Title:
Postmitotic
nuclear pore assembly proceeds by radial dilation of small ER
membrane openings
Authors/Affiliations:Shotaro
Otsuka,Anna M. Steyer,Martin Schorb,Jean-Karim Hériché,M. Julius
Hossain,Suruchi Sethi,Moritz Kueblbeck,Yannick Schwab,Martin Beck,
Jan
Ellenberg. Cell Biology and Biophysics Unit, European Molecular
Biology Laboratory, . Structural and Computational Biology Unit,
European Molecular Biology Laboratory, Electron Microscopy Core
Facility, European Molecular Biology Laboratory, Meyerhofstrasse 1,
69117 Heidelberg, Germany. *Corresponding author Contact:
jan.ellenberg AT
embl.de
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Abstract:
The
nuclear envelope has to be reformed after mitosis to create viable
daughter cells with closed nuclei. How membrane sealing of DNA and
assembly of nuclear pore complexes (NPCs) are achieved and
coordinated is poorly understood.
Here,
we reconstructed nuclear membrane topology and structure of
assembling NPCs in a correlative three dimensional electron
microscopy time-course of dividing human cells. Our quantitative
ultrastructural analysis shows that nuclear membranes form from
highly fenestrated ER sheets, whose shrinking holes are stabilized
and then dilated into NPCs during inner ring complex assembly,
forming thousands of transport channels within minutes. This
mechanism is fundamentally different from interphase NPC assembly and
explains how mitotic cells can rapidly establish a closed nuclear
compartment while making it transport-competent at the same time..
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4.0 International license
not
peer-reviewed) is the author/funder. It is made available under a
The
copyright holder for this preprint (which was
.
http://dx.doi.org/10.1101/141150doi: bioRxiv preprint first posted
online May. 23, 2017;
Keywords:
NPC,
nuclear pore complexes
Nups,
nucleoporins
NE,
nuclear envelope
ER
endoplasmic reticulum
ELYS,
nuclear pore components
reticulons,
ER-shaping proteins Reticulons
are a group of evolutionary
conservative proteins
residing predominantly in endoplasmic
reticulum,
Ran,
Ras
-related nuclear protein, GTP binding nuclear protein Ran
AO,
anaphase onset
GFP,
green
fluorescent protein
Introduction
The
nuclear pore complex
(NPC) is the
largest non-polymeric protein complex in eukaryotic cells and
composed of multiple
copies of around 30 different proteins
termed nucleoporins
(Nups) . NPCs are
the sole gates of macromolecular transport across the double membrane
of the nuclear envelope (NE).In higher eukaryotes, NPCs and the NE
disassemble at the beginning of mitosis and their rapid reformation
during mitotic exit is essential for establishing a functional
nucleus in the daughter cell
The
process of postmitotic assembly of the NPC
and the nuclear
membranes from
mitotic ER has been studied in vitro using nuclei assembled in
Xenopus egg extract and by live cell imaging using fluorescence
microscopy. Several molecular players regulating the process have
been identified, including inner nuclear membrane proteins, ER
shaping proteins such as reticulons, nuclear pore components ELYS and
Nup107‒160 complex, nuclear transport receptors and Ran.
In addition, kinetic observations of the bulk NPC formation across
the NE has shown that postmitotic assembly proceeds by sequential
addition of Nups in a clear temporal progression, that is almost
identical between ro
dent
and human cells.
Despite
these important insights, the mechanism of NPC assembly after mitosis
has
remained
unclear and is highly debated .
-
Whether postmitotic NPC assembly is initiated in an already sealed NE and the NPC is inserted into this double membrane by a de novo fusion event similar to NPC assembly during interphase , or if it starts already on the naked DNA and the membrane only later engulfs assembling NPC from the side , has remained unanswered.
-
How thousands of NPCs can assemble within a few minutes without interfering with the rapid sealing of NEs during mitotic exit thus has remained mysterious.
A
major reason for this gap in our knowledge was that individual NPCs
and ER topology are below the resolution of live cell fluorescence
microscopy that is needed to capture the dynamic process of
postmitotic nuclear assembly, precluding reliable and quantitative
observation of NPC assembly and the sealing of NE membranes.
Here,
we combine live cell imaging with high resolution 3D electron
microscopy to ultrastructurally reconstruct the dynamic process of
postmitotic NPC and NE assembly.
Results:
Nuclear
membranes form from highly fenestrated ER sheets
To
measure how nuclear membrane sealing around DNA relates to NPC
formation in space and time, we reconstructed whole dividing human
cells with a time resolution of approximately one minute after the
beginning of mitotic chromosome segregation by correlating single
cell live
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bioRxiv preprint first posted online May. 23, 2017;
imaging
with focused ion beam scanning electron microscopy (FIB-SEM) (Fig. 1a
and Supplementary Fig. 1).
Segmentation
of membranes in close proximity to chromosomes showed that the layer
of mitotic ER that contacts chromosomes exhibits a high degree of
fenestrations (Fig. 1b,c and Supplementary Movie 1) as reported
previously.
At
early times only about 10% of the chromosome surface was associated
with ER, but starting at about 5 min after anaphase onset (AO), the
surface of ER-chromosome contacts increased rapidly covering the
chromosomes with newly formed NE within 2 min (Fig. 1b,d and
Supplementary Table 1).
Fine
3D segmentation of ER/NE membranes (Supplementary Movie 2) in the
large volume EM data showed that at early times (up to 3.9 min)
variably sized holes made up 43% of the surface of the ER sheets
contacting chromosomes (Fig. 1c,e) and that 59% of these
discontinuities displayed a diameter below 100 nm (Fig. 1f), i.e. on
the order of NPCs. The degree of fenestration and hole size in the
ER sheets contacting chromosomes decreased rapidly (Fig. 1c), with
holes making up only 16% of the surface two minutes later and now 75%
of them having a diameter below 100 nm (Fig. e,f; 6.3 min). This data
demonstrates that the NE forms from highly fenestrated ER sheets that
contain a very large number of discontinuities whose diameter
shrinks as the ER-derived NE covers the chromosomes (
Fig.
1d). Coverage of chromosomes by nuclear membranes is closely linked
to pre-pore formation. As ER fenestrae started to shrink
significantly as early as 4.3 min after AO (
Fig.
1f), many of the pore sized discontinuities started to contain
electron dense material
(Fig.
1a‒c and SupplementaryTable 1) and could therefore be classified as
pre-pores. From their first appearance, the number of such pre-pores
increased rapidly to 2000 with the local density of 15 pores/m2
within
only 3 min(Fig. 1d). Kinetic analysis of chromosome coverage by newly
forming NEs and the appearance of pre-pores showed that both
processes display sigmoidal kinetics and are closely linked in time
with pre-pore appearance reaching its half-maximum within less than
one minute after chromosome coverage (Fig. 1d).
NPC
assembly proceeds by dilation of small membrane holes. Knowing when
exactly pre-pores start to form during NE formation, enabled us to
examine the architecture of assembling NPCs at an even higher
resolution. We performed correlative live imaging with electron
tomography, in cells captured every 1‒2 min after AO (Fig. 2a and
Supplementary Movie 3), starting at 4.8 min when pre-pores first
appear (Fig. 1d) until 15 min when NE formation is completed (Fig.
1b,d). Since NE sealing is delayed in the so called ‘core-regions’
due to clearance of dense spindle microtubules (Fig. 1b), we focused
our
.
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analysis
on the non-core regions of the NE (Fig. 2a). In a total of 27.8 m2
reconstructed
NE surface area, we identified 360 particles consistent with
pre-pores (i.e. displaying a NE discontinuity containing regular
electron dense material) captured at different times of 100
postmitotic
assembly (Fig. 2b, Supplementary Fig. 2a and SupplementaryTable 2).
At
early times we also found 50 small NE discontinuities which were very
similar to holes present in the ER not yet in touch with the
chromosome surface (Supplementary
Fig.
3). We classified these as “small holes” in NE or ER whose
associated density is too low to be detectable as a distinct regular
structure, although they might contain smaller amounts of proteins.
We
first focused our analysis on changes in NE topology. Tracing of the
pre-pore
membrane
profiles in the 3D tomograms and their quantitative analysis
revealed that pre
-pore
diameter increased rapidly from 39 nm (4.8 min after AO) to 63 nm
(10.2 min) at which size they stabilized (Fig. 2b,c). The profile
analysis also revealed other interesting NE topology changes
(Supplementary Fig. 2b,c).
The
pre-pore dilation showed sigmoidal kinetics, reaching its
half-maximum within 1.2 min after pre-pore appearance (Figs.
1dand 2c), predicting that it represents a maturation step into fully
assembled NPCs. Detailed analysis of the distribution of pre-pore
diameters at different postmitotic times allowed classification into
two groups, smaller and larger pre-pores with a
mean diameter of 42 and 62 nm, respectively (Supplementary Fig. 4).
As predicted, the combined abundance of smaller and larger pre-pores
matched the number
of
mature NPCs found after completion of nuclear reformation
(Fig. 2d, Supplementary Fig. 5 and Supplementary Table 2).
Interestingly,
at the beginning of pre-pore appearance (4.8 min), the slightly
lower than expected density of smaller pre-pores was made up by the
presence of similarly sized small NE holes, which disappeared
at later times ( Fig. 2d). Overall, this data indicates that
pre-pores mature by membrane hole dilation into fully-assembled NPCs
and that small NE holes are likely to be precursors of pre-pores
that have not yet accumulated a significant amount of
dense material. NPC assembly proceeds by centrifugal formation
of a membrane associated ring
We
next analyzed how the distribution of dense material inside
pre-pores changes during their maturation. We first radially
averaged all density inside the membrane hole in the NE plane of
pre-pores (Fig. 3a). The change in mean radial intensity profiles
over time showed that initially (4.8 min), pre-pores contained
material in the center of the membrane hole; From 4.8
to 10 min, material progressively accumulated next to the membrane,
resulting in a growing intensity peak that was close to the
expanding wall of the membrane channel. After this peripheral
accumulation of material, the center of the channel
accumulated additional density from 10 min
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to
interphase, leading to a second central peak in the radial profiles (
Fig.
3a and Supplementary Fig. 6).
Subtomogram
averaging revealed a clear structural maturation of pre-pores
To
obtain better insight into the structural changes during NPC
assembly, subtomogram
averaging
of single pre-pores in the same state of assembly is necessary. Since
the distributions of membrane hole diameters and profiles of dense
material indicated that individual pre-pores sampled at the same
time-point can vary in structure (
Supplementary
Figs. 4 and 6), we ordered them independent of time based on
structural similarity using spectral seriation (Fig. 3b,c). Such
spectral ordering of pre- and mature pores overall
recapitulated their temporal sampling during anaphase, with pores at
early (4.8 and 6.1 min), middle (7.7 min), and late
(10, 15 min and interphase) time points ranked together (Fig. 3c and
Supplementary Fig. 7), showing that postmitotic NPC assembly
is indeed a progressive process.
Based
on their structural similarity, we partitioned pores into five
assembly states (Supplementary Fig. 7) and performed subtomogram
averaging. The averages revealed a striking progression of structural
changes during postmitotic NPC assembly (Fig. 4a).
Early
and smaller pre-pores (cluster 1), exhibited dense material in
the center of a narrow membrane gap, which subsequently shifted
centrifugally towards the membrane (cluster 2) and then
dilated into a clear peripheral ring with a first sign of the 8-fold
rotational symmetry of the NPC inner ring complex (cluster 3).
Inner ring complex formation was then completed with clear 8-fold
symmetry (cluster 4), which was followed by maturation of the
central channel density (cluster 5, Fig. 4a).
Below
the double nuclear membranes, density consistent with the nuclear
ring was present from the beginning (cluster 1), whereas cytoplasmic
ring-like density above the NE appeared only later (cluster 3, Fig.
4a). The same order of inner ring formation and dilation on top of an
early assembled nucleoplasmic ring, followed by cytoplasmic ring
assembly and central channel maturation, was also observed if
pre-pores were clustered only
according to experimental time
(Supplementary
Fig. 8a,b), showing that the pore maturation process is largely
synchronous. Analysis of the increase in inner ring complex
intensity over time in time
-clustered
averages furthermore showed that its sigmoidal rise coincides with
the process of membrane dilation (Supplementary Fig. 8c), suggesting
that inner ring complex self-assembly could drive pore dilation.
The
structural progression we observed is consistent with previous
observations in live cells that nuclear/cytoplasmic ring components
(Nup107 and Nup133) start to accumulate in the NE early, followed by
the inner ring component Nup93 .
We
revisited these observations, by
.
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creating
genome-edited cells in which nucleoporins are endogenously tagged
with GFP to avoid effects of non-physiological expression levels .
High time resolution live cell imaging and kinetic analysis of
postmitotic NE accumulation showed that Nup205, a component of the
inner ring complex, is incorporated after Nup107, a component of the
nuclear and cytoplasmic ring complexes (Supplementary Figs. 9 and
10), consistent with our previous observations
Interestingly,
the concentration of Nup107 on the NE had reached its half-maximum
already at 6 min after AO when pre-pores contain only the nuclear
ring, and increased further until 8 min when the cytoplasmic ring
appears (Supplementary Figs. 8B and 10b). The live cell kinetics
support the notion that the observed nuclear ring in early pre-pores
contains Nup107-160 subcomplex members, and the long accumulation of
Nup107 is furthermore overall consistent with the EM finding that the
nuclear ring assembles first followed by the later appearance of the
cytoplasmic ring.
Finally,the
live cell accumulation of Nup205 shows similar kinetics to the
increase in inner ring complex density over time observed by EM
(Supplementary Fig. 10c), consistent with the idea that Nup205
contributes to inner ring assembly observed in EM. It should be noted
that postmitotic NPC assembly is not perfectly synchronous at the
single pore level as seen by the structural variability of
pre-pores at each time point (Supplementary Figs. 4 and 6), and
therefore the live bulk measurement of NE protein accumulation cannot
provide the precise molecular assembly choreography of single pores.
Discussion:
How
thousands of NPCs assemble into the reforming NE during mitosis exit
has remained
unclear
due to the resolution limitation of conventional microscopy typically
used to observe this dynamic process. Pioneering studies that used
in vitro assembled nuclei with Xenopus egg extract and isolated
nuclei from Drosophila embryos could unfortunately not establish the
physiological nature of assembling NEs and NPCs, because the native
membrane structure was disrupted during the egg extract preparation
and nuclear isolation, and the observation was done on the outer
surface of nuclei as they used scanning electron microscopy.
Our
temporally staged ultrastructural analysis for the first time
resolved postmitotic NE and NPC assembly in situ in intact
human cells at nanometer resolution, enabling us to formulate
a data-driven model of its mechanism (Fig. 4b,c). The fact that
ER sheets that form the NE contain a sufficient number of small
discontinuities for NPC assembly already at early time points (3.1,
3.9, and 4.3 min; Fig. 1f), and that we did not observe holes smaller
than 20 nm in the NE although our resolution is sufficient to
resolve them (Fig. 2 and SupplementaryFig. 4), strongly suggests that
postmitotic NPC assembly starts in pre-existing small NE openings
rather than by de novo
.
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fusion
into already sealed double membranes. While our EM data cannot
exclude that individual nucleoporins, that are not yet assembled into
higher order structures, are already present in these small holes,
they are morphologically clearly different from pre
-pores
and we cannot detect reproducibly positioned or ring-like densities
in them (Fig. 2
and
SupplementaryFig. 3). Since the first pre-pores containing such
densities have a similar small size of around 40 nm diameter (Fig.
2b,c), it is likely that the ER/NE hole shrinkage is stalled and
stabilized by protein accumulation in the center of the membrane
hole. At later time points, pre-pores then dilate the membrane hole
to normal NPC size of about 60 nm during inner ring complex
formation, the cytoplasmic ring assembles and the central channel
matures (Fig. 4b,c). The fact that an inner ring component Nup205
starts to be incorporated at 6 min after AO in live cells
(Supplementary Fig. 10), argues that the density in the center of
pre-pores at 4.8 min may not be explained by Nup205 and
according
to our earlier work also not much Nup93- The most likely candidate
protein to
explain
this early density would therefore by exclusion be Nup155, which
forms the innermost layer of the inner ring complex and has indeed
been shown to be
required
for recruiting other inner ring components Nups205, 188 and 93 .
We
have previously shown that de novo assembly of NPCs into intact
nuclei during
interphase
proceeds via inside-out extrusion of the INM and fusion with the ONM
, which is
fundamentally
distinct from the dilation mechanism of pre-existing membrane holes
during
postmitotic
assembly reported here. While interphase NPC assembly takes about 45
min and
is
sporadic and rare , the rapid radial dilation of small membrane holes
concomitant with inner ring complex formation supports the assembly
of ~2000 postmitotic NPCs in only 3 min during NE sealing after
mitosis. One of the reasons for this very high efficiency could be
that assembly into the holes of highly fe
nestrated
mitotic ER sheets does not require a new membrane fusion at the
assembly site
that
is needed for interphase NPC assembly and could represent a rate-
limiting
step.
In
addition, the mitotic cell contains a high concentration of “assembly
ready”
NPC
subcomplexes, that become permissive for assembly synchronously by
the reduction in mitotic kinase activity , while an interphase cell
has to synthesize nucleoporins for assembly.
Combined,
this could explain the high efficiency of postmitotic NPC assembly
that is essential for the rapid establishment of functional nuclei to
exit mitosis. Our finding that the NPC assembles via a fundamentally
different mechanism in mitosis than in interphase provides the basis
to dissect the key structural and molecular transitions and
regulatory steps in the future.
CC-BY
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not
peer-reviewed) is the author/funder. It is made available under a
The
copyright holder for this preprint (which was
.
http://dx.doi.org/10.1101/141150
doi:
bioRxiv
preprint first posted online May. 23, 2017;
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