We used field emission in-lens scanning electron microscopy to examine newly-assembled, growing nuclear envelopes in Xenopus egg extracts. Scattered among nuclear pore complexes were rare ‘dimples’ (outer membrane depressions, 5–35 nm diameter), more abundant holes (pores) with a variety of edge geometries (35–45 nm diameter; 3.3% of structures), pores containing one to eight triangular ‘star-ring’ subunits (2.1% of total), and more complicated structures. Neither mature complexes, nor these novel structures, formed when wheat germ agglutinin (which binds O-glycosylated nucleoporins) was added at high concentrations (>500 microg/ml) directly to the assembly reaction; low concentrations (10 microg/ml) had no effect. However at intermediate concentrations (50–100 microg/ml), wheat germ agglutinin caused a dramatic, sugar-reversible accumulation of ‘empty’ pores, and other structures; this effect correlated with the lectin-induced precipitation of a variable proportion of each major Xenopus wheat-germ-agglutinin-binding nucleoporin. Another inhibitor, dibromo-BAPTA (5,5′-dibromo-1,2-bis[o-aminophenoxylethane-N,N,N′,N′-tetraacetic acid), had different effects depending on its time of addition to the assembly reaction. When 1 mM dibromo-BAPTA was added at time zero, no pore-related structures formed. However, when dibromo-BAPTA was added to growing nuclei 40–45 minutes after initiating assembly, star-rings and other structures accumulated, suggesting that dibromo-BAPTA can inhibit multiple stages in pore complex assembly. We propose that assembly begins with the formation and stabilization of a hole (pore) through the nuclear envelope, and that dimples, pores, star-rings, and thin rings are structural intermediates in nuclear pore complex assembly.

Akey
C. W.
(
1989
).
Interactions and structure of the nuclear pore complex revealed by cryo-electron microscopy.
J. Cell Biol
109
,
955
970
Akey
C. W.
,
Radermacher
M.
(
1993
).
Architecture of Xenopus nuclear pore complex revealed by three dimensional cryo-electron microscopy.
J. Cell Biol
122
,
1
19
Akey
C. W.
(
1995
).
Conformational plasticity of the nuclear pore complex.
J. Mol. Biol
248
,
273
293
Allan
V. J.
,
Vale
R.
(
1994
).
Movement of membrane tubules along microtubules in vitro: evidence for specialized sites of motor attachment.
J. Cell Sci
107
,
1885
1895
Byrd
D. A.
,
Sweet
D. J.
,
Pante
N.
,
Konstantinov
K. N.
,
Guan
T.
,
Saphire
A. C. S.
,
Mitchell
P. J.
,
Cooper
C. S.
,
Aebi
U.
,
Gerace
L.
(
1994
).
Tpr, a large coiled coil protein whose amino terminus is involved in activation of oncogenic kinases, is localized to the cytoplasmic surface of the nuclear pore complex.
J. Cell Biol
127
,
1515
1526
Cordes
V. C.
,
Reidenbach
S.
,
Franke
W. W.
(
1995
).
High content of a nuclear pore complex protein in cytoplasmic annulate lamellae of Xenopus oocytes.
Eur. J. Cell Biol
68
,
240
255
Dabauvalle
M. C.
,
Loos
K.
,
Scheer
U.
(
1990
).
Identification of a soluble precursor complex essential for nuclear pore complex assembly in vitro.
Chromosoma
100
,
56
66
Dabauvalle
M. C.
,
Loos
K.
,
Merkert
H.
,
Scheer
U.
(
1991
).
Spontaneous assembly of pore complex-containing membranes (“Annulate Lamellae”) in Xenopus egg extract in the absence of chromatin.
J. Cell Biol
112
,
1073
1082
Davis
L. I.
(
1995
).
The nuclear pore complex.
Annu. Rev. Biochem
64
,
865
896
Feldherr
C. M.
,
Akin
D.
(
1990
).
The permeability of the nuclear envelope in dividing and nondividing cell cultures.
J. Cell Biol
111
,
1
8
Finlay
D. R.
,
Forbes
D. J.
(
1990
).
Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored.
Cell
60
,
17
29
Finlay
D. R.
,
Meier
E.
,
Bradley
P.
,
Horecka
J.
,
Forbes
D. J.
(
1991
).
A complex of nuclear pore proteins required for pore function.
J. Cell Biol
114
,
169
183
Georgatos
S. D.
,
Meier
J.
,
Simos
G.
(
1994
).
Lamins and lamin-associated proteins.
Curr. Opin. Cell Biol
6
,
347
353
Gerace
L.
,
Ottaviano
Y.
,
Kondor-Koch
C.
(
1982
).
Identification of a major polypeptide of the nuclear pore complex.
J. Cell Biol
95
,
826
837
Greber
U. F.
,
Senior
A.
,
Gerace
L.
(
1990
).
A major glycoprotein of the nuclear pore complex is a membrane-spanning polypeptide with a large lumenal domain and a small cytoplasmic tail.
EMBO J
9
,
1495
1502
Greber
U. F.
,
Gerace
L.
(
1995
).
Depletion of calcium from the lumen of endoplasmic reticulum reversibly inhibits passive diffusion and signal-mediated transport into the nucleus.
J. Cell Biol
128
,
5
14
Goldberg
M. W.
,
Allen
T. D.
(
1992
).
High resolution scanning electron microscopy of the nuclear envelope: demonstration of a new, regular fibrous lattice attached to the baskets of the nucleoplasmic face of the nuclear pores.
J. Cell Biol
119
,
1429
1440
Goldberg
M. W.
,
Allen
T. D.
(
1993
).
The nuclear pore complex: three dimensional surface structure revealed by field emission, in-lens scanning electron microscopy, with underlying structure uncovered by proteolysis.
J. Cell Sci
106
,
261
274
Goldberg
M. W.
,
Allen
T. D.
(
1995
).
Structural and functional organisation of the nuclear envelope.
Curr. Opin. Cell Biol
7
,
301
307
Goldberg
M. W.
,
Jenkins
H.
,
Allen
T. D.
,
Whitfield
W. G. F.
,
Hutchison
C. J.
(
1995
).
Xenopus lamin B3has a direct role in the assembly of a replication competent nucleus: evidence from cell-free egg extracts.
J. Cell Sci
108
,
3451
3461
Goldberg
M. W.
,
Allen
T. D.
(
1996
).
The nuclear pore complex and lamina: three dimensional structures and interactions determined by Field Emission In-lens Scanning Electron Microscopy.
J. Mol. Biol
257
,
848
865
Görlich
D.
,
Mattaj
I. W.
(
1996
).
Nucleocytoplasmic transport.
Science
271
,
1513
1518
Guan
T.
,
Muller
S.
,
Klier
G.
,
Pante
N.
,
Blevitt
J. M.
,
Haner
M.
,
Paschal
B.
,
Aebi
U.
,
Gerace
L.
(
1995
).
Structural analysis of the p62 complex, an assembly of O-linked glycoproteins that localizes near the central gated channel of the nuclear pore complex.
Mol. Biol. Cell
6
,
1591
1603
Hallberg
E.
,
Wozniak
R. W.
,
Blobel
G.
(
1993
).
An integral membrane protein of the pore membrane domain of the nuclear envelope contains a nucleoporin-like region.
J. Cell Biol
122
,
513
521
Hart
G. W.
,
Haltiwanger
R. S.
,
Holt
G. D.
,
Kelly
W. G.
(
1989
).
Glycosylation in the nucleus and cytoplasm.
Annu. Rev. Biochem
58
,
842
874
Hicks
G. R.
,
Raikhel
N. V.
(
1995
).
Protein import into the nucleus: an integrated view.
Annu. Rev. Cell Dev. Biol
11
,
155
188
Hinshaw
J. E.
,
Carragher
B. O.
,
Milligan
R. A.
(
1992
).
Architecture and design of the nuclear pore complex.
Cell
69
,
1133
1141
Hutchison
C. J.
,
Bridger
J. M.
,
Cox
L. S.
,
Kill
I. R.
(
1994
).
Weaving a pattern from disparate threads: lamin function in nuclear assembly and DNA replication.
J. Cell Sci
107
,
3259
3269
Jarnik
M.
,
Aebi
U.
(
1991
).
Toward a more complete 3-D structure of the nuclear pore complex.
J. Struct. Biol
107
,
291
308
Kessel
R. G.
(
1992
).
Annulate lamellae: a last frontier in cellular organelles.
Int. Rev. Cytol
133
,
43
120
Kiseleva
E.
,
Goldberg
M. W.
,
Daneholt
B.
,
Allen
T. D.
(
1996
).
RNP export is mediated by structural reorganization of the nuclear pore basket.
J. Mol. Biol
260
,
304
311
Leno
G. H.
,
Downes
C. S.
,
Laskey
R. A.
(
1992
).
The nuclear membrane prevents the replication of human G2 nuclei but not G1 nuclei in Xenopus egg extract.
Cell
69
,
151
158
Lohka
M. J.
(
1988
).
The reconstitution of nuclear envelopes in cell-free systems.
Cell Biol. Int. Rep
12
,
833
848
Lohka
M. J.
,
Masui
Y.
(
1983
).
Formation in vitro of sperm pronuclei andmitotic chromosomes induced by amphibian ooplasmic components.
Science
220
,
719
721
Lohka
M. J.
,
Masui
Y.
(
1984
).
Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs.
J. Cell Biol
98
,
1222
1230
Macaulay
C.
,
Meier
E.
,
Forbes
D. J.
(
1995
).
Differential mitotic phosphorylation of proteins of the nuclear pore complex.
J. Biol. Chem
270
,
254
262
Macaulay
C.
,
Forbes
D. J.
(
1996
).
Assembly of the nuclear pore: biochemically distinct steps revealed with NEM, GTPS, and BAPTA.
J. Cell Biol
132
,
5
20
Madine
M. A.
,
Khoo
C. Y.
,
Mills
A. D.
,
Musahl
C.
,
Laskey
R. A.
(
1995
).
The nuclear envelope prevents reinitiation of replication by regulating the binding of MCM3 to chromatin in Xenopus egg extracts.
Curr. Biol
5
,
1270
1279
Maul
G. G.
(
1977
).
The nuclear and cytoplasmic pore complex: structure, dynamics, distribution, and evolution.
Int. Rev. Cytol. Suppl
6
,
75
186
Maul
G. G.
(
1977
).
Nuclear pore complexes: elimination and reconstruction during mitosis.
J. Cell Biol
74
,
492
500
Maul
G. G.
,
Maul
H. M.
,
Scogna
J. E.
,
Lieberman
M. W.
,
Stein
G. S.
,
Hsu
B. Y. L.
,
Borun
T. W.
(
1972
).
Time sequence of nuclear pore formation in phytohemagglutinin-stimulated lymphocytes and in HeLa cells during the cell cycle.
J. Cell Biol
55
,
433
447
Maul
H. M.
,
Hsu
B. Y. L.
,
Borun
T. M.
,
Maul
G. G.
(
1973
).
Effect of metabolic inhibitors on nuclear pore formation during the HeLa S3cell cycle.
J. Cell Biol
59
,
669
676
Meier
E.
,
Miller
B. R.
,
Forbes
D. J.
(
1995
).
Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation.
J. Cell Biol
129
,
1459
1472
Newmeyer
D. D.
,
Wilson
K. L.
(
1991
).
Egg extracts for nuclear import and nuclear assembly reactions.
Meth. Cell Biol
36
,
607
634
Pante
N.
,
Aebi
U.
(
1993
).
The nuclear pore complex.
J. Cell Biol
122
,
977
984
Reichelt
R.
,
Holzenburg
A.
,
Buhle
E. L.
,
Jarnick
M.
,
Engel
A.
,
Aebi
U.
(
1990
).
Correlation between structure and mass distribution of the nuclearpore complex and of distinct pore complex proteins.
J. Cell Biol
110
,
883
894
Rout
M. P.
,
Blobel
G.
(
1993
).
Isolation of the yeast nuclear pore complex.
J. Cell Biol
123
,
771
783
Sheehan
M. A.
,
Mills
A. D.
,
Sleeman
A. M.
,
Laskey
R. A.
,
Blow
J. J.
(
1988
).
Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs.
J. Cell Biol
106
,
1
12
Strambio-de-Castillia
C.
,
Blobel
G.
,
Rout
M. P.
(
1995
).
Isolation and characterization of nuclear envelopes from the yeast Saccharomyces.
J. Cell Biol
131
,
19
31
Sullivan
K. M. C.
,
Busa
W. B.
,
Wilson
K. L.
(
1993
).
Calcium mobilization is required for nuclear vesicle fusion in vitro: implications for membrane traffic and IP3receptor function.
Cell
73
,
1411
1422
Sullivan
K. M. C.
,
Lin
D. D.
,
Agnew
W.
,
Wilson
K. L.
(
1995
).
Inhibition of nuclear vesicle fusion by antibodies that block activation of 1,4,5-trisphosphate receptors.
Proc. Nat. Acad. Sci. USA
92
,
8611
8615
Ullman
K. S.
,
Forbes
D. J.
(
1995
).
RNA polymerase III transcription in synthetic nuclei assembled in vitro from defined DNA templates.
Mol. Cell. Biol
15
,
4873
4883
Unwin
P. N.
,
Milligan
R. A.
(
1982
).
A large particle associated with the perimeter of the nuclear pore complex.
J. Cell Biol
93
,
63
75
White
J. M.
(
1990
).
Viral and cellular membrane fusion proteins.
Annu. Rev. Physiol
52
,
675
697
Wilson
K. L.
,
Newport
J.
(
1988
).
A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro.
J. Cell Biol
10
,
7
–.
Wozniak
R. W.
,
Bartnik
E.
,
Blobel
G.
(
1989
).
Primary structure analysis of an integral membrane glycoprotein of the nuclear pore.
J. Cell Biol
108
,
2083
2092
Wozniak
R. W.
,
Blobel
G.
,
Rout
M. P.
(
1994
).
POM152 is an integral protein of the pore membrane domain of the yeast nuclear envelope.
J. Cell Biol
125
,
31
42
This content is only available via PDF.