Chapter 1: General Principles of Cellular Organization

Unlike Eukaryotes (protist, fungi, plants and animals), Prokaryotes (Eubacteria and Archaea) lack
nucleus and more generally the concept of compartmentalization (membrane-bound organelles),
along with cytoskeleton (actin, intermediate filaments and microtubules).

Compartmentalization
Lysosomes serve as a good example of compartmentalization (which serves in splitting the
functions), as they contain hydrolytic enzymes which break down cell components, which poses
the need of a membrane-bound structure to separate the function of the lysosome from the cytosol
(in order not to destroy needed cell structures). These hydrolytic enzymes require an acidic pH
which makes their activity optimal. This is maintained in the lysosome, however, this can’t be
present in the cytosol or else the cell would die. This acidity of the lysosome comes from protons,
through pumps that will keep pumping ions (protons in this case) against the concentration
gradient, in order to acidify the lysosome. The environment of the lysosome is separate from the
cytosol (as well as the function), which gives an idea about the selectivity of the membranes,
through which a proton can’t escape. Nothing moves through the membranes of membrane-bound
organelles without being regulated.
Eukaryotes are more sophisticated, and they have regulatory systems. Compartments are
important for regulation. An example would be the synthesis of mRNA in the nucleus and its
transfer to the cytosol, where it will get translated to proteins. One mRNA with a mistake is
actually problematic, as it will lead to the production of several proteins, by different ribosomes
and different translation centers. Thus, the cell checks if the mRNA contains any mistake before
it leaves the nucleus to the cytosol.
In prokaryotes, there is no pause to check for the quality of the mRNA, as both transcription and
translation occur in the same area, due to the lack of compartmentalization.
In eukaryotic cells, mRNA comes first as a precursor which is the hnRNA (heterogeneous nuclear
RNA), from which we need to splice the introns. In some cases though, some introns double as
nuclear retention sequence (NRS), which makes the mRNA unable to leave the nucleus, until the
splicing occurs and NRS is removed.
HnRNA stands for heterogeneous nuclear RNA. As its name suggests, hnRNA is a term that
encompasses various types and sizes of RNAs found in the eukaryotic cell nucleus. RNAs exist in
many forms and carry out a wide range of functions. Messenger RNA (mRNA) is the only type of
RNA that codes for proteins. Other types of RNA in eukaryotic cells include: ribosomal RNA
(rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), micro RNA (miRNA), small
interfering RNA (siRNA), and small nucleolar RNA (snoRNA). The majority of hnRNAs are pre-
mRNAs, newly synthesized mRNAs often made up of two types of segments: exons and introns.
The exon segments are joined together to produce a mature mRNA that encodes a protein; the
non-coding intron segments are removed by splicing. From the time new mRNAs are synthesized
by RNA polymerase II, they are associated with RNA-binding proteins called heterogeneous
ribonucleoprotein particle (hnRNP) proteins. HnRNP proteins stabilize pre-mRNAs by allowing
them to form a unique secondary structure. Different hnRNP proteins recognize and bind to
different sequences along the pre-mRNA. HnRNP-bound pre-mRNAs are then spliced by
spliceosomes (large macromolecular structures made up of polypeptides and RNA) to produce
mature mRNAs. And the mature mRNAs in turn serve as the templates translation machinery use
to produce their encoded polypeptides.
Another famous example of regulation through compartmentalization is the transcription factors,
which regulate transcription. Regulation of a lot of transcription factors occur by keeping them in
the cytosol.
Proteins are generally regulated at different levels. One way is by regulating it at the level of
expression (the amount of protein) through regulating synthesis and degradation. Another one is
by regulating its activation status, so even if the level of expression of a lot of proteins do not
change, regulation can occur through post-transcriptional modification. The protein cyclin is
regulated by nucleic synthesis, and it was called cyclin due to the fact that its level goes up and
down. Another protein is P53 protein, which is controlled by deciding whether the cell wants to
break it down or not. A third way to regulate proteins is by sub-cellular localization, which isn’t
possible without compartmentalization.
When the mRNA reaches the cytosol, translation starts in the cytosol, and may proceed at the
surface of the rough ER. Seeing a still microscopy image showing the ribosome at the surface of
the ER doesn’t mean that two types of ribosomes exist, one of which stays at the ER’s surface.
Ribosomes are made up from rRNA and polypeptides. The latter are made in the cytosol and then
go to the nucleolus, where they will assemble with the rRNA to form the small and large
ribosomal subunits. In fact, there is only one pole of ribosomes. These translate mRNA into
proteins, which in case they have a signal that destines them to the ER will move the whole
translation machinery to the ER. A live image by microscopy would show some ribosomal
subunits coming to the surface of ER and others leaving.
In addition of separating the functions and environmental conditions and regulation,
compartmentalization allows the cell the ability to establish electrochemical gradients. In
eukaryotes, the cell doesn’t only establish electrochemical gradients with its surroundings, but it’s
able to do so intracellularly. In biological systems, potential energy exists in two forms: chemical
bonds and electrochemical gradients. In case of chemical bonds, breaking bonds will provide the
cell with energy. However, in the case of the electrochemical gradient, dissipating the gradient
provides energy. All living energy comes from the sun, which then will pass through different
natural layers. The first layer in nature to translate this energy is producers, which undergo
photosynthesis, by taking row elements and sun rays to give more complex molecules
(carbohydrates), which are broken in cellular respiration (in the mitochondria) to form ATP,
which is another form of chemical bonds to store potential energy.
Bacteria keep pumping protons across the plasma membrane to establish an electrochemical
gradient, which can be dissipated to make ATP. This is also the case in eukaryotic cells.
Electrochemical gradients also provide us with Action Potential.

Cytoskeletons
Exclusive to eukaryotes, cytoskeletons are actin filaments (the thinnest), intermediate filaments
and microtubules (which are the thickest). These structures function in shaping and support.
Underneath the plasma membrane, there is a highly branched actin network that can support the
cell shape, and underneath the nuclear envelope, there is a very elaborate matrix of intermediate
filaments that can support this envelope. Thus, these filaments provide mechanical strength and
support. However, they shouldn’t be very rigid, as the cell needs to be flexible and change in
shape depending on its function and the needs imposed by the environment. For example, a
neutrophil (immune cell) needs to be flexible to trace a bacterium, and flexibility can be shown in
the example of phagocytosis. To sum up, a cell needs to be flexible in changing its shape,
depending on its function. In this context, every single differentiated cell has different function,
thus a different shape or form. For example, epithelial cells are naturally not moving around, so
they have a shape that fits this function. These cells, however, can move if they express the
proteins responsible for their movement. So, there is regulation of gene expression. This is due to
different differentiation patterns of cells, which is associated with genes’ expression.
This regulation of gene expression is absent in cancer cells, so they express genes regardless of
the organism, thus expressing the genes, that are normally regulated in normal cells.
Flexibility of the cell is needed for cell migration, phagocytosis, a response to the environment,
cell division, apoptosis, signaling (when receptors are signaling, they need to cluster), etc. All of
these flexible events are mediated by the cytoskeleton. Therefore, the cytoskeleton needs to be
flexible, which occurs by polymerization and de-polymerization, to allow the cell’s flexibility.
The third function of cytoskeleton is particular to microtubules. These microtubules are tracks
through which proteins can move. This happens through proteins called Microtubules Associated
Proteins (or MAPs). These proteins hydrolyze ATP and undergo conformational changes, to be
able to “walk” on the microtubules, exactly the same way myosin hydrolyses ATP to undergo a
conformational change for the contraction to happen. In doing so, MAPs carry proteins on the top
of the microtubules. They can also carry vesicles and whole organelles. Proteins translocation in
the cell is an intracellular trafficking or intracellular translocation, where in most cases
microtubules are being involved, which adds the value of regulation by these tracks, instead of
proteins just swimming in the cell.

Universal Principles of Living Cells

1. Information needed for cell growth, function and multiplication is
found in DNA. DNA is duplicated and passed on to daughter
cells.

DNA provides the starting material, from which the functions in

the cell are encoded. However, there’s the factor of epigenetics.
This can be supported by the fact that although the Human
Genome Project was finished by 2000, we don’t know the answer
to everything, as we don’t know what every protein does nor the
differential expression between different cells and the information
associated with every protein. This can be explained by the added complexity, through
regulation of gene expression, regulation by epigenetic factors, etc. Thus, different layers
of regulation are added, such as differential expression of a certain gene based on
different types of cells, or within the same type of cells based on the signal and cascades
 of signals. Some layers that would add complexity also include the interference of some
other cells, mutations, etc.
2. One dimensional chemical sequences in DNA code for both the linear sequences and the
3 dimensional structures of
proteins.

The 3D folding of proteins is

governed by hydrogen bonds,
hydrophobicity, disulfide bridges
& electrostatic interaction.
If other factors (than DNA)
would intervene in this folding
of proteins, such as the solvent,
then everything would fall apart
in the cell, in case it encounters
any problem.
On this basis, bioinformatics
programs predict the 3D structure of some proteins. However, the field of structural
biology (using NMR, crystallography…) is still needed, despite all the algorithms, since
the latter can’t develop the structures of large proteins.

Proteins having multiple subunits will form the quaternary structure, which dictates how
multiple subunits will bind to each other. This is regulated. For example, protein PKA
has a catalytic subunit and regulatory subunits, thus having the quaternary structure.
When the protein is inactivated, these subunits are together, however, when it gets
activated, they separate.

Regulation of proteins would also include cleavage of proteins (within the same
supplement) by certain enzymes, within a cascade, for them to get activated.

3. Macromolecular structures assemble from subunits.

Many cell components self-assemble without enzymes, they contain the information
required to assemble into complex structures. Molecules get together by diffusion, lock
and key (exclusion of water), hydrogen bonds and electrostatic bonds hold subunits
together. Ex: DNA to form chromatin, RNA to form ribosomes, polymerization to form
actin.

Hydrophobicity is an active process, where water molecules get excluded from the
hydrophobic molecules (that don’t interact with water, as they’re non-polar or
uncharged). Hydrophilic compounds have poles of charges or partial charges, which
enable them to interact with water (as it has a partial positive charge and a partial
negative charge). In fact, molecules are pushed to assemble together, by the exclusion of
water.

4. Membranes grow by expansion of pre-existing membranes.

This includes components being cut off a compartment and fusing with a receptor from
another compartment. This involves processes of endocytosis and exocytosis as well. In
 exocytosis, the vesical fuses with the membrane to dump its components out of the cell,
such as in the case of neurotransmitters’ secretion. This occurs in the opposite way in
endocytosis, where the membrane invaginates and takes in certain components, then buds
in and form vesicles.

The membranes formed intracellularly aren’t exactly the same in terms of structure as the
plasma membrane. Although they all have the same basis of phospholipid bilayer, they
differ in their composition of proteins. In mitochondria, for example, H+ pumps are
needed to pump H+ ions out, however, these proteins
aren’t present in other organelles. Additionally,
polarized cells have different compositions of proteins in
the membrane of different poles, as these cells have an
apical side and a basolateral side.

5. Signals target cell constituents (proteins) to their

destined locations.
After translation, a signal, which is part of the protein
coding sequence, will direct the protein to its destination.
For example, proteins destined to the nucleus have a
NLS (nuclear localization sequence) that binds receptors
which facilitate passing through nuclear pores. We need
other accessory
proteins, carriers and
receptors to recognize
the signal for the protein to be carried to its destination.

6. Cell constituents move by diffusion, pumps and motors.

A lot of components move in and out of the cell or its organelles via integral proteins,
such as pumps, channels...

7.

Receptors and signal transduction relay extracellular environmental clues to the cell.
These receptors are present at the surface of the cell, for it to communicate with other
cells and its external environment. This is called signal transduction, which starts at the
level of the cell surface, through the receptor which receives a stimulus, then relays the
message intracellularly, without physically allowing molecules to get transmitted through
the membrane. On the other hand, pumps and channels transmit physical molecules
across the membrane.
 Signal transduction is a pathway with several steps with several proteins being involved,
to lead to an effect that starts at the surface and can end in the nucleus. In this context, the
cell can get signals from its environment that can affect the cell’s gene expression.

8. Feedback mechanisms.
Any pathway inside the cell has feedback
mechanisms. This can be explained by the product
affecting the reaction. For example, the accumulation
of the product makes the reaction not favorable
energetically, when the product is no longer needed.
This concept of energy preservation can be found in
all living organisms, starting from prokaryotes. The
feedback mechanisms are, however, more
sophisticated in eukaryotic cells.
This happens in every step in signal transduction,
where the product feeds into the reaction, affects the
enzyme and controls whether the reaction proceeds
or not.
Feedback mechanisms are classified as positive and
negative loops. In a positive feedback loop, the
product activates the reaction. For example, an
activated receptor activates a protein, which once activated has the ability to further
activate the receptor. Another example is where a receptor is activated in a response to a
ligand, then the activated receptor will positively activate the expression of the protein
that will further positively activate the receptor. Other examples also include an activated
receptor that activates a transcription factor that will positively regulate the expression of
more receptor to amplify the response.
The cell cycle is a perfect example of positive and negative feedback loops. The cell
cycle is the cycle the cell goes through when undergoing interphase and mitosis (shortest
period within the cycle). Interphase, which is the longest period, is the period in which
the cell is preparing itself for the division, with checkpoints (should the cell does this or
not? For hours). The cell cycle is made of stages, G1, S and G2. To go from one stage to
the other, the cell has developed checking mechanisms, called checkpoints. This is a
molecular barrier, which is a series of events that need to occur for the cell to be able and
prepared to go into the next stage that is defined by other physiological characteristics.
These checkpoints consist of a collection of proteins that will either inhibit or allow the
cell to proceed into the next stage. If anything goes wrong in the next stage, this will
feedback an inhibition of the cell going into the next stage in a negative feedback
mechanism.

Overview of Cellular Constituents

The nucleus is the powerhouse of the cell, and it houses the chromosomes. In most cases, the
nucleus contains the chromatin material, which is less condensed than chromosomes. In fact, both
chromosomes and chromatin material have DNA-binding proteins, which have nothing to do with
condensation, as they are present in the least stage of folding, the nucleosome stage. Packaging,
 in this sense, differs from condensation
(which is more at the macro-level).
Packaging occurs for the DNA to fit in the
nucleus (DNA of 1 meter fitting in a nucleus
with a diameter of around 2 microns).
Nucleic acid is made of nucleotides Adenine,
Thymine, Cytosine and Guanine, that bond to
each other through phosphodiester bonds,
and wrap themselves around a nucleosome
core particle, which is made of eight proteins,
called histones. In the nucleosome core
particle, we have H2A, H2B, H3 and H4
proteins, each present as a dimer. This is the first level of organization and packaging, and it’s
called a string of beads, with linker DNA. When the cell is vulnerable and undergoing apoptosis,
we shock off the DNA in these vulnerable places in this structure, which are the DNA linkers.
The next level of organization is the thicker axis, and the last one is the loops. These levels of
organizations are all packaging, and not condensation, and that’s how chromatin fibers look like.
Actually, this chromatin can’t be seen under light microscopy. However, chromosomes can be
visualized under light microscopy (it’s condensed and viewed as sister chromatids). Chromatin
looks like flimsy little fibers that are not organized, and with this structure, mitosis can’t occur, as
the homology search won’t be possible. For this reason, we have condensation. Non-condensed
packaged DNA is active in terms of transcription and replication, as proteins will have access to
the DNA. In fact, DNA duplication for cell division happens when DNA is in its chromatin-form,
before condensation and the occurrence of mitosis. DNA in chromosomes, in their condensed
forms, isn’t active.
To sum it up, going from chromatin to chromosomes is for organizational purposes and
homology search, and going back to chromatin is for the activity of the DNA.
With respect to the gene constitution of the nucleus, only 5 % of the 3 billion nucleotides in
human DNA constitutes genes, and as we go to a higher level of organism (from C. elegans to
Drosophila), not only do we find more genes, but also an exponential increase in the non-coding
regions. This proposes a very important role of these non-coding regions, in accordance with
evolution. Scientists suspect that these regions have a regulatory function, as reports show that
many patients who correlate diseases show mutations in the introns. This serves as a proof that
the introns get spliced, and before being spliced in the mRNA, they regulate transcription.
The heterochromatin region in the middle is called centromere, and that at the end is called a
telomere. The latter functions in stabilizing chromosomes. As the somatic cells divide, telomeres
shorten with every round of division. That way, telomeres are molecular rulers of age and of how
many rounds of divisions the cell still has, as the biological definition of aging is telomere
shortening. Biologically, aging is when cells become older and has divided many times, with
telomeres being shortened. Although the cells grown in the lab are immortalized cells, they start
dying after several rounds of divisions. When the telomeres shorten beyond a certain measure,
where the cell won’t divide anymore, and that’s why aging occurs. However, most people die of a
disease and not of a biological old age. When the telomeres shorten, the cells gradually start to
die and the organs start to fail.
Telomerase is an enzyme that has the ability to replenish telomeres. Unfortunately, telomerase is
not expressed in somatic cells. It’s only expressed in three types of cells: stem cells, and that’s
why they have the ability to divide all the time, germ cells and cancer cells. The latter don’t create
anything new, they just lead to an inappropriate expression or a dysregulation of expression of a
protein that shouldn’t be expressed. When inappropriately expressing telomerase, cancer cells can
divide and become immortal.
Research on telomerase in the US is divided into one focusing on the relation between telomerase
and aging, and the other focusing on cancer. The former would suggest an expression of
telomerase in somatic cells, with the latter suggesting stopping its expression in cancer cells.
However, expressing telomerase in normal somatic cells can make them cancerous.
Opposite to the myth that cancer has increased drastically, the ability to diagnose it has improved.
• The Nucleolus is where ribosomal units are made.
• The nuclear envelope has nuclear pores that function in the continuous communication
between the cytoplasm and the nucleoplasm. For example, mRNA leaves the nucleus to
the cytosol through these pores. These nuclear pores are not passive channels and they
are heavily regulated, and this is at the basis of regulation of the mRNA before it leaves
the nucleus. There are a system of carriers and accessory proteins involved in this
process.

The Endoplasmic Reticulum (ER) has the rough ER and the smooth
ER. It covers the outer part of the nuclear envelope and controls
Ca2+, as there are many Ca2+ sequestering proteins in the ER with
many Ca2+ pumps at the surface of the ER as well, which keep
pumping calcium into the ER. In this context, the ER plays the role
of an internal store of calcium, and whenever it’s needed, it can be
released to the cytosol.
Proteins containing signal sequences will direct the ribosome to the
surface of the rough ER.

The plasma membrane Impermeable to ions and water due to the

hydrophobic interior of the lipid bilayer, which is derived from
triglycerides. So they have glycerol, two fatty acid chains and a
phosphate group. These phosphoglycerides have a negative charge
on the phosphate group (hydrophilic), and have the non-polar and
uncharged fatty acid part (hydrophobic). These phospholipids, when present in aqueous medium,
will form spheres hiding the hydrophobic part inside. In biological membranes, the
phospholipids form a bilayer sandwiching the hydrophobic part inside, with the hydrophilic part
being exposed both intracellularly and extracellularly.
The impermeability of the plasma membrane protects the cell from lysing, by ensuring that
nothing is moving in and out freely, creating an osmotic pressure. Nevertheless, the plasma
membrane is also extremely available for exchange, through the proteins that allow the cells to
either import or export molecules depending on their needs. Passive diffusion is usually a free
diffusion with no vehicle. When we have a carrier, we call it facilitated diffusion, where there is

regulation and control over the process.

Some of the Microtubule Associated Proteins always go to the negative end and some of them
always go to the positive end.

 There are analogous proteins for actin and for tubulin in prokaryotes. And bacteria (E.
coli) have proteins that’s analogous to a protein that regulates actin, but the function is
still unknown. One of these proteins was found to regulate one type of secretion to
regulate polarity. The question remains on how this protein regulates polarity, when it has
a cytoskeleton component. Even though in this proteins we don’t have filaments, but
they’re still considered cytoskeleton for their homology.
 Bacteria also have DNA-binding proteins.