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Nuclear Envelope
The nuclear envelope has two membranes, each with the typical unit membrane
structure. They enclose a flattened sac and are connected at the nuclear pore sites. The
outermost membrane is continuous with the rough endoplasmic reticulum (ER) and
has ribosomes attached (see figure to the left). The space between the outer and inner
membranes is also continuous with rough endoplasmic reticulum space. It can fill with
newly synthesized proteins just as the rough endoplasmic reticulum does. The nuclear
envelope is enmeshed in a network of filaments for stability.
The nuclear envelope is shown in an electron micrograph in the figure on the
right. The filaments outside the envelope are not visualized with these protocols. Also,
the nuclear lamina just inside the nuclear envelope is not shown well (see paragraph
below for description). However, one can see ribosomes on the outer membrane and the
sac enclosed by the two membranes. Dense patches of heterochromatin are seen just
inside the inner membrane
Nuclear lamina
The inner membrane of the nuclear envelope lies next to a layer of thin filaments
which surrounds the nucleus except at the nuclear pores. These may also serve as
stabilizing filaments. This structure is called the "nuclear lamina". It has the following
structural and functional characteristics.
 Consists of "intermediate filaments", 30-100 nm thick.
 These intermediate filaments are polymers of lamin, ranging from 60-75 kD
 A-type lamins are inside, next to nucleoplasm; B-type lamins are near the nuclear
membrane (inner). They may bind to integral proteins inside that membrane.
 The lamins may be involved in the functional organization of the nucleus.
 They may play a role in assembly and disassembly before and after mitosis.
After they are phosphorylated, this triggers the disassembly of the lamina and
causes the nuclear envelope to break up into vesicles. Dephosphorylation reverses
this and allows the nucleus to reform.
Nuclear Pore Complex: Structure
Nuclear pores are formed at sites where the inner and
outer membranes of the nuclear envelope are joined. The figure
to the left shows an electron micrograph of a nuclear pore. It
appears as if the two membranes are pinched at that site, leaving
a space filled with filamentous material. Sometimes a thin
diaphragm may be seen running horizontally through the pore.
Also, the chromatin which carries the genetic material is
organized so that a space or "pathway" is created to the nuclear
pore. The following figure illustrates a model for the structure of
the components of the nuclear pore complex.
The above figure on the left
shows a view of the nuclear pore
from the top. It contains 8 subunits that "clamp" over
region of the inner and outer membrane where they join.
Actually, they form a ring of subunits 15-20 nm in
diameter. Each subunit projects a spoke-like unit into the
center so that the pore looks like a wheel with 8 spokes
from the top. Inside is a central "plug". The next (left
below) figure shows a cross section of the pore with the
clamp-like complex adjacent to the membranes. The
projected spoke is directed towards the central "plug' or
granule.
How can you visualize the nuclear pore complex?
Negative Staining Technique
One of the techniques used to study nuclear pore
complexes is called "negative staining". This protocol
deposits heavy metal stains around structures and delineates
their surface structure. When placed in an electron
microscope, the heavy metal around the structure retards
the electron beam. The structure itself allows the electron
beam to pass and this activates the photographic emulsion.
Thus, a "negative" image is created in the photograph.
The figure to the right illustrates a preparation of
nuclear pore complexes that were isolated from an oocyte
and spread on plastic. Then, the heavy metal stain was
applied to delineate their structure. Note that one can
visualize the 8 subunits, the spokes of the wheel and the
central granule. Figure modified from Bloom and Fawcett,
A Textbook of Histology, Chapter 1, Figure 1-10, Chapman
and Hall, Publishers, 1994.
Freeze-fracture/freeze-etch
Another way of visualizing
nuclear pores is via freezefracture/freeze etch. This protocol
involves the rapid freezing of
structures followed by fracturing.
The membranes are cleaved along
their lipid bilayer and either the
face next to the cytoplasm
(protoplasmic or P face) or the
extracellular (E) face of the
membrane is shown. Then, a
replica is made of the membrane by
evaporating heavy metal over the
surface.
This replica is what is viewed in the transmission electron microscope.
The above figure shows a surface view of nuclear pores scattered in the inner
nuclear envelope membrane. The subunits cannot be appreciated with this preparation.
However, it can be used to study formation of
nuclear pore complexes. This varies with the
physiological state of the cell.
Another preparation shows more details
of the structure of the nuclear pore complex.
Here we see the subunits forming the rings and
their spokes. Note that one of the pores appears
to be open in the center, forming a channel.
Figures modified from Bloom and
Fawcett, A Textbook of Histology, Chapter 1,
Figure 1-9, Chapman and Hall, Publishers, 1994
How does the nuclear pore
complex work to transport material in and out of the nucleus?
The pore serves as a water filled channel and has an effective diameter of 10 nm.
Therefore, transport in and out of the nucleus can occur in several ways.
Diffusion
This can be tested by adding different sized molecules to the cytosol and watching
the rate of transport of each group. For example, molecules of: 5,000 MW are freely
diffusable, 17,000 MW-- take 2 min to establish equilibrium, 44,000 MW--take 30 min to
establish equilibrium, 60,000 MW--cannot move in by diffusion.
This concept is important because it means that mature ribosomes (with both
subunits joined) cannot reenter the nucleus. Therefore, protein synthesis (translation of
mRNA) must occur outside the nucleus.
Active Transport
This form of transport is assumed when molecules larger than the pore diameter
(10 nm) get into the nucleus. Studies with colloidal gold markers show that the pore can
actually dilate up to 26 nm when it gets the appropriate signal. The active transport
requires energy (of ATP).
Studies have shown that the nuclear localization signal (NLS) is in the peptide
sequences. These recognition sequences are rich in lysine, arginine, and proline.
The signal controls direction of transport: protein and nucleic acid nuclear
export signals also exists. Gold-labeled tRNA or 5S RNA may leave the nucleus, but may
not come back. Also, transport of mRNA is inhibited by alteration of the 3' end or the 5'
cap structure.
What tests can be used to prove a particular signal?
A peptide sequence, called nucleoplasmin, was isolated and
linked to colloidal gold. It was then injected into an oocyte and
traced with electron microscopy. As shown in the figure to the left,
the gold particles mark the site of transport of the nucleoplasmin
and the studies showed that it was transported into the nucleus.
Small gold markers are evident inside the nucleus. Figure was
taken from Alberts et al., Molecular Biology of the Cell, Garland
Pub., N.Y.
In a test of T antigens of the SV40 virus, if the sequence is
altered by even one amino acid (see figure below), the peptide is no
longer transported to the nucleus. The studies used immunofluorescence to detect either the native or the altered sequences
which were conjugated to fluorescein, to make the protein
fluorescent, allowing detection. The fluorescence micrograph on
the left (A) shows entry of the native peptide into the ovoid nuclei. The micrograph on
the right (B) shows no entry into nuclei. The altered peptide stays in the cytoplasm.
Figure was taken from Alberts et al., Molecular Biology of the Cell, Garland Pub., N.Y.
How does one prove that transport requires energy (or ATP)?
Transport of mRNA can be inhibited by cooling the cells (placing them at 4 C).
ATP hydrolysis is required to import a protein into the nucleus. In the absence of ATP,
the proteins bind specifically at the cytosolic face of isolated frog oocyte nuclei. When
ATP is added, the proteins are allowed to enter. This can be traced with colloidal gold
labeling of the proteins. Studies show that ATP is needed for entry, but not for binding to
specific receptors.