Exploring the nucleus

Semester I
Botany Core Course 2 (Group B)
Dr. Riddhi Datta

Nucleus
 The presence of a nucleus is the principal feature that distinguishes eukaryotic from
prokaryotic cells.
 The nucleus serves both as the repository of genetic information and as the cell's
control center.
 DNA replication, transcription, and RNA processing all take place within the nucleus,
with only the final stage of gene expression (translation) localized to the cytoplasm.
Dr. Riddhi Datta

Nucleus
 Nuclear envelope
 Nucleolus
 Nucleoplasm
 Chromatin reticulum
Dr. Riddhi Datta

Nuclear envelope
Dr. Riddhi Datta

Nuclear envelope
 The nuclear envelope separates the contents of the nucleus from the cytoplasm and
provides the structural framework of the nucleus.
 The two envelope membranes act as barriers that prevent the free passage of
molecules between nucleus and cytoplasm thus maintaining the nucleus as a distinct
biochemical compartment.
 The nuclear pore complexes allow regulated exchange of molecules between nucleus
and cytoplasm.
Dr. Riddhi Datta

Structure of nuclear envelope
 The nuclear envelope has a complex structure consisting of two nuclear membranes, an
underlying nuclear lamina, and nuclear pore complexes.
 The nucleus is surrounded by a system of two concentric membranes, called the inner
and outer nuclear membranes. Both the membranes shows unit membrane model, i.e.
they are made up of phospholipid bilayer.
 The outer nuclear membrane is continuous with the endoplasmic reticulum, so the space
between the inner and outer nuclear membranes is directly connected with the lumen of
the endoplasmic reticulum. The outer membrane also has ribosomes bound to its
cytoplasmic surface.
 The outer and inner membranes are separated by a space, called perinuclear space
which is 10-50 nm wide.
 The inner nuclear membrane carries proteins that are specific to the nucleus, such as
those that bind the nuclear lamina.
Dr. Riddhi Datta

Permeability
 The nuclear membranes are permeable to small non-polar molecules
(like O2, CO2, etc.). Other molecules can not pass.
 The inner and outer nuclear membranes are joined at nuclear pore
complexes, the sole channels through which small polar molecules,
macromolecules (proteins, RNA, etc.) or ions (Ca++, K+, etc.) pass
through the nuclear envelope.
Dr. Riddhi Datta

Nuclear lamina
 Underlying the inner nuclear membrane is the nuclear lamina, a fibrous meshwork that
provides structural support to the nucleus.
 The nuclear lamina is composed of 60 to 80 kd fibrous proteins called lamins.
 Lamins are a class of intermediate filament proteins. Mammalian cells have three lamin
genes, designated A, B and C, which code for at least seven distinct proteins.
 A-type and C-type lamins are inside, next to the
nucleoplasm, forming the lamina network, but
are not associated with the inner nuclear
membrane.
 B-type lamins are near the inner nuclear
membrane and may bind to integral proteins
inside this membrane. Specific lipids (prenyl
groups) are attached to the end of the
polypeptide chains of B-type lamins which helps
in attaching the B-type lamins to the nuclear
membrane.
Dr. Riddhi Datta

Intermediate filaments
 Intermediate filaments have diameters between 8-11 nm, which is intermediate between
the diameters of actin filaments (about 7 nm) and microtubules (about 25 nm).
 They play a structural role by providing mechanical strength to cells and tissues.
 More than 65 different intermediate filament proteins have been identified.
 All of the intermediate filament proteins have a central α-helical rod domain of
approximately 310 amino acids (350 amino acids in the nuclear lamins).
 This central rod domain is flanked by amino- and carboxy-terminal domains, which vary
among the different intermediate filament proteins in size, sequence, and secondary
structure.
 They are apolar structures.
Dr. Riddhi Datta

Lamin assembly
 The lamins associate with each other to form higher order structures, filaments.
 The lamin polypeptides form dimers in which the central α-helical regions of two polypeptide
chains are wound around each other, and form a structure called a coiled coil.
 Further assembly may involve the head-to-tail association of dimers to form linear polymers
and the side-by-side association of polymers to form higher order structures.
Dr. Riddhi Datta

 The association of lamins with the inner nuclear
membrane is facilitated by the posttranslational
addition of lipid, in particular, prenylation of C-
terminal cysteine residues.
 In addition, the lamins bind to specific inner
nuclear membrane proteins such as emerin and
the lamin B receptor, mediating their attachment
to the nuclear envelope and localizing and
organizing them within the nucleus.
 The nuclear lamina also binds to chromatin
through histones H2A and H2B as well as other
chromatin proteins.
Lamin assembly Dr. Riddhi Datta

Prenylation of nuclear lamins
 These proteins terminate with a
cysteine residue (Cys) followed by
two aliphatic amino acids (A) and any
other amino acid (X) at the C
terminus.
 The first step in their modification is
addition of the 15-carbon farnesyl
group to the side chain of cysteine
(farnesylation).
 This step is followed by proteolytic
removal of the three C-terminal
amino acids and methylation of the
cysteine, which is now at the C
terminus.
Dr. Riddhi Datta

Disappearance of nuclear envelope
 Lamins play a role in
assembly and disassembly
of nuclear envelope during
cell division.
 At the end of prophase, a
phosphate group attaches
to the cystein residue of
the lamin polypeptide. This
phosphorylation triggers
disassembly of lamina and
breakdown of nuclear
envelope into vesicles.
Phosphorylation is
catalyzed by the Cdc2
protein kinase.
 Dephosphorylation reverse
these changes and allows
the nucleus to re-form.
Dr. Riddhi Datta

 Disassembly of nuclear envelope involves
changes in all its 3 components.
 The nuclear membranes are fragmented
into vesicles, nuclear pore complexes
dissociates and nuclear lamina
depolymerizes into individual lamin dimers.
 The B-type lamins remain associated with
the vesicles, but A- and C-type lamins
dissociate from the nuclear membrane and
are released into the cytosol as free
dimers.
 The nuclear pore complexes appear to
dissociate into subunits and
phosphorylation of one or more nuclear
pore proteins may play a role in this
disassembly.
 Integral nuclear membrane proteins are
also phosphorylated during cell division.
Dr. Riddhi Datta

 During the completion of mitosis (telophase), two daughter nuclei form around the
separated sets of daughter chromosomes.
 The progression from metaphase to anaphase involves the activation of a ubiquitin-
mediated proteolysis system that inactivates Cdc2 by degrading its regulatory subunit,
Cyclin B.
 Inactivation of Cdc2 leads to dephosphorylation of the proteins that were
phosphorylated at the initiation of mitosis, resulting in exit from mitosis and the
reformation of interphase nuclei.
Reappearance of nuclear envelope Dr. Riddhi Datta

 The initial step in reformation of nuclear
envelope is the binding of the vesicles formed
during nuclear membrane breakdown to the
surface of chromosomes.
 These vesicles then fuse to form a double
membrane around the chromosomes.
 This is followed by reassembly of the nuclear
pore complexes, reformation of nuclear lamina
and chromosome de-condensation.
 The double membranes surrounding the
chromosomes then fuse to with each other to
form a complete nuclear membrane.
Reappearance of nuclear
envelope
Dr. Riddhi Datta

Functions of lamins
 Provides structural support and maintains integrity of nuclear
envelope.
 Plays important role in assembly and disassembly of nuclear
envelope during cell division.
 Serves as site for chromosome end attachment.
Dr. Riddhi Datta

Diseases related to lamin defect
 Defects in lamin gene causes some diseases.
 LMN1 defect causes muscular dystrophy which results in production
of fragile nucleus in muscle cells.
 Defect in lamin gene also causes a disease called progerias, i.e.
premature aging. An individual looks like an aged person at an age
of 12 – 14 years. This disease can also result from defects in the
telomerase enzyme.
Dr. Riddhi Datta

Nuclear pore complex
Dr. Riddhi Datta

Nuclear Pore Complex
 The nuclear pore complexes are the only channels through
which small polar molecules, ions, and macromolecules
(proteins and RNAs) can travel between the nucleus and the
cytoplasm.
 The nuclear pore complex is an extremely large structure with
a diameter of about 120 nm and an estimated molecular mass
of approximately 125 million dalton -about 30 times the size of
a ribosome.
 By controlling the traffic of molecules between the nucleus and
the cytoplasm, the nuclear pore complex plays a fundamental
role in the physiology of all eukaryotic cells.
 Nucleoplasmin is the basic constituent of nuclear pore
complex.
Dr. Riddhi Datta

Structure under electron microscope
 Visualization of nuclear pore complexes by
electron microscopy reveals a structure with
eightfold symmetry organized around a large
central channel.
 The complex consists of 8 ring-like structures at
the periphery and one ring at the center.
 From the central ring, 8 spoke like structures arise
and the structure resembles the spokes of a
bicycle tire.
 Under higher resolution, the pore complex shows 2 concentric rings superimposed upon
one another.
 Each ring consists of 8 individual granules called annuli.
 The outer ring remains attached to the outer nuclear membrane while the inner ring
remains attached to the inner nuclear membrane.
Dr. Riddhi Datta

3D structure
 Detailed structural studies, including computer-based image analysis, have led to the
development of three-dimensional models of the nuclear pore complex.
 The nuclear pore complex consists of 3 concentric rings superimposed upon one another.
Dr. Riddhi Datta

3D structure
 The outer most ring is the cytoplasmic ring
which lies towards the cytoplasm and
remains attached to the outer nuclear
membrane.
 The inner most ring is the nucleoplasmic
ring which lies towards the nucleoplasm
and remains attached to the inner nuclear
membrane.
 Each of these two rings are composed of
8 annuli.
 8 protein filaments extends from the cytoplasmic ring towards the cytoplasm.
 8 protein filaments also projects from the nucleoplasmic ring towards the nucleoplasm
where they form a nuclear basket.
 These filaments send signals to open the pore complex to the central transporter.
Dr. Riddhi Datta

3D structure
 The central ring is composed of the spoke-ring
assembly.
 It consists of an assembly of 8 spokes around a
central channel.
 Each of these rings bifurcates and attaches itself to
the cytoplasmic as well as the nucleoplasmic rings.
 This spoke-ring assembly is anchored within the
nuclear envelope at sites of fusion between the
outer and inner nuclear membranes.
 The outsides of the cytoplasmic and nucleoplasmic
rings are connected to radial arms. The interior is
connected to spokes that project towards the
transporter that contains the central pore.
 The 8 radial arms outside the ring may be
responsible for anchoring the pore complex in the
nuclear envelope; they penetrate the membrane.
Dr. Riddhi Datta

3D structure
 The central channel formed by the spoke-ring assembly is
approximately 10 nm in diameter and can open upto at
least 25 nm, which is wide enough to accommodate the
macromolecules.
 The central channel often appears to contain a structure
called the central plug, the nature of which is unclear.
 The assembly of spoke-ring complex creates 8 smaller
channels between the spokes which have diameter of
about 10 nm. They form open aqueous channels through
which small molecules diffuse and continuity of cytoplasm
and nucleus is maintained.
 This model of nuclear pore complex resembles the
structure of a waste paper basket and hence is called
‘Basket model’.
Dr. Riddhi Datta

Transport through nuclear pore complex
 Depending on their sizes, molecules can travel through the nuclear pore complex.
 Small molecules (ions, nucleotides, and other small molecules) with molecular mass
<20-40 kD pass rapidly across the nuclear envelope in either directions. They diffuse
passively through open aqueous channels in the pore complex. The diameter of these
channels is approximately 10 nm.
 Proteins having molecular mass <50 kD can pass through the central pore (passive
diffusion).
 Most proteins and RNAs, however, are >50kD and are unable to pass through these
open channels. Instead, these macromolecules pass through the central pore by an
active process in which appropriate proteins and RNAs are recognized and selectively
transported in a specific direction (nucleus to cytoplasm or cytoplasm to nucleus).
 Proteins require signals to be transported through the pore. The NILS and NES consist
of short sequences that are necessary and sufficient for proteins to be transported
through the pores into or out of the nucleus.
Dr. Riddhi Datta

Nucleolus
 The most prominent nuclear body is the nucleolus, which is the site of rRNA
transcription and processing as well as aspects of ribosome assembly.
 It is a sub-organelle of the nucleus, which itself is an organelle.
 The nucleolus is roughly spherical and is surrounded by a layer of condensed
chromatin. No membrane separates the nucleolus from the rest of the nucleus.
 The nucleolus is visible in the interphase
(G1 and G2) of the cell cycle. On the
onset of division, it disappears and
again reappears at the end of the
division. It is not a permanent structure
of the cell.
 Existence of nucleolus was first reported
by Fontana in 1781.
Dr. Riddhi Datta

Structure under light microscope
 Under light microscope, nucleolus is a dense body of variable size and shape.
 It is a highly refringent body.
 It is feulgen-negative after fixation, indicating absence of DNA and stains with
pyronin and absorbs UV light at 260nm which indicates presence of RNA
components.
 It may be surrounded by a ring of feulgen-positive chromatin which represents the
heterochromatic regions of nucleolar chromosome.
 The 3 main components of isolated nucleolar fractions are:
 DNA
 RNA
 Protein
 Chemically nucleolus is made up of ribonucleoprotein fibrils (RNP fibrils), i.e.
RNA+Proteins.
Dr. Riddhi Datta

 Under electron microscope, nucleolus shows some electron opaque and some electron
transparent zones. The granular and fibrilar zones are electron opaque.
 The following components can be distinguished:
 Granular component: Contains ribonucleoprotein granules of 15-18 nm diameter,
usually distributed towards the periphery (pars granulosa). Although the nucleolus is
not bound by any membrane, due to presence of the granular components the
border with the surrounding chromatin and nucleoplasm is usually distinct.
 Fibrillar component: It includes a fibrillar region of a meshwork of filaments of 4-6
nm diameter containing RNA and a considerable amount of protein (pars fibrosa).
The fibrillar component includes:
 Fibrillar center
 Dense fibrillar component
Structure under electron microscope Dr. Riddhi Datta

 Fibrillar center: Made up of a network of fine (4-5 nm thick) fibrils. The fibrillar center
is typically globular with diameter ranging from 50 – 1000nm. Cells with lower cellular
activity usually has fewer fibrillar center than others.
 Dense fibrillar component: Made up of fine (3-5 nm thick) fibrils which are densely
packed. It usually surround the fibrillar component when they are present in a
meshwork. The amount of dense fibrillar component roughly reflects the nucleolar
engagement in ribosomal biogenesis. The are now considered as sites where rDNA
cistrons are located.
 DNA binding proteins: Composed of DNA binding proteins which extends into the
interstices of nucleolonema (pars chromosoma).
 Matrix: It is a heterogeneous proteinaceous matrix.
 Nucleolonema: it is a course thread composed either of pars fibrosa or pars granulosa
or both. The ribonucleoproteins involved are the precursors of cytoplasmic ribosomes.
Structure under electron microscope Dr. Riddhi Datta

MOLECULAR ORGANIZATION
OF CHROMATIN
Dr. Riddhi Datta

Chromosomes and chromatin
 The genomes of eukaryotes are composed of multiple chromosomes, each containing a
linear molecule of DNA.
 The DNA of eukaryotic cells is tightly bound to small basic proteins (histones) that pack-
age the DNA in an orderly way in the cell nucleus.
 For example, the total extended length of DNA in a human cell is nearly 2 meters, but
this DNA must fit into a nucleus with a diameter of only 5 to 10 μm.
 The degree of compaction of DNA, i.e. how many turns of DNA is required to
accommodate it into the cell is denoted by an index called Packaging ratio.
Length of DNA molecule
Packaging ratio=
Length of the unit that accommodates DNA
Dr. Riddhi Datta

Chromatin
 The complexes between eukaryotic DNA and proteins are called chromatin.
 The major constituents of eukaryotic chromatins are DNA, RNA, basic proteins called
histones and non-histone proteins.
 The contents of RNA and non-histone proteins is variable but DNA and histones are
always present in a fixed ratio of 1:1.
 Non-histone proteins are heterogenous.
 Histones are small basic proteins containing a high proportion of basic amino acids
(arginine and lysine) that facilitate binding to the negatively charged DNA molecule.

 The 4 major histones – H2A, H2B, H3 and H4 – are among the most conserved
proteins known. These 4 ‘core histones’ are present in equimolar amounts.
 Histone H1 is not conserved and is rather loosely associated with the chromatin. It is
involved in maintaining higher order folding of chromatin.
Dr. Riddhi Datta

DNA PACKAGING
Dr. Riddhi Datta

Nucleosome model: 1st stage of DNA compaction
 The basic structural unit of chromatin, the nucleosome,
was described by Roger Kornberg in 1974. Two types of
experiments led to his proposals:
 Digestion of chromatin by micrococcal enzyme (which
digests DNA)
 Electron microscopic study
 When interphase was isolated and observed under
electron microscope, it revealed a structure containing
huge number of beads on a string of DNA.
 This is the ‘beads on a string configuration’ proposed by
Kornberg. It represents the 10 nm chromatin fiber.
 Each bead represented a nucleosome.
Dr. Riddhi Datta

 Nucleosome is flat disc-shaped particle, 11 nm in diameter and 5.7 nm in height.
 Neighboring nucleosomes are connected by linker DNA.
 The DNA coils 1 th turn around each nucleosome and these turns are sealed off by an
H1 (histone) molecule.
 After leaving a nucleosome and before entering the next nucleosome, the length of the
linker DNA varies from 8 – 114 bp (avg. 50 bp).
¾
Dr. Riddhi Datta

 Studies of digestion of chromatin by micrococcal nuclease showed
that the DNA was cut into multiples of a unit size which was 200 bp.
This 200 bp fragment is called mononucleosome.
 The nucleosome model proposes that the 4 core histones – H2A,
H2B, H3 and H4 – are arranged in octamers containing 2 copies of
each of them, every 200 bp of DNA.
 This histone octamer is coiled by DNA from outside.
 If digestion by micrococcal enzyme continues further, 166 bp
fragments are formed. These are called trimmed nucleosomes or
chromatosomes. It accommodates 166 bp DNA wrapped around
H2A, H2B, H3 and H4 and sealed by H1.
 Further digestion yields nucleosome core particles which has 147
bp DNA along with H2A, H2B, H3 and H4.
Dr. Riddhi Datta

 Two molecules of each of H3 and H4 associate with each other to form a tetramer with
a horse-shoe shape. This is the ‘kernel structure’ of the nucleosome.
 The H2A-H2B dimers remain associated with this kernel structure – one on the upper
side and one below the kernel. This constitutes the structure of core nucleosome.
 Several tail-like structures arise from the histones of the nucleosome core. These are
histone tails which plays important functions.
 The histone tails interact with those of its neighboring nucleosome cores and thus
helps in compaction. Histone tail modification plays crucial role in controlling gene
activity.
 The DNA of a 10 nm fiber has a packing that is about 6-fold.
Dr. Riddhi Datta

 The ‘solenoid model’ was proposed by Klug.
 This 30 nm fiber of chromatin probably arises from the folding of the nucleosome chain in
a solenoid structure having about 6 nucleosomes per turn.
 This represents a model of higher level packaging to explain the chromatin compaction
during metaphase.
 The whole structure is stabilized by interactions between H1 molecules in the neighboring
nucleosomes. The H1 molecules can interact with each other because they are located in
the ‘central hole’ of the solenoid.
 H1 is thought to play a fundamental role in this condensation because solenoids can not
be formed when H1-depleted chromatin is treated in the same way.
 The DNA of a 30 nm solenoid has a packing that is about 40-fold.
Solenoid model: 2nd stage of DNA compaction
Dr. Riddhi Datta

 When metaphase chromosomes are treated with dextran sulphate (a polyanion), it
starts to bind the cations and the histones are separated from the complex.
 This is the ‘histone depleted structure’.
 It consists of a pair of central axis (scaffold) made up of non-histone proteins from
which a number of DNA loops generate. Each non-histone axis bearing DNA loops
from a single DNA molecule corresponds to one chromatid of the chromosome.
 The axis was discovered by Laemii.
Histone depleted structure Dr. Riddhi Datta

 The next model that depicts the maximum condensation of chromatin fiber is the radial
loop scaffold model.
 This model propose that certain non-histone proteins including topoisomerase II bind to
chromatin every 60-100 kb and tether the supercoiled nucleosome-studded 30 nm fiber
into structural loops (due to formation of knots on a straight chromatin fiber, its length can
be reduced).
 Other non-histone proteins may, in turn, gather the loops into daisy-like rosettes and
additional non-histone proteins may then compress the rosette centers into a compact
bundle.
 A range of non-histones thus form the condensation scaffold. This proposal of looping and
gathering is known as the ‘radial loop scaffold model’ of compaction.
Radial loop scaffold model: 3rd stage of DNA compaction
Dr. Riddhi Datta

 Recent analysis of DNA indicates that special, irregularly spaced, repetitive base
sequences associate with non-histone proteins to define the chromatin loops. These
stretches of DNA are known as scaffold associated regions or SARs. SARs are most
likely the sites at which the DNA is anchored to the condensation scaffold.
 Other forms of SARs are called matrix attachment regions or MARs.
 The non-histone proteins exists as matrix proteins in the interphase and give rise to
scaffolds. The sequence of DNA that binds to SAR at metaphase, binds with MAR
during interphase. Hence, both SAR and MAR have same DNA sequence bound to
them.
 Now it achieves a 250-fold compaction of roughly 40-fold compacted 30nm fiber.
 This radial loop scaffold model is a hypothetical structure.
Radial loop scaffold model: 3rd stage of DNA compaction
Dr. Riddhi Datta

Exploring the nucleus

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