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Biochemical Society Transactions (2011) Volume 39, part 6
Microtubule-associated nuclear envelope proteins
in interphase and mitosis
Ricardo A. Figueroa*, Santhosh Gudise*† and Einar Hallberg*1
*Department of Neurochemistry, Stockholm University, SE-106 91 Stockholm, Sweden and †Department of Biosciences and Nutrition, Karolinska Institute
(NOVUM), SE-141 89 Huddinge, Sweden
Abstract
The LINC (linker of nucleoskeleton and cytoskeleton) complex forms a transcisternal bridge across the NE
(nuclear envelope) that connects the cytoskeleton with the nuclear interior. This enables some proteins of the
NE to communicate with the centrosome and the microtubule cytoskeleton. The position of the centrosome
relative to the NE is of vital importance for many cell functions, such as cell migration and division, and
centrosomal dislocation is a frequent phenotype in laminopathic disorders. Also in mitosis, a small group
of transmembrane NE proteins associate with microtubules when they concentrate in a specific membrane
domain associated with the mitotic spindle. The present review discusses structural and functional aspects
of microtubule association with NE proteins and how this association may be maintained over the cell cycle.
Introduction
The nucleus is enclosed by the NE (nuclear envelope), which
is an extension of the endomembrane system (for a review,
see [1,2]). The NE consists of two concentric membranes
separated by the PNS (perinuclear space). The membranes
are perforated by nuclear pores joining the two membranes.
The pores in turn are occupied by the NPCs (nuclear pore
complexes), enabling selective nucleocytoplasmic transport
of macromolecules. The NPC also allows transport of
integral membrane proteins between the ONM (outer nuclear
membrane) and the INM (inner nuclear membrane) [3–5].
The ONM is continuous with the rough ER (endoplasmic
reticulum) and the PNS in turn forms a continuum with the
lumen of the ER. In higher eukaryotes, the proteins of the
INM connect to the nuclear lamina, an intermediate filament
structure that gives the nucleus physical rigidity and protects
against mechanical DNA damage.
The endomembrane system is well known to interact with
the cytoskeleton, and it has long been known that the ER
closely follows the microtubule network (for a recent review,
see [6]). In addition, the Golgi apparatus is organized by
the microtubules [7] and often localizes in close proximity
to the centrosome. This interaction is strikingly apparent
during mitosis, when the genetic material has to be properly
segregated. At the same time, the endomembrane system is
also segregated. In fact, the Golgi apparatus is a licensing
factor for mitotic progression [8].
Key words: centrosome, integral inner nuclear membrane protein, linker of nucleoskeleton and
cytoskeleton complex (LINC complex), mitosis, nuclear envelope, spindle endomembrane.
Abbreviations used: ER, endoplasmic reticulum; INM, inner nuclear membrane; KASH,
Klarsicht/ANC-1/SYNE homology; LINC, linker of nucleoskeleton and cytoskeleton; MNC,
microtubule-nucleating centre; NE, nuclear envelope; NEBD, nuclear envelope breakdown; NPC,
nuclear pore complex; ONM, outer nuclear membrane; PNS, perinuclear space; SE, spindle
endomembrane.
1
To whom correspondence should be addressed (email [email protected]).
C The
C 2011 Biochemical Society
Authors Journal compilation In mitosis of higher eukaryotes, the defining step
separating prophase from metaphase is NEBD (nuclear
envelope breakdown). During NEBD, the NE ruptures due
to the minus-end-directed pulling of centrosomes combined
with loss of interactions between the NE and structural
components of the nucleus [1,9]. According to the traditional
view, this rupture is suggested to allow the NE to be efficiently
resorbed into the ER, leaving the mitotic machinery devoid
of membranes and accessible for the required mitotic
components. In lower eukaryotes, mitosis is a closed process,
and NEBD does not occur, although partial rearrangement
of the NPC are known to occur [10,11].
Given the multitude of interactions between the endomembrane system and the cytoskeleton, it is perhaps not surprising
that nucleoplasmically localized NE proteins also interact
with the cytoskeleton during interphase. These interactions
are mediated by the recently discovered family of LINC
(linker of nucleoskeleton and cytoskeleton) complexes [12–
14]. The LINC complex consists of SUN domain-containing
integral membrane proteins situated in the INM and KASH
(Klarsicht/ANC-1/SYNE homology) domain containing
transmembrane proteins (in humans termed nesprins) in the
ONM. A physical link forms by the interaction of the SUN
domain with the KASH domain in the PNS. This mechanical
link between the INM and the ONM is extended on the
cytoplasmic side by interactions with the cytoskeleton and
on the nucleoplasmic side by interactions with other INM
proteins and the nuclear lamina. The LINC complexes have
been shown to be involved in vital processes, such as cell
migration and division.
It has been shown that centrosome attachment to nuclei
is mediated by adaptor-dependent interactions between
microtubules and nesprins and is affected by the presence
of other NE proteins. Emerin [15], lamin A/C [16], nesprin
1 and 2 [12–14] and Samp1 [17] (Figure 1) have been
shown to be essential for the proximal localization of the
Biochem. Soc. Trans. (2011) 39, 1786–1789; doi:10.1042/BST20110680
Nuclear Envelope Disease and Chromatin Organization
Figure 1 Detachment of centrosome from the NE after
post-transcriptional silencing of Samp1
Phase-contrast micrographs of HeLa cells treated with control siRNA
(small interfering RNA) (a) and specific siRNA against Samp1 (b–d).
Centrosomes are marked with white asterisks. Note the increased
distance between the centrosome and the nuclear boundary after
silencing of Samp1 expression. Scale bar, 10 μm.
of SUN2 and nesprin2 assembled on actin cables, during
nuclear migration in polarized fibroblasts.
The LINC complex can vary in its composition, allowing
for a multitude of interactions with cytoskeletal and
nucleoskeletal components. In humans, to date, four nesprins
with several isoforms have been characterized, allowing
for interactions with microtubules, actin and intermediate
filaments [12–14]. Nucleoskeletal interactions of the LINC
complex, in turn, depend on the identity of the different SUN
proteins, of which five have been characterized in humans to
date. Furthermore, NE proteins (e.g. lamin proteins, emerin
or Samp1) and proteins of the PNS (e.g. torsin A) may also
influence LINC complex composition. Full understanding
of NE protein patterning and its functional significance
may have to include studies of all physiologically relevant
permutations of the LINC complex at nanoscopic resolution.
Microtubule-associated NE proteins in
mitosis
centrosome to the NE, suggesting that these proteins are part
of a common functional network. Centrosome dislocation is
observed in Emery–Dreifuss muscular dystrophy, suggesting
a disease mechanism involving aspects of microtubule–NE
interaction [15]. In addition, the cytoskeleton is suggested
to be of importance for chromosome organization [18] and
INM proteins have been shown to affect radial positioning
of chromosomes [19]. Furthermore, mechanosensation and
the tensegral structure of the cytoskeleton are suggested
to depend on the interactions between the NE and the
cytoskeleton [20–22].
Protein networks in the NE in interphase
The relatively small number of studies of NE protein
distribution that have been performed using high-resolution
fluorescence microscopy show that INM proteins are not
uniformly distributed, but rather form distinct patterns with
variable degrees of overlap [23–26]. A- and B-type lamins
form separate, but interconnected, networks [24]. SUN1 and
SUN2 are partially co-localized and partially form separate
networks [25,26], where a significant population of SUN1 colocalized with nuclear pores. In HeLa cells, Samp1 displayed
a low degree of overlap with a number of NE markers, but
a partial co-localization with SUN1 [23]. Because relative
nuclear pore positions as well as bleach marks in the nuclear
lamina were maintained despite elastic deformations of the
NE observed in live cells [27], the NE protein distribution
pattern is likely to be relatively stable and may serve as
anchoring sites for the cytoskeleton. Support for this idea
was recently provided by Luxton et al. [28], using live-cell
imaging to show concerted retrograde movements of TAN
(transmembrane actin-associated nuclear) lines, linear arrays
Recently, transmembrane NE proteins have been shown
to associate with microtubules of the mitotic spindle
[17,29]. Using specific antibodies and expression of chimaeric
fusion protein, we have demonstrated the existence of
a novel membrane domain associated with the mitotic
spindle containing the integral INM protein Samp1 [17]
(Figure 2). We term this membrane domain the SE (spindle
endomembrane). The existence of membranes along the
spindle microtubules was totally unexpected in view of
the traditional model where the metazoan mitosis is
completely open and the spindle devoid of membranes.
More recently, the existence of SEs attained further support
when Lu et al. [30] were able to detect GFP (green
fluorescent protein)-tagged integral ER proteins along
spindle microtubules. These transmembrane ER proteins did
not accumulate at the SE, but rather were present relative
to their concentrations in the ER. Very recently, two novel
integral INM proteins, WFS1 (Wolfram syndrome 1) and
Tmem214 (transmembrane protein 214), were shown to be
able to concentrate to the SE [29], like Samp1 [17].
The SE has to be accounted for when we view the evolution
of the nuclear membrane and mitosis in metazoan cells and
raises questions concerning the function of the SE and its
relationship to other spindle components. The existence of
an elusive structure known as the ‘spindle matrix’ in the
mitotic spindle has been suggested by several different groups
(reviewed in [31]). The spindle matrix is a non-microtubule
matrix-like structure located in the central spindle region,
but absent from the subcentrosomal region. The spindle
matrix is envisioned to contribute in force generation and
stability during chromosome segregation and/or recruitment
of motor proteins and SAFs (spindle-assembly factors). The
spindle matrix and the SE are two different structures that
may have different functions in the mitotic process, and
it is possible that the SE may serve as a scaffold for the
spindle matrix. It is also well established that components of
the nucleocytoplasmic transport system have been found to
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Biochemical Society Transactions (2011) Volume 39, part 6
Figure 2 The SE in metaphase
Figure 3 Hypothetical models of SE organization
Fixed and permeabilized HeLa cells were immunostained using
antibodies specific for Samp1 (green) and tubulin (red). A confocal optical
(A) Closed mitosis in lower eukaryotes with intact NE and the
microtubule-organizing centre (MTOC) (green circle) attaching to the
slice shows the distribution of the SE in relation to spindle microtubules.
Scale bar, 10 μm.
MNCs (grey circle) on the NE. (B) Mitosis in higher eukaryotes. Either
the SE (purple) can be organized as tubular membrane protrusions
(a) associating to kinetochore microtubules (green), which are naked
in traditional models of open mitosis (b), or the SE can enclose the
microtubules (c), thereby conserving the topology of closed mitosis (A).
In the latter case, MNCs (grey circle) are situated on the tips of SE fingers.
localize to the mitotic spindle. For example, the nucleoporins
Nup98/Rae1, RanBP2 (Ran-binding protein 2), the Nup107–
160 complex and nucleocytoplasmic transport receptors
have been shown to play important role in assembly and
maturation of the mitotic spindle [32–34]. Many questions
remain concerning the function and origin of the SE.
It is possible that the SE is formed from the collapsing NE
during NEBD and that the SE is one of the first membranes
to come in contact with chromosomes of the daughter
nuclei during post-mitotic reformation of their NEs. The
SE may thus be retained to ensure the proper partition of
NE membranes and proteins to the daughter nuclei. Another
possibility, as hinted above, would be conservation of some
aspects of the functional protein networks of the interphase
NE in order to transfer some structural information across
the mitotic process. This may perhaps aid in the organization
of chromatin and ensure the propagation of architectural
epigenetic cues [35].
In order to seek an answer to the question of a function of
the SE and how it is organized along the spindle microtubules,
let us consider the evolution of the eukaryotic cell and
the processes of mitosis and cytokinesis. Eukaryogenesis
is still an unresolved mystery. For an extensive review of
the origin of the nucleus and mitosis, see [36]. During the
evolution of eukaryotes, novel methods to properly segregate
a membrane-enclosed genome had to be formed, but also
ways to ensure the proper segregation of various organelles
and division of the endomembrane system. Most probably,
a system for the segregation of the endomembrane system
preceded the development of a nucleus. In early eukaryotes
and in lower eukaryotes today (Figure 3A), the NE remains
intact during mitosis and segregation depends on association
of the genome to intranuclear spindles emanating from
MNCs (microtubule-nucleating centres). The MNCs, in
turn, are attached to the cytoplasmic microtubule network
emanating from the centrosomes. There are several variations
C The
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Authors Journal compilation on the mitotic system present today [10], one being open
mitosis that developed in metazoa.
With the discovery of the SE, some steps in the process
of mitosis in higher eukaryotes might have to be revised.
At the moment, it is not known how the SE is organized
(Figure 3B). In the present review, we consider two major
possible scenarios. First (Figure 3B, a), during mitosis after
NEBD, tubular SE/ER membrane protrusions may either be
caught or extended de novo along the spindle microtubule in
much the same way that ER does along microtubules during
interphase. Components of the SE, including a subset of INM
proteins, might then be recruited by interactions with the
mitotic spindles, thereby setting up the observed selective
accumulation of spindle-associated membrane proteins. A
second, and somewhat speculative, possibility is that, right
after NEBD, the spindle microtubules projecting from the
spindle pole start interacting with the residual NE, thus
pushing and deforming the endomembrane now lacking the
supportive nuclear lamina. This would lead to complete
encasement of the spindle microtubules on the cytoplasmic
side, thereby conserving the topology of closed mitosis in
lower eukaryotes (Figure 3B, c). In this model, the points of
microtubule interaction at the end of the SE cones might act
as MNCs. This would be by analogy to the process of closed
mitosis (Figure 3A), except that there has to be an MNC
for each kinetochore microtubule. From the evolutionary
perspective, this is an attractive model. Whichever of these
two models that will come closest to reality has to await
supportive evidence at the ultrastructural level or confirmed
presence of specific markers at the MNCs. In either case,
some NE protein may stay associated to the microtubule
cytoskeleton throughout the cell cycle.
Nuclear Envelope Disease and Chromatin Organization
Funding
This work was supported by grants to E.H. from the Swedish
Research Council [grant number 2010-4481] and the Swedish Cancer
Foundation [grant number 10 0277].
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doi:10.1042/BST20110680
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