Cell Biology Lectures

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Cell Biology Lectures

• 1. Molecular organisation of cell membranes
• 2. Intracellular Organelles and Protein
  Trafficking
• 3. Cell Proliferation cell cycle
• 4. Cell Death

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Cell Biology

• 1. Molecular organisation of cell membranes

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mitochondria
nucleus
Bonus Mark
mitochondria
Endoplasmic reticulum
nucleolus
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Cell Membranes - Introduction

• Plasma Membrane
• Intracellular membranes
• Function of membranes is diverse, e.g
• Act as a barrier, i.e. physically separates the
  intracellular components from the extracellular
  environment.
• Regulate and facilitate the transport of
  materials.
• Play a vital role in cell signalling.

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Membrane Structure

• Structural basis for all animal cell
  membranes is the Lipid Bilayer interspersed with
  proteins

Bonus Mark
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Lipid composition of cell membranes

• Varies between different membranes
• Composition often reflects function
• Plasma membranes - 50 lipid, 50 protein
• Inner mitochondrial membrane - 75 protein
• 3 classes of membrane lipids in animal cells
• Phospholipids
• Cholesterol - 15 of lipid content
• Glycolipids - 5 of lipid content

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Phospholipids
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Amphiphatic
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Fatty Acid Structure
O O-
Carboxyl group Hydrophilic can form covalent
bonds with other molecules
C CH2 CH2 CH2 CH2 CH2 CH3
Hydrocarbon tail Hydrophobic
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Fatty Acid nomenclatureOne system of
classification is based on the number of double
bonds
O O-
O O-
C CH2 CH2 CH2 CH2 CH2 CH2
C CH2 CH CH
Free rotation
double bond is rigid and creates a kink
CH2
CH2
CH2
Unsaturated eg. Oleic acid/ Linoleic acid
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Saturated e.g. Stearic Acid (CH17COOH)
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4 major Phospholipids
Phosphotidylethanolamine Phosphotidylserine
Phosphoytidylcholine Sphingomyelin

Concentrated on cytosolic side
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Fifth Phospholipid

• phosphatidylinositol
• localised to the inner half of the plasma
  membrane
• Present in only small amounts
• Important in cell signalling

OH
OH
OH
OH
OH
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Phospholipids contd

• Phospholipids are amphiphatic molecules
• Spontaneously form bilayers in aqueous solutions
• Bilayer is the most thermodynamic stable
  structure for phospholipids

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Additional Lipids Glycolipids
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Structure of a glycolipid
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Additional Lipids Glycolipids

• Found exclusively on the extracellular portion of
  the bilayer
• Form a carbohydrate coat on the surface
• Glycocalyx

CELL COAT
EXTRACELLULAR
Phospholipid bilayer
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CYTOSOL
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Cell Coat or Glycocalyx
Electron micrograph of the surface of a
lymphocyte stained with ruthenium red emphasizes
the thick carbohydrate layer surrounding the cell
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Function of Glycocalyx

• Cell protection from injury
• Transplant compatability
• Cell Adhesion

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Additional Lipids cholesterol

• Provides cell membranes rigidity
• Present in large quantities in plasma membrane

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Membrane fluidity phospholipids
Membrane Fluidity depends on its composition

• Membranes are dynamic fluid like structures
• Lateral movement of lipids (flip-flop is rare)
• Rotational movement

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• Membrane fluidity contd
• Length of phospholipid hydrocarbon chains
• Shorter chains interact less well - provide
  greater fluidity that longer chains
• Degree of saturation (no. of double bonds)
• The presence of double bonds induces a small kink
  in the HC chain
• Chains with kinks pack less efficiently more
  fluid
• Greater the unsaturation the more fluid the
  membrane
• Cholesterol content
• the greater the cholesterol content, the more
  rigid the membrane

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The lipid bilayer is assymetrical

• The two faces of the bilayer consist of very
  different
• Phospholipids
• Glycolipids

Bonus Mark
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Assymetry is important for function

• Cytosolic proteins bind to specific phospholipid
  head groups
• e.g. Protein Kinase C in cell signalling
• Binds to the inner cytosolic head
• Requires a ve charge
• Cell Death
• Phosphotidylserine translocates to the
  outer surface
• Acts as a signal to induce macrophages to
  phagocytose cell

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Membrane Proteins

• Membrane proteins perform most of the functions
  of membranes
• - Myelin membrane (electrical insulation of
  nerve axon) lt25
• - Mitochondrial membrane (ATP synthesis) gt75
• Proteins can also have carbohydrate chains
  attached
• glycoproteins (glycocalyx)

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Protein linked
Transmembrane proteins
Lipid linked
Located entirely on the outside covalently
attached to lipid/glycolipids
Bound indirectly to either face of the membrane
by weak interactions
Extend through the bilayer consist of both
hydrophobic and hydrophilic domains
Integral protein Integral protein
peripheral protein
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Function of Membrane Proteins

• Transport e.g. glucose transporter protein
• Enzymes e.g. Adenylate Cyclase (plasma
  membrane bound enzyme)
• Receptor e.g. Insulin receptor
• Recognition cell-cell recognition, immune
  recognition
• Adhesion cell adhesion proteins
• linkage to
  the ECM

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Fluidity of Membrane Proteins

• Like membrane lipids, membrane proteins do not
  flip-flop
• Rotational diffusion
• Lateral diffusion

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Cells can confine lipids and proteins to
particular domains of the membranee.g. Gut
epithelium membrane proteins restricted to
particular domains of the plasma membrane
GUT LUMEN
Tight junction
protein A (transport of nutrients from the gut)
Apical membrane
Basal membrane
protein B (transport to tissues/bloodstream
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Traffick across Membranes

• The plasma membrane acts as a barrier to most
  hydrophilic molecules
• Permeable to
• Small non-polar molecules e.g. O2 and CO2
• Water molecules readily cross lipid bilayers
• Impermeable to
• Ions and charged molecules
• Specialised transport systems are required to
  transport ions, sugars, aminoacids, nucleotides
  etc.

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The traffick of small molecules

• Diffusion
• molecules to pass through the membrane directly
  e.g. H20, C02
• Facilitated transport
• membrane proteins transport down a concentration
  gradient
• E.g. Charged ions Na , Cl-
• E.g. Glucose transport (Glut-1 transport protein)
  in rbcs
• Active Transport
• membrane proteins requires energy (ATP)
• can transport up or down concentration gradients
• E.g. Na K pump

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The traffick of large molecules - Membrane
assisted transport
Endocytosis
Exocytosis
2. Fusion of vesicle with membrane
cytosol
1. Invagination of membrane 2. Fusion of
membrane to form vesicles
1. Vesicles bud off ER or Golgi
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e.g. insulin
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Cytosol

• Area outside organelles
• 50 of the cell content
• Metabolic processes take place
• Contains the cytoskeleton

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Molecular Organisation of cell membranes- Summary
1

• Membrane Lipids
• Phospholipids form the basic structure of
  membranes- lipid bilayer
• Amphiphatic
• Glycerol backbone covalently linked toa. two
  long, non-polar fatty acid hydrocarbon chainsb.
  variable phosphate-containing polar group
• Other lipids
• Cholesterol (provides rigidity) glycolipids
  (exterior of the cell only)
• Membrane Proteins
• Three types, Integral, transmembrane or lipid
  linked
• Function as enzymes, receptors, transporters, in
  communication and adhesion

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Molecular Organisation of cell membranes- Summary
-2

• Membrane Assymetry
• Phospholipids and proteins are asymmetrically
  distributed
• Glycolipids and glycoproteins are exclusively
  found on outer half of membrane
• Lipid mobility
• Lateral and rotational movement only within the
  plane of the membrane
• Cholesterol increases rigidity
• Greater number of unsaturated chains increases
  fluidity
• Greater number of shorter chains increases
  fluidity
• Protein mobility
• Lateral and rotational movement only within the
  plane of the membrane
• Movement of some proteins is restricted

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Molecular Organisation of cell membranes- Summary
-3

• Selective barrier function
• Transport of small molecules
• Passive transport (no ATP requirement)
• Diffusion
• Facilitated transport via membrane transport
  proteins
• Active transport (ATP required)
• Membrane transport proteins
• Transport of large molecules (ATP required)
• Endocytosis/phagocytosis
• Exocytosis
• Pinocytosis

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2. Intracellular organelles and protein
trafficking
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Nucleolus
Lysosomes
Nuclear membrane
Rough ER
Mitochondria
Ribosomes
Golgi vesicle
Golgi apparatus
Smooth Endoplasmic Reticulum
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Plasma membrane
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Protein trafficking

• The process of directing each newly made
  polypeptide to a particular destination
• Most proteins are encoded by nuclear DNA, and
  synthesized on ribosomes in the cytosol
• Distributed to their correct destinations via the
  action of several sorting signals

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Signal sequences

• Signal or sorting sequences direct delivery of
  proteins to specific organelles
• Many have no sorting signal and remain in cytosol
• Receptor proteins on the organelle surface
  recognize specific signal/sorting sequences in
  the new proteins

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• Cellular Organelle 1 Nucleus
• Present in all cells of the body
• exception erythrocytes
• Contains DNA
• Site of mRNA synthesis
• Enclosed by two membranes

Nuclear pore
Nuclear membrane
nucleolus
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Nuclear pore
Nuclear membrane
nucleolus
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• Nucleus sub-organelle Nucleolus
• Site of synthesis of ribosomal RNAs (rRNAs)
• rRNAs complex with proteins to form ribosomes

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Examples of macromolecules that traffick between
the nucleus and cytosol
Nucleus
RNA polymerases DNA polymerases Histones
mRNAs Ribosomal proteins
Cytosol
Nuclear Localisation Signal directs proteins
from the cytosol to the nucleus
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• Cellular Organelle 2 Ribosomes
• Ribosomes are the sites of protein synthesis
• Consist of large complex of proteins and
  ribosomal RNAs (rRNAs)
• Eukaryotic ribosomes consist of 2 subunits
• 40S and 60S (complete ribosome 80S)
• Can be bound ribosomes (endoplasmic reticulum)
  or free ribosomes (cytosol)

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Cellular Organelle 3 Endoplasmic reticulum (ER)

• Network of membrane-enclosed tubules and sacs
  (cisternae) that extends from the nuclear
  membrane throughout the cytoplasm
• Rough ER - covered by ribosomes on its outer
  surface
• Protein synthesis
• Smooth ER is not associated with ribosomes
• involved in lipid, metabolism

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Structure of the Rough Endoplasmic Reticulum (ER)
ribosomes
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Protein synthesis on the Rough Endoplasmic
Reticulum

• All protein synthesis starts on free ribosomes
• If the growing protein has a certain ER signal
  sequence" the ribosome attaches to the ER
• As polypeptide chain grows it passes through the
  ER membrane into the lumen
• Newly synthesised proteins
• accumulate in the lumen or
• Are embedded in the ER membrane

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In the RER proteins undergo post translation
modifications

• Formation of disulfide bonds
• Proper folding
• Addition and processing of carbohydrates
• Specific proteolytic cleavages
• Assembly into multimeric proteins
• occur exclusively in the RER

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Post translational modification - Proper folding

• Chaperone Proteins direct the folding of proteins
  into their proper three-dimensional structure
• Main proteins are heat shock proteins, hsp60 and
  hsp70
• Uses an ATP dependent mechanism that is poorly
  understood
• Within the Golgi, protein undergo further
  modifications
• Addition of carbohydrate groups
• Proteolytic cleavage

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Misfolded proteins

• Misfolded proteins in the ER are exported into
  the cytoplasm and degraded
• Degradation takes place within a multiprotein
  protease complex called the proteosome

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Functions of the Smooth Endoplasmic Reticulum

• Detoxification of drugs/toxins
• Synthesis of lipids and carbohydrates

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Structure of Mitochondria
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Cellular Organelle 5 Mitochondria

• Consists of two separate membranes (inner outer
  membrane) and an inter-membrane space
• The inner mitochondrial membrane is highly
  convoluted -cisternae
• Harness energy from the oxidation of food
  molecules to produce energy (ATP)
• Many metabolic process
• oxidative phosphorylation
• electron transport

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Traffick of proteins into mitochondria

• Mitochondria have their own DNA
• Most mitochondrial proteins are encoded by
  nuclear DNA
• Synthesised of free ribosomes
• Proteins have a specific signal sequence that
  allows entry to mitochondria
• Proteins are unfolded and transported across
  membrane
• Chaperone proteins assist to help the protein
  refold
• Requires energy

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Intracellular Organelles and Protein Trafficking-
Summary 1

• Nucleus
• Contains DNA
• Contains nucleolus- synthesises ribosomal RNAs
  and ribosomes
• Endoplasmic Reticulum Ribosomes
• Synthesise proteins for secretion plasma
  membrane
• Proteins destined for other organelles incl.
  Golgi and lysosomes
• Rough Endoplasmic Reticulum
• Performs post translation modifications of
  proteins
• Packages proteins in vesicles and delivers
  proteins to Golgi complex

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Intracellular Organelles and Protein Trafficking-
Summary 2

• Cytosolic ribosomes
• Synthesise cytosolic proteins
• Proteins for other organelles incl. nucleus,
  mitochondria, peroxisomes
• Smooth Endoplasmic reticulum
• Synthesises lipids and detoxification function
• Mitochondria- Powerhouse of the cell (ATP)
• Signal/ Sorting sequences
• Specific aminoacid sequences present within
  proteins
• Direct proteins to organelles within the cell

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3. The Mammalian Cell Cycle
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Mammalian Cell Cycle

• Cells grow by
• 1. Replicating DNA (DNA synthesis) S phase
• 2. Cell Division (Mitosis) M phase
• Cycle of duplication and division is termed The
  Cell Cycle

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The mammalian cell cycle consists of 4 phases
M phase (Mitosis) 1 hour S phase (DNA
synthesis) 8 hours G1 (Gap phase) variable G2
(Gap Phase) 2 hours Interphase G1, S, G2
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Rapidly dividing cells
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Cells divide at different rates

• Some cells never divide
• Nerve, lens and cardiac muscle cells
• Most cells are non dividing but can divide to
  replace dead /injured cells
• Skin fibroblasts
• Smooth muscle cells
• Endothelial cells
• Some cells divide rapidly
• Epithelial skin and intestinal cells

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Non dividing cells are in the G0 phase of the
cell cycle
G0

• G0 cells are termed quiescent
• e.g. -Neurons and skeletal muscle cells, are in a
  terminally
• differentiated G0 state

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Properties of G0

• Non-dividing state
• Metabolically and functionally active state
• Permanent e.g. neurons
• Temporary e.g. Liver cells, lymphocytes
• Most adult cells are in G0

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Regulation of the cell cycle by extracellular
signals

• 1. Mitogens
• a. Growth factors
• platelet derived growth factor
• Insulin growth factor
• Epidermal growth factor
• b. Hormones
• Growth hormone
• Estrogen
• c. Cell cell interactions
• Cells are only responsive to mitogens in G1

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Regulation of the cell cycle by extracellular
signals
2. Nutrient supply and cell size
M (mitosis)
G2
CELL CYCLE
S (DNA synthesis)
G1
Once cell passes R, cell is committed to at
least one cycle Controlled by the availability
of nutrients, cell size If nutrient supply is
low cell enters G0
R point
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Control of progression through the cell cycle

• Progress through the cell cycle is controlled by
  protein kinases
• Progress is monitored at various checkpoints

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Control of the cell cycle- the players

• 1. Cyclin dependant kinases (CDK)
• Enzymes that cause phosphorylation
• Each phase of the cell cycle has a specific CDK
  which is activated
• Present in an inactive state in cells
• CDKs on their own are not active
• Require a specific protein termed a cyclin to
  be active

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Control of the cell cycle- the players

• 2. Cyclin proteins
• Cyclins are synthesised and degraded in a phase
  specific manner
• G1 specific
• G2 specific
• S phase specific
• When synthesised, cyclins complex with and
  activate CDKs

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Control of the cell cycle- the players

• 3. Active CDK holoenzyme (Cdk and kinase)
• Causes the phosphorylation of threonine and
  serine residues of target proteins
• Transfer of the terminal phosphate of ATP to a
  hydroxyl group of specific
• aminoacids

ATP
ADP
kinase
Pi
P
OH
ATP
phosphatase
ADP
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Control of the cell cycle - the players

• 3. Cyclin dependant kinase Inhibitors
• Inhibit the action of CDKs-cyclin complexes
• e.g. p21 protein

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Activity of mammalian Cdk-cyclin complexes
through the course of the cell cycle
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Cyclin and CDK complexes regulate G1-S
transcription Restriction point control

• Cyclin D is the first cyclin synthesised in
  response to mitogens
• Cyclin D complexes with CDK-4/CDK-6
• CDK holoenzyme phosphorylates the retinoblastoma
  protein (pRb)
• pRb is the molecular device that serves as the R
  point switch
• First identified tumour suppressor protein

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Retinoblastoma protein
E2F
E2F Transcription factor
Unphosphorylated pRB is inhibitory Associates
with E2F protein
CDK 4/6
Cyclin D
E2F
Active CDK complex
P
Retinoblastoma protein
P
phosphorylated pRB is stimulatory E2F protein
dissociates
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Retinoblastoma protein
E2F
E2F Transcription factor
Unphosphorylated pRB is inhibitory Associates
with E2F protein
CDK 4/6
Cyclin D
Initiates transcription of Genes involved
in Cell cycle progression
E2F
Active CDK complex
P
Retinoblastoma protein
G1 S
P
phosphorylated pRB is stimulatory E2F protein
dissociates
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Cell cycle checkpoints- intrinsic control

• G1 checkpoint
• Recognises DNA damage
• G2 checkpoint
• Recognises if DNA has not been replicated
• M checkpoint
• Monitors alignment of chromosomes on mitosis
  spindle
• Ensures complete set of chromosomes are
  distrubuted accurately

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Cell cycle checkpoints-DNA damage checkpoint in G1

• If damaged DNA is replicated then daughter cells
  will also contain damaged DNA
• Cells with damaged DNA are genetically unstable
• Cells must be able to monitor for the presence of
  DNA damage
• Repair the damage or kill the cell
• Prevent daughter cells from acquiring the damage

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DNA Damage
Cell cycle arrest
Is the damage repairable? DNA repair mechanisms?
P53 can activate transcription
Stabilisation of p53 protein
Apoptosis/cell death
Damage is to not repairable Cell must die!
G1 DNA damage Checkpoint role of the p53 tumour
suppressor protein
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DNA Damage
P53 can activate P21 CDK inhibitor
P21 CdK inhibitor
G1 S
Stabilisation of p53 protein
S phase CDK complex
S phase CDK complex
active
inactive
G1 DNA damage Checkpoint activation of CDK
inhibitor p21
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Loss of cell cycle control and cancer

• Hallmark of cancer is abnormal unregulated cell
  proliferation
• Loss of cell cycle checkpoints allows for
  uncontrolled cell growth
• p53 function is lost in over 50 of all cancers
• pRB function is also commonly lost in cancers
• CDKs, cyclins and CDK inhibitors can be
  deregulated

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Cellular Senescence

• Normal cells do not divide indefinitely
• Normal cells exhibit a limited replicative
  lifespan after which they enter senescence
• Irreversible arrest of cell growth
• Differs from G0

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Cell Cycle Summary-1

• Four Phases, G1, M, G2, S
• Fifth phase G0 (non-dividing state but
  functionally and metabolically active)
• Control is tightly regulated
• Cyclins are synthesised in a phase specific
  manner
• CDKs are present in the cell at all times
• CDKs complex with cyclins to form active
  holoenzyme
• Extrinsic regulation- R point- mediate by pRb
• Controls entry from G1 to S phase, depends of
  external factors (cell size, nutrients)
• Intrinsic regulation p53 DNA damage checkpoint
• prevents entry from G1 to S phase
• Commits the cells to exit in G0-repair the damage
  or enter cell death
• Other checkpoints- G2 Mitosis

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Cell Cycle Summary -2

• Senescence
• Aged cells that are permanently exit the cell
  cycle
• Controlled by telomere DNA
• Some cell types overcome senescence by activating
  telomerase
• Abnormalities in cell cycle regulation result in
  cancer
• Loss of checkpoint controls pRb and p53
• Cancer cells can overcome senescence by
  activating telomerase

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4. Cell death
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Necrosis

• Pathological response to injury
• Mechanical damage
• Exposure to toxic chemicals
• Hypoxia
• Ischemia
• Accidental form of cell death

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Cell Death
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Necrotic cells undergo a characteristic series of
changes

• Chromatin clumps
• Mitochondria swell and rupture
• Plasma membrane ruptures
• Cell contents spill out
• Affects neighbouring cells
• Stimulates an inflammatory response

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Cell Death
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Cell Death
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Apoptosis

• Programmed cell death or Cell Suicide
• Apoptosis is a normal physiological process
• Apoptosis is essential for development
• formation of the fingers and toes of the fetus
• sloughing off of the inner lining of the uterus
  at the start of menstruation

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Cell Death
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Apoptosis

• Apoptosis is needed to destroy cells that
  represent a threat to the integrity of the
  organism
• Cells with damaged DNA
• Cells infected with viruses

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Cell Death
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Apoptosis role of caspases

• Proteolytic enzymes stored as inactive zymogens
• Induce apoptosis via cell surface receptors
• Results in cleavage of key cellular substrates
• Activate other degradative enzymes
• Proteases, DNases, RNases
• Results in distinct morphological changes

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Cell Death
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Apoptotic cells undergo a characteristic series
of changes

• Chromatin condenses
• DNA fragments
• Cytoplasm shrinks and membrane blebs
• Contents are packed into membrane bound
  structures termed apoptotic bodies
• The phospholipid phosphatidylserine is exposed on
  the surface
• Receptors on phagocytic cells (macrophages) then
  engulf the cell fragments

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Cell Death
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Cell Death
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Apoptotic cells Note how the membrane blebs and
shrinks and cells round up
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Cell Death
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Apoptosis and disease

• 1. Reduced Apoptosis
• Cancers
• Viral Infections
• Some viruses can degrade p53 function
• E.g. Human Papillomavirus type 16 causative agent
  of Cervical Cancer

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Cell Death
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Apoptosis and disease

• 2. Increased Apoptosis
• Neurodegenerative diseases
• Alzheimers Disease
• Parkinsons Disease

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Cell Death
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Cell Death Summary

• Necrosis
• Pathological
• Cell swelling
• Membrane integrity lost
• Leak of cell contents
• No apoptotic bodies
• No DNA cleavage
• Dead cells not injested
• Inflammatory response
• Not regulated
• Caspases are not activated

• Apoptosis
• Physiological or pathological
• Cell shrinkage
• Membrane integrity maintained
• No leak of cell contents
• Apoptotic bodies
• DNA cleavage
• Dead cells injested
• No inflammatory response
• Regulated process
• Activates caspases

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Cell Death