Part 2 - THE CELL

The-cell.
pdf
Zaira_beluna5
Morfología Humana
1º Grado en Ingeniería Biomédica
Escuela Técnica Superior de Ingeniería de Telecomunicación.

Campus de Fuenlabrada
Universidad Rey Juan Carlos
Reservados todos los derechos.

No se permite la explotación económica ni la transformación de esta obra. Queda permitida la impresión en su totalidad.
Biomedical Engineering Zaira Benítez Luna
UNIT 1: CELL AND METHODS OF STUDY
1-ALL LIVING CREATURES ARE COMPOSED OF CELLS
● Prokaryotes: bacteria and cyanobacteria: primitive
● Eukaryotes: more complex and evolved
● Animal
● Plants
● Fungi
● Microorganisms
● Multicellular
● Unicellular
● Complex and organized systems capable of using matter and energy to grow and

self-replicate (living beings): they consist of cells
● The cell is the fundamental unit of living things
● In plants and animals → cells organized in tissues, organs- apparatus or systems
● (embryonic / adult)
● The activities of an organism are the consequence of the sum of its cellular activities
● The disease is the result of cellular alterations
2-CELL THEORY
2.1- EXPLAINS THE BASIC FEATURES OF CELLS STRUCTURE
● All surrounded by a membrane that separates their content from their exterior
● Cells contain genetic material 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 of the cell’s
activities
● They reproduce - new cells are produced from existing cells
2.2-THE SCIENTISTS BEHIND IT

How was it possible for scientists to discover cells?
● The invention of the 1st microscope (1590) was what made it possible for cells to be
discovered but Van Leeuwenhoek improved it tremendously (1673)
Who discovered them?
● Robert Hooke in 1665.
● Matthias Schleiden - plants are made by cells 1838/ Theodor Schwann-animals too
● Rudolf Virchow - cells can be produced from other cells by cell division 1855
● S. Ramón y Cajal: Doctrine of the neuron: nerve cells are independent elements, and
the propagation of nerve impulses is through contacts between them, 1889. It forms
the basis of modern neuroscience → Universality of Cell Theory
a64b0469ff35958ef4ab887a898bd50bdfbbe91a-5138864
Sutton, Boveri and Morgan - theory of chromosomes as carriers of genetic material and
genes.
Watson and Crick - discovered the double helix molecular structure of DNA, the carrier
molecule of the genetic program (1962 Nobel Prize)
Lynn Margulis - creator of the Endosymbiotic Theory, also called Serial Endosymbiosis
Theory.
3-ORGANIZATION AND STRUCTURE OF CELLS

Prokaryotes vs Eukaryotes
• Mycoplasmas (125-150 nm microorganisms), typical bacteria (E. coli), blue-green algae
(cyanobacteria).
• Viruses / Viroids - infectious living things but NOT cellular organisms.
• Prions - other infectious agents NOT living things.
4-ULTRASTRUCTURE OF PROKARYOTIC CELLS

● Prokaryotic cells (0.1 – 2 µm)
○ No nucleus or organelles, only Ribosomes 70S
○ Entirely filled with cytoplasm
○ Nucleoid -one lineal/circular DNA molecule not associated with proteins
○ Cell division by binary fission
○ A single (plasma) membrane
○ Cell wall-outer covering of most cells that
protects the bacterial cell and gives it shape.
5-CELLS ARE THE FUNDAMENTAL BUILDING BLOCKS OF ALL LIVING CREATURES

In unicellular organisms one cell carries out all life functions (Paramecium)
● Nutrition
● Metabolism
● Growth
● Response
● Excretion
● Homeostasis
● Reproduction
Abre tu cuenta N26 y llévate 10 € en 10 minutos ¡Clic aquí!

6-MULTICELLULAR ORGANISMS-CELL DIFFERENTIATION/

SPECIALIZED TISSUES
• In humans 220 different and highly specialized cell types
• Gene expression-production of specific proteins & cell differentiation
• The control of gene expression is the key to development
• Cells organized to interact and cooperate-Emergent properties
7-CELLS COME IN DIFFERENT SHAPES & SIZES

Very large: some neurons > 1 m (body and axon)
Normal size: 4 - 100 micrometers (mm)
1 mm = 10-3 mm = 10-6 m
Subcellular structures: 10 -100 nanometers (nm)
1 nm = 10-9 m
Visible scale: anatomy (macroscopic)
<10 nm: Molecular Biology
8-MICROSCOPE HAS REVOLUTIONIZED THE CELL SCIENCE

●Light microscope (1590)
●Improvements in the second half of the 19th
century allowed the discovery of bacteria and study

of chromosomes, basis of sexual behavior (mitosis
& meiosis)
●Fluorescent microscope (fluorescent markers)
●Laser scanning microscope ,with laser
illumination centered on a plane of preparation
(Minsky 1957) 2000x
●Electronic microscopes (1930) use electrons as
sources of illumination. Magnification is more than
500,000x
● Magnification is important but also the resolution
9-FLUORESCENCE 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 centered 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
10-ELECTRON MICROSCOPE
● EM (1930) – uses an electron beam to produce the image of the object
High vacuum tube (→ no living cells) → l de 0,005 nm → d = 3 Å (0,3 nm)
● (in practice, d = 1 nm = 0.001 mm)
● Magnification produced by magnetic fields that act as condensator and objective.
Magnification x 500.000
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 or photographic film. Image below: Nucleus
10% de descuento en toda la web con el código WUOLAH

Scanning electron microscope (SEM): The electrons are dispersed on the sample without
crossing it. Ultra thin samples are not necessary → 3D image Image above: blood cells.
12-SAMPLES PREPARATION FOR MICROSCOPY
Tissue fixation: in general, chemical treatment to preserve the sample; it may also improve
sample affinity for certain stains. Formaldehyde, Bouin's Fixative, Glutaraldehyde). Physical
fixation: by rapid heating / freezing
Dehydration: Alcohols of increasing concentration Organic solvents (xylene, toluene)
-miscible transition fluids

Embedding: Paraffin, resins, agar
Tissue cutting: with microtomes / ultramicrotomes
Dewaxing/rehydration: Solvents and alcohols of decreasing concentration
Staining: Different dyes depending on desired outcome
Mounting: Glass slides with coverslip
Frozen samples: Freezing tissue is a quick way to harden it without need for embedding.
For traditional studies – e.g. enzymatic assays and intraoperative biopsies
Traditional freezing or liquid N2
Cryostat cutting
Fixing, staining, mounting on slides ...
13-SAMPLE STAINING FOR LIGHT MICROSCOPY

Histochemistry:
Wide range of color solutions
Objective: to reveal a molecule or family of molecules present in a
histological section and to study its tissue distribution "in situ".
Hematoxylin-Eosin:
Hematoxylin: basic dye → dyes structures / acid elements (basophilic):
dark blue
Eosin: acid dye → basic elements (acidophilic): pink
Periodic Acid-Schiff (PAS, panel A) Labels Carbohydrates – used in
medical diagnosis e.g. adenocarcinoma
Feulgen stain (by Robert Feulgen, panel B) Chromatin localization –
depends on the acid hydrolysis of DNA
Detection of lipids (Sudan stain, panel C) It binds triacylglycerols (red) and other lipids
like myelin (black)
¿Qué música o podcast han marcado tu 2022? ¡Descúbrelo en Spotify!

Enzyme histochemistry or histoenzymology - Localization of enzymatic activities by
adding a substrate that reacts with the enzyme, that then precipitates and is colored in the
presence of a reagent.
Enzymes detected: NADH, ATPase, Phosphatase, Peroxidase, etc
Cold processing is usually required – sections obtained by freezing and cut on cryostat.
14-TECHNIQUES FOR IMMUNOLABELING

● Immunocytochemistry is a group of
techniques for the localization of
molecules in tissues by means of
antibodies.
● It allows the detection of the
products of gene expression
(proteins) by taking advantage of

the specificity of the antigen-
antibody binding.
15-ANTIBODY PRODUCTION
Antibody production/generation
- It is produced in an animal after being injected with a molecule that it recognizes as foreign
body.
- Stimulation of the immune system with immunogens: Substances that induce a specific
immune response against an epitope → structural element recognized by antibodies.
Immunizations
Injection with / without Freud's adjuvant in rabbits / goats / pigs or purified proteins. → large
amounts of serum → direct use.
Types of antibodies:
- Polyclonal antibodies – by recognizing several antigenic determinants allow for rapid
production and amplification of antigen-antibody reaction
- Monoclonal - by recognizing a single epitope, its specificity is higher, reducing the
probability of cross reactions and background noise in the assays.
16-VISUALIZATION OF ANTIGEN-ANTIBODY COMPLEX

Conjugation of antibodies
Detection of antigen-antibody complexes:
1. Direct labeling: antibody-bound chromogen (I AB conjugated to a chromogen)
2. Indirect labeling: chromogen coupled to the II AB → amplification of the signal with
respect to the direct marking.
17-TYPES OF CHROMOGENS
Proteins and enzymes
They induce the colored reaction of a substrate Horseradish peroxidase (HRP): in the
presence of H2O2 it catalyzes the oxidation of the diaminobenzidine (DAB) → brown/black
staining (panel A)
Fluorochromes:
Substances that emit light when excited by a different light FITC, Alexa Fluor, Rhodamine
(panel B)
18-IMMUNOHISTOCHEMISTRY
Immunohistochemistry
Protein detection – light or electron microscopy.
The samples (tissue sections) must go through a series of steps before using the antibodies.
1.- Tissue permeabilization
2.- Blocking nonspecific antigen binding
3.- I AB – followed by wash
4.- II AB – followed by wash
5.- Reaction/labeling revealed
Panel A. - a triple staining with antibodies conjugated to fluorescent molecules
Panel B. - a reaction with HRP-DAB

19-CELL CULTURE
● In vitro maintenance of isolated cells
● A routine laboratory technique since 1950.
Application/Utility
● Study of the properties, nutritional requirements, and functions of specific cell types
● Organ culture
● Therapeutic management of cells prior to transplantation
Cell origin:
Primary cultures: of body fluids or solid tissues: → tissue/cell disintegration by enzymatic
mechanics
→ Explants
→ Cell lines
20-FLOW CYTOMETRY
- Biophysical technology that allows for quantification and separation of fluorescently labeled
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.
¿El resumen de tu año en Spotify? ¡Clic aquí para más!

21-STEM CELLS
21.1- CELL CULTURE
● 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
● Embryonic stem cells – from embryo 4-5 days post fertilization
● Adult stem cells – from adult tissues
○ Umbilical cord
○ Bone marrow
○ Muscle
○ Mesenchymal cells
● Therapeutic use
● Diseases treated with Stem Cells:
Diabetes.
Osteoarthritis
Neurodegenerative diseases
Acute Leukemia
Chronic Leukemia
Myelodysplastic Syndromes
Lymphomas Anemias - deficiencies or

malformations of the red blood
● Ethical controversy - still divided opinions
21.2- MEDIUM
● Buffered liquid nutrient medium
○ Undefined: varying serum concentrations
○ Defined: known components and concentrations
Components:
H2O and mineral salts (K +, Na + and phosphate)
Bicarbonate buffer (HEPES)
Phenol red Glucose (pyruvate and / or lactate)
Albumin Antibiotics (Growth factors, interleukins, etc.) (Variable serum concentrations:
5-20%)
21.3-SEEDING/CELL PLATING
Cell seeding/cell plating:
- Plastic dishes: flasks or multiple wells plates to which they will attach themselves
Nutrient mediums used:
- Normal medium
- Treated for culture adhesion
- Coated with extracellular matrix elements
- With nutritious monolayer (feeder layer)
21.4-GROWTH
Culture growth:
- Frequent change of nutrient medium
- Constant temperature conditions (incubator)
- Homeostasis
Llévate 1 año de WUOLAH PRO con BBVA. ¿Cómo? ¡+Info aquí!

- Cell culture growth is limited by the nutrient availability and cell density
Precautions to consider:
- Homeostasis
- Avoid contamination → work in sterile conditions
- Pressurized air cabins (Microflow).

UNIT 2: CELL MEMBRANE
1-CELL MEMBRANE
● Composition and structure:
Lipids: Barrier
Proteins: Transport of substances and communication
Carbohydrates: Recognition of other cells/extracellular environment (interactions
with the environment)
● Functions :
○ External limit of the cell and organelles
○ Responsible for internal cellular organization
○ Control of substance exchange
○ Regulates cellular interactions with the external environment
○ Communication with other cells
○ Capacity of invagination: exocytosis, endocytosis, pinocytosis
*Support, transport, signaling and recognition functions
● Fluid mosaic model of cell membrane by Singer and Nicolson, 1972: still in use
and it applies to all cell membranes and to those of organelles.
1.1-FREEZE FRACTURE
● The membrane is a phospholipid bi-layer consisting of
a polar head group, phosphate, and hydrocarbon tails.
● Hydrophobic compounds can diffuse across the
membrane
● Hydrophilic compounds will not diffuse across the
membrane
2-LIPIDS
- Amphipathic molecules
- Variable composition: according to cell type and organelle
- The lipid bilayer gives the membrane fluidity and elasticity.
● Main classes of membrane lipids
Neutral fats (minor component)
Phospholipids (phosphoglycerides)
- Phosphatidic acid
- Phosphatidyl-choline, -ethanolamine, -serine,
- inositol, -glycerol
Sphingolipids: sphingosine derivatives
- Ceramides

- Cerebrosides (galactocerebroside abounds in myelin
- Gangliosides
Sterols
- Cholesterol (<25%)
3-CELL MEMBRANE IS FLUID

Fluidity is the ability of a molecule that is part of a membrane to move through it. Lipids have
fluidity. Depends on:
●Temperature
●Fatty acids saturated / unsaturated Cholesterol → Decreases
fluidity
Lipid movements
- Translation (lateral diffusion within the same layer)
- Rotation & flexion (around itself, in the same layer)
- Translocation: flip-flop (between two layers) (flippases,
floppases, scramblases-ATP independent but Ca-dependent)
●Cholesterol has the ability to flip-flop with relative ease
(mediated by floppases).
4-CELL MEMBRANE IS SEMIPERMEABLE

The membrane is not permeable to all molecules.
Permeable to: water soluble molecules, nonpolar molecules (O2), polar covalent bonds
(CO2), Urea, H2O (small size, no charge)
Impervious to: large uncharged molecules, charged polar molecules, ions.
Integral membrane proteins - selectively allow the passage of molecules whose movements
are restricted.
● The permeability of the membranes also depends on the composition of lipids,
particularly cholesterol.
10

5-CELL MEMBRANE IS ASYMMETRIC

● The plasma membrane is an asymmetric structure
● Both proteins and lipids contribute
● Lipids: different distribution of saturated and unsaturated lipids between the two
layers
● Proteins: Different distribution of integral and peripheral proteins
● Lateral asymmetry - lipids and/or proteins clump together in a plane / zone of the
membrane
● Transverse asymmetry - from outer side to inner side
6-LIPID RAFTS
Lipid rafts: subdomains of the plasma membrane that contain high concentrations of
cholesterol, glyco- and sphingolipids.
Serve as organizing centers for the assembly of signaling molecules, influencing membrane
fluidity and membrane protein trafficking, and regulating receptor trafficking and
neurotransmission. → facilitate reception of signals
● Known to intervene in cellular functions as the response to the invasion of
pathogens, cholesterol homeostasis, angiogenesis, signal transduction.
Often grouped in caveolae: Invaginations of membrane (60 nm), coated with caveolin.

7-PROTEINS
● Amphipathic
● Integral (Transmembrane) Peripheral- (exterior, interior)
● Attached to lipids by covalent bond
● Bound to protein by electrostatic attraction
● Protein distribution is asymmetric
● Protein movements (translation, no translocation)
● Function: receptors, recognition, enzymes, adhesion proteins, channels,
transporters, pumps, etc.
● On the outside layer, proteins (and lipids) bind to carbohydrates - forming glycocalyx:
cell recognition and interaction
○ Selectins (formation of lymphocytes)
○ Integrins
○ Blood groups (A, B, 0)
● Structure
○ Primary –chain of amino acids
○ Secondary
○ Tertiary
○ Quaternary – when more than 1 polypeptide is involved
8-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 oxidoreduction, transfer of active
groups, or hydrolysis.
11

- Cell adhesion molecules allow cells to identify each other and interact. For example,
proteins involved in immune response.
9-TRANSPORT PROTEINS
- A membrane transport protein (or simply transporter) is a membrane protein involved in the
movement of ions, small molecules, or macromolecules, across a biological membrane.
- They are integral membrane proteins - they exist permanently within and span the
membrane
- They may assist in the movement of
substances by: facilitated diffusion (no need for
ATP as molecules and ions move down their
concentration gradient ) or active transport (the
movement of molecules across a membrane
from a region of their lower concentration to a
region of their higher concentration—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
Approximately 10% of the genes in a cell are related to membrane transporters.
10-CHANNEL PROTEINS/ION CHANNELS

Channel proteins
- Allow the passage of ions or specific molecules in favor of gradient (passive transport)
- Closed and open state
Closed in resting state (majority) while opening is regulated by stimuli:
● Membrane potential change - voltage gated (K, Na, Ca channels)
● Covalent modifications (e.g. phosphorylation)
● Ligand binding –ligand gated directly gated (neurotransmitter recognition) indirectly
gated neurotransmitter recognition + regulatory molecule (G protein, cGMP)
Responsible for maintaining Ionic concentration across the membrane
Responsible for resting and action potentials: nervous impulse conduction, muscle
contraction
11-LIGAND AND G-PROTEIN GATED

● Ligand gated- direct gating Ligand-gated ion channels - also commonly referred
as ionotropic receptors, are a group of transmembrane ion-channel 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 Gprotein subunits and the channel protein.
The GTP-bound α-subunit in some cases can directly activate the ion channel. In
12

other cases, the activated βγ-complex of the G protein may interact with the ion
channel.
12-ACTION POTENTIAL: MUSCLE CONTRACTION

13-CARRIER/TRANSPORTER PROTEINS
● Proteins which bind one particular ion/molecule and assist 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 conc. gradient (e.g amino acids).
● Carry out specific transports in favor or or against gradient (Pumps)
● They can simultaneously transport 2 substances (coupled transport, cotransport)
● Cotransport: symport or antiport.
14-PUMPS (ATP-asas)
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.
15-SODIUM/POTASSIUM PUMP
13
16-MEMBRANE TRANSPORT
● Particles move across the membrane by simple diffusion, facilitated diffusion,
osmosis and active transport
● Fluidity of the membrane also allows materials to be taken into cells by endocytosis
(liquids by pinocytosis) or released by exocytosis
● Vesicles also move materials within cells
17-EXOCITOSIS
● A form of active transport in which a cell transports molecules (e.g.,
neurotransmitters and proteins) out of the cell by expelling them through an energy-
dependent process.
18-ENDOCITOSIS
● Endocytosis – a form of active transport, imports extracellular molecules by forming
vesicles from the plasma membrane.
● Two types: pinocytosis and phagocytosis

19-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.
Immunoglobulin Superfamily
CAM. Calcium independent.
NCAM, ICAM, NgCAM, VCAM (CD106), CD2, Nectin,
Cadherins
CAM. Calcium-mediated
K-Cadherin, E-Cadherin, Desmogleins and Desmocolinas
Selectins
CAM. Calcium dependent
Extracellular lectin domain: protein that recognizes carbohydrates
E-selectin (CD62), L-selectin and P-selectin
Integrins
transmembrane receptors that facilitate cell-extracellular matrix (ECM) adhesion Calcium-
dependent
Up to 24 types
Collagen receptor (a1b1): Connective
Fibronectin receptor (a5b1): general
14

Laminin receptor (a6b1): basal lamina

Some mediate cell-cell binding:
LFA-1 (aLb2) (CD11a / CD18): lymphocytes, recognized by ICAM
VLA-4 (a4b1) (CD29): leukocytes VCAM ligand
15

UNIT 3: THE NUCLEUS

1-CELL THEORY - EXPLAINS THE BASIC FEATURES OF CELLS STRUCTURE
● All surrounded by a membrane that separates their content from their exterior
● Cells contain genetic material 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 of the cell’s
activities
● They reproduce - new cells are produced from existing cells
2-NUCLEUS- EUKARYOTIC CELL
● The largest cell organelle
● Discovered by A. Leeuwenhoek, R. Brown in 1831.
● Stores coded information DNA-nuclear genome-genes
● It is essential for life of cells
○ As it contains all the necessary instructions for their survival and the activities
that are carried out.
● Main function is to maintain the integrity of the genes
● By regulating gene expression, it controls cell differentiation and all the cellular
activities

- Present in most all eukaryotic cells - except erythrocytes and keratinocytes of the stratum
corneum.
- Normally there is 1 nucleus/cell but some cell contain several nuclei i.e. skeletal muscles
Also, cell under pathological conditions-metastasis of tumor cells
- Shape and position varies depending on the cell type
- The interphasic nucleus - characterized by a visible nuclear membrane, nucleolus,
chromatin, and nucleoplasm
- During mitosis the nuclear organization is lost
3-NUCLEOLEMMA
3.1-NUCLEAR MEMBRANE
- Double membrane (2x7nm) with perinuclear cistern (20-40 nm).
16

- Separates the interior content of the nucleus from the cytoplasm – regulatory point
(customs) in the transport of molecules - the nuclear pore
- The outer membrane is continuous with that of the endoplasmic reticulum and has
ribosomes attached - it allows the intermembrane space and the interior of the endoplasmic
reticulum to communicate directly
- The inner membrane contains a different molecular composition and has transmembrane
proteins that interact with chromatin and with the nuclear lamina (III component of the
nuclear membrane)
- Inside the nucleus - the chromosomes that store genetic information; the accessory
proteins involved in condensation and decondensation of the chromosomes during the cell
cycle; 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
3.2-COMPOSITION
● Definite structure revealed by electron microscope and freeze-fracture
technique Composition: lipid bilayer
● 30% lipids:
○ similar to ER but more saturated
○ 90% phospholipids
○ neutral lipids: triglycerides and sterol esters (cholesterol)
● 70% protein:
○ outer membrane: similar to ER: with ribosomes and vimentin binding proteins.
○ internal membrane: lamins - binding proteins,
○ between membranes: Nuclear Pore Complex
* The nuclear envelope disappears during mitosis and reappears again at the end of
it.
4-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; B1: 65kDa; B2: 78kDa and C: 72kDa
- Structural function: confers mechanical stability to the nuclear envelope, interacts with
chromatin and participates with 3D organization of interphasic nucleus – i.e. cell cycle
organization, cell differentiation and apoptosis.
- The deficiencies in the laminar proteins produce different pathologies - laminopathies
5-NUCLEAR PORE
5.1-COMPLEX
- Point of entrance and exit of different soluble substances.
- Diameter: 50-80 nm
- Variable number (3000-4000 / nucleus) –it depends on the
cell type and differentiation (i.e. it depends on the number
of cell transcriptions).
- It consists of a large protein complex - about 100 different
proteins (nucleoporins) arranged in octagonal structures
17
projecting into nucleoplasm and cytoplasm.

- Allows for passive diffusion of molecules <50 kDa and in favor of the concentration
gradient.
-The larger molecules are recognized by specific signal sequence and transported via
active, chaperon-mediated translocation linked with the Ran-GTPase gradient – RAN cycle.
5.2-TRANSPORT
- Molecules transported to the interior must have a Nuclear Localization Sequence (NLS).
- What has to come out, a Nuclear Export Sequence (NES) -known sequences of amino
acids
- Karyopherins (importins and exportins): receptors 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 GTPase called Ran – RAN cycle
- Step in favor of gradient favored 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.

6-NUCLEOPLASM OR KARYOPLASM
● Aqueous phase with:
○ Cofactors and precursor molecules: ATP, NAD +, AcetylCoA, steroid
hormones - cortisol, estrogen, progesterone, testosterone, fatty acids,
phospholipids
○ Proteins and enzymes: 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 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 – found a small number of structural genes encode
all our proteins (approx. 90000) but large parts of noncoding DNA has
18

regulatory function-may be linked to diseases and may be used as

therapeutic targets.
7-NUCLEOLUS
● The nucleolus is a component of the nucleus.
○ It has no membrane that limits it.
○ In the nucleolus lies the region of chromosomes (DNA) containing the highly
repeating 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
8-CHROMATIN
● Chromatin is a complex of macromolecules found in cells, consisting of DNA and
proteins - the basic substance of the chromosomes.
● DNA wraps around histone proteins forming nucleosomes; the "beads on a string"
structure
● The basic structural units of chromatin are nucleosomes - "pearl neckless“
● Nucleosome – disk shaped octamer core (2 copies of each of the four histones H3,

H4, H2A and H2B) around which the DNA helix is wound
2 possible configurations
- Heterochromatin -condensed, in principle, transcriptionally inactive (DNA which codes
inactive genes ("turned off"), it is more condensed and associated with structural proteins
- Euchromatin - decondensed, in principle, 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.
9-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: Non-genetic
factors that modify gene expression. The histone configuration alters the accessibility of the
transcription factors:
- 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.
- Acetylations: in the tails of histones at the level of lysine and arginine: modify the
chromatin to be transcription- ready.
- Deacetylations: chromatin condensation mutes transcriptional activity.
10-KARYOTYPE
● The chromosome pattern of a species - number, size and shape of chromosomes
that defines a species.
○ In humans - 23 pairs in the nucleus of each cell.
Euploidy: number of chromosomes as a multiple of the number n (number of
chromosomes): haploid, diploid, triploid, etc.
19

Aneuploidy: numbers that are not multiples of the basic: nullisomy, trisomy, Abnormal
number of chromosomes , more than 90% of all cancers: unknown mechanism Most tumors
have accumulation of univocal mutations.
Haploid - is the quality of a cell or organism having a single set of chromosomes.
By means of the karyotyping –we can analyze numerical and structural chromosomal
abnormalities
11-CHROMOSOME CLASSIFICATION -karyotyping
- The autosomal chromosomes designated by numbers, the sexual ones with X or Y.
- They have 2 arms: p- short arm, q- long arm and a centromere in the middle.
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. Sub-metacentric, medium and small.
F: 19 and 20. Small metacentric.
G: 21-22 and Y. Small and acrocentric.
They allow detecting translocations, inversions, aneuploidia
etc. e.g. Down, Philadelphia chromosome (chronic myeloid
leukemia)

• 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. Employed
to detect and localize the presence or absence of specific DNA sequences on
chromosomes.
20

UNIT 4: THE CYTOPLASM
1-CYTOPLASM
● The cytoplasm is 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 substructures.
● 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.
2-ENDOPLASMIC RETICULUM -ER

● Cell is viewed as a multitude of membranes
● ER sure fits with this description
● ~ 50% of the total membrane surface in an animal
cell is provided by ER

● Two types of endoplasmic reticulum: rough
endoplasmic reticulum (RER) and 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
3-ROUGH ENDOPLASMIC RETICULUM - RER

● Extensive organelle composed of greatly convoluted but flattish sealed sacs
(cisternae), which are continuous with the nuclear membrane
● Called ‘rough’ because 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.
● Differential centrifugation – method for separating RER from the rest of the cell.
3.1-STRUCTURE
● 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
○ 30% lipids phospholipids are shorter and less saturated than of the
plasmalemma
○ 70% proteins specific for translocation & 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.
The general structure of the endoplasmic reticulum is a network of membranes called
cisternae i.e. –sacs-like structures.
21
3.2-FUNCTION
● Serves many functions including: - protein synthesis, storage, processing, quality
control) and trafficking
○ 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.

3.2.1-RER FUNCTION – PROTEIN SYNTHESIS
● Transcription – in the nucleus –
synthesis of mRNA
● During translation, the mRNA works
with a ribosome and tRNA to
synthesize proteins.
● Newly synthetized proteins have a
‘destination label’ (signal peptide or
sequence) that is recognized by the
signal recognition particle (SRP)
4-SRP SEQUENCE & RIBOSOME – RER DOCKING
22

5-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 on the amino acid side chains (R) or at the proteins C- or N termini.
Phosphorylation is a common mechanism for regulating the activity of enzymes and is the
most common post-translational modification of proteins.
Some have metal groups added to them. It is in the rough ER for example that four
polypeptide chains are brought together to form hemoglobin.
● 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.
● At the -OH end of serine and threonine (glycosylations in O) - they take place In
Golgi Apparatus
● At the NH end of -asparagine NH (glycosylations in N) They take place in RER and
GA
● 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.

6-N-GLYCOSILATION
The biosynthesis of N-linked glycans occurs via 3 major steps (panel A):
1. Synthesis of dolichol-linked precursor oligosaccharide
2. En bloc transfer of precursor oligosaccharide to protein
3. Processing of the oligosaccharide Steps 1 and 2 occur in the RER, while step 3 takes
place in GA.
7-PROTEIN FOLDING AND CONTROL

● In the lumen of the rough ER that proteins are folded to produce the highly important
biochemical architecture which will provide ‘lock and key’ and other recognition and
linking sites.
● 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).
● Chaperone proteins: BiP (binding immunoglobulin protein), calnexin / calreticulin
active in quality control.
● 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).
23

UNIT 5: SMOOTH ER AND GOLGI COMPLEX

1-SMOOTH ENDOPLASMIC RETICULUM
● Smooth (SER) is more tubular than rough ER and forms an Interconnected series of
tubes that curves through the cytoplasm - sub-compartment of ER.
● Lacks membrane-bound ribosomes –hence the name ‘smooth’.
● It is found fairly evenly distributed throughout the cytoplasm.
● It has variable extension depending on cell type or function
Composition:
Lipids: similar to RER
Protein: some similar to the RER, others specific to its function
1.1-FUNCTION REL
participates in the following cellular processes:
- Lipid synthesis
- Cell transport
- In detoxification: thanks to detoxifying enzymes that metabolize alcohol and other
chemicals
- In glycogenolysis: essential process to maintain adequate glucose levels in the blood
- Calcium storage: acts as a reservoir of calcium.

1.2-LIPIDS SYNTHESIS
SER is devoted almost exclusively to manufacturing lipids (triglycerides, phospholipids,
steroids) and their metabolism, as well as of the associated products Synthesis of fatty
acids: in cytoplasm (cytosolic enzymes) + enzymes of the SER membrane (acyl
transferases, fosfatasas, choline phosphotransferases, scramblases)
Synthesis of phospholipids: Fatty acid-CoA (fatty acyl CoA) + glycerol P (cytoplasm) →
phosphatidic acid → diacylglycerol→ phospholipid → transfer to the interior. The GPAT
(glycerol-3-phosphate acyltransferase) is the rate limiting enzyme in the synthesis of
glycerolipids, it plays a pivotal role in the regulation of triglyceride and phospholipid
synthesis.
2-GOLGI APPARATUS/COMPLEX
Golgi apparatus: processes, packages, and secretes
modified proteins
● 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.
2.1-WHAT IS IT?
● Organelle present in eukaryotic cells
24

● They are located very near the rough endoplasmic reticulum and hence near the
nucleus.
● Formed by one or more stacks of flattened, disclike, 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 synthesize) and the
cell membrane (proteins incorporated)
2.2-COMPOSITION
● Similar to other cytoplasmic membranes:
○ 35% lipids (more cholesterol and saturated lipids than ER)
○ 65% proteins (phosphatases, glycosyl 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
2.3-FUNCTION
● 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.
● It mainly modifies the proteins that are prepared by the rough endoplasmic reticulum.
● Also involved in lipid transport around the cell.
● Involved in lysosomes formation.
● The Golgi complex is thus referred to as post office where the molecules are
packaged, labelled and sent to different parts of the cell.
● CHECKING, MODIFICATIONS & SORTING (packaging) OF GOODS DELIVERED
FROM ER that arrive by transport vesicles.
● In the Golgi apparatus the vesicles are delivered into the ‘unloading bay’ of the cis
Golgi network- the ‘goods received’ are checked over.
25
● 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.
● 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.
● Modifications takes place in the cisternae, sequentially while passing from CIS to
TRANS face
● The correct ‘labelling ‘of products is critical
● Lysosomal storage diseases –a large group of rare inherited metabolic disorders-
the large molecules accumulate within the cell, eventually killing it – produced by
incorrect labeling and lack of lysosomal enzymes
3-POST TRANSLATIONAL MODIFICATIONS IN GA:

3.1-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 cis-Golgi
● The next step involves further addition and removal of sugar residues by
glycosyltransferases & glycosidases, respectively) in the Golgi complex.
3.2-GLYCOSYLATION O-linked
Glycosylation O-linked: binding of of hydrates 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.
4-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. sulfated proteoglycans
heparin sulfate (involved in angiogenesis, blood coagulation, developmental
processes), chondroitin sulfate (anti inflammatory effect in osteoarthritis, synthesis
of hyaluronic acid)
● Phosphorylation of oligosaccharides on lysosomal proteins takes place in the
early Cis Golgi network
26

● Phosphorylation - may form a 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 Golgi-important in forming
outer plasma membrane lipid bilayer, cell recognition & signaling
5-DISTRIBUTION OF MODIFIED PRODUCTS IN 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.

6-VESICLE DESTINATION
● 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).
● 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.
● 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 SIGNAL .
7-VESICULAR COAT PROTEINS –VESICLE TRAFFIC

COATED VESICLES- classical vesicle transport "COP" - specific coat protein complex
(coatomer) that initiates the budding process on the cis-Golgi membrane. The coat consists
of large protein sub complexes 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 cisGolgi RER)- anterograde
Clathrin- coat proteins are used to build small vesicles to transport molecules within cells.
Clathrin-coated vesicles (CCV) selectively sort cargo at the cell membrane, trans-Golgi
27

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
8-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 docking and fusion depends on Rab and 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
4) Exosomes mediated secretion (IL1)
9-EXOSOMES MEDIATED SECRETION

● Exosomes are either released from the cell when multivesicular bodies fuse with the
plasma membrane or released directly from the plasma membrane. e.g. IL1, mRNA,
proteolytic enzymes
● 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.
28

UNIT 6: MITOCHONDRIA
- Described for the first time by Altmann in 1884.
- It was thought that they were living structures that parasitized the cell – bioblasts or
endosymbionts.
- Have their own independent genome that shows substantial
similarity to bacterial genomes. Contain DNA, which is organized
as several copies of a single, usually circular, chromosome.
- Endosymbiotic hypothesis (Lyn Marguilis, 1986) suggests
that mitochondria were originally prokaryotic cells
- They are dynamic structures: they move, they group and
ungroup, they merge and divide.
- They can be visualized with Janus green (1900, Michaelis).
Also with the acid fuchsin of Altmann and with the iron
hematoxylin of Regaud; by immunohistochemistry; EM detailed

structural studies.
- 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 - they contain enzymes of the Krebs cycle and
oxidative phosphorylation, as well as the components of the electron transport chain.
*Involved in the oxidation of carbohydrates, lipids, proteins and the production of ATP
- energy source for the cell
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:
- Porins family → permeable to small molecules ˂ 5 kDa (aqueous channels)
- Enzymes of fatty acid metabolism (acyl-CoA synthetase)
- Electron transporters (cytochrome b5 and NADH-cytochrome b5 reductase)
- Receptors and transport proteins
- Proteins of the Bcl-2 family (regulate apoptosis)
Intermembrane space (peri mitochondrial space)
- 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).
2.INTERNAL MEMBRANE
2.1.STRUCTURE AND COMPOSITION
Internal membrane forms invaginations (cristae) - increase of the surface i.e. available
working space at which enzymes are located. For a typical liver mitochondria, the area of the
inner membrane is about 5x as large as the outer membrane due to cristae.
29
20% lipids:
-Cholesterol free; With cardiolipins: a type of phosphatidylglycerol with 4 fatty acids →
renders inner membrane 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 ketoglutarate DH and succinate DH)
- Electron transport chain (complexes I, II, III and IV, electron transfer via redox reactions)
- ATPase (ATP synthetase) (complex F, F0F1)
3.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 (55S mitoribosomes)
- Mitochondrial RNA
- 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.
4.FUNCTIONS
4.1.OXIDATION OF SUBSTRATES
Mitochondria -> site of the ultimate aerobic oxidation of cell metabolism products
Without the mitochondria, the cells would depend on anaerobic glycolysis (degradation of
glucose to pyruvate) to obtain all of their ATP = 2ATPs
Oxidation of Carbohydrates (Hans Adolf Krebs, Nobel Prize in Physiology in 1953):
- In cytosol: I phase - Glucose → pyruvate (enzyme pyruvate dehydrogenase) → acetyl CoA
- In matrix: II phase- Acetyl CoA enters the Krebs cycle → oxidized to CO2 and energy
conserved in NADH and FADH2
- III phase Electron transfer - NADH and FADH2 release electrons-which are driven by the
electron transport chain (separated from protons) until they reunite with them and with the
O2 to form water (oxidative phosphorylation).
For each glucose molecule degraded 36 molecules of ATP are formed in the mitochondria.
30

4.2.OXIDATION OF LIPIDS
Lipolysis – takes place in the cytoplasm
Free fatty acids cannot penetrate any biological membrane due to their negative charge.
They must cross the mitochondrial membrane through specific transport proteins.
Fatty acid catabolism consists of:
- Activation and membrane transport of free fatty acids by binding to coenzyme A (Fatty acyl-
CoA).
- Oxidation of the beta carbon to a carbonyl group.
- Cleavage of two-carbon segments resulting in acetylCoA.
- Oxidation of acetyl-CoA to CO2 in the Krebs cycle.
- Electron transfer from electron carriers to the electron transport chain in oxidative
phosphorylation. Fatty acyl-CoA + carnitine → acylcarnitine
→ pass by outer membrane (enzyme CPT1)
→ acylcarnitine passes to matrix (enzyme CAT)
→ acylcarnitine converted in acyl-CoA(enzyme CPT2)
→ b-oxidation (as many rounds/turns as carbon pairs )
→ acetyl-CoA (one molecule produced in each loop) (+ FADH2 + NADH)
→ Krebs cycle
Total acetyl CoA enters the Krebs cycle acetyl
CoA →→→→ 2 CO2 + 3 NADH + FADH2 + GTP

4.3.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 (Peter D. Mitchell, 1961, Nobel prize 1978)- 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 to ATP in the presence of inorganic phosphate.
• The key site of ATP production in oxidative phosphorylation is the inner mitochondrial
membrane.
31

4.4.FUNCTION OF ATP SYNTHASE

The H + accumulated in intermembrane space return 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 FO and contains a ring of c subunits and
the proton channel. 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
-Thermogenin
-Poisons (dinitrophenol) - allowing protons to leak across the inner mitochondrial membrane
and thus bypass ATP synthase
4.5.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.
5.PROTEIN SYNTHESIS IN THE MITOCHONDRIA
Encoded by mitochondrial DNA (mtDNA)
- Multiple circular copies 16.5 kbp
- Protein subunits, 2 rRNA, 22 tRNA
mtDNA is transmitted by the mother
- Translated in the mitochondrial ribosomes
- Smaller than the cytosolic ribosomes ( 35S and 25S)
- Inhibited like bacterial
- First amino acid: N-formyl methionine
- Non universal genetic code
6.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
32

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: presequence translocase 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.
7.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 cytosolic 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)
- No synthesis of ATP
33
UNIT 7: THE CYTOSKELETON

The cytoskeleton is a dynamic 3D protein network that maintains the shape of the cell,
facilitates cellular mobility and plays an important role in intracellular traffic and in cell
division.
Composed of multiprotein elements dispersed in the cytosol
- Highly conserved in the evolution
- Stable or dynamic
In eukaryotic cells there are 3 types of elements:
- Microfilaments (actin): 7 nanometers (nm) Ø,
- Intermediate Filaments: 10-12 nm Ø
- Microtubules: 24 nm Ø
They have different functions that :
- Depend on specific cell type
- Are related to associated proteins.
1.CYTOSKELETON - VISUALIZATION
EM

IMMUNO-
CYTOCHEMISTRY
2.MICROFILAMENTS
Highest concentration found under the plasma membrane - one of its functions is to maintain
the shape of the cell.
Actin filaments:
- The most abundant protein in eukaryotes
- Highly conserved in the evolution
- 6 isoforms: 4 muscle actins: a-actin
2 non-muscular actins: b-actin; g-actin
To carry out their contractile activity, 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).
3.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.
34

Actin G - actin / monomeric actin molecule: globular, in free form, soluble (42 kDa)
Actin F - polymerized, forming linear polymers - microfilaments
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 actin in the filament is
bound to actin G-ADP.
The process of microfilament assembly is a strictly regulated process: the filaments are
formed by polymerization of actin G forming a double-stranded helix.
Various proteins that interact with actin regulate assembly and disassembly in the cell
Typical situation → dynamic stability
Balance between polymerization and depolymerization
Depends on Actin G: 0.1 mM on + end; 0.6 mM on - end
In vitro experiments demonstrate:
- Dynamic process
- Polymerization at both ends of the filament.
- Nucleation
- Elongation
- Steady state
- The polymer has polarity
- Suggest that the rate of ATP hydrolysis and the rate of monomer incorporation are strongly

coupled.
*Once the steady-state phase is reached, the equilibrium concentration of the pool of
unassembled subunits is called the critical concentration (Cc). Under typical in vitro
conditions, the Cc of G-actin is 0.1 µM. Above this value, a solution of G-actin will
polymerize; below this value, a solution of F-actin will depolymerize.
4.PROTEINS THAT REGULATE MICROFILAMENTS FORMATION

In non-muscle cells, actin filaments are formed proximal to membrane surfaces. Their
formation and turnover are regulated by many proteins.
Arp-Actin-Related Protein-2/3 -Arp2/3 complex , favor nucleation of the filaments when the
complex is activated by Ras GTP-ases
Profilin-actin monomer binding protein: binds to G actin favoring phosphorylation of ADP
that is bound to it. The actinATP monomers are incorporated at the ends (+) of the
microfilaments.
GTPase from the Ras family favor actin nucleation
Thymosin: binding to actin-G-ADP prevents polymerization to Factin and growth of the
polymer = sequestration
Gelsolin and CapZ: They cover end (+) → non-polymerization.
Cofilin: binds to the end (-) Inhibits actin phosphorylation of GADP → depolymerization.
Thymosin (in neutrophilic leukocytes and platelets): sequesters actin-G-ADP and acts in a
similar way
Tropomodulin: binds to ends (-) → no depolymerization, stabilizes actin F in myofibrils of
muscle sarcomeros
Arp2 / 3 complex: Stimulates actin branching (panel B).
Filamin: Cross links filaments → branching network (panel B).
a-Actinin: Joins microfilaments in parallel filaments → bundles formation
35

5.MICROFILAMENTS- FUNCTIONS IN NONMUSCLE CELLS

They shape the cell, relate it to the neighboring cells or the extracellular environment
(matrix) 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.
Organization of the microvilli skeleton - example of crosslinked, noncontractile actin bundles.
2. Involved in cell movements –actin based motility Cellular contraction, cell movement of
vesicles, assembly disassembly of actin filaments, formation of networks and bundles –
require association with Myosins
Ca- dependent (ATP) Myosin proteins
Myosin I (mini myosin) does not polymerize into filaments - with 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 mini myosin head would be attached to the
filament, and the tail, to the plasma membrane (A).
- Can move along an actin filament, dragging along a vesicle or membranous organelle (B).
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.
6.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/ lamellipodia)
–cellular locomotion
Ameboide movements
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.
7.MICROFILAMENT FUNCTION
They can join with Myosin II - Function in contractile cells- Muscle contraction.
36

Myosin V is involved in the transport of cargo (e.g. RNA, vesicles, organelles, mitochondria)
from the center 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.INTERMEDIATE FILAMENTS (IF)

Heterogeneous group – different in different cell types
They have structural function

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 a-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
- Generally more stable than the rest of the cytoskeleton
- Disassembly mediated by phosphorylation –e.g. nuclear lamins
- 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.
8.1.CLASSIFICATION
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)
Present 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)
37
- Used as epithelial markers

- Genetic alterations-several diseases: e.g.
Genetic mutations - Epidermolysis bullosa , Epidermolytic hyperkeratosis.
9.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 motor neurons and
produce muscle paralysis.

10.OTHER IFs
Glial filaments – so far only demonstrated in vertebrates, in the cell body and in the
cytoplasmic extensions of astrocytes and Schwann cells
- GFAP –glial fibrillary acidic protein - similar in all vertebrates
- They form more compact bundles than neurofilaments
Desmin – is a chief intermediate filament 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 to 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
11.MICROTUBULES
• A constant / uniform component in all cells
• They are distributed in the same way as the filaments of keratin and vimentin
Under the EM - tubules of 24 nm Ø Variable length polymers
38

Composed of highly conserved tubulin-protein Heterodimer: formed by a- and b-tubulin

Polymerizing tubulin - each monomer has a GTP linked 2 GTP binding sites
a-tubulin-GTP
b-tubulin-GTP / GDP (hydrolysis 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
11.1.POLYMERIZATION
They consist of tubulin dimers
Growth from b-tubulin end (extreme +) → polarity Lateral association of 13 protofilaments →
microtubule (24 nm)
b-tubulin GTP hydrolysis rapidly, while GTP from α-tubulin is trapped between dimers
The half-life of tubulin is 20h
Occasionally microtubules group together: forming microtubule doublets (cilia and flagella)
triplets (centrioles)
Tubulogenesis is facilitated by: MAPs, cAMP, Ca, centrioles, kinetochores and nuclear
pores.
39

UNIT 8: CELL CYCLE CONTROL

1.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, a
cell 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
• Cytokinesis is a part of the cell division process during which
the cytoplasm of a single eukaryotic cell divides into two daughter
cells.
2.THE CELL CYCLE CONTROL SYSTEM CAN ARREST THE CELL CYCLE AT SPECIFIC
CHECKPOINTS

• All the steps are strictly monitored so that if the
conditions to go to the next stage are not met, the cycle
stops.
• 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 unfavorable
environmental conditions).
• There are three main checkpoints: the G1 checkpoint
(Major Checkpoint); the G2/M checkpoint; and the mitotic
or the spindle checkpoint.
3.CELL CYCLE REGULATION

The progression of the cell division cycle is strictly controlled by two large groups of genes:
1. The genes that encode proteins necessary for cycle progression (e.g. enzymes and
precursors of DNA synthesis and enzymes for synthesis and assembly of spindle tubulins).
2. Genes that encode proteins that regulate the cycle in a positive or negative way.
- Positive cycle regulation - proto-oncogenes
Their products activate cell proliferation → cells leave G0 and enter the S phase and enter
division (those that encode cyclin system proteins and cyclin-dependent kinases).
Proliferating genes → positive regulation of the cycle
* Heterozygous mutation → oncogenes → cancer
Most important genes encoding the proteins of the cyclin and cyclin-dependent kinase (Cdk)
system
40

- Negative regulation - tumor suppressor genes and check proteins.
4.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)
- Four classes of cyclins - each defined by the stage of the cell cycle at which they bind
CDKs and function.
• Described more than 15 cyclins, the most important are (D, E, A, B), this being the order of
appearance
• G1 -cyclins, help promote passage through Start or the restriction point in late G1 – cyclin
D
• G1 /S-cyclins bind CDKs at the end of G1 and commit the cell to DNA replication – cyclin
E
•S-cyclins bind CDKs during S phase and are required for the initiation of DNA replication
cyclin A
• 3. M-cyclins promote the events of mitosis cyclin B
* Cyclical changes in cyclin levels result in the cyclic assembly and activation of the cyclin-
Cdk complexes; this activation in turn triggers cell-cycle events
5.POSITIVE REGULATION OF THE CELL CYCLE – CYCLIN-DEPENDENT KINASES

Cyclin-dependent kinases - Cdks are kinase enzymes, act by phosphorylating serine and
threonine residues of target proteins to trigger cellular processes.
Cyclin-dependent kinases (Cdks):
- Similar to other kinases
- Phosphorylated (activated) by binding to cyclins → Cyclin / Cdk active complex dependent
on concentration of cyclins.
• When the concentration of cyclins is low, the Cdk lack them and are inactive.
The only function of Cdks is the phosphorylation of other cellular proteins.
6.ACTIVATION AND DESTRUCTION OF THE CYCLINS-CDKs COMPLEXES
Activation by Cdk activating kinase (CAK)
41
The cyclin-Cdk binding eliminates the blockage produced by the T-loop over the catalytic
center 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.
• The CDK-cyclin complex can be inhibited by cyclin – dependent-kinase inhibitors (CKI) –
proteins (p27, p21) that interact at the same time with the cyclin and CDK blocking kinase
activity.
• Cyclin destruction occurs by a ubiquitin
-dependent mechanism- activated enzyme
recognizes specific sequences on the cyclin
and attaches multiple copies of ubiquitin to
it- marking the protein for complete
destruction in proteasomes.
• Ubiquitin ligases – cyclin degradation-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
7.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 - their products ensure the fidelity of the genome during its
replication / segregation - 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.
42

5. Proteins that induce the cycle exit - towards a differentiated cellular state or towards
apoptosis (Bad, Bax, Bak promote apoptosis)
8.THE RETINOBLASTOMA PROTEIN (RB)

Rb - nuclear phosphoprotein
It has a fundamental role in the negative control of the cell cycle and in tumor 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 checkpoints: Point G1 (G1 / S)
Complexes cyclin-Cdk
Cyclin D-Cdk4 / 6 → Phosphorylates pRb / E2F complex → Active E2F → synthesis of cyclin
E and cyclin A
And PCNA - proliferating cell nuclear
antigen, which speeds DNA replication
and repair
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-Cdk2 (S cyclin):
Activation of chromatin replication (entry to S phase)
9.THE CELL CYCLE CONTROL by p53 GENE

Cellular tumor antigen-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 the G1/S
checkpoint 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:
Activates CKI p21 → inhibits cyclin/CDK → no
phosphorylation (of Rb also) & cell cannot enter into the
next division
Activates FasR and bcl-2 → apoptosis
Active synthesis interferon → apoptosis
Inhibits Nanog expression (gene for ESC renewal) → no pluripotency → maintains genetic
stability
43

*ESC embryonic stem cells
10.CHECK POINT 1 - pRB and p53
11.CYCLINB-CDK1 (MPF)
Cyclin B / Cdk1 controls the passage from phase G2 to phase M.
- Induces mitosis until early anaphase
- When the cell enters the G2 phase, cyclin B is synthesized and binds to Cdk1 → cyclin B /
CDK1 complex (MPF) - whose activity is essential for the cells to pass to the M phase.

- CyclinB-Cdk1 - inactive in phosphorylated state
- Activation by dephosphorylation by phosphatase Cdc25
-The activated kinase phosphorylates several proteins involved in mitosis:
- Phosphorylates H1 → chromatin condensation
- Phosphorylates lamins → nucleus rapture
- Phosphorylates MAPs → mitotic spindle formation
- Phosphorylates Gwl → PP2A phosphatase inactivation → allows activation of Cdks
again
-Essential in the expression of survival signal survivin → proper formation of the
mitotic spindle
* MPF –maturation/mitosis promoting factor
* MAPs- microtubule associated proteins
44

12.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
→ increases cell volume (swelling / swelling)
→ change of metabolic pathways
→ cellular and organelle breakdown
→ destruction of lysosomes and release of enzymes
→ fragmentation of cellular and nuclear membrane
→ involvement of neighboring cells → inflammatory response
→ 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
13.APOPTOSIS: SIGNALING PATHWAYS

Regulated by external and internal factors:→ signal translation cascade from cell surface
to the nucleus) → Activation Caspases → affectation of: kinases (FAK-focal adhesion
kinase), lamins, cytoskeleton (intermediate filaments, actin, tubulin), DNAse.
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 (tumor necrosis factor receptors
-TNFR)
- Union ligand-TNF activates FADD / TRADD adapter proteins that binds to procaspase 8
(initiator) → activates numerous executing caspases.
14.APOPTOSIS: MORPHOLOGICAL AND MOLECULAR CHANGES

The 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
Fragmentation of DNA by three endonucleases
Activation of several enzymes:
- Proteases / caspases
45
- Calcium-dependent transglutaminases
- Hydrolases
- Phagocytosis: phagocytes recognize molecules of phosphatidylserine exposed on the
membrane of apoptotic bodies.
15.APOPTOSIS: INTRINSIC PATHWAY

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.
46

UNIT 9: NERVOUS SYSTEM-NERVOUS TISSUE

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 I
t consists of two main parts (anatomical division):
Central Nervous System (CNS)
- Brain
- Spinal cord
Peripheral Nervous System (PNS) –mostly nerves
- Somatic
- Autonomic (sympathetic and parasympathetic)
- Enteric (gastro-intestinal innervation)
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 nervous system
- Parasympathetic nervous system
- Enteric nervous system
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 behaviors.
The spinal cord is the main information highway that connects the brain and the PNS
3.THE SPINAL CORD CONTAINS NEURAL CIRCUITS THAT CONTROL SIMPLE

RESPONSES -REFLEXES
47

4.CELLULAR COMPOSITION
Neurons – highly specialized cells/nerve cells
- Connected to each other in a complex way (neural circuits/networks)
- 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)
5.NEURONS
They are similar to other cells in the body in that:
- They are surrounded by cellular membranes.
- They have a nucleus with DNA / genes and 1 nucleolus
- They contain cytoplasm with organelles (Nissle bodies - RER, SER, mitochondria,
lysosomes, 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
48

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
6.NEURONS – SPECIALISED PROJECTIONS

Dendrites
Short and numerous.
Content similar to pericarion but without Golgi apparatus
Characteristic distribution according to neuronal type
With lateral projections (spines)

Axon
1 per neuron
It transmits stimuliI can be branched (collateral branches)
Branches in terminal bulb (synaptic buttons)
Axolema = plasmalemma, may have myelin sheath: lipid
Axoplasma: with mitochondria and transport via neurofilaments
The dendrites enter information into the body, or soma, of the neuron while the axons
carry it outwards.
7.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)
8.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
49
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
Neurons communicate with each other by electrical impulses (action potential) →
electrochemical processes
Vesicles synthesized in the soma transported through axon
- Neurotransmitter packed into vesicles at terminal buttons
- Increase in [Ca2 +] induced by the arrival of membrane potential
- 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)
9.NEUROTRANSMITTER
Neurotransmitter -NT
- 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
(recycling-reuptake)
- NT of low molecular weight or: acetylcholine (cholinergic neurons), adrenaline
(epinephrine), noradrenaline (norepinephrine), dopamine, serotonin
50

Amino acids: glycine, GABA, glutamate, aspartate

- Opioid neuropeptides: endorphins, enkephalins
- Hormones: somatostatin, oxytocin, cholecystokinin
- Gases: NO and CO
10.GLIA
Cells more abundant than neurons. They divide in situ.
Functions
-Support
-Nutrition
-Osmotic regulation
-Immune defense
-Clean “debris” produced in the brain
Central nervous system glia (neuroglia)
→Astrocytes
→Oligodendrocytes
→Microglia
→Ependymal cells
Peripheral nervous system glia
→Schwann cells
→Ganglion Satellite Cells (Anficitos)
11.ASTROCYTES
They are the main and most numerous glial cells-astroglia. They form the so-called glia
limitans - the border between the organism and the central nervous system.
Its morphology (as its name suggests) resembles a star - a large number of cytoplasmic
processes called feet that radiate from the soma to neighboring cells.
They express in their membrane a large number of receptors of different transmitters -
thanks to this, they can respond to different neurotransmitters: glutamate, GABA, Ach, NA
(NE, NO), released by brain neurons.
-Derived from neural stem cells (NSC).
51

-Some make contacts with blood vessels (perivascular feet).

-The main component of its cytoskeleton - intermediate filaments with glial acidic protein
(GFAP).
Types:
• Protoplasmatic: in the gray matter, with numerous
projections.
◦ Contain less GFAP.
◦ They cover the surface of the brain and the
spinal cord (glia limitans - glial limiting
membrane).
• Fibrous: in the white matter, with fewer extensions.
Abundant GFAP.
Functions:
• Structural: support and separation
• Metabolic: provide nutrients to neurons
• They digest parts of the dead neurons
• Regulation of ion concentration in the extracellular space
• Vasomodulation: intermediaries in neuron regulation of blood flow
• CNS repair - increasing its size and sendung its projections to fill the damaged area

- glial scar
• Formation of the blood-brain barrier (limit between blood and SNC)
• Stimulate the formation of synapses
• Modulation of synaptic transmission
• Clean “waste” of the brain
Tripartite synapsis - connection between neurons and astrocytes: the NTs released by the
synaptic terminals can activate the receptors on the astrocytes membrane - triggering a
Ca2+ signal - that can produce a release of gliotransmitters (Glut…, ATP, prostaglandins,
etc)
12.OLIGODENDROCYTES - OLIGODENDROGLIA
Smaller and less branched than astrocytes.
Derived from neuronal stem cells (NSC).
Next to neuronal soma:
-In gray matter
-Supporting role, regulation of ionic homeostasis
-Provide also trophic support by the production of glial cell line-derived neurotrophic factor
(GDNF)
Interfascicular oligodendrocytes:
-They produce the myelin sheath in the CNS - myelinated axons.
-Each one myelinated several axons.
-In white substance.
13.MICROGLIA
Microglia - Del Rio Hortega cells (the first to call them that, 1920).
-Small, with thin extensions.
-With phagocytic capacity - form the immune system of the CNS.
52

-Important role during development - induction of controlled cell death in certain regions of
the CNS.
-Involved in the elimination of synaptic terminals.
*Monitor the CNS - approach lesioned areas - amoeboid form.
-Able to produce anti-inflammatory molecules - they manage to slow down the inflammatory
process and increase the survival of viable cells.
-Able to recognize foreign bodies, viruses, fungi and other microorganisms, swallow them-
function like part of the immune system.
14.EPENDIMAL CELLS (EPENDIMOCYTES)

-Neurologlial cells.
-Epithelial cells of cylindrical ro cuboid shape.
-They cover the ventricles of the brain and the central canal of the spinal cord.
-With cilia and apical microvilli - characteristic that facilitates the movement of the

cerebrospinal fluid.
-At the level of the cerebral ventricles - form the choroid plexuses - whose mission is to
secrete and conserve the chemical composition of the cerebrospinal fluid.
15.SCHWANN CELLS
Schwann cells (or neurolemmocytes) - named after physiologist Theodor Schwann, are
the principal glia of the peripheral nervous system (PNS).
Main support cells of the PNS:
• Myelinating Schwann cells wrap around axons of motor and sensory neurons to form
the myelin sheath.
• Elongated, parallel to the longitudinal axis of the axon they surround.
• Direct the regeneration of peripheral axons.
There are 2 types of Schwann cells - myelinating and
non myelinating.
Glia in the PNS also includes satellite cells - olfactory
ensheathing cells, enteric glia and glia that reside at
sensory nerve endings (e.g. the Pacinian corpuscle)
Schwann cells function - involved in many important
aspects of peripheral nerve biology: the conduction of
nerve impulses along axons, nerve development and
regeneration, trophic support for neurons, production
of the nerve extracellular matrix, modulation of
neuromuscular synaptic activity.
16.MYELINATING SCHWANN CELLS

In myelinated axons (large diameter). Schwann cells form the myelin sheath:
• Absent in synaptic buttons and terminal branches.
• Stabilized by adhesion proteins.
• Variable thickness proportional to the axon diameter.
• Schmidt-Lanterman incisors/cleft - cytoplasmic collar between layers of myelin (small
amount of cytoplasm that remains in the inner layer of the myelin sheath created by
Schwann cells wrapping tightly around a nerve.
• Schwann cells are an energy dependent process.
53
The myelin sheath is not continuous - individual myelinating Schwann cells cover about 100
micrometers of an axon.
Nodes of Ranvier - the gaps between adjacent Schwann cells:
• Without myelin.
• Place of occurrence of the saltatory impulse → increasing the conduction velocity of
APs.
• Thickened axon attached to a Schwann cell by zonula occludens i.e. tight junction
between 2 cells.
54


Part 2 - THE CELL

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Part 2 - THE CELL

Uploaded by

Copyright:

Available Formats

The-cell.

1º Grado en Ingeniería Biomédica

Escuela Técnica Superior de Ingeniería de Telecomunicación.

Reservados todos los derechos.

UNIT 1: CELL AND METHODS OF STUDY

Reservados todos los derechos.

2.2-THE SCIENTISTS BEHIND IT

3-ORGANIZATION AND STRUCTURE OF CELLS

4-ULTRASTRUCTURE OF PROKARYOTIC CELLS

5-CELLS ARE THE FUNDAMENTAL BUILDING BLOCKS OF ALL LIVING CREATURES

Abre tu cuenta N26 y llévate 10 € en 10 minutos ¡Clic aquí!

6-MULTICELLULAR ORGANISMS-CELL DIFFERENTIATION/

7-CELLS COME IN DIFFERENT SHAPES & SIZES

8-MICROSCOPE HAS REVOLUTIONIZED THE CELL SCIENCE

Reservados todos los derechos.

9-FLUORESCENCE CONFOCAL MICROSCOPE

10% de descuento en toda la web con el código WUOLAH

Reservados todos los derechos.

13-SAMPLE STAINING FOR LIGHT MICROSCOPY

¿Qué música o podcast han marcado tu 2022? ¡Descúbrelo en Spotify!

Enzyme histochemistry or histoenzymology - Localization of enzymatic activities by

14-TECHNIQUES FOR IMMUNOLABELING

Reservados todos los derechos.

16-VISUALIZATION OF ANTIGEN-ANTIBODY COMPLEX

Reservados todos los derechos.

¿El resumen de tu año en Spotify? ¡Clic aquí para más!

Reservados todos los derechos.

Llévate 1 año de WUOLAH PRO con BBVA. ¿Cómo? ¡+Info aquí!

Llévate 1 año de WUOLAH PRO con BBVA. ¿Cómo? ¡+Info aquí!

UNIT 2: CELL MEMBRANE

Reservados todos los derechos.

3-CELL MEMBRANE IS FLUID

4-CELL MEMBRANE IS SEMIPERMEABLE

Llévate 1 año de WUOLAH PRO con BBVA. ¿Cómo? ¡+Info aquí!

5-CELL MEMBRANE IS ASYMMETRIC

Reservados todos los derechos.

8-MEMBRANE PROTEINS FUNCTIONS

Llévate 1 año de WUOLAH PRO con BBVA. ¿Cómo? ¡+Info aquí!

Reservados todos los derechos.

10-CHANNEL PROTEINS/ION CHANNELS

11-LIGAND AND G-PROTEIN GATED

Llévate 1 año de WUOLAH PRO con BBVA. ¿Cómo? ¡+Info aquí!

12-ACTION POTENTIAL: MUSCLE CONTRACTION

Reservados todos los derechos.

Reservados todos los derechos.

Llévate 1 año de WUOLAH PRO con BBVA. ¿Cómo? ¡+Info aquí!

Laminin receptor (a6b1): basal lamina

Llévate 1 año de WUOLAH PRO con BBVA. ¿Cómo? ¡+Info aquí!

UNIT 3: THE NUCLEUS

Reservados todos los derechos.

Llévate 1 año de WUOLAH PRO con BBVA. ¿Cómo? ¡+Info aquí!

Reservados todos los derechos.

projecting into nucleoplasm and cytoplasm.

Reservados todos los derechos.

Llévate 1 año de WUOLAH PRO con BBVA. ¿Cómo? ¡+Info aquí!

regulatory function-may be linked to diseases and may be used as

Reservados todos los derechos.

Llévate 1 año de WUOLAH PRO con BBVA. ¿Cómo? ¡+Info aquí!

Reservados todos los derechos.

Llévate 1 año de WUOLAH PRO con BBVA. ¿Cómo? ¡+Info aquí!

UNIT 4: THE CYTOPLASM

2-ENDOPLASMIC RETICULUM -ER