TEXT
HISTORICAL BACKGROUND
The nucleolus (Fig.1) was first described in 1781 by Felice
Fontana (Fig.3), who noted its occurrence in the slime of an eel
(Fig.2), and reported it in a simple remark in his work on the
venom of vipers (Fontana, 1781). The name ‘‘nucleolus’’ was
coined by Gabriel Gustav Valentin in 1839 (Fig.4), who noticed
that most cells had a secondary nucleus or a ‘‘nucleus within a
nucleus.’’
Fig.1 Nucleolus
Fig.2 Eel
Some 100 years after Fontana’s discovery, the nucleolus became
one of the first intracellular structures to be described in detail.
Fig.3
Felice Fontana
Fig.4
Gabriel
Gustav
Valentin
The variability of both size and number of nucleoli, and their
disappearance and reappearance at mitosis, was described. These
remarkable conclusions still hold true today. Some of the most
important scientific contributions came as late as the early 20th
century, when Heitz related the formation of nucleoli with
chromosome (Fig.5) location, and Barbara McClintock (Fig.6)
studied X-ray induced chromosomal rearrangements.
Fig.5 Chromosome
Fig.
6
Barbara McClintock
Heitz (1931) observed a direct correlation between the number
and lengths of secondary constrictions, i.e. regions of mitotic
chromosomes with a thin appearance, where DNA (Fig.7) could
not be detected by the Feulgen reaction, and the number and size
of nucleoli. McClintock (1934) suggested that the chromatin at
the secondary constriction was the nucleolar organizing element
(now termed nucleolar organizing region “NOR” (Fig.8), because
this
Fig. 7 DNA Structure
Fig.8
Nucleolar
organizing region (NOR)
of 6 Phaseolus
polytene chromosomes
region alone, without the original secondary constriction, was
able to give rise to a separate nucleolus. These data definitively
established the nucleolus as a genetically determined element.
Brachet (1940) and Caspersson and Schultz (1940) demonstrated
that nucleoli are enriched in RNA (Fig.9).
Fig. 9 RNA Structure
Fig.
10
Nucleus
The nucleolus is a large, distinct, spheroidal subcompartment of
the nucleus (Fig.10) of eukaryote cells, that is the site of
ribosomal RNA (rRNA) synthesis and assembly of ribosomal
subunits. The nucleolus referred to as a "non-membraneous
organelle" or "nuclear membraneless organelle" in the broader
sense of the term organelle; however, nucleoli lack a membrane
and, thus, are not organelles in the more technical sense of
structures that are separately enclosed within their own lipid
membrane. Most plant and animal cells (Figs.11 &12) have one or
more nucleoli, but some cell types do not have any.
Fig.11 Plant Cell
Fig.
12
Animal
Cell
The
nucleoli
are
specific
nuclear
domains
present
in
all
eukaryotic cells. The nucleolus is the ribosome factory (Fig.13) of
the cell (Brown and Gurdon, 1964). In cycling cells, nucleoli
assemble at the exit from mitosis, they are functionally active
throughout interphase, and they disassemble at the beginning of
mitosis (Fig.14).
Fig.13 Ribosome (Protein Factory)
Fig. 14 Various Phases
of Mitosis
The nucleolus is the site where different steps of ribosome
biogenesis are grouped together, i.e. transcription of ribosomal
genes
(rDNAs),
(rRNAs),
and
maturation/processing
assembly
of
rRNAs
of
with
ribosomal
ribosomal
RNAs
protein
(Hadjiolov, 1985). It was proposed that the nucleolus is ‘an
organelle formed by the act of building a ribosome (Melese and
Xue,1995). Indeed, the organization and size of the nucleolus is
directly
related
to
ribosome
production
(Smetana
and
Busch,1974). Consequently, the size of the nucleolus is a
diagnostic marker of highly proliferative cancer cells (Montanaro
et al., 2008). The variability of the nucleolar organization has
been intensively examined in different biological contexts, such as
proliferation, differentiation, development and disease.
The comparison of the nucleolar organization in evolutionary
terms revealed both the conservation in the basic ‘building
blocks’, and a higher complexity in modern eukaryotes (Thiry and
Lafontaine,
2005).
The
nucleolus
constitutes
a
model
to
understand the principles of the organization of nuclear domains,
the dynamics of protein trafficking, as well as the interplay
between nuclear bodies dedicated to related functions (Cajal
body, promyelocytic leukemia body, and nuclear speckles).
Throughout
the
past
50
years,
nucleolar
complexity
was
deciphered using multiple approaches. Thus, it was discovered
that other ribonucleoproteins (RNPs), in addition to ribosomal
subunits, are assembled or processed in the nucleolus. The best
example is the nucleolar assembly proposed for the signal
recognition particle (Politz et al., 2002). In plant cells, but not in
animal cells, nucleoli have been implicated as sites of silencing
RNA biogenesis (Pontes et al., 2006). Today, the nucleolus is
considered a multifunctional domain. Extra ribosomal functions
assigned to the nucleolus include the involvement in cell cycle
and
cell
proliferation
control,
stress
sensing
and
tumor
surveillance pathways, apoptosis, telomere formation, transfer
RNA modifications, viral life-cycle, etc. Unsurprisingly, nucleolar
dysfunction has severe consequences for human health (Hiscox,
2002; Emmott and Hiscox, 2009).
STRUCTURAL ORGANIZATION
The nucleolus (Fig.15) initially offered more hope for a
meaningful interpretation of its architecture because it showed a
Fig.15. Structure of Nucleus
Structures of the 30nm
Fig.16
Two
showing Nucleolus
chromatin filament
clear structural heterogeneity that appeared related to its
function. Aside from various kinds of associated condensed
chromatin (Fig.16) and nucleoplasmic-like inclusions, which are
generally called nucleolar vacuoles, the nucleolus shows three
fairly distinct structural components. The two major components
seen with conventional thin-section electron microscopy are
called the dense fibrillar component (DFC), and the granular
component (GC) (Jordan, 1984). Another component, the fibrillar
center material (FC) (Jordan & Chapman 1971), accounts for only
a small fraction of the total nucleolar volume (Fig.17a, b)
a
b
Fig.17
(a,
b)
Structural
Components
of
Nucleolus
It has been proposed that this particular organization is only
observed in higher eukaryotes, and that it evolved from a
bipartite organization with the transition from anamniotes to
amniotes.
Reflecting
the
substantial
increase
in
the
DNA
intergenic region, an original fibrillar component would have
separated into the FC and the DFC (Thiry and Lafontaine, 2005).
Another structure identified within many nucleoli (particularly in
plants) is a clear area in the center of the structure referred to as
a nucleolar vacuole (Fig.18) (Beven et al., 1996). The appearance
of the
nucleolar
vacuole
is
subsequent
to
the
output
of
preribosomes (Fig.19) from nucleolus. These vacuoles might play
a role in condensation and decondensation of the chromatin.
Fig. 18 Nuclear Vacuole
Fig.
19
Preribosomes
Fibrillar center (FC)
Fibrillar center (FC) is made up of network of fine (4-5 nm
thick) fibrils. The shape of an FC is roughly spherical, with the
diameter ranging from about 50nm to 1 μm. The number and size
of FCs per nucleolus is variable, and changes with cellular activity
and the need for ribosome production. Cells with lower cellular
activity usually have fewer FC than others.
Dense Fibrillar Component (DFC)
Dense Fibrillar Component (DFC) is also made up of very
fine (3-5 nm) and densely packed fibrils. DFCs usually surround
FCs when they are present and form a meshwork. As this is
particularly true for activated states, the amount of DFC roughly
reflects
the
nucleolar
engagement
in
ribosome
biogenesis.
Sometimes this meshwork occupies large areas of the nucleolus,
occasionally interspersed with small FCs. During S phase of cell
cycle, the increase in upstream binding factor (UBF) association
may be due to the increase in its ability to compete with the
histones for binding to the rDNA.
Granular component (GC)
The granular component appears to consist of small granules
with a diameter of about 15 nm. They typically form a mass
surrounding the fibrillar complexes and embed the FCs and DFC.
Thus a transition zone between DFC and GC can be observed.
Although the nucleolus is not membrane-bound, due to the
presence of GC the border with the surrounding chromatin and
nucleoplasm is usually distinct.
Ribosomal DNA (r DNA):
rDNA is a set of tandemly-repeated genes coding for
preribosomal RNA. Because these genes have the ability to
initiate
the
formation
of
nucleoli
during
interphase,
these
segments of the chromosomes are called nucleolus organizer
regions or NORs. In the human genome, there are tandem
repeats of the rDNA sequence on the short arms of each of the
two copies of chromosomes 13, 14, 15, 21 and 22.
NUCLEOLAR PROTEINS
The nucleolus contains many different proteins, only a few of
which have been characterized in any detail (Olson 1990,
Hernandez-Verdun1991). They include the proteins of the preribosomes and those with specific nucleolar functions, such as
RNA
polymerase
I,
topoisomerases,
methylases,
nucleases,
protein kinases, and phosphatases.
GENERATION OF NUCLEOLAR STRUCTURE
The nucleolus undergoes breakdown and reformation during the
cell cycle, disappearing from late prophase until telophase (De La
Torre & Gimenez-Martin 1982; Bourgeois & Hubert 1988). At
prophase, the irregularly-shaped GC disappears first, followed by
the DFC (Lafontaine 1968), with various nucleolar proteins
leaving the nucleolus in an apparently ordered progression
(Gautier et al., 1992). The nucleolar-organizing regions (NORs) at
mitosis, the secondary constrictions, are ultrastructurally similar
to the fibrillar centers in EM staining. Components, such as RNA
polymerase I, topoisomerase I, and UBF, which are involved in
transcription, remain associated with the NORs in the condensed
chromosomes (Chan et al., 1991; Scheer et al., 1993); whereas
other components such as protein B23 are found around the
periphery of all the chromosomes or dispersed throughout the
cytoplasm (Hernandez-Verdun & Gautier 1994). A nucleolar
remnant
often
persists
through
mitosis,
remaining
in
the
cytoplasm or associated with the chromosomes, and ultimately
disappears after nuclear envelope reformation (Azumgelade et
aI.,
1994).
Reformation
of
the
nucleolus,
often
termed
nucleologenesis (De La Torre & Gimenez Martin 1982), takes
place in two stages: first, small, round prenucleolar bodies (PNBs)
are formed (Stevens 1965), that contain various nucleolar
proteins including fibrillarin, B23, and nucleolin. Second, if
transcription is initiated, the PNBs associate with transcriptionally
active NORs to form the complete nucleolus (Benavente et al.,
1987); furthermore, this process seems to be dependent on
transcription by the appropriate polymerase (Oakes et al.,1993).
FUNCTIONS
The main nucleolus function is production of subunits which
together form the ribosomes. The ribosomes are known to
produce proteins and, therefore, nucleolus plays an indirect role
in protein synthesis. Out of the total production of RNA that takes
place in cells, nucleolus is involved in 50% of the RNA synthesis.
This functionality of nucleolus is attributed to hundreds of rgenes. Besides, recent research pointed out that nucleolus is also
responsible for the trafficking of various prominent small RNA
species. Nucleolus helps them during their maturation process
and route to their final cellular destination. Moreover, although
nucleoli become invisible every time during cell division, more
recent studies found that they involved in cell cycle regulation. A
continuous chain between the nucleoplasm and the inner parts of
the nucleolus exists through a network of nucleolar channels. In
this way, macromolecules with a molecular weight up to 2000
kDa are easily distributed throughout the nucleolus.
The first clear proof of the function of the nucleolus came in the
mid
1960s,
when
two
groups
showed
that
the
organizer
contained the genes coding for the major (28S and 18S)
ribosomal RNAs. Ritossa & Spiegelman (1965), working with
Drosophila genotypes carrying differing numbers of organizers,
demonstrated a correlated difference in the numbers of genes for
ribosomal RNA. Bimstiel & Chipchase (1970) bred Xenopus
mutants without nucleoli, which were shown to be devoid of
ribosomal RNA synthesis, and thus unable to make ribosomes. It
was also shown at about this time that the RNAs of the ribosomes
were initially synthesized as one large molecule that was
processed to yield the smaller 18S and 28S rRNAs (Scherrer et
al.,1963).
Ribosomal Subunit Assembly
The assembly of ribosomal subunits takes place in the
following manner. Transcription of rRNA precursor molecule from
DNA takes place in the nucleolus. This long rRNA precursor
molecule is processed and 3 mature RNAs are formed. The next
step after formation of mature RNAs is that of carrying out the
packaging. These RNAs are packaged with certain specific forms
of proteins and finally, the ribosomal units are formed. These
ribosomal units can vary in size. The process of translation
requires ribosomal subunits as the raw material. The ribosomes
subunits which are assembled get transported to the cell
cytoplasm, i.e. out of the nucleolus, and then participate in the
process of translation (protein synthesis).
m RNA Biogenesis
The nucleoli are known to play an important role in mRNA
biogenesis. The nucleolus is also involved in RNA metabolism.
Events
such
as
telomerase
RNP
and
assembly
of
signal
recognition particle are known to be important. Nucleolus is also
involved in these RNP assembly events.
Nucleolus Organizer Region
The NOR is region in which formation of nucleolus takes
place around chromosomes. After the division of nucleus, this
region gets associated with the nucleus. Several copies of genes
of
ribosomal
RNAs
are
contained
in
this
area.
The structure and functioning of nucleolus is far more complicated
than what has been studied till date.