Download Lecture 2: Cellular signalling and cell division

1. Basic Cell Biology, major cellular functions: cell division, cellular
differentiation and cell death. History of Physiological Vs
pathological cell death.
Chemistry of life
Eukaryotic cells Vs Prokaryotic cells
Compartmentalization for better regulations of gene expression and
other complex biological reactions
Cell structure
Cell types
Major cellular components:
2. Cellular signaling and cell division
3. Cellular Differentiation and development, Cell death: history of progress
in cell death research, necrosis vs. apoptosis.
Basic Cell Biology, major cellular functions: cell division, cellular
differentiation and cell death. History of Physiological Vs pathological
cell death.
Chemistry of life
Eukaryotic cells Vs Prokaryotic cells
Compartmentalization for better regulations of gene expression and
other complex biological reactions
Cell structure
Cell types
Major cellular components:
Plasma membrane
An asymmetrical Lipid bi-layer
Phasphatidylethenolamine, phasphatidylserine, phasphatidylcholine,
sphingomylein, membrane proteins, glycolipids, glycoproteins
Cholesterol (important for provide structural rigidity to membrane)
Membrane proteins: Receptor proteins, ion channels and transport proteins
Functions: as barrier between cytoplasm and extra-cellular environment
Controlled transport of chemicals, ions and macromolecules
Exocytosis and endocytosis
Signal transduction
Generation of action potential
Endoplasmic reticulum:
Lipid bilayer forming a continuous sheet enclosing a single space called ER
lumen or cisternal space
Rough and smooth ER
Functions:
Protein synthesis
Post-translational modification (glycosylation
Glycolipd synthesis
Vesicular Protein transport and secretion
Detoxification (Smooth ER contain cytochrome P450), water insoluble toxic
compounds are converted into excretable non-toxic soluble compounds
Ca2++ sequestering: Examples- muscle cells (ER is called sarcoplamic
reticulum), nerve cells.
Golgi bodies:
Membrane bound flattened sacs stacked over each other. Functionally
distinct parts (cis and trans parts)
Contains protein modification enzymes e.g. glycosyl transferase, nucleoside
diphosphatase and acid phosphatase
Functions:
Posttranslational modifications (glycosylation, dephosphorylation,
phosphorylation)
Involved in sorting and packaging macromolecule for secretion or for delivery
to other organelles.
Proteins destined for delivery to lysosomes are labelled with mannose-6phospate in Golgi bodies. Defect in this process results in lysosomes without
hydrolytic enzymes and secretion of these enzyme in I-cell disease or
inclusion cell disease).
Lysosomes:
Membrane-bound vesicles containing hydrolytic enzymes
Function:
Involved in intracellular digestion
Peroxisomes:
Membrane-bound vesicles containing oxidative enzymes such as catalase
and urate oxidase, and long chain fatty acid oxidation during which there is
production of hydrogen peroxide.
Functions:
Vestige of an ancient organelle that carried out oxidative reactions
May play role in anti-oxidative defence
Mitochondria:
Double membrane-bound structures, power plants of eukaryotic cells
Site for citric acid cycle and oxidative phosphorylation and fatty acid
metabolism in energy generating biochemical pathway
Outer mitochondrial membrane
Inter-membrane space
Inner mitochondrial membrane (contains components of electron transport
chain, ATP synthase, and transport proteins)
Matrix space (contains enzymes and cofactors for citric acid cycle, fatty acid
metabolism, mitochondrial genome, and transcription and translation
machinery)
Inner mitochondrial potential is generated and maintained by proton export
out side
ATP is synthesis is driven by proton flow towards inside
Protein import into mitochondria
Nucleus:
Nuclear envelope
Chromatin
Replication, transcription
Protein import and RNP export across nuclear envelope
Lecture 2: Cellular signalling and cell division
Cellular signalling:
Evolution of social behaviour in cells
Cell to cell communication and responses are essential for the organism as
whole.
Different types of cell signalling: synaptic
Endocrine
Paracrine
Autocrine
Cell to cell signalling by direct contacts: a) via receptors b) via gap junctions
and plasmadesmata
Extra-cellular signals: Hormones
Cytokines
Growth factors
Signalling Mechanisms:
1. Receptor enzyme mediated
2. G-protein linked Receptor mediated
3. Ion-channel-linked Receptor mediated
4. Intra-cellular Receptor mediated
Receptor enzyme mediated:
Receptor tyrosine kinases
Examples- receptors for most of growth factors e.g. FGF, PDGF, Insulin, IGF1, CSF
Binding of ligand to RTK----activation of TK activity------autophosphorylation----binding of GTPase activating protein or PI3 kinase or phospholipase---Activation of PKC and/or Ras---activation of MAPKKK----activation of
MAPKK---Activation of MAPK----c-Jun---activation of transcription
Receptor serine/threonine kinases:
Examples- TGF-family of receptors
G-protein linked receptor mediated Signalling:
Examples- adrenalin,Calcitonin, oxytosin, acatylcholine, dopamine, histamine,
PTH, retinal, serotonin etc.
Binding to receptor -----conformational change in G-protein complex-----GGTP formation-----activation of adnylate cyclase-----production of cAMP--activation of protein kinase A (camp dependent kinase)-----
Ion channel-linked Receptors:
Neurotransmitter receptors
NMDA receptors, serotonin, acetylcholine receptors etc
Binding----opening of ion channel------influx of Na, or K or Ca ions---downstream events
Drugs: barbiturates, antidepressants used a blockers
Intra-cellular Receptors
Mostly steroid hormone receptors: Cortisol, estrogen, progesterone,
thyroid hormone, retinoic acid, vitaminD
Binding to receptor---- exposure of DNA-binding site, and NLS---activation of transcription
NO signalling:
Recently discovered as signalling molecule.
Diffuses through plasma membrane rapidly and binds to Gunylate cylase.
Binding to heme group of gunylate cyclase
Activation
production of
cGMP
activation of downstream enzymes
Causes muscle relaxation, involved in release of neurotransmitters, at
high level neurotoxic.
Cell division: The fundamental principle of life
Interphase: cells look normal and just grow in size as observed under
microscope. But many molecular events take place in this phase and this phase
further subdivided into following subdivisions:
G1 phase: Starts after completion of mitosis and ends before beginning of
DNA synthesis. Important decision to start cell cycle is made in the late G1
phase.
S phase: This phase is marked by the start and completion of DNA
replication.
G2 phase: Cells prepare for cytoplasmic events for cell division and make
sure that there are appropriate conditions before entering the M phase.
Mitotic phase: Visible changes in the cells
Check points in cell division cycle
Cell cycle control: M-phase promoting factor – a complex of ser/thr kinase
called cyclin-dependent protein kinase Cdk-2 or cdc2 and a mitotic cyclin
Synthesis and destruction of cyclins control the activity of MPF.
Re-replication block
Growth factors trigger cascade of intracellular signals
Growth factor binding---activation of TK-----activation of MAPKKK-------transcription of myc gene-----transcription of Cdk and cyclins
Mitosis
Prophase
Metaphase
Anaphase
Telophase
Cytokinasis
Remember that DNA replication takes place in prophase and DNA is packed into
a condensed form called chromosomes. Each chromosome has two chromatids,
and during metaphase and telophase, each chromatid separates and daughter
cells have the same number of chromosomes.
Meosis:
Prophase I---subdivided in to 5 stages
Leptotene---Chromatids are formed, Chromosomes look like treads
Zygotene----Homozygous chromosome come close forming pairs
Pachetene---further condensation of chromosomes
Diplotene---Completion of pairing of chromosomes (completely aligned
chromosomes)
Dikinasis---chromosomes separate, and chromatids are attached at the places
of crossing over or chiasma. (chromosomes look like garlands)
Metaphase I
Anaphase I
(separation of chromosomes and not the chromatids)
Telophase I
Meotic division II: same as usual mitosis
Lecture-3/4
Cellular Differentiation and development, Cell death: history of progress in cell
death research, necrosis vs apoptosis.
Control of gene expression:
All the cells in an organism contain the same genetic information. Cells differ
from each other not because they contain different genes but because they
express different genes.
Different steps of control of gene expression:
Transcriptional control
RNA processing control
RNA transport control
Translation control
MRNA degradation control
Protein activity control
Molecular regulators of gene expression at transcriptional level
Transcription activator
Special class of DNA binding proteins
Transcription repressors
Four common structural motifs:
Helix turn helix motif: Two  helices connected by a short extended chain of amino acids which makes the
turn.
Zn-finger motif: Consists of an -helix and a -sheet held together by Zn (coordination bond with four amino acid side chains)
Lucine Zipper motif: Two -helices (one from each monomer) joined to gather
to form a short coiled coil by hydrophobic interaction of hydrophobibic amino acid
side chains (mainly lucine) of two helices.
Helix loop helix motif: A short -helix connected by a loop to another long helix (loop provide flexibility of the two helices)
These proteins generally have two domains: one which binds to specific DNA
sequence and other which alters the activity of RNA polymerase (or transcription
assembly)
Example of prokaryotic genetic switch:
lac-operon: first gene in this operon encodes -galactosidase, an enzyme
required for digestion of lactose producing glucoase and galactose
Glucose
cAMP
inactive CAP ------no transcription
No glucose------low camp-------Active CAP----Gene is transcribed if lactose is
available in medium (lac repressor is not bound)
Lactose-----allolactose------binds to repressor protein------release of repressor
from DNA -----transcription is allowed if CAP is bound
Eukaryotic Transcription is complex: requires many transcription factors in
addition to RNA polymerase II
Control of gene expression during development:
In amphibian development early signals for differentiation are due to the
asymmetry of the zygote.
Transplantation experiment showed the dorsal lip of the blastopore initiates
gastrulation movement which organizes the body plan.
For example Vg1 mRNA is synthesized in the oocyte and become localized in
vegetal cortical region of egg. An injection of mRNA coding the active Vg1
protein in the ventral blastomere can induce an entire body axis.
Morphogens: the biochemical species responsible for differentian
The gradient of morphogen can organize complex pattern as cells respond
differently to different concentrations of morphogen
Mammalian Development: in protected uterine environment
Nutrient provided by host through placenta
The embryo proper is derived from inner cell mass
The trophectoderm is the precursor for placenta
Totipotent cells: cells of the early mammalian embryo (up to eight cell stage)
are identical and unrestricted in their capabilities. Capable of developing in
normal animal.
Mammalian embryonic stem cells: Cells of the inner cell mass can be
dispersed and grown in culture under appropriate condition. They divide
indefinitely without any change in character. They are used in making transgenic
animal as well gene-knockout transgenic mutants.
Caenorhabditis elegans:
Very simple multi-cellular organism
Transparent body, every cell has been studied in terms of its origin during
development and its function.
Total 1090 somatic cells
131 cell die by apoptosis during development
14 genes have been identified, which affect the programmed cell death when
mutated.
They are called Ced-1, Ced-2, Ced-3, Ced-4------Ced-14. CED stands for cell
death defective.
Mammalian Homologues of Ced-3, Ced-4 and Ced-9 have been discovered.
These three genes have been studied extensively and they play critically
important role in apoptotic cell death.
Ced-3= ICE or Caspase-1
Ced-4= APAF-1
Ced-9= Bcl-2
Cell death:
Pathological or necrotic cell death
Programmed cell death or Apoptosis
Initial experiments by Dr Kerr
Liver ischemia
Morphology: shrinking cells, membrane intactness, chromatin condensation
Apoptotic bodies
Change in lipid composition of plasma membrane (flipping of negatively charged
phosphoserine or negatively charged lipids from inner leaflet to outer leaflet).
Increase in transglutaminase activity-leading to cross-linking of proteins and
lipids
Necrosis: pathological, accidental and traumatic: cell swelling, membrane
breakage, release of lysosomal enzymes in the vicinity, causing inflammation,
damaging the nebouring cells.
Biochemical Feature:
DNA fragmentation: In apoptosis –oligonucleosomal fragmentation, leading to
DNA ladder
In necrosis: no damage or random fragmentation leading to smear
Apoptosis during development: metamorphosis, development of digits,
massive death of neurons, eye development
Apoptosis in adult tissue: Intestine, skin, thymocytes, uterus cells, infected,
transformed or damaged cells
Lecture5/6
Apoptotic cell death during developmental and during adult tissue
homeostasis:
A. Development: Genetic basis of cell death in C. elegens development,
examples of PCD in mammalian neuronal development, and inter-digital cell
death during development and metamorphosis.
B. Tissue homeostasis: Cellular homeostasis in blood cells, immune system,
epithelial
cells, hormone dependent changes in tissue architecture such as
those in breast and uterine tissues.
C. Physiological cell death as safeguard against growth of cells carrying
mutations (potentially capable of becoming cancerous)
Example of programmed cell death during development:
a) C.elegens development: ced-3 and ced-4 genes encode pro-apoptotic
proteins. These proteins are required for cell death. Mutation in these
genes led to the prevention of death of 131 cells of the c. elegeans and
the worm developed unusually but survived. But if the ced-9 gene was
defective, all the cells died and so did the embryo. The ced-9 encodes an
anti-apoptotic protein. It is required in the other cells to prevent cell death
(Ellis R E and Hoevitz (1986) Cell, 44: 817-819, Yuan, J and Horvitz, H. R.
(1990) Dev. Biol. 138: 33-41).
b) In vertebrates:
Metamorphosis: Cells in the tadpole tail are triggered to die by thyroid
hormone secreted by thyroid gland. Similarly the intestine of tadpole is totally
degenerated by apoptosis induced by Thyroid hormone. Apoptosis can be
induced by adding to the epithelial cultures from tadpole. TH responsive
gene expression----matrix metalloproteases were expressed in response to
TH hormone (Patterton et al. (1995) Devel. Biol. 167:252-262).
Neuronal cell death during development (Johnson EM and Deckwerth TL
(1993) Annual Rev. Neurosci. 16:31-46).
Peptide inhibitors of caspase-1 arrested apoptosis of motor neurons in vivo
(Milligan et al, 1995, Neuron, 15 : 385-393)
Transgenic caspase-3 knockout mice, died post-natally (Kuida et al (1996)
Nature, 384: 368-372.
Large scale astrocyte death in developing cerebellum
More than 50% oligodendrocytes die during developing rat optic nerves.
Culture of oligodendrites require factors secreted by optic nerve cells. They
could be rescued by PDGE or IGF-1(Barres et al 1992, Cell 70:31-46).
Other culture models: Differentiation of PC-12 cells into neuronal cells by
NGF. The differentiated cells depend on NGF for survival. Removal of NGF
triggers apoptosis in these cells.
Tissue homeostasis:
Requirement of survival signals: Prostrate cells require testosterone to
survive.
Cells in adrenal cortex depend on ACTH secreted by pituitary gland.
Interlukin-2 dependent T- lymphoblast cells require IL-2 for survival
Endothelial cells require fibroblast growth factors (FGF) for survival
Game of cell proliferation and cell death in Immune system:
T lymphocytes: Responsible for cell mediated immunity
Cytotoxic T cells or killer T cells
Helper T cells: produce IL-2, and activate other lymphocytes
B lymphocytes: Mount antibody response against antigens
Lymphocyte maturation in Thymus
Cells expressing no receptor and cells expressing high affinity to self antigens
are induced to undergo apoptosis
Cells expressing receptors for foreign antigen and low affinity for self antigens
survive and are released in circulation- resting T cells
When they encounter antigen ----they get actvated-----proliferate, synthesize
lymphokines, produce perforin and grazyme B as well as non-functional Fas
receptor and ligand
This leads to removal of antigen
After the antigen is gone---now unnecessary cells are induced to undergo
apoptosis by 1. Deprivation of IL-2 survival signal or 2. by making functional
fas receptor and ligand, and inducing cell death