides a force to

tasks in both yeasts, a more likely scenario is that a defect counteract microtubules, allowing the centrosomes to

in SPB tethering was overlooked in S. cerevisiae (see [23]). separate across its surface before NEBD. Disruption of

How these proteins effect NE remodeling is unknown. the centrosome-NE tethering delays or inhibits formation

Interestingly, they are absent from the genomes of of a bipolar spindle; for example, in cultured human cells,

metazoans and plants, possibly suggesting that their role disturbing Nup133-mediated or dynein-mediated centro-

in NE remodeling is speciﬁc to closed mitotic systems some tethering delays spindle positioning and assembly,

[60 ]. resulting in abnormal spindles and multinucleated cells

[69 ,70]. Furthermore, the centrosome may dictate the

Centrosomes and nuclear envelope site of NEBD. The Go¨nczy lab demonstrated a local loss

breakdown of nucleoporin NPP-3/Nup205 from the NE at mitotic

In contrast to yeast, higher eukaryotes employ an open entry that was speciﬁcally limited to the vicinity of the

mitotic system where disassembly of the NE, a process centrosomes [59 ]. The loss of NPP-3 appears to be an

referred to as nuclear envelope breakdown (NEBD), early event in NEBD, as the nuclear lamina had not yet

occurs during prophase (Figure 1) (reviewed in disassembled. The centrosome may serve as a regional

[64,65]). NEBD is initiated through NPC disassembly, hub to direct remodeling of the NE. This may include

beginning with the loss of the peripheral nucleoporin recruitment of membrane remodeling factors and lipid

Nup98. NE disassembly is facilitated by two mechan- enzymes. Just as the NPC and SPB insertion required the

isms: breakdown of the nuclear lamina and physical reticulons in lower eukaryotes, evidence from C. elegans

tearing of the membrane by the microtubule cytoskeleton embryos illustrates a requirement for the reticulons YOP-

[66]. 1 and RET-1, along with the GTPase RAB-5, in mitotic

NE and ER remodeling [77]. Additionally, the Pah1

Although centrosomes are not inserted in the NE, cen- homolog in C. elegans, Lpin-1, also plays an essential role

trosomes are tethered to the nucleus by multiple mech- in NEBD: lpin-1 RNAi resulted in defects in NEBD and

anisms. By disrupting this connection, a series of recent reassembly [78 ,79]. Specialized proteins in NE and lipid

studies has elegantly shown that association of the cen- remodeling are clearly essential in NEBD, as well as NE

trosome with the NE is important not only for NEBD but reassembly, although how these proteins may also affect

also for rapid assembly of a spindle and chromosome the mitotic centrosome is still unclear.

biorientation. In most cells, centrosome tethering relies

on dynein localization to the NE [56–58,67–70]. Multiple Open or closed? The in between

lines of evidence linking the SUN/KASH proteins to Variations to open or closed mitosis exist in many forms,

centrosome tethering come from different organisms, some more similar to the open type and vice versa.

including C. elegans. Developmental differences in Although the NE remains intact during mitosis in Asper-

anchoring to the microtubule cytoskeleton can also be gillus nidulans, the A. nidulans NPC partially disassembles,

seen in this organism: in embryos, the KASH protein allowing diffusion of proteins through a core NPC, com-

ZYG-12 is required for localization of dynein and centro- posed of the AnNup84-120 complex. This permeability

some tethering via SUN-1, but in epidermal hypodermal of the NE leads to a semi-open class of mitosis, since the

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6 Cell architecture

NE is still intact but the NPCs no longer restrict transport References and recommended reading

Papers of particular interest, published within the period of review,

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[80]. Lin Liu et al. suggested that the AnNup84-120

complex plays a role at the mitotic NE that partially of special interest

of outstanding interest

overlaps with AnNdc1; while the three pore membrane

proteins were dispensable in A. nidulans, deletion of a

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www.sciencedirect.com Current Opinion in Cell Biology 2014, 26:1–9