1.

CELL AND METHODS OF STUDY

1.1 INTRODUCTION
All living creatures are composed by cells. It is the fundamental unit of living things.

• Prokaryotes: bacteria and cyanobacteria (Primitive)

• Eukaryotes: more complex and evolved
• Animals
• Plants
• Fungi
• Microorganism

Complex and organized systems capable of using matter and energy to grow and self-replicate (living beings):
they consist of cells

1.2 CELL THEORY

• All are surrounded by a membrane that separates their content from their exterior.
• Cells contain genetic material (nucleus) that stores all the instructions needed for their survival and
activities they carry out.
• Have their own power plant-energy release system that powers all the cell’s activities. They perform
a lot of activities and they need a lot of energy, produced on the mitochondria.
• They reproduce new cells are produced from exiting cells. They can reproduce.

How was it possible for scientist to discover cells?

Invention of the 1st microscope (1590) was what made it possible for cells to be discovered but Van
Leeuwenhoek improved the microscope with which he became the first scientist to observe certain cells, such
as bacteria, protozoa, muscle and red blood cells (1673)

Who discovered them?

• Robert Hooke in 1665. While analysing a sheet of cork with a simple microscope, he observed that it
was made up of small, honeycomb-like compartments. He called them cells, from latin word “small
room”
• Matthias Schleiden, plants are made by cells (1838). Theodor Schwann discovered that animals too.
• Rudolf Virchow stated that cells can be produced from other plants by cell division in 1855.

After cell theory:

• Mendel (1860): study of inheritance

• Ramón y Cajal (1888): central nervous system made by unique cells, neurons. Nervous system is
a continuum, no interruptions.
• Sutton, Boveri and Morgan (1910): discovered chromosomes.
• Watson and Crick (1975): structure of DNA and RNA.
• L. Margullis (1981): stablish a new concept about how life is originated. Specials infection by other
organisms.
1.3 ANIMAL CELL ANATOMY

ORGANIZATION AND STRUCTURE OF CELLS

ULTRASTRUCTURE OF PROKARYOTIC CELLS (0.1 – 2 μm)

• No nucleus or organelles, only ribosomes 70S

• Entirely filled with cytoplasm
• Nucleoid-one circular DNA molecule not associated with
proteins
• Cells division by binary fission
• A single (plasma) membrane
• Cell wall-outer covering of most cells that protect the
bacterial cells and give it shape.

In unicellular organism one cells carry all life functions: nutrition,

metabolism, growth, response, excretion, homeostasis,
reproduction….

MULTICELULAR ORGANISM/SPECIALIZED TISSUES

• In humans 220 different and highly specialized cell types

• Gene expression: production of specific proteins and cell
differentiation
• The control of gene expression is the key to development
• Cells organized to interact and cooperate. Emergent
properties, they come up as a result of this cooperation.
Ex: our life.

Cells come in different shapes and sized.

• Some neurons greater than 1 m.

• Normal size: 4-100 micrometers (1 micro= 10^-3 mm = 10^-
6 m)
• Subcellular structures:10-100 nanometers (10^-9 m)
• Visible scale: anatomy (macroscopic) <10 nm: molecular
biology

1.4 MICROSCOPE
• Light microscope
• Improvements in the second half of the 19th century allowed the discovery of bacteria and study of
chromosomes, basis of sexual behavior (mitosis & meiosis)
• Laser scanning microscope, with laser illumination centred on a plane of preparation (Minsky 1957)
2000x
• Electronic microscopes (1930) use electrons as sources of illumination Magnification 500,000
FLUORENCE CONFOCAL MICROSCOPE

• Fluorescent microscope: to observe samples treated with

fluorescent markers. Each marker has l concrete (excitation
wavelength), emission light (l not absorbed)
• Confocal microscope: with laser illumination centred on a
preparation plane (Minsky 1957) 2000x; less "noise" and possibility
of 3D reconstruction
• Instead of illuminating the whole sample at once, laser light is
focused onto a defined spot at a specific depth within the sample. EXAMPLE: “BRAINBOW”

ELECTRON MICROSCOPE

• Scanning electron microscope (SEM): the electrons are

dispersed on the sample without crossing it. Ultra-thin samples
are not necessary: 3D image.

• Transmission electron microscope (TEM): the electrons go

through the preparation based on the higher/lower density of
the sample> necessary ultra-thin samples. They are collected on a fluorescent screen of photographic
film.

SAMPLE PREPARATION FOR MICROSCOPY

1. Fixing: Preserve the sample. Formaldehyde, Bouin fixative, Glutaraldehyde ...

2. Dehydration: Alcohols of increasing concentration. Organic solvents (xylene, toluene...)
3. Embedding: Paraffin, resins…
4. Cutting with microtomes / ultramicrotomes
5. Dewaxing/rehydration: Solvents and alcohols of decreasing concentration.
6. Staining: Varied depending on desired outcome.
7. Mounting: Slides with preparation + coverslip.
8. Frozen samples:
• For traditional studies, enzymatic assays, intraoperative biopsies...
• Traditional freezing or liquid N2 Cryostat cutting.
• Fixing, staining, mounting on slides…

SAMPLE STAINING FOR LIGHT MICROSCOPY

• Histochemistry:
o Wide range of colour solutions
o Mechanism of action not fully known: acid components vs basic
o Specific structures / cells.
• Hematoxylin-Eosin:
o Hematoxylin: basic dye → dyes structures/acid elements (basophilic): dark blue
o Eosin: acid dye → basic elements (acidophilic): pink
• Periodic Acid-Schiff (PAS): Labels Carbohydrates
• Feulgen stain: Chromatin localization
• Detection of lipids (Sudan): It binds triacylglycerols (red), myelin (black)
• Localization of enzymatic activities by adding a substrate that reacts with the enzyme, precipitates
and is coloured in the presence of a reagent: NADH, ATPase, Phosphatase, Peroxidase... Cold
processing is usually required
1.5 TECHNIQUES FOR IMMUNOLABELING
They allow the detection of the products of gene expression (proteins) by taking
advantage of the specificity of the antigen-antibody binding.

Antibody production/generation

Stimulation of the immune system with immunogens: Substances that induce a

specific immune response against an epitope (structural element recognized by
antibodies)

AntiBody PRODUCTION

Immunizations: Injection with / without Freud's adjuvant in rabbits / goats / pigs

of purified proteins. → Large amount of serum (non-plasma) → Direct use.

Concentration: Polyclonal antibodies rapid production and amplification of antigen-antibody reaction

VISUALIZATION OF ANTIGEN-ANTIBODY COMPLEX

Conjugation of antibodies: detection of antigen-antibody complexes

1. Direct labeling: antibody-bound chromogen (I AB conjugated to a chromagen)

2. Indirect labeling: chromogen coupled to a II AB → amplification of the signal with respect to the direct
marking.

TYPES OF CHROMOGENS

Fluorochromes: Substances that emit light when excited by a different light FITC, Alexa Fluor, Rhodamine

Proteins and enzymes: They induce the coloured reaction of a substrate.Example: Horse radish peroxidase
(HRP) in the presence of H2O2 it catalyzes the oxidation of the diaminobenzidine (DAB) → brown
1.6 IMMUNOHISTOCHEMSTRY
Protein detection – light or electron microscopy

1.- Permeabilization
2.- Blocking unspecific antigen binding
3.- I AB - wash
4.- II AB - wash
5.- Reaction/labeling revealed

1.7 CELL CULTURE

In vitro maintenance of isolated cells.

- Advantages: Study of properties, nutrient requirements and functions of specific cell types
- Management of cells before transplantation

Cell origin: Primary cultures: of body fluids of solid tissues: cell disintegration enzymatic mechanics; Explants;
Cell lines

FLOW CITOMETRY

Quantification and separation of labelled cells.

Based on the use of laser light, used in the cell count and classification according to their morphological
characteristics, presence of biomarkers, and in protein engineering.

STEM CELLS

• Unlimited capacity to replicate.

• Can be used to grow tissues or replace damaged/lost
cells.
• They are not fully differentiated and can differentiate to
produce different cell types:
1. Embryonic (4-5 days post fertilization)
2. Umbilical cord
3. Adult
1.8 CELL CULTURE MEDIUM
• Buffered liquid nutrient medium:
1. Undefined: varying serum concentrations
2. Defined: known components and concentrations
• Components:
o H2O and mineral salts (K +, Na + and phosphate)
o Bicarbonate buffer (HEPES)
o Phenol red
o Glucose (pyruvate and / or lactate)
o Albumin, Antibiotics
o Growth factors, interleukins, etc. And variable serum concentrations: 5-20%
• Cell seeding/cell plating
o Plastic dishes: into a flask or multiple wells plates to which they will attach themselves
o Normal medium
o Treated for adhesion culture
o Coated with extracellular matrix elements
o With nutritious monolayer (feeder layer)
• Culture growth:
o Regular media change
o Constant temperature conditions (incubator)
o Limited growth due to nutrient availability and density
• Precautions to consider avoid contamination → work in sterile conditions

2. CELL MEMBRANE
Functions:

- External limit of the cell and organelles

- Support, transport, signaling and recognition
- Controls substance exchange
- Regulates cellular interactions with the
external environment
- Responsible for internal cellular organization

Composition and structure:

• Fluid mosaic model (Singer and Nicolson, 1972): Proteins are

embedded into the lipid layer (internal proteins)
1. Lipids: Barrier
2. Proteins: Transport and Communication, signaling
3. Carbohydrates: Recognition of other cells, first from
similar cells to make tissues together, or second to
recognise the external environment. They can be attached to lipids and proteins
• Freeze-fracture technique (EM)-breaking the membrane along the line of least resistance.
• The membrane is a phospholipid bi-layer consisting of a polar head group, phosphate, and
hydrocarbon tails.
• Amphipathic (Semipermeable): Hydrophobic compounds can diffuse across the membrane;
Hydrophilic compounds will not diffuse across the membrane.
2.1 LIPIDS
Variable composition, depending on the cell /organelle type (60%lipids vs 40% proteins)

There are three major classes of membrane lipids – the phosphoglycerides, sphingolipids and sterols

• Phosphoglycerides: <50%
• Sphingolipids-derivatives of sphingosine
• Sterols: Cholesterol (<25%) keeps the membrane fluid membrane
permeability decreases with its abundance

The phosphoglycerides and sphingolipids can be combined as one class, the

phospholipids.

Membranes are asymmetrical:

- Different Integral and Extrinsic proteins

- Lipid movements:
o Translation
o Rotation
o Translocation: flip-flop (flippases, floppases, scramblases-ATP
dependent-exchange between 2 layer)

THE MEMBRANE IS FLUID AND SEMIPERMEABLE

Depends on:

• Temperature
• Fatty acids saturated/unsaturated and Cholesterol →
Decreases fluency

Simple diffusion

Osmosis - passive transport (concentration gradient)

Facilitated diffusion-a transporter (integral protein) is needed

Active transport (depends on energy)

RELATIVE PERMEABILITY OF A PHOSPHOLIPID BILAYER

2.2 PROTEINS
• Amphipathic
• Integral (Transmembrane)
• Peripheral- Attached to lipids by covalent bond
• Bound to protein by electrostatic attraction
• Protein distribution is asymmetric
• There is no translocation, if translation.

MEMBRANE PROTEINS FUNCTIONS

Membrane proteins perform a variety of functions vital to the survival of organisms:

• Membrane receptor proteins relay signals between

the cell's internal and external environment.
• Transporter proteins move molecules and ions across
the membrane. They can be categorized according to
the Transporter Classification database.
• Membrane enzymes may have many activities, such as
oxidoreductase, transferase or hydrolase.
• Cell adhesion molecules allow cells to identify each
other and interact. For example, proteins involved in
immune response.

LIPID RAFTS

Subdomains of the plasma membrane (increased thickness) that contain high concentrations of lipids and
proteins. Serve as organizing centers for the assembly of signalling molecules, influencing membrane fluidity
and membrane protein trafficking, and regulating neurotransmission and receptor trafficking.

Facilitate reception of signals. Often grouped in caveolae:

Invaginations of membrane (60 nm), coated with caveolin.
TRANSPORT PROTEINS

- Membrane transport protein (or simply transporter) is a

membrane protein involved in the movement of ions,
small molecules, or macromolecules, as another protein,
across a biological membrane.

- They are integral membrane proteins - they exist

permanently within and span the membrane across which
they transport substances.

- May assist in the movement of substances by facilitated

diffusion (as molecules and ions move down their
concentration gradient ) or active transport (in the
direction against the concentration gradient) or other
obstructing factor (requires ATP expense = pumps).

- The two main types of proteins involved in such transport

are broadly categorized as either channels or carriers.

CHANNEL PROTEINS / ION CHANELS

- Allow the passage of ions or specific molecules in favor of gradient.

- Open (e.g. K + channel responsible for the membrane resting potential)

Closed in resting state (majority) while opening is regulated by stimuli:

1. Ligand binding –ligand gated

- Directly gated
- Indirectly gated - binding to regulatory molecules (G protein, cGMP)

2. Membrane potential change - voltage gated (K, Na, Ca, channels)

- Responsible for maintaining

ionic concentration across
the membrane.
- Responsible for resting and
action potentials: nervous
impulse conduction.
LIGAND AND G-PROTEIN GATED

Ligand gated- direct gating

Ligand-gated ion channels - also commonly referred as

ionotropic receptors, are a group of transmembrane ionchannel
proteins which open to allow ions such as Na+, K+, Ca2+, and/or
Cl− to pass through the membrane in response to the binding
of a chemical messenger (i.e. a ligand), e.g. a neurotransmitter.

G-protein gated –indirect gating

Typically, the activated effector protein begins a signaling cascade

(second messenger and protein kinases), which leads to the
eventual opening of the ion channel.

G-protein can also mediate direct channel gating - via physical

interactions between G- protein subunits and the channel protein.

The GTP-bound α-subunit in some cases can directly activate the

ion channel. In other cases, the activated βγ-complex of the G
protein may interact with the ion channel.

ACTION POTENTIAL: MUSCLE CONTRACTION

CARRIER/TRANSPORTER PROTEINS

• Proteins which bind one particular ion/molecule and assist it in moving across a membrane.
• Facilitated Diffusion: Passive process allowing larger molecules to diffuse, through a process of
binding and being released from special proteins. Speed is limited by the number of binding proteins.
(e.g. glucose transport).
• Active Transport: Movement of substances across the membrane, requiring energy, against the
concentration gradient (E.g amino acids).
• Carry out specific transports in favor or or against gradient (Pumps).
• They can simultaneously transport 2 substances (coupled transport)-cotransporters.

2.3. MEMBRANE TRANSPORT

• Particles move across the membrane by simple diffusion,
facilitated diffusion, osmosis and active transport.
• Fluidity of the membrane allows materials to be taken
into cells by endocytosis or released by exocytosis.
• Vesicles also move materials within cells.

PUMPS (ATPasas)

Transport proteins driven by ATP. Essential in:

• Maintenance of ionic gradients (Na + / K + pump)

• Muscle relaxation (Ca2 + - ATPase)
• Ph maintenance (H + pump, H + / K + pump)
• Elimination of toxins and xenobiotics:

ABC pumps = ATP-binding cassettes (flippases)

ABC transporters often consist of multiple subunits, one or two of

which are transmembrane proteins and one or two of which are
membrane-associated ATPases
SODIUM/POTASSIUM PUMP

2.4 CONNEXION & ADHESION PROTEINS

- Responsible for tissue binding: Involved in binding cell-cell (CAM) or cell-substrate (SAM).
- Responsible for the internal cellular organization.
- They can form multiprotein complexes.
1. Immunoglobulin Superfamily:

CAM. Calcium independent. NCAM, ICAM, NgCAM, VCAM (CD106), CD2, Nectin…

2. Cadherins:

CAM. Calcium-mediated. K-Cadherin, E-Cadherin, Desmogleins and Desmocolinas

3. Selectins:

CAM. Calcium dependent. Extracellular lectin domain: protein that recognizes carbohydrates Eselectin (CD62),
L-selectin and P-selectin.

4. Integrins: Transmembrane receptors that facilitate cell-extracellular matrix (ECM) adhesion. Calcium-
dependent. Up to 24 types:
- Collagen receptor (a1b1): Connective
- Fibronectin receptor (a5b1): general
- Laminin receptor (a6b1): basal lamina

Some mediate cell-cell binding:

LFA-1 (aLb2) (CD11a / CD18): lymphocytes, recognized by ICAM

VLA-4 (a4b1) (CD29): leukocytesVCAM ligand
2.5 EXOCITOSIS
It is a form of active transport in which a cell
transports molecule (e.g., neurotransmitters and
proteins) go out of the cell by expelling them
through an energy-dependent process.
2.6 ENDOCITOSIS
Endocytosis Imports extracellular molecules by
forming vesicles from the plasma membrane.

3. THE NUCLEUS
3.1. NUCLEUS-EUKARYOTIC CELL
• It is the largest cell organelle.
• Membrane enclosed organelle-contains nucleolus
and nucleoplasm/karyoplasm.
• Discovered by A. Leeuwenhoek, R. Brown in 1831
• Stores coded information i.e. DNA-nuclear genome.
• It is essential for life of a cell - As it contains all the
necessary instructions for their survival and the
activities that carries out.
• Main function is to maintain the integrity of the
genetic material - i.e. genes.
• Through gene expression it controls cellular activities and cell differentiation.

• Present in all eukaryotic cells, (except erythrocytes and keratinocytes of the stratum corneum).
• Not differentiated in prokaryotes
• Usually 1 per cell but some cell contains several nuclei like:
o Syncytium (skeletal muscles, osteoclasts)
o Also, cells under pathological conditions - metastasis of tumor
cells.
• Shape and position vary depending on the cell type and its function.

3.2. NUCLEOLEMA (NUCLEAR MEMBRANE)

• Double membrane (2x7nm) with perinuclear cistern (20-40 nm).
• Separates the inside of the nucleus of eukaryotic cells from the cytoplasm - regulatory point (customs)
in the transport of molecules - the nuclear pore
• Inside the nucleus - the chromosomes that store the genetic information, the accessory proteins
involved in the condensation and decondensation of the chromosomes during the cell cycle.
• Also proteins responsible for the synthesis of RNA and DNA copy (RNA and DNA polymerases
respectively), which are accompanied by transcription factors and other proteins that regulate their
activity. Continues to the Rough Endoplasmic Reticulum (RER) → same origin: shares functions with
RER
• During mitosis nuclear membrane disappears and reappears at the end of it.
➢ COMPOSITION
• Definite structure revealed by Electron microscope and freeze-fracture technique
• The outer membrane is continuous in many places with the rough ER. Like the rough ER it is dotted
with ribosomes.
• The inner membrane is attached to the nucleoplasm and attached to it is a sheet-like structure of
protein filaments called the nuclear lamina ( thought to help give strength and support to the nuclear
envelope and possibly provide an anchor point for chromatin fibers)
• 30% lipids:
o Similar to ER but more saturated (90% phospholipids)
o Neutral lipids: triglycerides and cholesterol
• 70% protein:
o Outer membrane: similar to ER: with ribosomes, vimentin binding proteins
o Internal membrane: lamins - binding proteins,
o Between membranes: Nuclear Pore Complex

3.3. NUCLEAR PORE COMPLEX

• Point of entrance and exit of different soluble substances.
• Variable number depending on the cell type and
differentiation. 3000-4000 per nucleus, depends on the
number of cell transcriptions).
• Diameter: 50-80 nm.
• It consists of about 30 different proteins(nucleoporins).
• Passive diffusion of mols. <50 kDa. and in favor of gradient.
• The larger molecules enter actively, recognized by specific
signal sequence.

3.4. NUCLEAR PORE TRANSPORT (active)

• Molecules transported to the interior must
have a Nuclear Localization Sequence (NLS)
• Those that move out, a Nuclear Export
Sequence (NES) -known sequences of amino
acids.
• Karyopherins (importins and exportins):
receptor that recognize NLS or NES in proteins
and transport them through the pore.
• The capacity of both importins and exportins
to transport their cargo is regulated by the
small G protein called Ran. (small enough to
diffuse through the pore down the
concentration gradient).
• Ran can bind both GDP and GTP
• Step in favour of gradient favoured by the rapid dissociation of the complex protein-importin when
entering, or the Ran-GTP upon exiting.
• Several diseases have been linked to pathologies of nucleoporins, notably diabetes, primary biliary
cirrhosis, Parkinson’s and Alzheimer disease.
3.5. NUCLEAR LAMINA
• Two-dimensional network, located at the periphery of the nucleoplasm especially dense under the
inner nuclear membrane.
• Composed of proteins- lamins A, B, C: A: 72kDa; and C: 72kDa, B1: 65kDa; B2: 78kDa
• Structural character -mechanical support to the nucleus, chromatin organization, cell cycle
organization, DNA replication, DNA repair and cell differentiation and apoptosis(program death cell.
Cells implote, no explote.)

3.6. NUCLEOPLASM OR KARYOPLASM

• Aqueous phase with:

- Cofactors and precursor molecules: ATP, NAD +, Acetyl- CoA, steroid hormones - cortisol, estrogen,
progesterone, testosterone, fatty acids, phospholipids
- Proteins and enzymes: involved in replication, repair and transcription of nucleic acids, associated with
DNA and RNA, of the nucleoli.
- Nucleic acids: DNA and RNA

• Chromatin:

- DNA Complex and Associated Proteins (Histones).

- Genome: Genetic information (DNA) of the nucleus.
- Sets of genes: DNA region that produces a functional RNA
- In humans: 3 billions of these pairs of DNA bases, 23 pairs of chromosomes within the nucleus of all
our cells.
- Human Genome Project: small number of structural genes encode all our proteins(aprox90000) but
large parts of noncoding DNA has regulatory function-may be linked to diseases, and may be used as
therapeutic targets.
3.7. CHROMATIN
Chromatin is a complex of macromolecules found in cells, consisting of DNA,
protein, and RNA- The basic substance of chromosomes. There are 2 possible
configurations:

- Euchromatin (10%) - decondensed transcriptionally active, with a high

concentration of genes. DNA which codes genes that are actively
transcribed ("turned on") is more loosely packaged and associated with
RNA polymerases.
- Heterochromatin (90%)-condensed, in principle, transcriptionally
inactive (DNA which codes inactive genes ("turned off") is more
condensed and associated with structural proteins.

3.8.HISTONES
• The most common cellular proteins.
• They are the chief protein components of chromatin, acting as spools around which DNA winds, and
playing a role in gene regulation.
• Histones have structural role but are also involved in epigenetic regulation: i.e. Non-genetic factors
that modify gene expression.
• The histone configuration alters the accessibility of
the transcription factors:
o Methylations: Can increase or decrease genes
transcription depending on which amino acids
are methylated. Intended for the maintenance
of a particular type of gene expression
o Acetylations: in the tails of histones at the level
of lysine and arginine: modifies the chromatin
to be transcription- ready.
o Deacetylations: chromatin condensation
mutes transcriptional activity.

3.9. NUCLEOLUS
• The nucleolus is a component of the nucleus.
• It has no membrane that limits it.
• The nucleolus makes ribosomal subunits from proteins and ribosomal RNA (rRNA)
• In the nucleolus lies the region of chromosomes (DNA) containing the highly repetitive rRNA genes.
• In the nucleolus these genes are transcribed and coupled to ribosomal proteins to form the pre-
ribosomal units which will subsequently give rise to cytoplasmic ribosomes.
• The nucleolus can be found next to the nuclear envelope.

3.10. KARYOTYPE
• The chromosome pattern of a species - number, size and shape of chromosomes that defines a species.
• In humans : 23 pairs (22 autosomal and 1 sexual pair) in the nucleus of each cell.
- Euploidia: number of multiple chromosomes of the number n (number of chromosomes): haploid,
diploid, triploid, etc.
- Aneuploidy: numbers that are not multiples of the basic n number: nulisomy, monosomy, trisomy…
Abnormal number of chromosomes. More than 90% of all cancers: unknown mechanism. Most tumors
have accumulation of univocal mutations.
➢ CHROMOSOME CLASIFICATION

The autosomal chromosomes designated by numbers, the sexual ones with letters X or Y. They have 2 arms:
p (short arm), q (long arm)

A. 1-3. Very large and metacentric

B. 4 and 5. Large and submetacentric (two arms very different in size)
C. 6-12 and X: Metacentric and submetacentric.
D. 13-15. Acrocentric.
E. 16-18. Submetacentric, medium and small.
F. 19 and 20. Small metacentric.
G. 21-22 and Y. Small and acrocentric.

Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that
bind to only those parts of the chromosome with a high degree of sequence complementarity. Used to detect
and localize the presence or absence of specific DNA sequences on chromosomes.

They allow detecting translocations, inversions, aneuploidia etc. e.g. Down, Philadelphia chromosome (chronic
myeloid leukemia)
 4. THE CYTOPLASM, ROUGH ER & PROTEIN SYNTHESIS
4.1. CYTOPLASM
The cytoplasm is all the material within a living cell, excluding the nucleus.

• It comprises cytosol (the gel-like substance enclosed within the cell membrane) and the organelles –
the cell’s internal sub-structures.
• Cytosol- contains water, inorganic and organic molecules and enzymes.
• It is within the cytoplasm that most cellular activities occur, such as many metabolic pathways – e.g.
glycolysis and processes such as cell division.

4.2 ENDOPLASMIC RETICULUM – ER

• Cell-viewed as a multitude of membranes.
• ER – complex of membranes contiguous with this nuclear membrane.
• ~ 50% of the total membrane surface in an animal cell is provided by ER.
• Two types of endoplasmic reticulum:
o Rough endoplasmic reticulum (RER)
o Smooth endoplasmic reticulum (SER).
• Present in animal and plant cells (Eukaryotes).
• Important in manufacturing proteins and lipids.
• Many of these products are made for and exported to other organelles.

ROUGH ENDOPLASMIC RETICULUM - RER

• Extensive organelle composed of greatly convoluted, flattish sealed sacs (cisternae), which are
continuous with the outer nuclear membrane.
• Called ‘rough’ as it is studded on its outer surface (the surface in contact with the cytosol) with
ribosomes.
• RER-membrane bound ribosomes are firmly attached to the outer cytosolic side of the ER.
• ~ 13 million ribosomes are present on the RER in the average liver cell.

➢ STRUCTURE
• The general structure of the endoplasmic reticulum is a network of membranes called cisternae i.e. –
sac-like structures.
• These sac-like structures are held together by the cytoskeleton
• The phospholipid membrane encloses the cisternal space (i.e. lumen),
which is continuous with the perinuclear space but separate from the
cytosol.
• Lumen can be 20-40nm
• ER membrane (7nm) similar to nuclear membrane.

- 30% lipids: phospholipids shorter and less saturated than those of the plasmalema.

- 70% proteins: ribosome recognition protein, proteins specific for translocation and assembly of
proteins, glycosylation.

** Ribosome recognition protein is an integral ER membrane protein – mediates the attachment of ribosomes
to the membrane of the endoplasmic reticulum.
➢ FUNCTION
• Serves many functions including:
o Protein synthesis, storage, processing (folding,
sulfation, glycosylation), quality control and
trafficking.
o Calcium homeostasis and intracellular signaling.
• Proteins are produced for the plasma membrane, Golgi
apparatus, secretory vesicles, plant vacuoles, lysosomes,
endosomes and the endoplasmic reticulum itself.
• The rough ER works with membrane bound ribosomes -
takes polypeptides and amino acids from the cytosol and
continues protein assembly including, at an early stage,
recognizing a ‘destination label’ attached to each of
them.

➢ PROTEIN SYNTHESIS **
• Transcription – in the nucleus – synthesis of mRNA.
• Translation- in the cytoplasm- the mRNA works with a ribosome
and tRNA to synthesize proteins.
• Newly synthetized proteins have ‘destination label’ (signal peptide or signal
sequence) that is recognized by the signal recognition particle (SRP).
• Codon (messengerRNA)-anticodon (transferRNA> aminoacids(methionine))
•
➢ SRP SEQUENCE & RIBSOME – RER DOCKING
• Sequence recognition protein/particle (SRC)
• Energy>GTP
• Signal peptidase: it cuts the sequence of polypeptide; the cleaved signal sequence is not useful after
entering to the lumen.
➢ POST-TRANSLATIONAL MODIFICATIONS*

Post-translational modification (PTM) refers to the covalent and generally enzymatic

modification of proteins during or after protein synthesis.

• PTM can occur at the proteins C - or N termini or on the amino acid side chains
(R).
• Phosphorylation is a common mechanism for regulating the activity of
enzymes and is the most common posttranslational modification of proteins.
• Some proteins have metal groups added to them.

Example: it is in the rough ER that four polypeptide chains are brought together to
form haemoglobin

(Phosphate to activate the protein with kinase enzyme. Or other type is to inactivate)>

• Glycosylations - addition of sugars

- Short and simple (oligosaccharides)-these proteins are called glycoproteins.
- High proportion of carbohydrates – proteins called proteoglycans, very
abundant in the extracellular matrix.
- Promotes protein folding/improves stability.
o At the -OH end of serine and threonine (glycosylations in O) They take place In
Golgi Apparatus (GA).
o At the NH end of -asparagine NH (glycosylations in N). They take place in RER
and GA
o SPECIAL SITUATION: In the RER only one type of oligosaccharide is transferred
to the proteins synthesized, it is composed of 14 sugars:2 molecules of
Nacetyl-glucosamine, 9 molecules of mannose and 3 glucose- N-
oligosaccharides. >>

N-GLYCOSYLATION
➢ PROTEIN FOLDING AND CONTROL
• In the lumen of the rough ER proteins are folded to produce the highly important biochemical
architecture which will provide ‘lock and key’ and other recognition and linking sites.
• Folding enzymes and chaperones (BiP-binding immunoglobuline protein; calnexin/clreticulin)
• Also in the lumen, proteins are subjected to a quality control check and those found to be incorrectly
formed or incorrectly folded are rejected.
• These rejects are stored in the lumen or sent for recycling for eventual breakdown to amino acids
(degradation in cytoplasm/ proteasomes).
• Folding-formation of di-sulfide bridges exclusive to the RER. Catalyzed by disulfide isomerase (PDI) of
the lumen e.g. pancreatic hormones, immunoglobulins.

*Hereditary emphysema (a lung problem) is caused by the ER quality control section continually rejecting an
incorrectly folded protein (and its accumulation in the lumen).

SMOOTH ENDOPLASMATIC RETICULUM (SER)

SER has a tubular appearance than rough ER and forms a series of interconnected tubes that curves through
the cytoplasm.

- Considered as sub compartment of RER.

- It lacks ribosomes attached to the membrane - hence its name
'smooth'.
- It is found fairly uniformly distributed throughout the cytoplasm.
- It has variable extension depending on the cell type or function.

➢ COMPOSITION
- Lipids: similar to RER
- Proteins: some similar to the RER, others specific to its function
➢ FUNCTION

Almost exclusively: metabolism of

lipids, phospholipids and steroids.

- Synthesis of fatty acids: in the

cytoplasm + membrane enzymes
of SER.
- Synthesis of phospholipids:
cytoplasmatic fatty acids - CoA +
glycerol P.

→ phosphatidic acid → diacylglycerol

→ phospholipid → transfer to
interior

➢ SECRETION OF “SER” PRODUCTS

• Secretion mediated by the Golgi Apparatus, through vesicles:
- Luminal, membrane and lipid proteins.
• Secretion not mediated by the GA: to other organelles or outside:
- Phospholipids, cholesterol, bile acids, calcium.

➢ OTHER FUNCTIONS

1. Cellular detoxification: Metabolism of liposoluble toxic substances: they are inactivated and eliminated
many toxic products of extracellular or intracellular origin

- e.g. alcohols, drugs, herbicides, insecticides, plastics

- In the liver, intestine, kidney, lungs and skin
- Membrane enzymes (ex. Cytochrome P450): they perform conjugations and oxidations →
generating water-soluble molecules that can be easily excreted by kidneys

2. Calcium storage: In striated muscle: sarcoplasmic reticulum.

- Surrounding myofibrils.
- Associated with membrane invaginations (tubules T).
- Stores and/or releases calcium in response to changes in membrane potential.

Metabolism of carbohydrates: as glycogen according to the need of the cell.

 5. GOLGI APPARATUS/COMPLEX

The Golgi apparatus is the only cell organelle to be named after a scientist.

• The visible characteristics of the organelle were first reported by Camillo Golgi at a meeting of the
Medical Society of Pavia on 19 April 1898 when he named it the ‘internal reticular apparatus’.
• Debate about the existence of the apparatus continued until 1954 when the work in electron
microscopy finally put the seal of approval on the existence of the organelle and the eponym ‘the
Golgi’, was fully accepted.

WHAT IS IT?

• Organelle present in eukaryotic cells (plant & animal cells).

• They are located very near the rough endoplasmic reticulum and
hence near the nucleus.
• Formed by one or more stacks of flattened, disc- like, membrane-
bound cavities (cisternae) called dictyosomes.
• In a typical animal cell, there are about 40 to 100 stacks. In a stack
there are about four to eight cisternae.
• Sort of intermediate position between the ER (where proteins
synthetized) and the cell membrane (proteins incorporated).

COMPOSITION

• Similar to other cytoplasmic membranes:

- 35% lipids (more cholesterol and saturated lipids
than ER).
- 65% proteins (phosphatases, glycosil transferases
and sulfotransferases).
• The membrane has asymmetrical structure and varies in
thickness and structure from one end of the stack to the
other end – CIS and TRANS face.
• One end of the stack (closer to the nucleus) is known as
the CIS face, it is the 'receiving department" while the
other end is the TRANS face and is the "shipping
department".
• The CIS face of the Golgi apparatus is closely associated
with the endoplasmatic reticulum. In terms of cell
biology these sections, working from the rough
endoplasmic reticulum (RER) outwards, are as follows:
1. Cis Golgi network - Receiving department - Goods
inwards
2. Golgi stack- Main processing area
3. Trans Golgi network - Shipping area - Goods outwards
FUNCTION

1. The main function of the Golgi apparatus is to modify, sort

and package the macromolecules that are synthesized by
the cells for secretion purposes or for use within the cell.
2. It mainly modifies the proteins that are prepared by the
rough endoplasmic reticulum.
3. Also involved in lipid transport around the cell.
4. Involved in lysosomes formation.
5. The Golgi complex is thus referred as post office where the
molecules are packaged, labelled and sent to different
parts of the cell.
6. CHECKING, MODIFICATIONS & SORTING (packaging) OF
GOODS DELIVERED FROM ER that arrive by transport
vesicles
7. In the Golgi apparatus the vesicles are delivered into the
‘unloading bay’ of the cis Golgi network- the ‘goods
received’ are checked over.
8. Any goods that have been wrongly delivered, including chemicals that should have stayed in the
RER, are sent back, packed in vesicles to the rough endoplasmic reticulum.
9. The proteins and lipids that have been correctly delivered are then passed into the cisternae of
the Golgi stack and processed and sorted in an orderly sequence according to any ‘labels’ they
bear.
10. Modifications takes place in the cisternae, sequentially while passing from CIS to TRANS face.
11. correct ‘labelling ‘of products is critical-inclusion cell disease.

5.1. POST-TRANSLATIONAL MODIFICATIONS IN GA

GLYCOSYLATION N-linked

N-glycan processing is carried out in RER (attachment

of glycan to N end of Asparagine residues of nascent
polypeptides). Initial trimming of the precursor
molecule occurs in the ER and the subsequent
processing occurs in the Golgi.

• Once the protein is folded correctly, the three

glucose residues are removed by glucosidase I and
II - the removal of the final glucose residue signals
that the glycoprotein is ready for transit from the
ER to the cisGolgi
• The next step involves further addition and
removal of sugar residues by glycosyltransferases
& glycosidases, respectively) in the Golgi complex
GLYCOSYLATION O-linked

Glycosylation O-linked: binding of carbohydrates to -OH of serine or

threonine followed by other carbohydrates as galactose and sialic acid.

This process is important for certain types of proteins such as

proteoglycans - involves the addition of glycosaminoglycan chains to an
initially un-glycosylated proteoglycan core protein.

Function 1 - secretion to form components of the extracellular matrix -

adhering one cell to another by interactions between the large sugar
complexes of proteoglycans.

Function 2 - is to act as a component of mucosal secretions, and it is the high concentration of carbohydrates
that tends to give mucus its "slimy" feel.

Human blood groups-A, B, and O blood-group antigens illustrate the importance of specific
glycosyltransferases

OTHER POST-TRANSLATIONAL MODIFICATIONS IN GA

Other general post-translational modifications of proteins

include the addition of sulfo-group or phosphates to
carbohydrates

• Sulfation (sulfurylation, addition of sulfo group HSO3) of

tyrosine residues of core proteins or carbohydrates
occurs in Trans Golgi network-e.g. sulfonated
proteoglycans heparin sulfate (involved in angiogenesis,
blood coagulation, developmental processes),
chondroitin sulfate (antinflammatory effect in
osteoarthritis, synthesis of hyaluronic acid).
• Phosphorylation of oligosaccharides on lysosomal
proteins takes place in the early Cis Golgi network.
• Phosphorylation - may form signal sequence that
determines the final destination of the protein – e.g. the
Golgi apparatus adds a mannose-6-phosphate label to
proteins destined for lysosomes.
• Glycolipids and sphingomyelin are synthesized within
the GA – this is important in forming outer plasma
membrane lipid bilayer, cell recognition & signaling.
5.2. DISTRIBUTION OF MODIFIED PRODUCTS IN GA/VESICULAR TRANSPORT
The way in which chemicals move through the Golgi
apparatus from cisterna to cisterna is not fully resolved.

• One way - a new cisterna forms at the cis end (the

end nearest the rough endoplasmic reticulum) and
then changes as it moves away from the RER
becoming in time the trans end. • A more accepted
idea - chemicals being processed in the GA travel
from one cisterna to another in transport vesicles
or possibly along microtubules.
• There are three main destinations for the trans GA
released biochemicals : (1) inside the cell to the
lysosomes; (2) the plasma membrane and (3)
outside of the cell.
• All the biochemicals transported away from the
trans GA network have labels and barcodes built
into them and packed in vesicles.
• The construction of the vesicle or vessel is largely
related to the vesicle contents, its destination and
end use.

VESICLE DESTINATION

1. Destination 1: inside the cell, ‘the lysosome line’

About 40-50 different biochemicals dispatched from
the GA in vesicles are destined for delivery to the
lysosomes (in animals many lysosomes where digestion
of life expired organelles and other material).
2. Destination 2: the plasma membrane, ‘the continuous
secretion line’ - default pathway/constitutive pathway
Vesicles containing biochemicals flow to and fuse with
the plasma membrane. They contribute to the
extracellular matrix, act as chemical signals to other
cells, and provide proteins for the repair and
replacement of the plasma membrane. Products from the
GA not labelled for other routes use this line.
3. Destination 3: outside the cell, ‘the regulated secretion
line’
Vesicles and chemicals of this group are produced in
specialist secretory cells. They move from the trans GA
network towards the plasma membrane but accumulate
in number before reaching the membrane (proteins
secreted in response to a stimulus)
VESICULAR COAT PROTEINS –VESICLE TRAFFIC

"COP" - specific coat protein complex (coatmer) that initiates the budding process on the cis-Golgi membrane.
The coat consists of large protein subcomplexes that are made of eight different protein subunits (COP I) or
four protein subunits (COP II).

- COP I – vesicle trafficking from the cis-Golgi to the

rough endoplasmic reticulum (RER)- retrograde
- COP II –vesicle transport traffic from the RER to cis-
Golgi RER)- anterograde.
- Clathrin- coat protein are used to build small vesicles
to transport molecules within cells.

Clathrin-coated vesicles (CCV) selectively sort cargo at the

cell membrane, trans-Golgi network, and endosomal
compartments for multiple membrane traffic pathways –
receptor- mediated endocytosis.

After a vesicle buds into the cytoplasm, the coat rapidly

disassembles, allowing the clathrin to recycle while the
vesicle gets transported to a variety of locations.

Vesicle formation is energy- dependent process.

Specific amino acid sequence directs proteins to specific types of vesicles:

- KDEL KXXX- recognized by COPI

- Asp-X-Glu (DXE)- recognized by COPII
- Manosa 6-P y LL -recognized by clathrin

VESICLE FUSION -EXOCITOSIS

Vesicle fusion is the merging of a vesicle with other vesicles or a part

of a cell membrane.

• In the latter case, it is the end stage of the secretion process from
secretory vesicles, where their contents are expelled from the cell
through exocytosis .
• Vesicles can also fuse with other target cell compartments, such as
lysosomes .

Vesicle fusion depends on SNARE proteins (family of 60 proteins best

studied in neurons) in the presence of increased intracellular (Ca2+) concentration.

➢ NON classical protein secretion:

There are many proteins like FGF1, FGF2, interleukin 1 (IL1) etc. which do not have
a signal sequence. They do not use the classical ER-Golgi pathway.

1. Direct translocation of proteins across the plasma membrane likely

through ABC membrane transporters,
2. Lysosomal secretion
3. Direct secretion from RER without involvement of GA complex (some connexins)
4. Exosomes mediated secretion (IL1).
EXOSOMES MEDIATED SECRETION

Exosomes are either released from the cell

when multi-vesicular bodies fuse with the
plasma membrane or released directly from
the plasma membrane. e.g. IL1, mRNA,
proteolytic enzymes.

• Exosomes may have specialized functions

and play a key role in processes such as
coagulation, intercellular signaling, and
waste management.
• Growing interest in the clinical applications
of exosomes - can potentially be used for
prognosis, for therapy, and as biomarkers
for health and disease.

6. MITOCHONDRIA
The mitochondria is described for the first time by Altmann in 1884.

- It was thought that they were living structures that parasitized the cell - bioblasts. - They are not visible, but
we can be visualized with: Janus green (1900, Michaelis); the acid fuchsine of Altmann and with the iron
hematoxylin of Regaud; by immunohistochemistry; EM- detailed structural studies.

- Have their own independent genome that shows substantial similarity to bacterial genomes. Contains DNA,
organized as several copies of a single, usually circular, chromosome.

- Dynamic structures: they move, they group and ungroup, they merge and divide.

- Size, number and form is variable -depends on the cell type (also the shape and number of the cristae is
variable ). Its number depends on the cell’s energy needs.

GREAT METABOLIC IMPORTANCE – key role in the Krebs cycle and oxidative phosphorylation and the electron
transport chain. Also cell signaling, cellular differentiation, apoptosis, control of cell cycle and cell growth.

6.1. STRUCTURE AND COMPOSITION

They have elongated shape, between 0.75 and 3 μm in diameter and up to 7 μm in length. Surrounded by two
membranes (different in their functions and enzymatic activities) that separate three spaces: the cytosol (or
cytoplasmic matrix), the intermembrane space and the mitochondrial matrix.

External membrane

40% lipids: very unsaturated phospholipids (low cholesterol.)

60% proteins:

- Enzymes of fatty acid metabolism (acyl-CoA synthetase)

- Electron transporters (cytochrome b5 and NADH-cit b5
reductase)
- Receptors and transport proteins
- Proteins of the Bcl-2 family (regulate apoptosis)
- Porins family → permeable to small molecules ˂ 5 kDa
(aqueous channels)
Intermembrane space (perimitochondrial)

- Contains a liquid similar to the hialoplasma; with high concentration of protons - result of the activity
of the enzymatic complexes of the respiratory chain.
- Enzyme Adenylate kinase - phosphorylates AMP in ADP from ATP
- Carnitine - a molecule involved in the transport of fatty acids from the cytosol to the mitochondrial
matrix (where they will be oxidized)

Internal membrane

Internal membrane forms invaginations (cristae) - increase of the surface i.e. available working space at which
enzymes are located. For typical liver mitochondria, the area of the inner membrane is about 5x as large as
the outer membrane due to cristae.

20% lipids: - Cholesterol free; With cardiolipins:

diphosphatidylglycerols with 4 fatty acids (4acyl
groups) → impermeable The inner membrane is freely
permeable only to oxygen, carbon dioxide and water.

80% proteins:

- Enzymes of fatty acid metabolism

- Transport proteins
- Enzymes of the Krebs cycle (DH ketoglutarate
and DH succinate)
- Electron transport chain (complexes I, II, III
and IV)
- ATPase (ATP synthetase) (complex F, F0F1,
elementary particles)

MITOCHONDRIAL MATRIX

The matrix (mitosol) is the space within the inner membrane. There take place various key metabolic routes
of utmost importance. It contains less molecules than the cytosol although it contains:

- Ions and metabolites to be oxidized,

- Enzymes of the metabolism of fatty acids
- Enzymes that generate acetyl CoA from pyruvate
- Enzymes of the Krebs cycle
- Double-stranded circular DNA very similar to bacterial
- Ribosomes
- Chaperones and protein processing enzymes
- Lipid inclusions
- Enzymes involved in the replication, repair, transcription and translation of nucleic acids.

The enzymes in the matrix facilitate reactions responsible for the production of ATP, such as the Krebs cycle,
oxidative phosphorylation, oxidation of pyruvate and the beta oxidation of fatty acids.
6.2. FUNCTIONS
OXIDATIONS OF SUBSTRATES

Without the mitochondria, the cells would depend on anaerobic glycolysis (degradation of glucose to
pyruvate) to obtain all of their ATP = 2ATPs

Ultimate aerobic oxidation of cell metabolism products:

Carbohydrates:

• In cytosol: I phase, Glucose to pyruvate

• In matrix: II phase, Pyruvate (enzyme pyruvate dehydrogenase) →
acetyl CoA → Krebs cycle
oxidized to CO2 and - generating reduced intermediates: NADH +
H and FADH2
NADH2 and FADH2 release electrons-which are driven by the
electron transport chain (separated from protons) until they
reunite with them and the O2 to form water (oxidative
phosphorylation).

For each glucose molecule degraded 36 molecules of ATP are formed in

the mitochondria.

OXIDATION OF LIPIDS

Free fatty acids cannot penetrate any biological membrane due to their negative charge. They must cross the
cell membrane through specific transport proteins. Once in the mitochondrial matrix they form acyl-CoA
molecules.

Fatty acid catabolism consists of:

1. Activation and membrane transport of free fatty acids by binding to coenzyme A.

2. Oxidation of the beta carbon to a carbonyl group.
3. Cleavage of two-carbon segments resulting
in acetyl- CoA.
4. Oxidation of acetyl-CoA to CO2 in the Krebs
cycle. (*)
5. Electron transfer from electron carriers to
the electron transport chain in oxidative
phosphorylation.

(*) Fatty acid-CoA + carnitine → acylcarnitine

→ Pass by internal membrane → Fatty acid-
CoA (in matrix) → ß-oxidation (as many
rounds/turns as carbon pairs ) → Acetyl-CoA
(one molecule produced in each loop of
oxidation) (+ FADH2 + NADH) → Krebs cycle

Total acetyl CoA enters the Krebs cycle:

Acetyl CoA →→→→ 2 CO2 + 3 NADH + FADH2 + GTP

FUNCTION OF ATP SYNTHASE

The H + accumulated in intermembrane space returns to matrix by means of ATP synthase: F0 subunits and
F1 subunit. Mitochondrial ATP synthase complex consists of two large structural components:

• The portion embedded within the membrane is called F0 and contains a ring of c subunits and the
protonchannel.
• The stalk and the ball-shaped headpiece is called F1 and is the
site of ATP synthesis.

ATP synthase is using the chemiosmotic proton gradient to power ATP

synthesis through oxidative phosphorylation (Mitchell’s hypothesis).
Factors that decrease its activity: Membrane transporters:

o Thermogenin
o Poisons (dinitrophenol): allowing protons to leak
across the inner mitochondrial membrane and thus
bypass ATP synthase.

OXIDATIVE PHOSPHORYLATION

Synthesis of ATP thanks to the energy released in the

oxidation of NADH and FADH2 during glycolysis and/or lipid
metabolism.

• NADH + H + + ½ O2 → NAD + + H2O ((ΔG: -52.6 kcal / mol))

• FADH2 + ½ O2 → FAD + + H2O ((ΔG: -43.4 kcal / mol))

Complexes that intervene:

- I -NADH dehydrogenase
- II -FADH dehydrogenase
- III -Cytochromes b-c1
- IV -Cytochrome oxidase

ATP synthase reaction-the non-spontaneous reaction of binding ADP to inorganic phosphate to produce ATP
is coupled to the oxidation of NADH or FADH2.

The chemiosmotic hypothesis (Mitchell, 1961)- suggests that the action of ATP synthase is coupled with that
of a proton gradient. It is the action of the proton gradient that causes a proton motive force that allows ATP
synthase to phosphorylate ADP in the presence of inorganic phosphate to ATP. In mitochondria, the key site
of ATP production in oxidative phosphorylation is the inner mitochondrial membrane.

OTHER FUNCTIONS

Synthesis of intermediates of cellular metabolism

• Synthesis of steroid hormones together with smooth ER (adrenal cortex, testicular Leydig cells).
• Urea cycle (enzymes that detoxify ammonia): ornithine to citrulline in matrix of liver mitochondria

Calcium storage: related to apoptosis, cancer, aging, and diseases such as Parkinson's or diabetes.

Establishment of genealogies and in anthropology (comparative studies of mitochondrial DNA) -

mitochondrial genes come directly from the maternal line and are not subject to gene recombination due to
sexual reproduction
6.3. PROTEIN SYNTHESIS IN THE MITOCHONDRIA
• Encoded by mitochondrial DNA (mtDNA)
• Multiple circular copies 16.5 kpb
• Protein subunits, 2 rRNA, 22 tRNA

Transmitted by the mother

- Translated in the mitochondrial ribosomes

- Smaller than the cytosolic ribosomes (35S and 25S)
- Inhibited like bacterial (chloramphenicol)
- First amino acid: N-formyl methionine (as in bacteria)
- Non universal genetic code

MITOCHONDRIAL PROTEIN IMPORT

• Only about 1% of all mitochondrial proteins are encoded by

the mitochondrial genome.
• Most mitochondrial proteins are encoded by nuclear genes
and then synthesized as precursors on cytosolic ribosomes
after which they must be imported into the organelle.
• The mitochondrial membranes contain specific machinery
(translocases) for recognition, translocation, and
membrane insertion of precursor proteins.
• Cytosolic chaperones are involved in guiding the precursor
proteins to receptors on the mitochondrial surface.
• During import they must go to their target compartment -
Address Sequences on N terminal
➢ Protein importing complexes TOM and TIM: Chaperones
such as Hsp70 and Hsp90 prepare the polypeptides for their
uptake into the mitochondria -sequence signal deployed
• TOM complex proteins: receptors and translocases of the outer membrane
• TIM proteins: transfer channels of the inner membrane. The signal sequence is then
translocated into the matrix in a process that requires an electrochemical H+ gradient across
the inner membrane. Protein folding by matrix chaperons.

MITOCHONDRIAL DISEASES

• Human mitochondrial DNA contains genetic information for 13 mitochondrial proteins and some RNA.
• Most mitochondrial proteins come from genes located in the nuclear DNA and are synthesized by free
cytosol ribosomes.
• Both mutations of mitochondrial DNA and nuclear DNA give rise to mitochondrial genetic diseases -
malfunctioning processes that develop in mitochondria, such as alterations of enzymes, RNA, components
of the electron transport chain and transport systems of the internal mitochondrial membrane
• Many of them affect the skeletal muscle and the CNS.
• Leber hereditary optic neuropathy- degeneration of retinal ganglion cells and their axons - acute /
subacute loss of central vision.
• Neuropathies: Mitochondrial encephalopathies (Leigh's encephalopathy, Friedreich's ataxia).
• Degenerative diseases related to mitochondrial damage and free radical damage (Parkinson's,
Alzheimer's, heart disease).
• Metabolic disorders due to hypoxia or anoxia: excess of lactic acid (lactic acidosis) or no synthesis of ATP.
7.PEROXISOMES
• Spherical organelles 0.2-1 mm Ø
• Discovered in 1965 → Present in all cells, In erythrocytes not.
• Consist of a matrix surrounded by a membrane.
• Abundant in liver and kidney
• Membrane similar to RE
• Transport proteins
• Cytochrome P450
• Major function of the peroxisome- the breakdown of very long chain fatty acids through betaoxidation.
• Also plays a role in the production of bile acids important for the absorption of fats and fat-soluble
vitamins, such as vitamins A and K.

7.1. INTERIOR COMPOSITION

Enzymes responsible for the oxidation of substrates: flavinic
oxidases, D-amino oxidase, acyl CoA oxidase, urate oxidase,
glycolate oxidase.

Within the peroxisome: by using molecular oxygen, they remove

hydrogen atoms from specific organic substrates (labeled as R), in an
oxidative reaction, producing hydrogen peroxide (H2O2, itself toxic):

RH2 + O2 → R + H2O2

Enzyme catalase: always present. Catalyzes the decomposition of

peroxide (toxic):

2H2O2 → 2H2O + O2

Catalase is also capable of using peroxide to oxidize other substrates - e.g. oxidation of toxic substances such
as phenols, ethanol, formaldehyde, which are subsequently eliminated – e.g. the detoxification mechanism
present in the liver and kidneys.

7.2. FUNCTIONS
1. Lipid metabolism (Not associated with the
production of ATP)
2. Degradation of fatty acids (β-oxidation) in the
form of long-chain fatty acid-CoA (> 20C) <20C
also in mitochondria.
3. NADH to cytosol → reoxidation
4. Acetyl CoA to cytosol → another synthesis
5. FADH → oxidized
7.3. PEROXISOMAL DISORDERS
• Purine catabolism: oxidation of purine bases to uric acid →
excretion
• Detoxification: especially in liver. e.g. alcohol → acetaldehyde
• Peroxin (or peroxisomal/peroxisome biogenesis factor) is a
protein found in peroxisomes. Peroxins (Pex genes): Recognize
Address Sequence (PTS) on peroxisomal proteins synthetized in
cytosol → membrane binding and translocation
• Peroxin deficiencies → Peroxisomal Biogenesis disorders (PBDs)
PBDs:
o Zellweger syndrome: mutations in> 20 genes, almost
without matrix enzymes - affects brain, kidneys, muscles.
o Adreno-leukodystrophy: linked to the X mutation in fatty
acid transport proteins. It produces an intense
demyelination and premature death in children.
o Adreno-myeloneuropathy: adult onset of adrenoleukodystrophy associated with a mixed
motor/sensory symptoms and neuropathy - with spastic paraplegia in adults.

8. CYTOSKELETON
The cytoskeleton is a dynamic structure that:

- Maintains the shape of the cell

- Facilitates cellular mobility
- Plays an important role in intracellular traffic (e.g. the movements of
vesicles and organelles) and in cell division.

Composed of multiprotein elements dispersed in the cytosol: highly conserved

in the evolution and they can be stable or dynamic. In eukaryotic cells there are
3 types of elements:

- Microfilaments: 7 nanometers (nm) Ø

- Intermediate Filaments: 10-12 nm Ø
- Microtubules: 24 nm Ø

They have different functions that depend on specific cell type and these functions are related to associated
proteins.
8.1. MICROFILAMENTS
Highest concentration found under the plasma membrane. One function is to maintain the shape of the cell.

Actin filaments

- Actin - the most abundant protein in eukaryotes

- Highly conserved in the evolution
- 6 isoforms: 4 muscle actins: α-actin
- 2 non-muscular actins: β-actin; γ-actin
- Humans have 6 actin genes (encode 6 isoforms)

To carry out their contractile activity, actin microfilaments require another filamentous protein called myosin
(in muscle cells). Actin and myosin predominate in muscle cells where they make two types of myofilaments:

• Thin actin myofilaments

• Thick myosin myofilaments (14 nm in diameter or greater, depending on the type of muscle).

ACTIN STRUCTURE AND ASSEMBLY

The actin microfilaments of non-muscular cells are not

permanent structures, they polymerize and depolymerize
continuously according to the functional needs of the cell.

• Actin G - actin / monomeric actin mollecule:

globular, in free form, soluble (42 kDa)
• Actin F - polymerized, forming linear polymers
microfilaments/ each double helix

(Actin G, free form = actin G – ADP)

Phosphorylation of ADP to ATP (actin F) -> polymerization-> filament formation → Upon incorporation of
actin F-ATP into the filament ATP hydrolyzes → each filament contains actin G-ADP

• The process of microfilaments assembly is strictly regulated process: the filaments are formed by
polymerization (head-tail) of actin G forming a double-stranded helix.
• Various proteins that interact with actin regulate its assembly and disassembly in the cell Typical
situation → dynamic stability
• Balance between polymerization and
depolymerization depends on Actin G
conc.: 0,1 µM on (+) end; 0,6 µM on (–) end.

In vitro experiments demonstrate:

• Dynamic process
• Polymerization at both ends of the filament.
• Nucleation
• Elongation
• The polymer has polarity
• Suggest that the rate of ATP hydrolysis and the rate of monomer incorporation are strongly coupled.

Typical situation → dynamic stability Balance between polymerization and depolymerization Depends on Actin
G: Critical concentration = concentration of actin G in which the dynamics of addition and elimination of
monomers does not produce modification in the length of the microfilament. Cc = 0.1 mM
PROTEINS THAT REGULATE MICROFILAMENTS FORMATION

In non-muscle cells, actin filaments are in close proximity to the cell membrane. Their formation and turnover
are regulated by many proteins.

• Proteins that favour polymerization:

o GTPase of the Ras family - favour actin nucleation.
o Profilin: binds to actin G, favouring the phosphorylation of ADP attached to it. The resulting
actin-ATP monomers are incorporated into the (+) ends of the filaments
o Phaloidine: (non human) inhibits depolymerization
• Proteins that prevent polymerization:
o Cofilin: binds to actin-free molecules G-ADP → prevents them from passing to actin-ATP →
prevents polymerization.
o Thymosin (in neutrophilic leukocytes and platelets): binding to actin-G-ADP prevents
polymerization to F-actin and growth of the polymer = sequestration
o Latrunculin (in sponges) - acts similar to cofilin
• Proteins that favor depolymerization:
o Cofilin - also joins the ends (-) favoring depolymerization
o Gelsolin: Covers and fractionizes actin filaments at the end (+) → not extreme polymerization
(+) but allows depolymerization at (-)
o Cytochalasin D: binds to end (+) preventing elongation but continues depolymerization by end
(-)
• Proteins that stabilize actin filaments:
o Coronation proteins (cap-Z): two subunits, form a terminal cap at the end (+) so that new
actin-ATP units are not incorporated. They cover end (+) → blocks polymerization.
o Tropomodulin: forms a cap on the ends (-) → no depolymerization; stabilizes actin F in
myofibrils of muscular sarcomeres
• Other proteins:
o Arp2 / 3 complex: Stimulates branching of actin.
o Filamin: Cross links filaments → branching network.
o Tropomyosin and α-Actinin: Join parallel filaments → beam formation.
MICROFILAMENTS (FUNCTIONS IN NON- MUSCLE CELLS)

They shape the cell, relate it to the neighbors or the extracellular environment and are responsible for its
movements.

1. Form the cytoskeleton – dense, complex branched network under the cell
membrane in cortical cytoplasm- cortical actin.

Set of actin filaments and associated proteins (Filamin) form a dynamic 3D network
under the plasma membrane → important in membrane receptor anchoring and in
translation of exterior signals to cellular signaling cascades.

Form microfilament bundles - microfilaments arranged parallel (longer than those

forming branched networks) and in association with other proteins such as
Tropomyosin (attached uninterruptedly along actin).

These bundles can bind to Fimbrin and Minimiosin (the non- muscular
myosin, Miosina I) → bundles of non-contractile parallel filaments are
produced - they intervene in the displacement of substances at an
intracellular level.

2. Involved in cell movements –actin based motility

Cellular contraction, cell movement of vesicles/ organells, assembly-disassembly of actin filaments, formation
of networks and bundles, association with Myosins.

Ca- dependent (ATP) Myosin proteins

Myosin I (minimyosin) does not polymerize into filaments - has a globular head, which
binds to actin filaments, and a tail that binds to a phospholipid of a plasma membrane
(provides stability to the actin bundles).

They can also participate in cell movements:

• They can move the filaments of actin in the cytoplasmic

prolongation of a mobile cell producing the extension of
this prolongation - the minimyosin head would be
attached to the filament, and the tail, to the plasma
membrane.
• They can move along an actin filament, dragging along a
vesicle or membranous organelle.

3. Involved in cell rigidity, tensile strength and resilience,

cellular movement (e.g. pseudopodia and mesenchymal cell
migration). Pseudopodia are associated with actin near the
moving edge of the cell.
CELLULAR MOVEMENTS

Cell migration - a central process in the development and maintenance

of the multicellular organization. Formation of specialized protrusions of
the cell surface- temporary cytoplasmic extension (pseudopodia/
lamelipodia) – cellular locomotion

The surface of most cells have protrusions or extensions that intervene in

cell movement (amoeboid movements, cultured fibroblasts,
macrophages, etc.), phagocytosis or in specialized basic functions such as
nutrient absorption.

Filopodia - in developing neurons - are thin cytoplasmic projections

similar to lamellipodia that extend from the growth cone forming
adhesion points/contacts with the substrate.

MICROFILAMENT FUNCTION

They can join with Myosin II - Function in contractile cells- Muscle

contraction. Myosin V is involved in the transport of cargo (e.g. RNA,
vesicles, organelles, mitochondria) from the centre of the cell to the
periphery, but has been furthermore shown to act like a dynamic
tether, retaining vesicles and organelles in the actin-rich periphery of
cells.

Myosin VI is an unconventional myosin motor, it walks along actin

filaments, travelling towards the pointed end (- end) of the filaments,;
it is thought to transport endocytic vesicles into the cell.

• Formation of Adherens junction -contractile belt next to

zonula adherens- Stress fibers → focal contacts (cell-cell, cell-
extracellular matrix).
• Formation of contractile equatorial ring of cytokinesis (end of
mitosis) important for cytokinesis.

8.2. INTERMEDIATE FILAMENTS (IF)

• Heterogeneous group – different in different cell types They have structural function.
• More stable than the rest of the cytoskeleton
• Absent in plants and prokaryotes, early embryo cells Several types of IF can coexist In the same cell.
• Most types of IF are cytoplasmic but the lamins, are nuclear.
• Thicker than microfilaments - all have a similar structure (keratin): - Helical fibers of 10 nm (helical rod
part and globular ends.
• Monomers intertwine - united in α-helix - forming dimers.
• Dimers associate in antiparallel fashion → tetramers
• Associated tetramers form protofilaments (subfilaments).
• The final IF contains 8 protofilaments wound around each other in a ropelike structure8 → 1 intermediate
filament.
• Disassembly mediated by phosphorylation. Proteins associated with intermediate filaments (IFAP) - join
filaments with each other or with other structures.
• Plectin- acts as a link between the three main components of the cytoskeleton: actin microfilaments,
microtubules and intermediate filaments.
INTERMEDIATE FILAMENT-CLASIFICATION

Five main types of IF are known:

• Keratins - epithelial cells

• Neurofilaments - neurons
• Gliofilaments - glial cells
• Desmin - the smooth and striated muscle
• Vimentin - cells of mesodermal origin.

➢ KERATIN
• Type I -Acidic and Type II-neutral or basic
• Encoded by two large groups of genes
• Not only different between species but also between the different cells of the same individual (Type I and
II in the same proportion)
• In all epithelia: associated with desmosomes. Immunofluorescence demonstrates - bundles of keratin
filaments form a network that runs throughout the cytoplasm and is particularly dense under the plasma
membrane and surrounding the nucleus.
• Many isoforms:
- 10 in hard epithelial structures: nails, hair, wool
- 20 in epithelial cells (cytokeratins)
• Used as epithelial markers
• Genetic alterations-several diseases: e.g. Epidermolysis bullosa, Epidermolytic
hyperkeratosis

➢ NEUROFILAMENTS
• They provide cytoskeleton to the soma, dendrites and axons of the neurons - keeping their shape.
• Facilitate cellular transport-intervene micro tubules too.
• In the CNS and PNS of mammals - formed by three polypeptides (NF-L, 70 kDa), (NF-M, 150 kDa) and (NF-
H, 200 kDa).
• Present throughout the animal kingdom but its composition is not constant.
• All very vulnerable to proteolysis in the presence of Ca2 +.
• Genetic alterations in the enzyme superoxide dismutase -> degenerative neuromuscular diseases such as
amyotrophic lateral sclerosis (ALS) and infantile spinal muscular atrophy (Werding's disease) where large
amounts of neurofilaments accumulate in the spinal and cortical motorneurons and produce muscle
paralysis.

➢ Glial filaments

So far only demonstrated in vertebrates, in the cell body and in the cytoplasmic extensions of astrocytes and
Schwann cells (GFAP). GFAP –glial fibrillary acidic protein - similar in all vertebrates They form more compact
bundles than neurofilaments.
 ➢ Desmin

In smooth muscle cells, formed by a protein similar to GFAP - does not participate in contraction. In striated
muscle - integrates the sarcolemma, Z disk, and nuclear membrane in sarcomeres and regulates sarcomere
architecture. Desmin-related with myofibrillar myopathy. Desmin alterations observed in some congenital
cardiomyopathies.

➢ Vimentin
• Mesenchymal cells
• Composed by a protein similar to desmin filaments
• Similar in all vertebrates
• With desmin, it coexists in many cases and constitutes more extended intermediate filaments.
• → striated muscle marker sarcomas
• Distribution similar to keratin IFs and microtubules
• Gathered around the nucleus-radiating towards the plasma membrane
• → Fixes the position of organelles / Shapes the cell
• Associated with microtubules

8.3. MICROTUBULES
• A constant / uniform component in all cells.
• They are distributed in the same way as the filaments of keratin and
vimentin
• Under EM - tubules of 24 nm Ø Variable length polymers.
• Composed of highly conserved tubulin-protein.
• Heterodimer: formed by α- and β-tubulin.
• Polymerizing tubulin - each monomer has a GTP linked. 2 GTP binding sites:
o α-tubulin-GTP
o β-tubulin-GTP / GDP (hydrolyses to GDP when incorporated into the
microtubule
• g-tubulin: does not polymerize
• In microtubules there are also other proteins:
- Microtubules Associated Proteins.
- MAP proteins collaborate in the assembly of the
dimers

MICROTUBULES POLYMERIZATION

They consist of tubulin dimers.

Growth from β-tubulin end (extreme +) → polarity

Lateral association of 13 protofilaments → microtubule (24

nm)

Β-tubulin GTP hydrolyses rapidly GTP from α- tubulin is

trapped between dimers.

The half-life of tubulin 20h.

Occasionally: microtubule doublets (cilia and flagella)

triplets (centrioles).
9.CELL CYCLE CONTROL
9.1. THE CELL CYCLE- WHAT IS IT?
The cell cycle or cell-division cycle- the series of events that take
place in a cell leading to its division and duplication of its DNA to
produce two daughter cells.

• It is a multi-stage process in which the cell increases in size:

- G1 stage (gap 1, or growth;6-12h): protein synthesis
- S stage (synthesis:10-12h): histones and DNA
replication, prepares to divide
- G2 stage (gap 2, 3-4h: continues protein and RNA
synthesis
- M stage(mitosis): cell division
• The stages G1, S, and G2 make up interphase, which
accounts for the span between cell divisions.
• Thus, in eukaryotes, the cell cycle is divided into three
periods: interphase, the mitotic (M) phase, and cytokinesis.

9.2. CONTROL/REGULATION OF THE CELL CYCLE

The progression of the cell cycle is strictly controlled by two large groups of genes. Genes that encode proteins
that regulate the cell cycle in a positive or negative way.

1. Positive regulation of the cycle: Protooncogenes – proliferative genes, their products activate cell
proliferation → cells leave G0 and move to S phase and enter division.

- Heterozygous mutation → oncogenes → cancer

- More important are the genes that code for cyclins and cyclin-dependent kinases (Cdk).

2.Negative regulation - tumour suppressor genes and verification proteins.

POSITIVE REGULATION OF THE CELL CYCLE

➢ CYCLINS

Cyclins-most important CDK regulators – unless CDKs tightly bound to

them they have no protein kinase activity. Named for their cyclic
activity - their concentration rises and falls with a predictable pattern
in each cell cycle.

• Heterogeneous group of proteins (36 - 87 kDa)

• Variable concentration
• With specific sequence of binding to Cdks (cyclin box)
• Described more than 15 cyclins, the most important are (D, E, A, B), this being the order of appearance.
• Four classes of cyclins - each defined by the stage of the cell cycle at which they bind CDKs and function:
o G1-cyclins, help promote passage through Start or the restriction point in late G1 (cyclin D)
o G1/S-cyclins bind CDKs at the end of G1 and commit the cell to DNA replication (cyclin E)
o S-cyclins bind CDKs during S phase and are required for the initiation of DNA replication (cyclin A)
o M-cyclins promote the events of mitosis (cyclin B)
 ➢ CYCLIN-DEPENDENT KINASES

Cyclin-dependent kinases (Cdks) are kinase enzymes, act by

phosphorylating serine and threonine residues of target proteins to
trigger different cellular processes.

• Structure similar to other kinases (34kDa)

• Catalytic Center – T-Loop
• Phosphorylated (activated) by binding to cyclins → Cyclin -
Cdk active complex dependent on concentration of cyclins.
When the concentration of cyclins is low, the Cdk lacks them
and are inactive.
• The only function of Cdks is phosphorylation of other
proteins.

ACTIVATION AND DESTRUCTION OF THE CYCLINS- CDKs COMPLEXES

• Activation by Cdk activating kinase (CAK) : the cyclin-Cdk binding eliminates the blockage produced by the
T-loop over the catalytic cavity of the Cdk, and the threonine in the active site is accessible for
phosphorylation by the CAK.
• PP2a phosphatase: dephosphorylates this threonine, thus preventing the activation of Cdks.
• CDK-cyclin complex can be inhibited by cyclin – dependent-kinase inhibitors (CKI) –proteins (p27, p21) that
interacts at the same time with the cyclin and CDK blocking kinase activity.
• Cyclin destruction occurs by a ubiquitin -dependent mechanism- activated enzyme recognizes specific aa-
sequences on the cyclin and attaches multiple copies of ubiquitin
to it- marking the protein for complete destruction in
proteasomes.
• Ubiquitin ligases – two ubiquitin ligases are important in the
destruction of cyclins and other cell-cycle regulators.
- Enzyme complex called SCF (G1 and S phase)
- Anaphase-promoting complex (APC, M phase) -
responsible for the ubiquitination and proteolysis of M-
cyclins and other regulators of mitosis.
• Transcriptional control - provides an added level of regulation -
changes in cyclin gene transcription and cyclin synthesis.

The cell cycle control system can arrest the cell cycle at specific checkpoints
(imp)

• The central controller triggers each process in a set sequence.

• It ensures that the events are properly timed, occur in the correct order,
and occur only once per cell cycle.
• The system is responsive to various intracellular and extracellular
signals - so the cell-cycle progression can be halted (e.g. the cell either
fails to complete an essential cell- cycle process or encounters
unfavourable environmental conditions)
NEGATIVE REGULATION OF THE CELLULAR CYCLE

➢ TUMOR SUPPRESSOR GENES AND VERIFICATION PROTEINS

• Tumor-suppressor genes (TSGs) or anti-oncogenes are genes that protect the cell from a single event or
multiple events leading to cancer. When these genes mutate, the cell can progress to a cancerous state.
• They encode proteins that ensure genome fidelity during its replication and segregation:
- They ensure the dependence between two sequential processes of the cycle – i.e. the cycle does
not continue beyond a point if at this point there has been an alteration of the normal process.
- Normally homozygous mutation → cancer

Heterogeneous group of genes – encode proteins that ensure the genome fidelity during its replication /
segregation - verification proteins (verification routes):

1. Proteins that prevent mutations of regulatory genes of the cycle.

2. Proteins that inactivate the Cdks by phosphorylation / dephosphorylation: Wee1 kinase that
phosphorylates the amino acids Thr14 and Tyr15 of the Cdk1 causing their inactivation.
3. Cycle inhibitory proteins (CDKs inhibitory proteins: p53, p21 and p16 proteins) that act at the checkpoint
of G1
- Family of p21 (21, 27, 57) → inhibit by binding to the cyclin / CDK complex
- Family of p16 (15, 16, 18, 19) → compete with cyclins for the Cdks
4. The retinoblastoma protein (Rb) - a nuclear phosphoprotein - its genetic alteration is involved in the
development of the cancerous retinal tumor of the same name.
5. Proteins that induce the cycle exit - towards a differentiated cellular state or towards apoptosis (Bad,
Bax, Bak promote apoptosis)

THE RETINOMBLASTOMA PROTEIN (checkpoint G1) (imp)

Rb protein - nuclear phosphoprotein: It has a fundamental role in the negative control of the cell cycle and in
tumour progression.
- Its active form is the hypophosphorylated form and its function is to inhibit the E2F transcription factor
→ inhibit cell proliferation by inhibiting the E2F-DP complex (E2F dimers and DP-dimerization partner
dimers) during the G1 phase.
- When phosphorylated (inactive) stimulates the E2F transcription factor → the E2F proteins pass to the
nucleus to stimulate the synthesis of important proteins that allow the cell to progress through the
cycle → step G1-S.
Cycle check points: Point G1 (G1 / S)
Complexes cyclin-Cdk:
Cyclin D-Cdk4 / 6 → Phosphorylates pRb / E2F
complex
→ Active E2F → synthesis of cyclin E
Cyclin E-Cdk2: Total phosphorylation of pRb /
E2F → total release of E2F → transcription of
all important genes in S-phase progression.
pRb remains phosphorylated in S, G2 & M.
During M-G1 progressively dephosphorylated.
Cyclin A-Cdk 2: Activation of chromatin
replication
THE CELL CYCLE CONTROL by p53 GENE (checkpoint G1) (imp)

Cellular tumour antigene-p53 (Stops cycle in G1 if DNA is damaged)

- Can activate DNA repair proteins when DNA has sustained
damage.
- It can arrest growth by holding the cell cycle at theG1/S check
point on DNA damage recognition (if it holds the cell here for long
enough, the DNA repair proteins will have time to fix the damage
and the cell will be allowed to continue the cell cycle).
- It can initiate apoptosis (i.e., programmed cell death) if DNA
damage proves to be irreparable.
P53 protein pathway:
1. Activates CKI p21 → inhibits cyclin / CDK → no phosphorylation
(of Rb also) & cell cannot enter into the next division
2. Activates FasR and bcl-2 → apoptosis
3. Active synthesis interferon → apoptosis
4. Inhibits Nanog expression (gene for ESC [embryonic stem cells] renewal) → no pluripotency →
maintains genetic stability.

THE CELL CYCLE CHECK POINT 1

CYCLINB-CDK1 (MPF) (checkpoint G2)

Cyclin B / Cdk1 controls the passage from phase G2 to phase M.

1. Induces mitosis until early anaphase

2. When the cell enters the G2 phase, cyclin B is synthesized and
binds to Cdk1 → cyclin B / CDKI complex (MPF)- whose activity is
essential for the cells to pass to the M phase.
3. CyclinB-Cdk1 - inactive in phosphorylated state. Activation by
dephosphorylation by phosphatase Cdc25.
4. The activated kinase phosphorylates several proteins involved in mitosis:
- Phosphorylates H1(histones) → chromatin condensation-
- Phosphorylates lamins → nucleus rapture
- Phosphorylates MAPs → mitotic spindle formation
- Phosphorylates Gwl → PP2A phosphatase inactivation → allows activation of Cdks again

*MPF –maturation/mitosis promoting factor

*MAPs- microtubule associated proteins

SUMMARY OF CELL CYCLE
(just to understand)

9.3. CELLULAR DEATH

NECROSIS - traumatic death

It implies a pathological character. It is triggered after extreme cell damage

(e.g. lack of oxygen or poisoning) that irreversibly damages the functioning
of the cell. Rupture of osmotic equilibrium:

1. increases cell volume (swelling / swelling)

2. change of metabolic pathways
3. cellular and organelle breakdown
4. destruction of lysosomes and release of enzymes
5. fragmentation of cellular and nuclear membrane
6. involvement of neighboring cells → inflammatory response
7. spread of necrotic phenomenon

APOPTOSIS - programmed cell death / cell suicide

• Activated by a self-destruction program

• During embryonic development eliminates surplus cells
• Eliminates senescent or damaged cells
• Fundamental for the maintenance of homeostasis
• Plays role in the control of the cell cycle
• Provides a dynamic balance between survival and death
• Energy dependent
• There is no rupture or external release of cell content → neighboring
cells are not affected
 ➢ SIGNALING PATHWAYS

Regulated by external and internal factors: → Activation Caspases → affectation

of kinases (FAK- focal adhesion kinase), lamins, cytoskeleton (intermediate
filaments, actin, tubulin), DNAase.

• Extrinsic pathway: extracellular signals

- Induced by ionizing radiation, high temperature, viral infection, poisonous
chemicals.
- Response to external ligands (Ligand FAS, NF)
- Mediated by receptor with associated death domain (tumour necrosis
factor receptors- TNFR)
- Union ligand-TRNF activates FADD/TRADD adapter proteins that binds to
procaspase 8(initiator) → activates numerous executing caspases.

• Intrinsic pathway: mitochondrial pathway

Response to internal stimuli: Oxidative stress, hypoxia, glucocorticoids excess, growth factor withdrawal,
irreparable genetic damage, etc.

- These signals cause mitochondrial release of cytochrome C to the cytosol → union with Apaf-1 protein
→ formation of apoptosome → activation of caspase 9 (initiator) → activating caspases.
- Mediated by Bcl-2 family of proteins: pro / anti- apoptotic
members

The Bcl-2 / Bcl-XL proteins of the mitochondrial membrane inhibit

apoptosis by preventing the release of cytochrome c and by inactivating
Apaf-1 factor.

- Other members of the same family (Bad, Bax and Bak) promote
apoptosis.
- Bax / Bak stimulate the release of cytochrome c.
- Bad inactivates family members that act as inhibitors of
apoptosis.
*Both the extrinsic and the intrinsic path converge onto the same caspases.
➢ MORPHOLOGICAL AND MOLECULAR CHANGES

Cell changes its normal shape; its surface becomes irregular and loses contact with the cells
that surround it.

- Compaction of chromatin
- Condensation of the cytoplasm
- Nuclear fragmentation/chromosomal DNA fragmentation
- Cytoplasmic fragmentation → apoptotic bodies → phagocytosed by macrophages

MOLECULAR PROCESSES: 1.Fragmentation of DNA by three endonucleases 2.Activation of

several enzymes:

- proteases / caspases
- calcium-dependent transglutaminases……….hydrolases
- Phagocytosis: phagocytes recognize molecules of phosphatidylserine exposed on the
membrane of apoptotic bodies.
 ➢ APOPTOSIS CAUSES
• Physiological causes
o Programmed destruction of cells during embryogenesis. Many structures of the embryo have
regressed before birth
o Involution of hormone-dependent tissues:
▪ Endometrium - estrogens and progesterone.
▪ Prostate - male hormones
o Renewal of epithelia (skin, digestive tract etc)
o Elimination of cells that have already fulfilled their function (neutrophils in an acute inflammatory
response and lymphocytes at the end of an immunological reaction)
o Elimination of autoreactive T lymphocytes in the thymus. Elimination of cells infected by virus,
neoplastic cells and cells of transplanted organs (induced by cytotoxic T lymphocytes)
• Pathological causes:
o Apoptosis induced by DNA damage (affected by radiation and antineoplastic drugs).
o Apoptosis induced by misfolded proteins in the cell.
o Some viruses trigger apoptosis in the cells that they infect (viral hepatitis, AIDS).
o Atrophy of organs after ductal obstruction (pancreas, parotid and kidney).
o Cell death due to tumours.

10. MEIOSIS AND EMBRYONIC DEVELOPMENT

10.1. THE PREPARATORY STEPS
The preparatory steps that lead up to meiosis are identical in pattern
and name to the interphase of the mitotic cell cycle.

• Growth 1 (G1) phase: a very active period, where the cell

synthesizes its vast array of proteins, including the enzymes and
structural proteins it will need for growth. Each of the
chromosomes consists of a single (very long) molecule of DNA. In
humans, at this point cells have 46 chromosomes, 2N (i.e.
identical to somatic cells).
• Synthesis (S) phase: The genetic material is replicated: each of its
chromosomes duplicates, so that each of the 46 chromosomes
becomes a complex of two identical sister chromatids. The cell is
still considered diploid because it still contains the same number of centromeres.
• Growth 2 (G2) phase: G2 phase as seen before mitosis is not present in Meiosis. Actually, the first four
stages of prophase I in many respects correspond to the G2 phase of mitotic cell cycle.

*Interphase is followed by meiosis I and then meiosis II.

MEIOSIS – REDUCTIONAL DIVISION (sexual reproduction)

Meiosis: a specialized type of cell division that reduces the chromosome number by half, creating four haploid
cells, each genetically distinct from the parent cell that gave rise to them.

During S phase (pre-meiotic “S-phase" or "meiotic S-phase) - the DNA of each chromosome is replicated so
that it consists of two identical sister chromatids that remain held together through sister chromatid cohesion
→ 2n chromosomes, 4c DNA (4 chromatids).
Meiosis I and Meiosis II are each divided into prophase, metaphase, anaphase and telophase stages.

Meiosis I: separates homologous chromosomes into two daughter cells reducing the chromosome
number by half → 2n (diploid) to n (haplod) but each chromosome contains 2 chromatids (2c). Genetic
recombination happens in this phase.

Meiosis II → the reduction of the amount of DNA from 2c to c (which is what each gamete will have)
Essential for the formation of gametes - haploid cells

10.2. MEIOSIS I
Purpose of meiosis: Fidelity of genetic material and genetic variability

PROPHASE I

• Homologous chromosomes pair and exchange DNA (homologous recombination). This often results in
chromosomal crossover → source of genetic variations.
• In G2, a phenomenon occurs whereby the cell goes to meiosis instead of mitosis (hypothesis: a new type
of histone is synthesized that is added to the usual ones?)
• Typically, the longest phase of meiosis (years or decades): Leptotene, Zygotene, Pachytene, Diplotene and
Diakinesis
1. Leptotene (leptonema) from Greek words meaning "thin threads”:
- Individual chromosomes (each consisting of two sister
chromatids) become "individualized" to form visible strands
within the nucleus.
- The two sister chromatids closely associate - visually
indistinguishable from one another.
- Lateral elements of the synaptonemal complex assemble.
2. Zygotene (zygonema), from Greek words meaning "paired threads”:
- The chromosomes approximately line up with each other into homologous chromosome pairs
(paternal & maternal).
- The synapsis (pairing/coming together) of homologous chromosomes takes place.
- Thus, pairing is highly specific and exact. The paired chromosomes are called bivalent or tetrad
chromosomes.
3. Pachytene (pachynema) from Greek words meaning "thick threads”
- The stage when homologous recombination, including chromosomal
crossover (crossing over), occurs.
- Nonsister chromatids of homologous chromosomes may exchange
segments over regions of homology. -At the sites where exchange
happens, chiasmata form.
- Crossing over - mediated by the appearance of Recombination Nodule
between the two homologs - a protein structure of 90 nm in diameter
(contains the enzymes that mediate in the recombination process).
- During this phase a small DNA synthesis is produced - probably related
to DNA repair phenomena linked to the recombination process.
- It's the longest stage in
men.

4. Diplotene (diplonema), from Greek words meaning "two threads",

- The tetrads are visible – chromosomes continue condensing.
- The synaptonemal complex degrades and homologous chromosomes separate from one
another a little but the homologous chromosomes of each bivalent remain tightly bound at
chiasmata (the regions where crossing-over occurred).
- The chiasmata remain on the chromosomes until they are severed at the transition to
anaphase I.

Dichtyotene

In mammalian and human fetal oogenesis all developing oocytes develop to this stage and are
arrested before birth. It lasts until meiosis is resumed to prepare the oocyte for ovulation, which
happens at puberty or even later.

5. Diakinesis:
- Tetrads are clearly visible.
- Sites of crossing over entangle together, effectively
overlapping, making chiasmata clearly visible.
- Throughout the prophase I continuous synthesis of RNA
in the nucleus but it stops at the end of this phase.
- The rest of the stage closely resembles prometaphase
of mitosis → the nucleoli disappear, and the nuclear
membrane disintegrates into vesicles.
- The end of this stage the meiotic spindle is formed.
- One kinetochore is formed by each chromatid (2 per
each homologue and are on the same side) but they
behave as a functional unit.
PROMETAPHASE I

• Maximum condensation of the chromosomes.

• The microtubules invade the nuclear region and attach to the chromosomes at the kinetochore -
kinetochore functions as a motor, pulling the chromosome along the attached microtubule.
• Each homolog is bound by the microtubules to a pole (not to both poles as in mitosis).
• Both chromatids in each homologue behave as a functional unit.

METAPHASE I

• The meiotic spindle is fully developed, the bivalents are

located in the equatorial plane ready to be separated.
• Terminal chiasmas present.
• Degradation of cohesins except in centromeres.

ANAPHASE I: Kinetochore microtubules shorten, pulling homologous

chromosomes (which consist of a pair of sister chromatids) to opposite
poles.

TELOPHASE I

• The first meiotic division effectively ends when the chromosomes arrive at the
poles.
• Each daughter cell now has half the number of chromosomes (n) but each
chromosome consists of a pair of chromatids (2c).
• The microtubules that make up the spindle network disappear, and a new nuclear
membrane surrounds each haploid set.
• The chromosomes uncoil back into chromatin.
• Cytokinesis - the pinching of the cell membrane in animal cells (contractile ring)
occurs, completing the creation of two daughter cells.
• Sister chromatids remain attached during telophase I.
10.3. MEIOSIS II
After Telophase I cells may enter a period of rest known as
interkinesis or interphase II. No DNA replication occurs
during this stage. The chromosomes are in haploid number
(n), but the DNA content is 2c because each chromosome is
made up of two chromatids.

• Meiosis II will separate both chromatids from each

chromosome leaving two haploid cells (n) with a
DNA content equal to c).
• Meiosis II is like a mitosis to which cells arrive with a
haploid (instead of diploid) chromosome envelope.

11.GAMETOGENESIS
Gametogenesis is a biological process by which diploid or haploid precursor cells undergo cell division and
differentiation to form mature haploid gamets.

Sexual reproduction (a more complex mechanism of cellular reproduction) is mediated by gamets (haploid
cells (with a single set of chromosomes, n)

Meiosis: essential for the formation of gametes, not only because it is reducing the number of chromosomes
but also because of the exchange of the genetic material (crossing over) -> genetically distinct daughter cells.

Gametogenesis (Oogenesis and Spermatogenesis) is the formation of gametes by means of meiosis from
germinal precursor cells.

11.1. OVOGENESIS
Oogenesis or ovogenesis is the production/differentiation of the ovum (egg cell) into a cell competent to
further development when fertilized. It is developed from the primary oocyte by maturation in gonads
(ovaries).

Oogenesis starts with the process of developing oogonia. Occurs via the transformation of primordial folicules
into primary oocytes (oocites I), a process called oocytogenesis (is complete either before or shortly after
birth).

• Oocytes I (primary oocite) contain a nucleus (with loose chromatin and prominent nucleolus), the
cytoplasm has numerous ribosomes, ER-rough, ER smooth, developed Golgi apparatus, mitochondria
and lysosomes.
• Primordial follicles consist of:
- Oocyte I derived from oogonies.
- Surrounded by a single layer of flat epithelial cells (supporting granulosa cells) - follicular cells.

Follicular development (foliculogenesis): Begin in the 3rd month of fetal development, 5,000,000 oocytes
produced.

• Most degenerated by atresia (apoptosis).

• At the time of birth there are only about 400,000 primordial follicles.
• In the fertile woman there are follicles in different stages of development.
Ovogonia -> (oogenesis) -> Primary oocyte - (Meiosis I) -> First polar body (discarded later) +
Secondary oocyte - (Meiosis II) -> Second polar body (discarded later) + Ovule (or ovotidia-a
functional gamete )

• During embryonic development, oocytes from the primordial follicle complete prophase I (Meiosis I) till
the dichtyotene. They will be detained at that stage until puberty.
• During infancy, the primordial follicles continue in dichtyotene - most of them experience atresia
(apoptosis).
• At the beginning of puberty there are only about 40,000 primordial follicles

11.2. FOLICULOGENESIS
Oogenesis consists of several subprocesses: oocytogenesis, ootidogenesis and, finally, maturation to form an
ovule (oogenesis itself).

Folliculogenesis: during follicular development, primordial follicles undergo a series of critical changes in
character, both histologically and hormonally. It develops in parallel with oogenesis and during this process
the follicle passes through various stages.

1. Primordial follicle (prepubertal phase, no receptors for FSH).

2. Primary follicle, (which contains the oocyte and the follicular epithelium)
3. Secondary or preantral follicle (at this stage several rows of granulosa cells form the stratum
granulosum and secrete a
glycoprotein layer that will be the
zona pellucida,).
4. Tertiary or antral follicle (which
contains a cavity, the follicular
antrum and the oocyte is on a side
called cumulus oophorus.
5. Graafian follicle (the large follicle
ready for ovulation)
PRIMARY FOLLICLES

Formed by maturation of the primordial follicles stimulated by FSH (20-24 post-gestation week)

Follicular cells are cuboidal-unilaminar:

• Oocytes start growing (50-80µm)

• They enter the meiosis I - detained in prophase (primary oocyte).
• Formation of the membrane or zona pellucida
• Follicular cells proliferate (cells of the Granulosa)
• Primary follicles develop receptors to follicle stimulating hormone (FSH) at this time,
but they are gonadotropin-independent until the antral stage.

SECONDARY FOLLICLE

• Granulosa with 6-12 layers of cells

• Oocyte continues to grow (125µm)
• Appearance of fluid filled cavities in granulosa → Follicular Antrum (Antral Follicle),
growth factors, FSH.
• Stroma-like theca cells are recruited by oocyte-secreted signals. They surround the
follicle's outermost layer, the basal lamina, and undergo cyto differentiation to
become the Theca externa and Theca interna.
• An intricate network of capillary vessels forms between these two thecal layers and
begins to circulate blood to and from the follicle.
• It is thought that LH directs
the differentiation of the
thecal cells that surround
the follicle-.
• The oocyte maturation
inhibitor –OMI- a protein
that detains their growth
(secreted by the granulosa
into the antrum).
•

MATURE-PREOVULATORY (Graaf’s follicle) FOLLICLE

• Preovulatory stage
• It is the last stage of the maturation of the oocyte.
• Large increase in size to reach 10-20 mm.
• At this point, the majority of the of follicles that started growth have died.
(atresia)-radical apoptosis.
• The antrum becomes larger and the oocyte becomes eccentric in this cavity, as in
a lagoon of follicular fluid, supported only by a column of granulosa cells that constitute the Ooophore
Cluster (Cumulus oophorus) and surrounded by a single layer of granulosa cells - the corona radiata.
• FSH is secreted - in addition to inducing the proliferation of granulosa cells, it increases the number of FSH
receptors thus, enhancing its own effect.
• Eventually, only one follicle will be viable. This remaining follicle, called the dominant follicle, will grow
quickly and dramatically—up to 20 mm in diameter—to become the preovulatory follicle.
• Continuation of Meiosis I from oocyte I (leaves the dictyotene) 12-14h before the peak of LH
• Formation of the oocyte II and the first polar body.
11.3. HORMONAL REGULATION
Folliculogenesis is controlled by the endocrine system. Five
hormones regulate folliculogenesis:

1. Gonadotropin-releasing hormone (GnRH) secreted by

the hypothalamus
2. Two gonadotropins: follicle stimulating hormone (FSH)
and luteinizing hormone (LH)
3. Estrogen
4. Progesterone LH stimulates theca interna → secretion
of androgens that reach granulosa.
5. FSH stimulates granulosa for conversion of androgens
into estrogens.

In the middle of the cycle (a little before ovulation)

adenohypophysis induces LH to spike sharply. → no estrogen
production → resumption of meiosis-oocyte I comes out of
dictyotene I and meiosis I ends → formation of oocyte II and 1st
polar corpuscle → ovulation

At the time of ovulation, the Oocyte II is stopped in metaphase II of meiosis II. And it remains that way until
fertilization.

The arrest of meiosis is due to the cytostatic factor Mos, - a serine-threonine kinase that activates MAPK
kinases - threonine kinase that activates MAPK kinases

11.4. OVULATION
Liberation of the secondary oocyte from the follicle of Graaf

• Graafian follicle - is ready to release the oocyte in response to the LH peak that
occurs approximately in the middle of the ovarian / menstrual cycle.
• Increased volume and pressure of follicular fluid.
• Enzymatic proteolysis of the follicular wall.
• Contraction of smooth muscle of theca externa.
• The secondary oocyte comes out accompanied by the corona radiata.
• If it is fertilized → resumption of meiosis II, 2nd polar body formation.
CORPUS LUTEUM

After ovulation granulosa and theca cells are mixed and collapsed - forming the corpus luteum - which replaces
the follicle from which the ovule was released.

Both types of cells begin to secrete another hormone, progesterone → They stimulate the growth of the
uterine mucosa (endometrium) for egg implantation in anticipation of fertilization.

In the second part of the ovarian cycle, the levels of FSH and LH descend until they reach their lowest level at
the end of the cycle (day 28) - produced because progesterone acts on the pituitary gland inhibiting the
secretion of FSH and LH.

• If there is no pregnancy (9 days after ovulation) → autolytic degeneration of the Corpus Luteum
(transformed into Corpus Albicans) → The production of progesterone ceases and menstruation occurs.
• If there is fertilization (pregnancy):- the corpus luteum becomes the corpus luteum graviditatis, which
increases in size and continues to produce progesterone (until the end of the IV month of embryonic
development) → after that degenerates slowly → later replaced by placental secretions.

11.5. SPERMATOGENESIS
A process by which the male gametes, known as sperm are formed in gonads (testicles).

Interstitial cells of Leydig - found adjacent to the seminiferous

tubules in the testicle.

• Polyhedral, eosinophilic, large

• Responsible for the secretion of testosterone Inactive from 5
months of fetal life to puberty

No spermatogenesis occurs until puberty: Sertoli and germ cells

(gonocytes) are immature.

At the end of spermatogenesis 4 functunal cells are produced and

non of them degenerates.

12. NERVOUS TISSUE – NERVOUS SYSTEM

• The nervous system - set of structures and organs formed by nervous tissue.
• Highly specialized tissue/system
• Its main function is to quickly capture and process internal and external signals to achieve an adequate,
timely and effective interaction with the changing environment.
• Controls and integrates activities of organs and bodily systems → allows the organism to respond to
changes in the internal and external environment
• It consists of two main parts (anatomical division):
o Central Nervous System (CNS)
- Brain
- Spinal cord
o Peripheral Nervous System (PNS) –mostly nerves
- Somatic
- Autonomic (sympathetic and parasympathetic)
- Enteric (gastro-intestinal innervation)
12.1. PNS FUNCTIONAL DIVISION
Somatic Nervous System (voluntary control of body movements via skeletal muscles) → motor and sensory
innervation of the whole body except viscera, smooth muscle and glands.

Autonomic Nervous System (vegetative system- unconscious control) → motor innervation of the heart,
smooth muscles, glands and viscera (primary mechanism of the fight –or-flight control).

Consists of: sympathetic, parasympathetic and enteric nervous system.

12.2. INTEGRATIVE CAPACITY

- The skin / muscle (i.e. peripheral receptors send information to the spinal
cord through the spinal nerves.
- The nerve fibers enter the spinal cord and contact the neurons that are
there producing responses such as reflexes or behaviours.
- The spinal cord is the main information highway that connects the brain
and the SNP.
- THE SPINAL CORD CONTAINS NEURAL CIRCUITS THAT CONTROL SIMPLE
RESPONSES -REFLEXES
12.3. CELLULAR COMPOSITION
Neurons are highly specialized cells/nerve cells. They are connected to each other in a complex way (neural
circuits/networks). They also have the property of generating, propagating, coding and processing nerve
signals / impulses.

Glia (neuroglia) - main function of support and protection of neurons - accompanying cells

• CNS Glia
→ Astrocytes
→ Oligodendrocytes
→ Microglia
→ Ependymal cells
• PNS Glia
→ Schwann cells
→ Ganglion Satellite Cells (Anficitos)

12.4. NEURONS
They are similar to other cells in the body in that:

• They are surrounded by cellular membrane.

• They have a nucleus with DNA / genes and 1 nucleolus
• They contain cytoplasm with organelles (Nissle bodies - RER,
SER, mitochondria, lisosomes, Golgi apparatus
• Lipid and lipofuscin inclusions
• Neurofilaments → Intermediate filaments: main element of
cytoskeleton
• Microtubules (neurotubules): responsible for transport
• They carry out basic cellular processes such as protein
synthesis and energy production.

Cellular body = soma or pericarion- composed of the nucleus and the cytoplasm

They differ from other cells because:

• They have specialized projections → dendrites (short and

numerous) and axons (long and only one).
• They form specialized connections called "synapses" and produce
special substances called neurotransmitters, released at synapses.
• Neurons communicate with each other by electrical impulses
(action potential) → electrochemical processes

SPECIALISED PROJECTIONS

Dendrites: Axon:

• Short and numerous. • 1 per neuron

• Content similar to pericarion but without Golgi
apparatus Characteristic distribution according
to neuronal type
• With lateral projections (spines)
• It transmits stimuli
• It can be branched (collateral branches)
• Branches in terminal bulb (synaptic buttons)
• Axolema = plasmalemma, may have myelin
sheath: lipid
NEURON TYPES

According to the number of extensions:

- Bipolar
- Pseudo-unipolar
- multipolar

According to the axon length:

- Golgi type I: long axon

- Golgi type II: (of integration or association) short and branched axon

According to the function:

- afferent (sensory)
- efferent (motor)
- Interneurons: relate neurons in CNS

Other: Form of the pericarion -pyramidal - Purkinje cells (discoverer Jan Evangelista Purkyně in 1839)

SYNAPSE

Point/place of contact between 2 neurons where neurotransmitter-mediated transmission of nerve impulse

occurs.

- Anatomy: space of 20-30 nm between cells.

- Presynaptic region: Neurotransmitters in vesicles, Synaptic space (cleft).
- Postsynaptic Region: With neurotransmitter receptors

Classification:

• Based on contact location:

- Axodendritic
- Axosomatic
- Axoaxonics
• Based on form:
- Type I: asymmetric post and presynaptic
regions.
- Wide synaptic cleft, excitatory vesicles.
- Type II: symmetrical pre and post
synaptic regions.
- Synaptic cleft narrow inhibitory vesicles.
• Based on signal type:
- Chemical
- Electrical
 ➢ HOW SYNAPSE WORK?

Neurons communicate with each other by electrical impulses (action potential) → electrochemical processes.

1. Vesicles synthesized in the soma transported through axon

2. Neurotransmitter packed into vesicles at terminal buttons
3. Increase in [Ca2 +] induced by the arrival of membrane potential
4. Vesicle exocytosis

Mechanism → Kiss and run (ms) (porocitosis) →

transient fusion → Total fusion → collapse into the
membrane → Neurotransmitter-receptor junction in
postsynaptic area

Types of receptors

• Ionic channels → fast transmission

• Coupled to G protein (metabotropic receptors)
→ indirect activation of ion channel → slow
transmission
• its ligands: neuromodulators (neurohormones)

➢ NEUROTRANSMITTER
• Substance released by exocytosis at the synapse as a result of an action potential.
• Produces inhibitory and excitatory signals 80% returns to the presynaptic region for reuse
(recyclingreuptake)
• NT of low molecular weight or: acetylcholine (cholinergic neurons), adrenaline (epinephrine),
noradrenaline (norepinephrine), dopamine, serotonin.
• Amino acids: glycine, GABA, glutamate, aspartate.
• Opioid neuropeptides: endorphins, enkephalins.
• Hormones: somatostatin, oxytocin, cholecystokinin.
• Gases: NO and CO.