Midterm Flashcards
Central dogma of life
DNA->RNA->Protein
Cells are the simplest structure that can support the
process of the genetic information flows from DNA to
RNA (transcription) and from RNA to protein
(translation)
Viruses rely on the cell to reproduce themselves and
thus they are not considered living organisms
Model organisms of cell biology
• E.coli perfect for studies of DNA replication, transcription, translation
• Yeast Saccharomyces cerevisiae – simple model of the eukaryotic
cell
• Drosophila melanogaster developmental biology and genetics
• Snails, zebrafish – neuroscience
• Mice, rats, pigs etc…
Cells biology scale
<1 micron to ~100 microns
Light Microscopy
Advantages: simple and the
least invasive.
Disadvantage: can mostly be used for morphological studies, can tell what kind of processes are happening inside the cell
Transmission Electron microscopy
Advantage: highest resolution possible, broad range;
Disadvantage: only fixed cells, takes a long time to prepare sample, possible artifacts of preparation
Fluorescence microscopy
The most versatile and widely used method. Can be applied to both live and fixed cells. Allows to investigate structure as well as biological activity of the cells.
Cell electrophysiology
Direct measurement of the electrical activity of the cell.
Particularly useful for studies of excitable cells: Neurons, Cardiac, Muscle cells.
Can measure: membrane potential; integral cell ion current; single-channel ion current.
Respirometry
Real time measurement of cell energy
metabolism by measuring consumption of the
oxygen and production of the carbon dioxide.
Allows to measure: rates of ATP production;
energetics capacity of the cell; rates of glucose and
fatty acids metabolism; overall mitochondrial health
and fitness.
Role of Cell Membrane
Barrier between the cell and the environment or cell organelles
- Maintain Ionic and molecular homeostasis
- Signal transduction
- Energy production
- Cell growth and motility
- Molecular synthesis
Different membranes of the cell
• Plasma (cell) membrane: signal transduction; cell
movement; import/export of small molecules
• Nucleus: storage of DNA material
• Mitochondria: energy production; cell death; calcium
signalling
• Endoplasmic reticulum: protein synthesis; calcium
signalling
• Golgi: protein and membrane assembly and trafficking
• Peroxisome, lysosome, vesicles:
compartmentalization
Fluid Mosaic Model
Fluid lipid bilayer forms a matrix dependent on the amphipathic nature of lipids
• Lipids can diffuse laterally and rotate
• Proteins are either peripheral or integral and may diffuse laterally and rotate
accounted for:
• Varied protein (20-75%) & lipid content
• Asymmetry of both protein & lipid (head size)
• Rapid diffusion of proteins in membrane (modulated by cholesterol which increases packing to decrease fluidity)
• High electrical resistance
• Impermeability of polar substances
Membrane Lipids
Amphipathic: has both hydrophobic &
hydrophilic regions, as in phospholipids or detergents
• Spontaneously form micelles or bilayers & reseal
• 109 molecules/small cell
– Phospholipids: 14-24 C tail; asymmetrical; unsaturated
(C=C) increase fluidity by decreasing packing
– Cholesterol: modifies fluidity by increasing packing
– Glycolipids: only on external face
• Asymmetrical with little flip-flop across bilayer
• Primary diffusion barrier
• High electrical resistance: 106 Ω/cm2
MOST ENERGETICALLY STABLE IN SPHERICAL FORM - NOT PLANAR
Phosphatidylcholine
most common phospholipid in cell membranes
Lipid Classification
Glycerolipids – based on glycerol:
- Phosphatidylcholine (PC), - Phosphatidylethanolamine (PE) - Phosphatidylserine (PS) - Phosphatidylinositol (PI)
Sphingolipids – based of sphingosine (can be phospholipids, but not only, can be glycolipids)
-Sphingomyelin
Glycosphingolipids (contain sugar)
Sterols
-Cholesterol
Unique for mitochondria – cardiolipin (4 tails)
Lipid Bilayer Movements
- Lateral diffusion
- Flexion
- Rotation
- Flip-flop (rare)
Increases to Membrane Fluidity
1) INCREASE in unsaturation of lipids
2) DECREASE in lipid chain length
3) DECREASE in Cholesterol
4) INCREASE in Protein Content
Asymmetric lipid distribution in the membranes
Phospholipids and glycolipids are distributed asymmetrically in the plasma membrane
bilayer
Hexagonal head groups are sugars on glycolipids, which are restricted to the outer leaflet.
Cholesterol is distributed almost equally in both monolayers.
Scramblase – random transfer of lipids
Flippase – specific transfer of lipids
Membrane Lipid Rafts
Induced by Cholesterol
a lateral segregation in a lipid mixture
• a more ordered structure
• helps to segregate proteins lateral distribution
Membrane proteins perform several different functions
Transporters
Anchors
Enzymes
Receptors
Cell Cortex
Formed by Spectrin & actin
Provide mechanical strength for plasma membrane
Allows cells to change actively shape and to move
Restrict the diffusion of proteins within the membrane
Glycocalyx
•Many extracellular membrane proteins & lipids have sugars (Glycocalyx) that protect and lubricate surfaces & involved in cell–cell recognition.
Membrane Permeability
Only permeable to SMALL, NONPOLAR molecules (such as oxygen, CO2 etc.) but most other molecules
require participation of the specialized transport systems
General classification of the transport mechanisms
Simple diffusion – does not require any protein – just going through the lipid bilayer
Channel – the protein which forms the pore, that allows free (but selective!) flow of the molecule(s)
Transporter – protein which takes the molecule on the one side and releases on the other (never opens as a “pore”)
ACTIVE transporter – can carry molecules against the concentration and/or electrical gradient.
Transport Mechanisms
Uniport – unidirectional transport of specific molecule (ion)
Symport – co-transport of two different molecules
Antiport – two molecules are transported in the opposite directions
Active Transporters (also called “Pumps”)
Coupled pump uses the energy gradient of the “coupled” molecule
-
ATP driven – uses energy released during the hydrolysis of ATP into ADP and phosphate*
- ATP driven pump is responsible for establishing ion gradients in excitable cells such as Na+ and K+ or Ca+
Light driven – direct use of light energy
Ion Channels
passive selective transport of ions driven
by electrochemical gradient
Ion channels are integral proteins in the lipid bilayer, can be open or closed.
Open channel is characterized by specific selectivity.
Channels can be classified by selectivity (what ion they allow to go through) and gating (what makes them
transfer from open to closed state and back).
EXAMPLE IS NEURAL COMMUNICATION
Ion channels – general classification
• By gating
Ligand gated
Voltage gated
Mechanosensitive
• By selectivity
Sodium
Potassium
Calcium
Chloride
Cationic
Anionic
Large pores (technically not “channels”)
Nernst equation
the force tending to drive an ion across a membrane is made up of two components:
one due to the electrical membrane potential and one due to the electrical membrane potential and one due to the concentration gradient of the ion.
At equilibrium, the two forces are balanced and satisfy a simple math relationship give by the Nerst Equation:
V = 62 log10 (Co/Ci)
V= membrane potential in milivolts Co = concentration outside Ci = concentration inside
Action Potential
Na conductance shows a fast,
transient increase in response
to depolarization
K conductance shows a slower,
more sustained increase
Functional studies of ion channels
can be performed using patch-clamp approach
Channel structure Investigation
using X-ray crystallography or cryoelectron microscopy
Targeted Protein synthesis
Ribosomes are targeted to the ER only
after protein signal (targeting) sequence
becomes synthesized.
SO…
There is a common pool of ribosomal subunits sitting in the cytosol, If there is no targeting sequence synthesized, protein synthesis will occur via the free ribosomal cycle. HOWEVER, if there is a targetting signal, the ribosomal subunits will bind to the rough ER for protein synthesis.
Coordinated with protein translocation - mainly on the
Ribosomes bound to rough ER
Soluble protein synthesis on ER
Following synthesis, the soluble proteins are released into the lumen of the ER
Signal peptide (signal sequence) is cleaved by signal peptidase and remains in the ER membrane
Membrane protein synthesis on ER
Following synthesis proteins remain in the ER membrane***
Stop transfer sequence (hydrophobic sequence) keeps protein imbedded in membrane, seperating lumen and cytoplasmic sides of the protein
Posttranslational protein modification in ER
Posttranslational modification – chemical
modification of the protein that is not coded
by DNA-RNA. (COVALENT BONDING OF DIFFERENT MOLECULES AFTER PROTEIN IS ALREADY MADE)
Many dozens exist and can
involve many amino acids and many types of
“attachments”
ex. Glycosylation – attachment of a sugar
residue like with the Glycocalyx
Golgi Apparatus
the main destination for the proteins
made in ER
Protein synthesized in ER, then they need to be processed in the golgi apparatus and get
there via vesicles. Golgi vesicles then take finished proteins for either exocytosis or endocytosis
Golgi apparatus structure
Golgi consists of many flatten membrane enclosed sacs: “cisternas” (3 to 20 cisternas in every stack of Golgi)
Has two sides: cis (entry side) and trans (exit side)
Cis – close to the ER; Trans – close to the plasma membrane
Golgi apparatus vesicular transport
Proteins entering ER (cis) are either moved
trough the stack or returned back to the ER (if
they have ER retention signal)
Proteins exiting from Golgi (trans) either go to
the lysosomes or cell surface
Golgi is the place where further sugar protein
modification occurs
SO…it can either undergo UNREGULATED exocytosis in which newly snythesized soluble proteins are transported via a transport vesicle made up of newly synthesized plasma mem. lipids/proteinsto the cell membrane for constitutive secretion
OR…
it can go through REGULATED exocytosis where the secretory where the proteins are transported into a SECRETORY vesicle - which stores the proteins until an extracellular signal (hormone or neurotransmitter) signals for the secretion of these proteins. thus, the signal transduction allows for the regulated secretion of the proteins out of the cell.
Lysosomes
Primary cites of intracellular digestion -
Includes acid hydrolases nucleases proteases glycosidases lipases phosphatases sulfatases phosolipases
The acidic environment of the lysosome is maintained by ATP dependent proton pump
There’s transporters on the membrane of lysosome that also transport metabolites out of the lysosome after the enzymes have broken them down.
o Nearly all of these enzymes in the lysosome are protected from being broken down themselves because they got lots of glycosyl modification; the
(mannose 6-phosphate) that protectthem from being
broken down by other lysosomal enzymes.
Lysosomal digestion pathways
Phagocytosis – digestion of
bacteria
Endocytosis – digestion of the
extracellular compounds
Autophagy – digestion of the
intracellular compounds
ER and Calcium Signaling
- ER plays a critical role in calcium signalling during muscle contraction
- ER in muscle is called SR (Sarcoplasmic Reticulum)
- SR is highly specialised to perform calcium pumping and release cycles
MUSCLE CONTRACTION CAN NOT PROCEED WITHOUT PARTICIPATION OF ER!!!!!!!!
Also, essential role of ER in neuronal calcium signalling
(TG-thapsigargin - drug that blocks calcium uptake in the ER)
Mitochondrial protein synthesis and import
• ~2000 proteins make mitochondrial
proteome
• Only 20-30 are coded by the mitochondrial
DNA
• Most of the mitochondrial proteins are
made in cytoplasm and imported into the
mitochondria
Mitochondria have their
own complete machinery for protein synthesis but only for vey few proteins
Mitochondrial DNA is maternally inherited:
it allows us to track different
inheritance because there is almost no DNA material mixing. You can track by mother inheritance and parental information in terms of origin of
different genomic lines.
Mitochondrial protein import machinery
The whole protein is made in the
cytoplasm and then it gets imported into mitochondria in an unfolded form through the protein translocator in the outer membrane and protein translocator in inner membrane.
• Proteins that are destined to the mitochondria generally have this signal sequence which can be recognized by this transporting protein. Then
can be translocated and unfolded and get into the matrix where they complete protein folding and the signal peptide gets cleaved and recycled.
• But what is not really well understood if you look at mitochondrial protein only a few proteins have this signal protein. By looking at the
protein we cannot determine if its mitochondrial or not. If we have a signal sequence we can but if it doesn’t, you cannot be for sure whether its
mitochondria targeted or not.
- proteins targeted to the (other than matrix) mitochondrial compartments have different targeting sequences
- many mitochondrial proteins do not have any obviously recognizable mito-targeting sequence.
Mitochondria (definition)
structures within cells that convert the energy from food into a form that cells can use” (NIH-2009)
- Mitochondria produce energy in form of ATP
- Mitochondria and calcium signaling
- Mitochondria – storage of pro-apoptotic molecules (they are key master regulator of cell death which is apoptosis.)
-Mitochondria – major source of ROS (Reaction Oxygen Species)
»_space;> Oxygen species are major signaling molecules that guide a lot of intracellular processes. Mitochondria are the place where most of these species are synthesized which can signal throughout the cell. Mitochondria are critical for that. Nobody knows how they do this.)
Diseases/Disorders/Conditions involving Mitochondria
• Diabetes Type II • Cardiomyopathy • Aging • Cancer • Parkinson’s Disease • Alzheimer's • Huntington’s
Cellular Energy Metabolism
Mitochondria are central in producing energy in eukaryotic cells especially in cells that rely on oxidative phosphorylation for energy.
• Two major pathways: glucose utilization and pathway for fatty acid utilization. Those are two types of food our cells can receive and process. A
majority of foods when it comes to the tissues come in the form of glucose or fat associated with proteins.
• One pathway goes from glucose, which is sugar, through pyruvate and then into the mitochondria. Then is the critic acid cycle (TCA) producing NADH and then end up in ETC which utilizes oxygen and eventually leads to the production of ATP
• The other pathway also eventually leads either to TCA, producing NADH or directly producing NADH and then to electron transport chain and again the
synthesis of ATP.
KEY METABOLITES
• GLUCOSE and FATTY ACIDS are the starting points.
- Then PYRUVATE is the central end product of glycolysis.
- ACETYL CO A is the major intermediate entering the TCA cycle from either side.
- NADH is major donor of protons and electrons to the electron transport chain and the end product is ATP.
Energy Production by Mitochondria
Outer membrane is permeably to the ions and small molecules
**98% of the body’s oxygen is used***
1) NADH is produced by TCA cycle
2) NADH donates electron and proton to the respiratory chain
3) Electrons travel through the respiratory chain towards complex IV and this is
coordinated with proton exiting from the matrix
4) Membrane potential is generated as a result of proton efflux
5) ATP is produced in ATP synthase when protons flow back into the matrix
Coupling of the Oxidation and Phosphorylation (Oxidative Phosphorylation)
- Oxidation – movement of the electrons from NADH to the oxygen which results in generation of the membrane potential
- Oxidation can be coupled to the Phosphorylation: Membrane potential is used for attachment of the phosphate to ADP and production of ATP
- Membrane potential can be used for:
1) ion transport
2) heat generation
3) pathological and physiological “leak” of the membrane
Mitochondrial Ion Transport
Roles of the mitochondrial ion channels:
• calcium uniporter – regulation of energy metabolism rates
• Katp channel – (proposed) to be involved in mitochondrial volume regulation
• PTP large pore – pathological channel involved in cell death
• NCX – (Sodium-calcium exchanger) helps to recycle calcium
• UCP – uncoupling protein – heat generation.
• VDAC – passage for metabolites across the outer membrane
• MAC – apoptosis channel
Investigating Mitochondrial Function
In most cases mitochondria
needs to be intact since integrity of the membrane is required
What can be measured in the intact mitochondria:
• NADH levels (imaging)
• Membrane potential (imaging)
• ATP levels (imaging)
• Oxygen consumption (respirometry)
-Place cells (mitochondria in air tight chamber) and
See how quickly oxygen is taken up (how the cells
Are “breathing”)
Flavonoid Quercetin
Addition of the flavonoid Quercetin to the neuronal culture leads to the increase of the mitochondrial membrane
• So the drugs that we believed improved mirochondrial function also helps
protect the brain.
• Flavonoids (E+Q) improve mitochondrial function by increasing the
spare respiratory capacity of the mitochondria
E+Q protect brain from stroke damage
Mitochondria and Pathology
• Cardiac cells heavily rely on ATP, but as soon as you depolarize the
mitochondria you will remove that membrane potential and ATP will
no longer be produced.
• Without ATP supply the cardiac cell is dead within a few minutes
Respirometry Example
(Initial control: rate they take up oxygen at first)
Then you add Oligomycin and it drops
then FCCP was added
And then Rotenone test and Antimycin test
* Oligomycin blocks ability of mitochondria to produce ATP (ATP synthase)
* FCCP resents membrane potential (does not allow a gradient to be formed) leads to increase in Oxygen because the only step that is occurring is the oxidation NO PHOSPHORALIZATION
* Rotenone stops complex I
* Antimycin stops complex III
Mitochondrial function during Stress
• What happens during stress, is that you have a lot of influx of calcium. Stress is for example, in case of heart attack, lack of oxygen. So mitochondria cannot function well. You have this gigantic influx of calcium which enters the mitochondria.
• This leads to the phenomenon called calcium induced permeability transition pore. Which is condition that occurs during cell death, stroke, or
heart attack.
- This porin resets membrane potential. The pore will open, it will depolarize and will cause massive tissue damage.
- This pathological pore which opens and kills mitochondria. Apparently what is important about it is that if you block this channel during this condition like in stroke you can significantly protect and diminish the tissue damage.
**• PTP – Permeability Transition Pore opens during calcium overload; leads to the loss of the membrane potential****
Mitochondria control the programmed cell death – apoptosis
- mitochondrial intermembrane space is not only space for energy metabolites but also space for pro apoptotic molecules like Cytochrome C, AIF; SMAC/DIABLO molecules
- They are stored there and are harmless for the cell unless apoptosis signal comes
• Then apoptosis signal leads to the formation of the mitochondrial apoptosis channel (MAC), which is large pore. Even larger than your traditional pore. The channel leads to the leak of these molecules into the
cytoplasm which trigger apoptosis.
Peroxisomes
- Membrane bound organelles (single membrane)
- Break down alcohol; toxins and fatty acids
• Note the outcome of the fatty acid hydrolysis is production of energy rich molecules of FADH2 and NADH (same enzymes as the mitochondria are
producing the same energy rich molecules except they are breaking down different things)
Cytosol components
- Water (85%) = semi-gel
- Proteins
- Carbohydrates
- Lipids
- Nucleic acids
- Buffers
- Ions
- Trace elements
Cytosolic and Cytoskeletal Functions
Maintenance of cell shape
- Every cell in the body or different types of cells in the body all have a different shape. That maintenance of shape comes from the cytoskeleton.
Whole cell movement
- Not all cells just sit there. Many of them move around. The ones that are moving around a lot are the white blood cells. They function usually in
connective tissue not blood vessels.
Contraction
Changes in cell shape
-There is a lot of places where cells can change shape and its important for
them to change shape. Somewhat during development.
Structural integrity of cell
-The cytoskeleton places an important role in structural integrity; in many cases, especially the intermediate filaments.
Organization and movement of organelles:
- mitochondria and the secretory granules and other organelles are organized and they move because of the cytoskeleton on the inside.
Cytosol Components
three types of filaments in the cytosol
• Mircofilaments – they are the smallest, 7-8 nm (will not ask numbers on the exam)
- Microtubules – largest filaments, about 24 nm
- Intermediate filaments – as electron microscopy got better, they discovered an intermediate filament
• Plus accessory and regulatory proteins of each are also part of the cytoskeleton
Actin
• It’s the component of MICROFILAMENTS
• It’s globular protein
• The monomer of the globular actin is G ACTIN, it will polymerize to form a filamentous chain which is called F-ACTIN
>The polymerization requires ATP and Magnesium.
> Microtubules and microfilaments have a polarity to
them. They form from a minus end towards the
plus end.
>If they’re not capped or maintained in some way,
they disassemble. The disassembly takes place
from the minus end of the filament in actin filament.
> Bundles of F-actin are referred as microfilaments.
> It’s really F-actin that is stabilized, it’s capped on
both ends so that it’s not continuing to
disassemble or to fall apart. It plays a role in
whatever the function of that cell might be.
• Different tissue in the body have different isoforms of actin. They’re basically the same but they have physiology functions that vary depending
on the isoforms of actin.
Different isoforms of actin (not going to talk a whole lot about these)
- α-skeletal - skeletal muscle
- α-cardiac - cardiac muscle
- α-vascular - vascular smooth muscle
- γ-enteric - visceral smooth muscle
- β-cytoplasmic - non-muscle cells
- γ-cytoplasmic - non-muscle cells
Muscle Cells
actin filaments are anchored in a very
regimented way in skeletal or cardiac muscle into these areas called the Z DISC
They interact with bundles of another cytoskeletal protein, MYOSIN
They induce a sliding of the filament. The sliding filaments take place in the non-muscle cells as much as they do in the muscle cells.
Non-Muscle Cells
They play the role in function related to cell shape and motility.
- Most of the functions in the non-muscle cells involve attachment to the cell membrane – they do that through interacting with proteins that are transmembrane proteins in the cell membrane.
- There is a bunch of actin binding proteins that are necessary for the actin to function within the cell.
Actin Binding Proteins
- Tropomyosin is important to skeletal and cardiac muscle.
- Alpha actin is important in muscle and non-muscle cells. These two are important in bundling filaments and something called microvilli.
- Spectrin is very important in crosslinking the membrane skeleton and something that is called terminal web.
- Myosin is important in sliding filaments in muscle and non-muscle cells.
- There are various Caps, the various capping proteins keep the filaments from depolarizing.
Actin Filaments function in Cell Shape Change
( 4 examples)
- Epithilial cell – are polarized, have a base and an apex, they can have cell surface and lateral specializations, can have finger-like projections called MICROVILI. They increase cell surface area. So you really see that these cells are involved in absorption. They absorb area of the GI tract (small intestine mostly as well as salivary gland ducts and kidney)
- Stress fibers - They position organelles and move organelles in very distinct patterns within the cell
• Cell motility – lamellipodium forms and you polymerize actin. Actin
myosin interaction then pulls up the rear end of the cell leaving bits and pieces of cell membrane and extracellular matrix and maybe integrins behind.
• Actin forms the contractile ring that separates the two daughter cells during Mitosis
Cell Motility
Cells form these LAMELLIPODIUM/Filapodia.
As the lamellipodium forms and you polymerize actin, it protrudes the front of the cell out further and further. It forms, because there are actin filaments in there and they are binding to integrins on the cell membrane, those integrins then bind to the extracellular matrix.
Cells don’t get infinitely long and skinny. What ends up happening is that the rear part of the cell has a actin
myosin interaction and it PULLS UP the rear end of the cell leaving bits and pieces of cell membrane and extracellular matrix and maybe integrins behind.
Contractile Ring in Mitosis
When cells go through mitosis and they divide, actin forms the contractile ring that separates the two daughter cells.
The contractile ring and the cleavage furrow is very important actin component in mitosis. Microtubules play a role in chromosome movement and separation.
Actin Attachment in Cell Membrane
Actin has to attach to the cell membrane to effectuate any of those sorts of cell shape changes.
ACTIN FILAMENTS are bundled together by protein ALPHA ACTININ
It comes through an ADAPTER COMPLEX that is made up of VINCULIN, and TALIN and a bunch of other specific proteins. They BIND TO A TRANSMEMBRANE
RECEPTORS. In this case it’s the INTEGRINS. Those integrins either bind to the EXTRACELLULAR MATRIX and something called the FOCAL ADHESION.
Actin can also bind to the same structures embedded into the membrane. As the actin interacts with myosin and it contracts, it can cause a contractile ring.
Terminal Web
In RBC, the terminal web includes GLYCOPROTEINS
There is a series of proteins that are very well studied ANCHORING BAND 3, PROTEIN 4.1, that organize this
altogether. What binds to ANKYRIN in this is transmembrane complex is a protein called SPECTRIN.
Spectrin is a super family. Spectrin binds to the
transmembrane protein at various spots all around this cell. ACTIN and protein 4.1 help crosslink and bind these together as a certain amount of tension is put on the system that gives you this biconcave disc. Every cell has a TERMINAL WEB.
Spectrin Gene Superfamily
- Spectrin I is in the erthrocytes (RBC) only
- Spectrin II is in every other cell in the body just underneath the cell membrane in various spots.
- Alpha Actinin - going to see it in skeletal muscle, see it in cardiac muscle and other muscles.
- Dystrophin – found in skeletal muscles. It is called dystrophin because it was discovered, in mostly boys at the time, that had duchenne and becker muscular dystrophy. Dystrophin was missing. Muscular weakness results and eventually death because even diaphragm doesn’t work.
Microvilli
Bundled actin filament have a structural function within microvilli
- Actin bundles are attached at their plus ends to the tip of the microvilli
- The actin filaments are bundled by the proteins VILLIN and FIMBRIN
- The actin bundles are attached to the sides walls of the microvilli plasma membrane via myosin I and calmodulin
- Within the terminal web, nonerythroid specturin (SPECTRIN II) and MYOSIN II links adjacent actin bundles to eachother and to intermediate filaments.
Microtubules**
MICROTUBULES
- Microtubules are made up of monomers or dimers of alpha and beta tubulin. Independent gene family products.
- They require GTP to polymerize.
- They assemble from a minus end, which is where things tend to disassemble.
- They have a growing end which is the plus end.
- The minus is always bound at something called microtubule organizing center and there are bunch of them inside cells.
• When the microtubules start to form, they start to form at an organizing center which is where the minus end tends to attach. It require GTP and the
GTP is on the terminal dimer
- But if you hydrolyze or dephosphorylate the terminal dimer, it leads to something called dynamic instability and the microtubule collaspes
- Microtubules continually grow from the centrosome, added to a cell extract. Quite suddenly however, some microtubules stop growing and then shrink back rapidly. A behavior called DYNAMIC INSTABILITY.
- Microtubules are the 13 protofilaments.
• Outside diameter is about 24 nanometers
.
• Inside, it is really a hollow tubule, its about 14 nanomenters.
• You add them on beta to alpha. They’re added on at the plus end and they peel off at the minus end unless you dephosphorylate these at the terminal end then the microtubules will collapse.
Microtubules and Cell Division
In the cell they all tend to startout at something called the CENTROSOME = when two centrioles line up perpendicular to each other.
Then there is a cloud of associated proteins that from these things called microtubule organizing center, these microtubules pull chromsomes apart
during cell division.
There are other microtubules organizing center. The one that is very well known and studied is the CILIA (long projections off the cell surface) in epithilial cells.
They form off of the basal body which binds the negative end.
Cilia function to move material across the cell surface. (EX. particles trapped in mucous on the surface of the trachea and lung)
Cilia
Cilia have a power stroke that takes place when the cilia are extended up into the mucus and moving into the direction they are organized to.
They drop off the mucus down into the liquid layer, recoil back, come up again around, hit the mucus and move it forward. They do this very fast and in an
organized fashion.
CILIA DO NOT MOVE WHEREAS FLAGELLA DO MOVE ( one of the major
differences between the two)
Centrosome
I. Contains two centrioles
II. Centrioles composed of 9 triplets of microtubules in a circle
III. Centrioles are perpendicular to each other
IV. Surrounded by a cloud of microtubule binding proteins (microtubule organizing center that binds minus ends)
Microtubule Movement
Vesicles in microtubules and there are a couple of different motors.
There is a motor that moves organelles from the minus end of the microtubule to the positive end. That motor protein called KINESIN.
There are those that move from positive
end back to where the centrosome is or cell center called DYNEIN.
• Where this is really magnified is in an axon on a motor neuron where these motor proteins dragging things
• dynein moves things towards the minus end (towards the cell center)
• kinesin goes towards the plus end (periphery of the cell)
Intermediate Filaments
Intermediate filaments are much more structural – are holding the cell into its shape.
They don’t move things, they don’t have motor proteins associated, they don’t polymerize.
- Intermediate filaments have a number of gene products.
- The proteins in every type of cell are different but the general function is the same.
- EPETHIAL cells have keratins.
- The gene family which is the vimentin and vimentin related genes are in CONNECTIVE TISSUES
- Then there is a special class in nerves called neurofilaments in NERVE cells.
- So although there are different proteins in different tissues, the general structure is the same.
- It’s an alpha helical monomer, it’s got an amino terminus and a carboxy terminus.
It forms a coiled dimer that work together»_space;> forms
tetramer»_space;» form two tetramers »_space;» they pack together putting 8 tetramers together »_space;> that gets coiled into a rope like filament that is 10 nm across.
- It’s a very structurally rigid, strong, filament.
- Does not do dynamic instability like microtubules do. They don’t spontaneously disassemble if you remove a regulatory protein.
- Intermediate filaments attach to desmisomes and hemidesmisomes
Neuroglia
Cellular connective tissue of the CNS
Astrocytes (Astroglia)
- Pure connective tissue cells - Vimentin and GFAP
Oligodendrocytes (Oligodendroglia)
- Contractile and produce myelin
- Vimentin only
Microglia (Mesoglia)
- Macrophages - Vimentin only
Dynamic Instability
Microtubules are highly dynamic and will frequently grow and shrink at a rapid yet constant rate. During this phenomenon, known as ‘dynamic instability’, tubulin subunits will both associate and dissociate from the plus end of the protofilament
the primary determinant of whether microtubules grow or shrink is the rate of GTP hydrolysis,
Adhesion Molecules
Have a transmembrane domain that anchors the protein in the cell membrane, and a cytoplasmic domain that mediates attachment to the
cytoskeleton.
Most cell adhesion molecules belong to one of fourmajor gene families:
- Cadherin - Are Calcium-Dependent Cell-Cell Adhesion Molecules
- Ig family (immunoglobulin) - The Immunoglobulin Family Contains Many Important Cell Adhesion Molecules
- Selectin - Are Carbohydrate-Binding Adhesion Receptors
- Integrin - Are Dimeric Receptors for Cell-Cell and Cell-Matrix Adhesion, heterodimers with alpha and beta subunits
Binding by Cell Adhesion
Molecules can be either HOMOPHILIC or HETEROPHILIC BINDING.
In homophilic adhesion a molecule on one cell surface binds to another identical molecule on the opposide surface.
In heterophilic adhesion, the molecule of
a different type of molecule on the other cell.
CELL JUNCTIONS
• Tight junction – seals neighboring cells together in an epithelial sheet to prevent leakage of molecules between them
-At the cell surface prohibits things from leaving the cell/lipids from moving around
- Adherens junction – joins an actin bundle in one cell to a similar bundle in a neighboring cell
- Desmosome – joins the intermediate filaments in one cell to those in a neighbor
- Gap junction – forms channels that allow small water-soluble molecules, including ions, to pass from cell to cell; communication between cells
• Hemidesmosome – anchors intermediate filaments in a cell to the basal lamina
-Receptors bind at the base of the cell (looks like half of the desmoosome)
Junction Morphology
each of those junctions have different type of
morphology
Zona (Zonula) – belt
-Zona Occludens & Adherens
Fascia - patches (mainly found in intercalated disk cardiac muscle) - they are not spots but they are not whole belts either
- Occludens & Adherens - Isolated
Macula - spot
- Adherens only
> Desmosomes – between cells
> Hemidesmosomes – between cells and
extracellilar matrix
Occludens Junctions
(Aka Tight junction)
Physical barrier to passage of molecules between cells (important in areas like the GI tract)
It’s Homophilic binding of Claudins to claudins (proteins) and Occludins to occludins (proteins) to form sealing strands
Prevents diffusion of membrane proteins in the apex of cells.
Plays a role cellular polarity
Tight junctions are formed when you have homotypic binding between the claudins to themselves or the occludens to themselves. They form these
structures called sealing strands.
More sealing strands, the tighter the seal.
BLOOD BRAIN BARRIER = TIGHEST ADHERENCE IN BODY
Adherens Junctions
(Spot and Zonula)
Cadhedrin mediated (transmembrane glycoprotein) – cadhedrins are homotypically bound
Calcium-dependent binding
Homophilic binding – two like molecules
Cytoskeletal element – Actin filaments to cytoskeleton
Potentially contractile may have a role in morphogenesis
Example of Zonula Adherins
embryology of glands – you get an invagination of an epithelial sheet and that can be just a permanent gland or just a deep invagination and nothing happens. But
there are sometimes where the epithelial tube pinches off and gives you an independent epithelial tube that has its own independent function.
Two common examples:
In a developing embryo – this is the neural tube and eventually that neural tube fuses and buds off and gives rise to the spinal cord
Another one would be the eye– would be developing corneal epithelium. But the epithelium here has budded off and totally encapsulates this structure
through this mechanism.
Desmosomes
(Anchoring junctions)
Cadhedrin mediated (transmembrane glycoprotein)
Calcium-dependent binding
Homophilic binding – two like molecules
Cytoskeletal element – intermediate filaments attached to an actin-spectrin intracellular plaque
Many together impart great tensile strength to epithelial cells
So in cells that get a lot of stress there are many of them and in cells that don’t get a lot there are few (generally referring to mechanical stress)
There’s lots of stuff here that’s anchoring the intermediate filaments.These plaques are composed of a lot of different proteins. They play a role in anchoring the keratin filaments if it’s an epithelial cell. They’re also inmuscle and in the desmosome in the heart it would be vimentin that would be attaching into these intermediate filaments in cardiac muscle.
Why you rarely get blisters unless you do a whole lot of mechanical force and friction to dislodge, and there you’re usually dislodging, if it’s a big blister, you’re dislodging from the basement membrane and not from the individual cells.
Hemidesmosomes
it attaches through integrins to LAMININ –
which is your extracellular matrix protein that is located in the LAMINA DENSA.
LAMINA LUCIDA AND LAMINA DENSA = BASAL LAMINA
In the lamina densa we also have TYPE 4 COLLAGEN, which attaches to COLLAGEN TYPE 7.
Those then interact with COLLAGEN TYPE 1 and 3 FILAMENTS. These are in what we call a RETICULAR LAYER.
Why is that important? If cells separate
here, or here, or here, it causes a serious breech of integrity, especially for skin.
Your first line of defense from the world is your skin.
Epidermolysis bullosa
It’s not a single phenotype to this disease
Epidermolysis bullosa simplex - keratin 5 or keratin 14
-Those keratin intermediate filaments aren’t quite attaching to the desmosomes; you’ll get cells to fall apart.
.Junctional epidermolysis bullosa - blister formation within the lamina lucida of the basement membrane.
Mutations in laminins or Col IV or A6/B4 integrin
.
Dystrophic epidermolysis bullosa – Mutation in Collagen VII .
this is collagen Type 7. It rips off and you get huge tears down into the connective tissue. Here you still have a little bit of a barrier for healing.
Gap Junctions
Communication junctions
Connexon – mediated = hexagonal array of connexins
Homophilic binding
Calcium independent
Pass ions and small molecules (< 1500d) – such as intracellular messengers
Gated channel (Calcium, pH or 2nd Messenger gated)
NO ADHESION
The main role is COMMUNICATION.
protein in the gap junction is a protein called the connexin. You get a hexagonal array of those to make
a half of a pore called a connexon.
The connexon on one side binds to the connexon on the other side, that’s homotypic or hemophilic binding.
It does not require calcium to bind. That forms a water-
filled pore that lets small ions and molecules pass in between.
example) you have the 6 connexins making half
of a connexon. And a connexon binds to the connexon on the other side. There’s a small gap between the cells. That’s why they’re called gap junctions. And molecules can get between these, very important in the function.
Integrins
Transmembrane Receptors
Heterodimers of α and β Chains
18 Alpha and 8 Beta Chains.
> 24 αβ receptors with varied functions.
Receptors are Tissue and Ligand Specific
Interact with the Cytoskeleton.
Integrin Functions
Cell Adhesion to the ECM
Cell-Cell Adhesion in some Cells
Cell Motility
Cell Proliferation
Cell Differentiation
Apoptosis
Intergrin Binding to Fibronectin
Cytoplasmic domain can then hook up with adapter proteins vanculin, tailin, and alpha-actin and
they go into the actin cytoskeleton.
Then in this case it’s showing how this
binding to a domain on fibronectin can pull these two arms together. And fibronectin, which is a big molecule can then bind to collagen fibrils out inside the
connective tissue.
And so what this depicts is that mechanical forces pulling on collagen, moving things transmit information into the cell.
And in the case of the heart it transmits through the integrin and can trigger hypertrophy, which is an increase in cell size, which is how cardiac muscle responds to increased load.
If you uncouple that, and there’s no mechanical tension, many cell types go through apoptosis. And then they play an important role in organizing that cytoskeleton. Organizing the cytoskeleton is an important component in whatever the cell function is. And in many cell types that interaction between the ECM and the actin cytoskeleton is how cells maintain their function
Actin Polymerization and Cell Movement
Most cell move due to actin polymerization
push out this little foot process and that grows by adding actin to polymerizing filament, or bundles of filaments in the perfusion, or lamelapodia or filapodia
But in human cells, or mammalian cells, that crawl around during development to function, what happens is that those lamelopodia, or pseudopods, that go out, they push out due to the actin polymerization and when they touch down they make a focal adhesion, , and attach.
What also happens is they don’t push out forever, they have to contract and pull up the rear of the cell. So the cells protrude out and they pull up the back end of
the cell through usually an actin/myosin interaction.
And many times the leave bits and pieces behind, and laying down the track as pioneer cells for all the cells coming behind. That’s important in neurogenesis and for cells to follow cells
Why Cells Move?
Two reasons that cells move: one is a chemo attractant.
Why do epithelial cells
migrate? They migrate in wound healing. We’ve all skin a shin or something and the scab forms, but what’s happening underneath is epithelium is migrating across and covering that extracellular matrix. Some of it is the chemo attraction, it’s a chemical that causes the cell to go towards it, but lots of it is the extracellular matrix interaction.
How did they go those huge distances?
Some of it is chemo attraction, but the majority of it is pioneer cells and other cells following.
Program Cell Death (PCD)
Program cell death (PCD) – cell suicide – PCD are orchestrated, programmed within each nucleated cell and ready to act, cells reach a point of no return after
which they will die
PCD PATHWAYS:
I. Apoptosis: induced to commit suicide - the primary PCD pathway
II. Autophagy: self consume
III. Necrosis: death induced by injurious agents (can be programmed cell death
– necroptosis or not programmed cell death - ordinary necrosis)
IV. Pyroptosis: PCD as part of an inflammatory reaction.
Why Die?
• During development, to clear away unwanted cells
(webs between toes)
- To maintain tissue homeostasis
- To eliminate damaged cells
- To prevent cancer
- To eliminat infected cell
- To have control over the death process to either prevent or induce inflammatory responses
Sometimes a cell dies quietly and contains it’s content = APOPTOSIS and AUTOPHAGY
Sometimes a cell spills its guts and stimulates and inflammatory response =
Necroptosis and pyroptosis and non-PCD necrosis
PCD is necessary for tissue homeostasis
- Our tissues do not continually expand because there is a balance between cell division and death
- 70 Billion cells die by apoptosis in the normal adult every day
- T cells that would react against the body are eliminated in the thymus before they enter circulation.
- Immune responses lead to a great expansion of antigen-specific T and B cells, which must die off after the infection is controlled.
In the vertebrate immune system more than half of many types of nerve cells die soon
after they are formed. (Positive selection for those that are chosen and negative by those that are destroyed (like for example, the autoimmune b-cells)
Regulation of Cell Death
Cell death is the default setting for most cells, which require stimulation to prevent death, such as a cytokine or contact with neighboring cells – keep
cells alive.
• If a cell is deprived of this stimulus, it undergoes PCD.
• Thus, maintaining tissue homeostasis requires constant intracellular communication involving “stay alive” signals received only when the right
cell is in the right place.
Cancers:
• turn off these requirements for extracellular factors and contact, allowing
cells to constantly grow indefinitely and spread to improper locations in the
body. (so no PCD)
PCD can be physiological or pathological
DNA damage caused by chemicals, radiation, etc. induces DNA repair mechanisms, or if repair is impossible, apoptosis. By inducing apoptosis, cells that cannot be saved are induced to die in a way that prevents them from damaging surrounding tissues and to be rapidly cleared away by phagocytic cells such as macrophages.
Cancers:
Cell death is a normal physiological thing, but it can also be something pathological. You don’t want too much apoptosis because this can cause disease. You also don’t want too little apoptosis – cancers.
- Cancer turns off PCD in response to DNA damage and senescence
- Suppress immune surveillance: suppress cytotoxic T cell induction of apoptosis – cancers also turn off those sensing mechanisms in the body where a killer T-cell would normally come along and kill the cancer cell.
Cancer therapy: many anti-cancer drugs turn apoptosis back on.
Ischemia
Ischemia: Heart attack, brain injury, stroke
Cells die by necrosis or apoptosis
Ischemia reperfusion injury: when the oxygen floods back into the area you can have a couple of
things go wrong, 1) you can have too much oxygen causing free or reactive oxygen species, and 2) you can have too many white blood cells coming to the
area. And these convulse and induce the cell to undergo necroptosis, or apoptosis and you can have a secondary injury.
Drugs that block PCD reduce tissue injury in heart attacks and strokes
So what they often will do is give you drugs that can reduce programmed cell death, prevent further injury after the incident.
Viruses
Viruses
• Virus infected cells often go into apoptosis as a defense mechanism.
• Turning off apoptosis is a major goal of many viruses, which have genes encoding proteins that inhibit this process. Increase or mimic Bcl-2 (stay
alive signal), degrade p53 (triggers cell death), stimulate anti-apoptotic kinases (stay alive signals), etc.
• Necroptosis can act as a fail-safe mechanism for an infected cell to die, and this mechanism of cell death releases the contents of the cell into the local
environment, stimulating an immune response.
• If a cell can’t undergo apoptosis and it is infected sometimes it will die by necrosis. Well it seems like one of the tricks that a cell has is when the apoptosis pathways have been turned off but it really wants to die
it can undergo necroptosis as a backup plan for killing itself. And this can stimulate an immune response which can be good or bad depending on the
situation, and then finally pyroptosis will come to at the very end.
• Pyroptosis can be induced by HIV-1 when it infects resting T cells, resulting
in release of inflammatory mediators.
Apoptosis
it entails cell shrinkage, loss of membrane potential, the mitochondria which is important to the whole signaling pathway, and then the nucleus breaks up
Most common PCD
• Intrinsic and Extrinsic mechanisms of induction
• Activation of caspases is a hallmark of apoptosis
• Physical changes:
• Loss of electrical potential across the inner mitochondrial membrane →release of cytochrome C from mitochondria to cytoplasm
• Cell shrinks and condenses
• Cytoskeleton collapses
• Nuclear envelope disassembles
• Nuclear chromatin condenses and fragments
• Cell surface blebs
• Large cells break into apoptotic bodies
Surface of cell becomes chemically altered so that phagocytic cells such as macrophages can engulf them before they spill their contents.
Phosphatidylserine moves from inner leaflet of plasma membrane to cell surface. ‘eat me signal’
Apoptotic bodies form and are engulfed. Rapid clearance to prevent an inflammatory reaction –
This is in contrast with necrosis, where the cell’s
contents spill out and induce localized inflammatory responses.
Endonucleases
your nuclear DNA is digested, and this is also a specific process triggered by caspases
But the point is that at the lowest level you have the DNA wrapped around nucleosomes in a repeating pattern.
Nucleases are triggered which cleave in between these nucleosomes randomly, not everyone is cleaved so you would have a single size, but at various spaces so what you end up if you run the DNA of an apoptotic cell on a gel it separates into a ladder.
Whereas if a cell is dying by necrosis and all hell has broken loose you don’t have that ladder, it just got digested randomly. So this ladder is a hallmark of
apoptosis and there are assay (tests) for apoptosis that look for this ladder or look for the ends of the DNA that have been exposed.
Autophagy
natural process where’s there’s always self-digestion to recycle things but it can get out of control and lead to cell death or it can just be part of cell death –
- A normal, non-pathological process by which a cell digests cell components (proteins, organelles) that are dysfunctional or otherwise unnecessary and recycles them.
- Cytoplasmic constituents are incorporated into an autophagosome which then fuses with a lysosome for degradation of the components.
- Is particularly active after cell injury as the cells tries to save itself and during times of starvation when resources are limiting.
- Autophagy can also be a programmed cell death pathway - Autophagic PCD.
However, it is not clear if cell death by autophagy is intentional or if the dead cell was trying save itself but failed.
Necrosis
Necrosis is when the membrane integrity is lost and fluids flood into the cell, swell it, and burst it. –
- Conventional Necrosis - NOT programmed cell death - is killing of cells by an outside agent, toxins, trauma, loss of blood flow (Ischemia), etc.
- The injury to the cell induces cell autolysis (self digestion)
• Membranes become permeable, water enters, cells swell and burst, releasing cellular contents into the extracellular space – creates a big mess in the
extracellular space
- Triggers inflammatory responses
- Is usually detrimental to the host blocks clearance of dead cells by phagocytes
- Myocardial infarction (heart attack)
- Ischemia-reperfusion injury
Pyroptosis
Pyroptosis
• A PCD in which caspase 1 is the initiator.
- Induces membrane rupture - cell lysis, release of IL-1β and inflammatory responses
- Primarily exists to fight off pathogens
- HIV infection of resting T cells in lymphoid tissues induces pyroptosis, while HIV infection of T cells in blood induces apoptosis.
- Improper caspase 1 activity is associated with several diseases including myocardial infarction (heart attack), inflammatory bowel disease and endotoxic shock.
- Inhibitors of caspase 1 can treat symptoms of these diseases.
Summary of Different Morphologies
Summary of different morphologies, mechanisms and outcomes of the 3 forms of cell death
Charact. Apoptosis Pyroptosis Necrosis
Morphology
Cell lysis NO YES YES
Cell swelling NO YES YES
Pore formation NO YES YES
Membrane blebbing YES NO NO
DNA fragmentation YES YES YES
Mechanism
Caspase 1 NO YES NO
Caspase 3 YES NO NO
Cytochrome C release YES NO NO
Outcome
Inflammation NO (anti) YES YES
Programmed cell death YES YES NO
Caspases
Caspase (Cysteine and aspartic acid -ase (enzyme))
Synthesized as inactive pro-caspase precursors that are subsequently cleaved by other caspases.
This results in a cascading reaction that is the commitment of a cell to death.
The Intrinsic Apoptosis Pathway
The intrinsic pathway is triggered by the p53 tumor suppressor in response to DNA damage and other types of severe cell stress
Conventional anticancer therapies (chemo, radiotherapy) activate this pathway via p53
p53 activates the intrinsic pathway through transcriptional upregulation of pro-apoptotic members of the BCL2 family of proteins such as PUMA and BAX
p53 is inactivated by mutations in more than half of human cancers
Cell-extrinsic apoptosis
- Cell surface receptors on cells are part of the TNF family (tumor necrosis factor)
o TNF receptor and Fas receptor are death receptors - If TNF is going to induce cell death, it binds the receptor and induces the activation of a death domain on that receptor
- Death domain then activates an initiator caspase (caspase 8 or 10), which dimerizes (just like caspase 9 does), and activates through cleavage the executioner caspases
Thus, both the intrinsic and extrinsic pathways have initiator caspases that are activated by dimerizing, and activate effector/executioner caspases by cleavage
Detailed Intrinsic Apoptosis
Cell-intrinsic apoptosis — intrinsic apoptosis (mitochondrial pathway)
DNA damage activates p53
- p53 is the guardian of the genome — decides if the cell can survive DNA damage (if the cell can repair itself properly or if the cell is too damaged and a risk to the body)
Cell cycle is stopped and cell tries to repair DNA
If this fails, p53 activates PUMA and NOXA
- These are transcriptionally activated if p53 gives the signal for cell death
PUMA and NOXA bind to and inhibit Bcl-2 and BCL-XL, and activate Bax and Bak
- Bcl2 family proteins usually inhibit BAX and BAK from forming channels in mitochondria that lead to the release of CytC
- Whether or not a cell dies is determined by the balance between Bcl2 holding BAX and BAK in check
- When p53 decides the cell will die, it transcriptionally activates PUMA and NOXA, which then suppress the activity of Bcl2 and stimulate the activation of Bax and Bak
Bax and Bak want to form channels in the outer mitochondrial membrane to release cytochrome C
Guardians normally prevent Bax and Bak from forming channels
- Bcl2 is regulated externally to maintain the supply of Bcl2 to a cell and keep Bax and Bak in check
Sensors prevent Guardians from inhibiting Bax and Bak → release of cytochrome C → activation of APAF1 →
formation of the apoptosome → activation of caspase 9 →
activation of caspases 3, 6, 7 → caspase positive feedback cascade → cleavage of cell DNA and lamin → cell destruction by apoptosis
- CytC combines with APAF1 protein to form an apoptosome
- This structure then triggers the caspase cascade
o Caspases are all zymogens — proteins that are inactive and need to be modified (usually cleaved or dimerized) to become active
o Apoptosome binds to caspase 9, which is then activated
o Once caspase 9 is in active form, it cleaves pro-caspases (3 or 7), creating active caspase 3 or 7
o Caspase 3/7 are the executioner caspases, and are the ones that do the real damage (the killer caspases)
Survival signals — cytokines, growth factors, etc.
Gaucher disease
a lysosomal storage disease, due to a defect in ß-glucocerebrosidase enzyme
- This disease demonstrates the relevance of the lysosomal system
- Causes large holes/lesions in the alveolar bone, thus affecting roots of teeth
- Affects Ashkenazi Jews (1 of 12)
Two main types of endocytosis
Pinocytosis — “cell drinking” (like pino noir)
- Small vesicles <150nm
- Two types of pinocytosis
> Indiscriminate pinocytosis
where the cell invaginates its membrane
and sucks up whatever is dissolved in the
extracellular fluid.
> Receptor-mediated endocytosis (1000x
greater concentration than indiscriminate
pinocytosis)
Phagocytosis — “cell eating”
- Large vesicles ≥250nm
- Consume microorganisms and large cell debris
Neutrophils and macrophages are the professional phagocytic cells in our bodies
- Consume bacteria by putting out these pseudopods and then eventually wraps all the way around the bacterium and then brings it inside the cell, and it gets degraded.
- Consume RBCs — turnover 1011¬ RBCs/day in our body; important function of macrophages
Cells import through endocytosis, export through exocytosis
General process of endocytosis
1) Vesicles bud off from the plasma membrane and fuse with an early endosome
2) Then early endosome matures into late endosome or fuses with existing late endosome
3) Then late endosome matures into lysosome or fuses with existing lysosome
4) Contents within lysosome are then degraded
Possible Routes for Protein Excretion
Classical protein secretion
Direct transit across the plasma membrane independently of a cell surface transporter
Unconventional secretion pathways by organelle carriers
- Secretory lysosome-mediated plasma membrane fusion
- Exosomes generated in MVBs and discharged by fusion at the plasma membrane
o Organelle between early and late endosome
o Characterized by multi-vesicular bodies
- Direct fusion of autophagosomes with the plasma membrane
o Responsible for breakdown of intracellular organelles
- Amphisome-mediated secretion
- Both the autophagosome and MVB can fuse to form an amphisome or autolysosome
- Direct fusion of autolysosomes with the plasma membrane
- Plasma membrane budding or shedding
Pinocytosis
So the cells continuously pinocytose the plasma membrane.
o Macrophages turn over 100% of their plasma membrane every 30 minutes.
o Fibroblasts are slower but it’s balanced, so as much membrane added to cell surface by exocytosis is removed by endocytosis.
o So two things are happening in a balance, otherwise you’d have too much of this membrane turned over or accumulating.
Endocytosis
Phagocytosis — engulfs microorganisms and large cell debris
- Makes large vesicles (phagosomes), which fuse with the lysosome
Indiscriminate pinocytosis — traps whatever is in the extracellular space
Receptor mediated endocytosis — molecules bind to receptors on the cell surface to become engulfed
Clathrin-coated pits and vesicles
E.g. cholesterol removing LDL particles
- LDL binds to specific LDL receptors on the cell surface
- Adaptin 2 then binds the receptor, and clathrin binds adaptin 2
- Then get invagination near this receptor binding
- Invagination forms an invaginated pit that then
buds off as a vesicle
Type of coated vesicle:
1) Clathrin-coated with coating proteins Clathrin + adaptin 1 from the Golgi apparatus destined to the Lysosome (via endosomes)
2) Clathrin-coated with Clathrin + adaptin 2 from the Plasma membrane destined to go to the endosomes
3) COP-coated with COP proteins from the ER
Golgi cisterna, Golgi apparatus, and Golgi apparatus destined to go to the Golgi cisterna and ER
Clathrin coated vesicles have clathrin cage molecules surrounding the vesicles (making a cage)
- Clathrin cage molecules are proteins with a heavy and light chain, which form a triskelion structure
- These triskelion structures come together to form a cage around the vesicles
Endocytosis Mechanisms with Clathrin Coated Vesicles
It is an example of Receptor Mediated Endocytosis
- Extracellular cargo molecules bind their receptor
- Receptor then binds adaptin 2 on the inner surface, which causes clathrin to bind
- the receptors all start to aggregate and the membrane starts to invaginate, curve inwards. You now have this vesicle forming, This process starts to cause curvature of the membrane
- So the vesicle forms with a clathrin coat
- Dynamin wraps around the neck of this vesicle and that cleaves that, and now you have this clathrin-coated vesicle inside the cell - separate from the plasma membrane
- Once it is inside the cell some enzyme reactions occur and the adaptin 2 and the clathrin fall off (the vesicle then gets uncoated)
- Finally, you are left with a naked transport vesicle
which can now fuse with the early endosome.
When this vesicle fuses with the early endosome, that lower pH causes the LDL to dissociate from its
receptor. Then the receptor buds off from tubular regions of the early endosome and recycles back to the cell surface, while the LDL gets transferred to the lysosome and gets degraded by the hydrolytic enzymes that are in the lysosome and the free
cholesterol gets transported across the lysosomal membrane into the cytosol.
Receptor mediated endocytosis
Specific receptors for specific macromolecules
Associated with clatherin-coated vesicles
1000-fold concentration compared wth simple pinocytosis
One example is cholesterol, others are vitamin B12, iron, and viruses (Influenza A) which use this as well, and there are many hormones as well that participate in thismechanism too, and growth factors.
Endocytosis of cholesterol
- LDL with cholesterol binds LDL-R
- LDLR binds adaptin 2, and clathrin binds adaptin
2, forming a clathrin coated vesicle - Uncoating results in a naked vesicle
- Naked vesicle fuses with endosome
o Endosome pH is lower than cytosol pH, so acidic
environment causes LDL to dissociated from the
receptor
o Receptor buds off in vesicles and is transported
back to the membrane, while the LDL is
transported to the lysosome for degradation
Antibodies that target PCSK9 (drug) prevent the rerouting and degradation of LDL receptors, thereby reducing plasma concentrations of LDL. EGF, epidermal growth factor
Types of Endocytosis
- RECYCLING: like with LDL and its
receptor, it comes in the cell with these transport vesicles, fuses with the early endosome and the LDL goes to the lysosome but the receptor can recycle back to the cell surface. - TRANSCYTOSIS – You can have vesicles that bud off from the early endosome and take whatever they are carrying across the cell and get expelled and then
can be transferred to an adjacent cell. - DEGRADATION - you can then have all contents going through degradation in the lysosome.
Different Responses based on pH
Early endosome pH ~6.5
Early/late endosome pH ~4.5
LDL dissociates most easily from its receptor around pH 6 — thus dissociates in the endosome
Iron is the next easiest
- Dissociates from the transferrin receptor
- Transferrin is the transport protein that carries iron
- Iron binds the transferrin receptor at neutral pH
- Once internalized, iron dissociates immediately
- The transferrin receptor returns to the plasma membrane, and transferrin returns to the plasma
Next, M6P (mannose-6-phosphate) dissociates from its receptor in the late endosome
- M6P-R can bud off vesicles and be transported to the trans golgi network
Last, EGF — epidermal growth factor
- EGF binds very tightly to its receptor, so it needs a very low pH to dissociate
- Thus, it is degraded in the lysosome along with its receptor
- This is a way for the cell to control growth factor levels
Lysosome formation
The acid hydrolases are actually made in the cis-Golgi.
They have mannose groups added to them, which are then phosphorylated
They then go through the Golgi and bud off in clathrin-coated vesicles with a different adaptin than used in receptor-mediated endocytosis.
The proteins associated with mannose 6-phosphate receptor. They are transported in these clathrin-coated vesicles. The clathrin dissociates and then these transport vesicles fuse with the late endosome where the phosphates are removed
Now the enzyme gets transported to the lysosome and that forms the lysosome.
Autophagy
Occurs when a cell is dead, or the cell is under starvation and desperately needs amino acids for metabolism
- First, a membrane made by the ER is formed and starts to surround the organelle, forming an early autophagic body
The early autophagic body then fuses with a lysosome, and lysosomal hydrolases break down the organelle
- This results in a tubular vesicular body that is broken down more into the late autophagic body, and then a residual body
Types of autophagy
Macroautophagy — membrane comes from ER and forms around fairly large organelles
- Mitophagy — when autophagy is around mitochondria
Autophagosome — autophagy around microorganisms or multiple organelles
Multivesicular bodies can also undergo endosomal microautophagy
Or, can just have a chaperone that takes a protein to the lysosome
These all fuse with the lysosome where degradation occurs
Summary of 3 Major Endocytic Pathways
Phagocytosis — form a large vesicle around microorganisms/large cell debris to form a phagosome that fuses with the lysosome, where contents are degraded
Pinocytosis
- indiscriminate — captures extracellular fluid
- Receptor mediated — naked vesicles fuse with the early endosome, and then either fuse with a late endosome or mature into a late endosome, which then fuses with a lysosome or matures into a lysosome, where degradation of contents occurs
Autophagy — ER membrane forms around cargo (e.g. mitochondria), forming an autophagosome that then fuses with the lysosome, where degradation occurs
Nucleus
- Most prominent organelle in a eukaryotic cell
- Nuclear envelope, which has 2 concentric membranes they are continous with the ER
- Contains chromosomes, nuclear DNA
• 1-5 nucleoli per nucleus (cancer cells can sometimes contain more) – the site where rRNA gets synthesized – so it is where 5 chromosomes that’s got the
genes for ribosomal RNA are in the DNA, they cluster there, and that’s why you can see the nucleoli.
Nuclear Envelope
The nuclear envelope of the nucleus dissolves at mitosis
-As the chrosomes condense, more of the nuclear
envelope dissolves and you end up with no
nuclear envelope or membrane and the cell can
now now undergo mitosis
It is thought that the nuclear envelope originated from invagination of the plasma membrane
This membrane is made up of inner and outer membranes (outer mem. continuous with ER)
-nuclear lamina attach to inner membrane
You also have nuclear pores
- To get things in and out of the nucleus you have
nuclear pores.
INTO NUCLEUS: newly made proteins from ctyosol
OUT NUCLEUS: ribosomal subunits which are assembled in nucleus as well as polA tailed and 5’capped mRNA
Signal Sequences
To get proteins in and out of the nucleus the proteins have specific sequences.
To get into the nucleus cells have nucleus-localization sequence and you can see they are very basic amino acids, positively-charged amino acids.
Export from the nucleus is a different sequence with a number of hydrophobic amino acids.
Nuclear Pores
• Have a very complex arrangement.
• You can see on the cytosol the nuclear pore has cytosolic fibrils so it is made up of many proteins and a whole set of proteins in the actual pore crossing
the nuclear envelope.
• On the inside of the nucleus is the nuclear basket (circle at the pore’s base that is thought to control
Import and export of molecules in/outside nucleus)
• When the proteins translocate into the nucleus they have the basic nuclear- localization signal and bind to and import to a receptor. The receptor binds to the cytosolic fibrils and is able to cross through the meshwork of fibrils through the basket and into the nucleus.
Steps into Nucleus
STEPS:
Protein with nuclear-localization signal binds to receptor
crosses the nuclear pore into the nucleus and in the nucleus Ran-GTP binding protein binds to the receptor.
That causes dissociation of the nuclear protein from the receptor protein.
Ran-GTP is bound to this receptor protein and that translocates out of the cell through a nuclear pore
In the cytosol Ran-GTP has a GTPase activity and can hydrolyze its own GTP and give off a phosphate and is now Ran-GDP and can assist in the process again.
Nuclear Lamin
These are proteins in the intermediate filament family.
and are associated with the inner nuclear membrane.
Upon initiation of mitosis, you get phosphorylation of nulcear pore proteins and lamins»_space; and you get dissolution of the nuclear envelope»_space; after mitosis occurs they get dephosphorylated and start to reform lamins with some of the membrane proteins and lipids»_space;> the new nucleus starts to form and you have the nuclear envelope.
Hutchinson-Gilford Progeria
Disease associated with mutation of just one lamin, nuclear lamin protein, lamin A.
• With this mutation they age really fast, they lose their hair, have osteoporosis, heart disease.
• In patients with progeria, the nuclear lamina does not completely form. The heterochromatin is not associated with the nuclear lamina and starts to be
transcribed when it shouldn’t be.
• It seems that the more laminae you have, the more ridged the cell is. That seems to occur in bone. If you have less lamin A, you have less ridged nucleus
or nuclear membrane. This ends up with the cell becoming really adipose.
And that may be why with these patients they particularly have osteoporosis and problems with their craniofacial development and teeth development.
Nucleolus
- It is made up of three domains, what’s called fibrillar centers of cores, dense, fibrillar region, and granular component or region.
- The nucleolus also dissolves during mitosis, although there is still some of it seen, which is the nucleolar organizing region.
• It is where all 5 chromosomes all aggregate and where the ribosomal RNA genes are associated there together. The nucleolus is the site of ribosomal
RNA synthesis.
- Fibrillar cores or centers are loaded with RNA polymerase I, which is responsible for transcribing ribosomal RNA.
- The granular regions have a lot of the ribosomes and there are some modifications of the RNA.
rRNA Processing
The first ribosomal RNA is 45S ribosomal RNA.
The 45S rRNA is processed to 30S rRNA and 18S rRNA.
30S is then further processed to 28S rRNA and 5.8S rRNA.
Non-nucleolar 5S rRNA form the ribosome particles together with a lot of protein OUTSIDE to creat final form of ribosome used for translation
OUT OF THE NUCLEUS
mRNA made in the nucleus by the euchromatin and is transported out of the nuclear pores with nuclear transport receptors
in Nucleolus you’re making rRNA. This is processed with proteins associated with the appropriate rRNA and you get the ribosomes, small and the large subunits. These also have to be transported out of the nuclear pore and into the cytoplasm and do so WITHOUT UNFOLDING and traveling by squeezing through pore.
Division of Cells
Labile cells, for example skin, and oral mucosa.
multiply constantly.
Stable cells.
they don’t normally divide, but when there
is a need, they may divide. Rentry into the cell cycle.
ex. liver cells
Permanent cells
they completely lose the prolifation and cell dividing
capacity like neurons, and cardiac myocytes, these
differentiated cells are incapable of dividing.
CANCER, EXCESS SKIN - may need bariatric surgery = are both a result of cell proliferation
Cyclosporine-induced gingival overgrowth – due to excessive proliferation in the gingiva of teeth, side affect of immunosupressent medication
Leukemia results from the proliferation of a clone of abnormal hematopoietic cells with impaired differentiation, regulation, and programmed cell death (apoptosis).
Replicative Senescence - Aging/ TELOMERES
Normal cells are not immortal and have a finite division numbers
Telomere erosion- the Hayflick limit - roughly 200 base pairs lost per turn at the telomere until it is so short it signals cell stop
TELOMERE EROSION AND ADDITION
• Telomere length prevents age-related decline
• Telomere- repetitive DNA that caps the end of each chromosome
- With each cell division, some of the telomere is
lost, but Telomerase- telomere terminal
transferase, is an enzyme that elongates
chromosomes
(2009 Nobel Prize in Physiology or Medicine for Telomere Research)
Phases in Cell Cycle
• G1- Gap between M and S-phase
increase in cell size
RNA and protein synthesis
• S- DNA Synthesis
2N to 4N DNA CONTENT
duplication of centrosomes (centrioles)
• G2- Gap between S and M
Synthesis of proteins (like tubulin for mitotic
spindles)
Synthesis of maturation promoting factor (MPF) for
inducing mitosis
THE PHASES ABOVE ARE PART OF INTERPHASE
• M- Mitotic phase
- Mitosis and cytokinesis
Cell-Cycle Control System
To ensure that they replicate all their DNA and organelles, and divide in an orderly manner, eukaryotic cells possess a complex network of regulatory proteins known as Cell-cycle control system
Guarantee a set sequence that each process has be completed before the next one begins
Regulated at certain critical points of the cycle by feedback from the process currently been performed
G0 PHASE- which is the cell is resting/dormant, and most of the stable cells are in this stage, the stable cells they don’t usually proliferate but they can come back
when there is a need, the liver cells after hepatectomy there is a need to proliferate, so they go from G0 to cell cycle.
CHECK POINTS
G1 aka restriction point – between G1 and S1 phase
“is the environment favorable?”
G2 check point – between G2 and M phase
“Is all the DNA replicated? Is there any damage? Has the damage been fixed?”
M check point
“Are all the chromosomes properly attached to mitotic spindle?”
There are others but G1 and G2 are the most important
Cellular Language -Signal Transduction
o Ligand receptor binding
- Ligand binds to this receptor, then the receptor
will make certain conformation change and
recruit certain downstream signaling
> Ex: growth factors
o Enzymes
- they can make modification, for example kinase or phosphorylase – can be on and off signals
o Transcription factor – DNA binding
Proteins that bind to DNA regulate
expression
Rb Protein - tumor suppressor
-binds E2F-E2F cannot bind DNA ans
transcription is blocked
- cell growth phosphorylates Rb and
releases E2F; E2F binds DNA and
turns on transcription advancing cell
cycle
o Signal transduction cascade
these signal transduction cascade, they are small signals that triggers a network of signals - apoptosis
Cyclin-Ddks
factors that guide the cells entering from one phase into the next.
CYCLIN concentration varies through the cell cycle, so it rises, and goes down
through the cell cycle, and during each phase of the cell cycle, different types of
cyclins are involved. The cyclins don’t work alone, they need to cooperate with
another group of proteins called cyclin dependant protein kinases, so it is a
kinase, and the function of kinase is to put a phosphate, they are called Cdks.
• Cyclin concentration varies through cell cycle
– No enzymatic activity by itself
• Cyclin dependent protein kinase (Cdk) concentration remains constant
• Cyclin-cdk is active
– Cyclin-dependent protein kinase
G1-Cdk = Cyclin D (partnered with Cdk4, Cdk 6)
G1/S = cyclin E (partnered with Cdk2)
S-Cdk = cyclin A (Cdk 2)
M-Cdk = cyclin B (Cdk 1)
cyclin-cdk phosphorylation
there is an inhibitory phosphate site and there is an active phosphate site. So you
put on phosphate here it turns on, is active, but also you need phosphatase to
remove this inhibitory phosphate to eventually have an active complex.
CYCLIN-CDK controls cell cycle checkpoints
- Control system monitors status and stops cell cycle progression if errors/problems
- Phosphorylation-dephosphorylation of cyclin-Cdks
- CDK inhibitor proteins
cyclin-dependant kinase inhibitors (Cdk1)
are a group of inhibitors that can block the activity of cyclin and Cdk complex, so this is why it is called inhibitors.
P16 - Tumor Suppressor - often lost in oral/head and neck SCC
loss of 9p21 (locus) - common in oral
cancer
results in activation of cyclin d-cdks
phosphorylation of Rb
amp of 11q13-CCND1 - common oral cancers
P21
blocks cyclin-cdk
blocks G1 to S and S Phase Progression
P53 - Guardian of the genome
Tumor Suprpressor Gene - commonly lost in cancers
G1 to S Transition: checkpoint to stop dividing or commit apoptosis
Activates Cdk Inhibitor Protein (p21) n
DNA damage and p53
arrrest in G1
activate p53
transcript. factor or p21 - p21 inhibits cyclin-cdk
cell repairs or dies
p14 , p15, p19, p27, p57,
cycle control failures lead to uncontrolled proliferation
HPV
Human papillomavirus (HPV) • it’s a known risk factor to oral cancer
• Infectious disease, is sexually transmitted
• This is well studied and the reason why HPV leads to oral cancer is because it
controls cell cycle, and the molecules this virus attacks include P53, Rb and
P16.
> One of the mechanisms that this HPV targets P53, to trick the cells to continue
proliferation. So the virus just mimics the proteins that can bind with P53, and once
it binds to P53 it can make the cells mistake. Makes it think that p53 is ready to get degraded, so it recruits the ubiquitination and then guides the p53 to proteasome and once p53 is degraded there will be no guardians in our genome to sense/detect DNA damage, so this is how smart the HPV virus can be to utilize this system and to trick the cells to make uncontrolled proliferation
oncogenes: c-Myc
- c-Myc is called an oncogene, so the more you have, the more the cells have a tendancy to proliferate
- c-Myc transcripts for a transcription factor
• A mutated version of Myc is found in many cancers, which causes Myc to be
constitutively (persistently) expressed.
• c-Myc drives cell proliferation through upregulation of cyclins,
downregulation of p21
STAGES OF MITOSIS
Prophase
– Chromosomes condense
– Centrosomes begin to separate and spindle assembles
Prometaphase
– Breakdown of nuclear envelope
– Chromosomes attach to spindle via kinetochores
Metaphase
– Chromosomes aligned in equator (metaphase plate)
– Homologous chromosomes line up independently
– Sister chromatids of chromosome attached to spindle on opposite
poles
Anaphase
– Sister chromatids separate and are pulled to opposite poles
– Anaphase-promoting complex (APC)-triggers chromatid separation;
degrades M-cyclins
Telophase – Chromosomes arrive at poles – Nuclear envelope reassembles – Chromosomes decondense – Division of cytoplasm with formation of contractile ring
Cytokinesis
– cell divided in half by contractile ring
Sexual Reproduction
They all go through mitotic proliferation, so the germ-line cells from the zygote proliferate.
• In female, these cells will immediately go into meiosis.
»_space; Then they go into arrest, so they stop there, and some of the cells during this phase will go through apoptosis»_space; Some cells will be circled with some somatic cells»_space; they are here to protect and support the germ-line cells. (this is called a follicle. This is right after birth) »_space; after puberty, each month, usually one, occasionally multiple, eggs will become mature and ovulation happens»_space; this iis how germ-line cells develop into germ cells in females.
• The males, they also start with mitotic proliferation, but they immediately go into mitotic arrest, so they stop in the cell cycle»_space;> after birth, they start to proliferate again. Upon puberty, they start the meiosis process
Take home message: they both have zygote proliferation in the beginning but immediately in females the germ-line cells enter meiosis and meiotic arrest. In
males, it is mitotic arrest then after birth is mitotic proliferation and upon puberty is
start of meiosis.
Asexual Reproduction
All offspring from asexual reproduction are identical to the parents and identical to each other. They are simple and uniform.
The advantage of sexual reproduction is mixing our genome. Each time the germ-line cells from parents mix. The offspring have much more diversity.
Evolution Selection
3 main competitive advantages
- Reshuffling the genes to help a species survive in an unpredictably variable environment
- Elimination of deleterious mutant alleles and help to prevent them from accumulating in the population
- Selection of survivals from parasites
Meiosis
- Strong analogy to mitosis
- Increases genetic variation
- Human male- 24 days and happens after puberty
- Human female- decades – whole proccess starts before birth and continues for decades until individualis depeleted of eggs
• One doubling of DNA plus two cell divisions
– Meiosis 1
– Meiosis 2
Haploid egg + haplois sperm = diploid zygote (fertilization)
• This goes through mitosis to make more somatic cells and a small portion remain as germ-line cells.
• 2N → 4N → 2N → N
• Every gamete is genetically unique
• Chromosomes, maternal vs paternal, randomly sorted due to independent segregation
• Human: 23 pairs chromosomes lead to 2n genetically distinct gametes
– 8.4 x 106 different gametes
STAGES OF MEIOSIS I AND II
Prophase 1
• chromatin condenses and there is a homologue chromatid pair. This happens in early prophase to mid prophase.
Formation of Bivalent - structure formed when a duplicated chromosome pairs with its homolog at the beginning of meiosis; contains four sister chromatids.
Metaphase 1
• The homologue pairs line in the middle.
• Recall that in mitosis chromosomes line up at the metaphase plate independently WHEREAS in meiosis homologous chromsomes are paired at the metaphase plate – they only need to make sure daughter cell get one copy from each parent
Anaphase and Telophase I
• They are paired and there is separation and go through the first division, meiosis 1.
MEIOSIS 2 –
Prophase II
• cells are still diploid because only one duplication and one division happened.
Metaphase 2 and Anaphase 2
- The chromosomes always align in the middle. You can see here it is called equatorial plate. They line here in metaphase 2.
- In anaphase 2, they start to separate. As you can see the chromosomes some of them are red with a little bit of blue.
Telophase II
• And then eventually in the second division, they become 4 haploid cells.
In meiosis 2, the cohesin protein that used to glue the chromatids together is degraded and the sister chromatids can be separated in the second division
and make haploid cells. So in this stage, in the females’ development of germ cells, there is meiotic arrest and
the chromosomes are already paired. During the long rest, when the female reaches age 40-45, the glue between the chromatids may be less and not as strong, and this is one of the reasons why abnormal chromosome children happen more frequently in
females who gave birth after 45 years of age.
Recombination
• Exchange of DNA between maternal and paternal chromosomes- Crossing- Over
– Increases genetic variation
• Occurs in prophase 1*****
• Recombination
– Synaptonemal complex-protein machinery for recombination
– Disassembled by end of prophase
• Chiasma (χ)
– Connection between two chromatids from crossing-over
> Each gene on chromosome we have a way to
locate. It is like zooming in an address. First
we have a short arm we call “p” and a long arm
we call “q” and in between these two is a
place where centrosome stays and we have
different band, a sub-band
Crossing Over
• Bivalent chromosome»_space; chiasma»_space; Then they exchange certain genes and cross over.
- Several crossing over can happen in one bivalent.
- This crossing over structure has a special term, chiasmata.
• The chiasmata is a physical structure that links the homologue chromosome during meiosis 1. The bivalent starts at prophase 1 and continues to metaphase 1 and during that phase, crossing over happens so that is when we can observe chiasmata under the microscope.
GENES THAT ARE A GREATER DISTANCE AWAY FROM EACHOTHER ARE LESS LIKELY TO CROSS OVER THAN GENES THAT ARE CLOSER APART – less roam to cross over
Errors in Meiosis
• Nondisjunction- homologous chromosomes fail to separate
– Gamete lacks a specific chromosome
– Gamete has an extra chromosome
• 10% meiosis in human oocytes have nondisjunction
– Leads to aneuploidy
– Increases with age
Nondisjunction.
• In normal meiosis you have two chromsoms in a cell, but in abnormal you
end up with one with three and the other with one. And if normal gametes are fertilized with a gamete with extra or less, the individual will have extra or less chromosomes.
• Trisomy chromosome 21: an example of non disjunction
Different disease is monosomy, missing a chromosome. This is also called Turner Syndrome. (One X chromosome)
DNA Packaging
- 3.2x109 base pairs of DNA x 2 of each chromosome = 6.4x109 base pairs total in each cell
o = 2 meters in length - 2 meters x 50 million million (trillion) cells in the human body = 100 billion km long!
o Distance from the sun to Pluto is 6 billion km (average)
Chromosomes = - 10,000:1 packing ratio
DNA must be packed down to fit into the cell
- DNA is organized first into nucleosomes (eukaryotes only)
- The core of nucleosomes consists of 8 histone proteins (Positively charged (basic) to bind the negatively charged DNA (acidic) — 11nm fibers)
- The DNA strand (147bp) wraps around the histone core, and then the nucleosomes assemble into helical coils called 30nm fibers
- Nucleosome = beads on a string
o Nucleosomes are connected by linker proteins (the string)
o Linker DNA Is 10-90bp
- The 11nm fiber is the most open form of the histone organization
- Histone core is an octamer of 8 proteins plus DNA
o H2A, H2B, H3, H4 (2 of each of these)
- Histone H1 is a linker protein added to generate
the 30nm fiber
o Needed to pack down from the 11nm fiber to the
30nm fiber
- Chromatin = DNA + proteins that produce chromosomes
Chromosome scaffold proteins
structural, non-histone proteins that help bind together the higher order structure of chromosomes
To form the chromosome, we then have to add scaffold proteins (Something for the DNA to hang onto)
Karotyping
Karyotyping
Humans have 46 chromosomes — 22 pairs of autosomes, and 2 sex chromosomes (XX in females, XY in males)
- Both autosomes are transcriptionally active, so sets/copies of genes are “on” on both chromosomes, with the notable exception of the Xi chromosome in females
- The Y gene is much smaller than the X chromosome
Karyotype = the full set of 46 human chromosomes
G (Giemsa) banding of metaphase chromosomes is used to identify individual chromosomes, to assess the karyotype and identify genetic abnormalities
- Banding pattern enables us to identify each chromosome
o Dark bands are AT-rich regions
We have 2 copies of each chromosome, and both are active
- Thus we can compensate for a mutation in one gene (unless it’s a dominant gene)
FISH - Fluorescence in situ hybridization
Chromosome painting via FISH provides a way to specifically identify chromosomal regions
DNA oligos can hybridize to target DNA, so we can tag the DNA with different colors
- this allows us to identify chromosomes and their defects (more easily than karyotyping in just black and white)
- More complex than Giemsa
Chromosomes
- Provide a way of organizing genes so that their expression can be regulated
- Provide structures to assist DNA replication and partitioning during cell division
- a. Origin of DNA replication (not strictly part of the chromosome, but is on the DNA)
- b. Centromere for mitotic segregation
- c. Telomere allows replication of linear DNA without loss of ends - Allow occasional transfers of genetic information and duplications of genes — evolution vs. genetic diseases
Although the genes in related species will be very similar, their arrangement on chromosomes and even the organization of the chromosomes themselves has a lot of plasticity
- Two closely related species may have radically different sets of chromosomes
- Thus karyotyping is not everything
Chromosome
p arm — short arm (petite)
q arm — long arm
Centromeres
- Point of contact between sister chromatids
- Site of kinetochore formation — where the spindle fibers connect to the sister chromatids
o Composed of 2-4mb (mega-bases) of multiple repeats of simple sequences
171bp repeats, “alphoid DNA” which is bound by nucleosomes, or other types of highly repetitive DNA
• Together called “satellite DNA”
o Centromeric chromatin is in the form of constitutive heterochromatin
It is always heterochromatin and has little or no expressed genes on it
Kinetochore assembles at centromeres and associates with mitotic spindles during mitosis and meiosis
- Microtubule organizing center (MTOC) is the anchor that is used to pull the two cells apart
Telomeres
Found at each end of chromosomes
Protect the ends of the chromosome from degradation by cellular nucleases
Telomeres contain repetitive oligomers of high GC content for a few thousand bp: TTAGGG
This must be replaced each cell division by telomerase in germ cells and stem cells, or shortening of the chromosome will eventually make the cell non-viable
- shortening is a signal for the cell to go into senescence and apoptosis
- Our cells have a finite number of divisions
Mutant mice which cannot repair telomeres can only reproduce a few generations
- Dolly, the first cloned sheep, had problems with this too
Telomerase, a riboprotein (RNA-protein complex) uses RNA as a primer for DNA synthesis (thus it’s a reverse transcriptase) to extend the telomere and restore its length
Telomerase
Telomerase preserves chromosome ends
- Telomerase is a reverse transcriptase — DNA synthesis using an RNA template
Telomerase has an RNA template that ligates with a few bps to the end of the DNA, and is used to extend the DNA
- Extend the RNA far enough so that when you back fill, the bit that’s lost is the bit that’s been added prior
- Thus in total, you don’t lose anything, and your DNA is preserved
Telomerase is ACTIVE in germ cells and many stem cells
- Need stem cells to divide over and over again in order to develop a large repertoire of cells
Telomerase is INACTIVE in cells that divide a limited number of times (most cells in the body), and in cells that never need to replicate again — post-mitotic cells
But telomerase is turned back on again in cancers —
thus telomerase is a possible target of treatment
In chronic HIV infection, CD8+ T lymphocytes over-replicate and lose telomere length, contributing to immune dysfunction
- If you’re constantly making killer T cells, they don’t have telomerase, so they keep getting shorter and shorter
- Thus you can tell how well or bad off someone is by looking at the length of their telomeres
Barr body
the inactive X chromosome which is highly methylated, condensed and located away from active transcription areas of the nucleus.
• In females, one X in each cell is randomly turned off
during the ~1000 cell stage of development, which keeps the number of expressed X-linked genes equal for males and females (Dosage compensation)
Calico cats are not a separate species or breed. They are a variant where a gene for coat color is on the X chromosomes, so XY males have one gene and so are uniform in color, while the XX females are mosaics based on which X is active in which part of their coat.
Gene Regulation
Almost all cells have exactly the same DNA, but different cell types look and function very differently.
This is result of expression of different sets of proteins
Protein expression is controlled primarily by
Regulation of transcription of mRNA by RNA polymerase II (pol II)
Protein expression is also regulated by splicing, translation and posttranslational modifications (phosphorylation, degradation, etc.)
Gene regulation determines cell fate during development/regulates how a cell functions and responds to the environment.
Transcription of a gene results from bringing pol II to the promoter and allowing it to travel down the DNA
This is regulated by:
• The sequence of the DNA (genetics) to which transcription factors bind
• Which transcription factors are expressed in a cell
• Modifications to DNA and to histones (epigenetics)
• Whether the DNA is in a heterochromatin or euchromatin configuration
All of these factors interact with each other to determine which genes are transcribed and which are not.
Control of Transcription
- Genetics: DNA sequence to which transcription factors bind.
- Epigenetics: Chemical modifications to DNA or to histones that alter transcription rate but don’t modify the DNA sequence.
Euchromatin vs. Heterochromatin
Euchromatin (light staining) Transcriptionally available 11 nM fibers Not complexed to histone H1 • Histones acetylated • Histones methylated in ways that favor transcription • DNA not methylated
Heterochromatin (dark staining) Transcriptionally repressed condensed 30 nM strands Complexed to histone H1 • Histones deacetylated • Histones methylated in ways that repress transcription • DNA methylated
Gene expression occurs on open chromatin
The location of a gene in the nucleus can change to a location in the nucleus that is more favorable to transcription.
Heterochromatin – condensed chromatin
• Usually at the periphery
• Mostly transcriptionally inert
Constitutive chromatin – never converted into euchromatin
• Centromeres and telomeres, most of the Y chromosomes
• Doesn’t have genes
• Always compacted so no transcription
• Contains several satellite sequences – repeat sequences
Facultative heterochromatin – interconverted between euchromatin and heterochromatin
Cell Differentiation
The differentiation of cells is the result of switching on
of cell type-specific genes to make proteins needed in that cell type.
The switches for differentiation are usually transcription factors.
MyoD turns on many other genes that make muscle-specific proteins
The response of a cell to environmental stimuli is controlled in part by pathways from the outside of the cell down to the level of transcriptional regulation
Initiation of Transcription
Initiation of transcription
Many promoters have a TATA (or TATAA) box, this is what we focus on here.
TATA binding protein (TBP) bind the TATA box.
TFIID/TBP = A complex of proteins including TBP.
1. TFIID/TBP binds TATA
2. TFIIH binds
3. pol II is recruited
1-3 = Transcription Pre-initiation Complex (also includes other proteins not shown)
4. Mediator complex binds to the tail of pol II blocks transcription starting
5. TFIIH phosphorylates pol II on its tail
this activates pol II, freeing it from Mediator, and it now can start transcription at a
basal level.
—> Other transcription factors, some general and some gene-specific, bind to DNA sequences upstream.
Some are in the enhancer.
Gene-specific transcription factors increase transcription above the basal level and determine overall transcription rate.
Each cell type and each state (replicating or not, activated or not, stressed or not, etc.)
expresses different set of transcription factors at varying levels.
DNA folding allows transcription factors bound to enhancers to interact with the promoter
Work of Transcription Factors
Example: MyoD: the master regulator for differentiation into muscle cells turns on many other genes that make muscle-specific proteins specialization into a muscle cell
o MyoD binds to muscle-specific promoters to turn on muscle-specific genes differentiation into a mature muscle cell (and stops replicating)
o MyoD must be made all the time it is involved in a feedback mechanism that allows for myoD to be produced and expressed all the time.
HOW?
Induces p21
shuts down cell replication, as well as represses a kinase (CDK, cyclin dependent kinase) that would phosphorylate myoD and inhibit it (“repressing a repressor”)
• The RESPONSE of a cell to environmental stimuli is controlled in part by pathways from the outside of the cell down to the level of transcriptional regulation
Thus MyoD sustains its own expression in a positive
feedback loop to maintain muscle cell differentiation
Epigenetics
Epigenetics (“epi,” meaning “on” “upon” or “above”); chemical changes in the DNA or histones that regulate gene expression without altering of the DNA sequence
- While the DNA sequence remains unchanged, how it will be read will be changed by epigenetics.
- Epigenetic modifications are HERITABLE
as cells divide, the daughter cells inherit these modifications
• If epigenetic changes occur early in an organism’s life, they are not inherited
Epigenetic mechanisms
Histone modifications:
are reversible and the result of a dynamic interplay
between enzymes that install the modifications and those which remove them.
Acetylation of the histone TAILS
acetylation is favorable to transcription (remember: acetylation & activation)
opens up nucleosome into free chromatin
- Performed by histone acetyltransferases (HATs) that work in tandem with histone methylation
- Removal of histone acetyl groups: histone deacetylases (HDACs) which repress transcription
- HATs and HDACs are regulated RAPIDLY and their concentration regulates histone acetylation
o NOTE: LYSINE is in high concentration on histone tails = target for methylation and acetylations
changes the histone’s charge from positive to neutral,
allowing their dissociation from DNA, the opening up of chromatin
30 nm fibers»_space;> 10 nm fibers, and transcription to begin
Methylation favorable or repressive to transcription, depending on the amino acid that is methylated (histone code)
- Methylation of histones can involve single, double, or triple methyl groups
- Performed by histone methyltransferases (HMT); histone methyl groups are removed by histone demethylases .
• Sometimes, histone methylation contributes to chromatin condensation
generate heterochromatin/inhibit transcription
• Other times, histone methylation contributes to maintain euchromatin (open chromatin) and recruit transcription factors.
Histone methylation provides longer term regulation than histone acetylation
DNA Methylation
Most common on cytosine units that are followed by a GCG or CpG
Most stable of epigenetic modifications
Is found throughout the genome and is essential
for viability; 60-80% of CpG are methylated in the
human genome (regions of genome are always
repressed, such as at retrotransposons)
Some promoters are highly methylated
Rarely activated, whereas others are never
methylated and thus are deemed metabolically
active. • CpG Islands; regions in promoters where there is an unusually high frequency of CGs
DNA methylation is highly repressive for transcription; characteristic of heterochromatin formation; inhibits binding of transcription factors
Catalyzed by DNA methyltransferase
CpG methylation is recognized by proteins that recruit histone deacetylases and other chromatin remodeling factors
turn euchromatin into heterochromatin repress gene expression.
CpG methylation patterns are maintained during cell replication
Tumor suppressor genes are turned off by DNA methylation
DO NOT CONFUSE HISTONE METHYLATION WITH DNA METHYLATION
DNA METHYLATION INHIBITS TRANSCRIPTION, BUT IT IS ALSO EPIGENETIC AND CAN BE INHERITED BY DAUGHTER CELLS
HIV-1 (Levy’s Research)
The HIV-1 promoter is repressed by nucleosomes and
needs the HAT p300 to be recruited to the promoter to acetylate them
Infected T cell is activated by an antigen this increases expression of transcription factors and HATs
Nucleosomes that are in contact with DNA near promoters act as a physical barrier to pol II binding and moving along the DNA.
Transcription factors bind the promoter and recruit the HAT p300
p300 banishes HDACs and recruits HATs that acetylate histones.
Histone acetylation weakens the binding of nucleosomes to DNA and more transcription factors can bind and pol II can travel past the location of the nucleosomes.
Embryonic Development and gene repression
In embryonic development the repression of gene expression must be removed so that zygotes regain the potential to express all tissue-specific genes - totipotency.
This reset allows fertilized eggs to differentiate into all the tissues in the body
There are two waves of CpG methylation removal:
1. During gametogenesis - formation of sperm and egg
2. Soon after fertilization during early embryogenesis
Non-gene DNA products
non-gene products are NON-CODING for proteins
• Some have function (ex: centromeres, telomeres) structural purposes
• Some are non-functional (“junk DNA” and other immaterial, noncoding sequences; not called “junk” DNA anymore because they actually serve a purpose)
Gene
A gene determines a heritable trait
- a “unit of selection” in evolution.
A gene encodes a functional product - either protein or RNA.
Variations in DNA sequence among copies of a gene are called alleles.
The sequence of all the DNA is the genome.
A gene is transcribed, but the modern definition includes all the DNA
elements that go into regulating transcription - both the coding and the
non-coding elements - promoters, enhancers, introns, etc.
These elements are all subject to evolution and selection.
A gene is found on DNA.
Protein Coding Genes
Protein Synthesis»_space;> Transcription (in the Nucleus) and Translation (at the ribosome between assembled subunits);
in Eukaryotes, transcription and translation are physically separated from one another.
• Protein Coding Genes»_space; first make messenger RNA (mRNA) via transcription by means of RNA polymerase II; synthesis of mRNA strand occurs in the 5’»_space; 3’ direction.
o Protein coding genes consist of:
Promoter: upstream from the initiation site of transcription; binds to the RNA polymerase II with the help of transcription factors.
Enhancers: can be far away from the promoter region
Coding regions: “ORFs” are OPEN READING FRAMES, AKA the CODING REGIONS; Coding regions are within EXONS and are not spliced out during alternative splicing
Introns: noncoding regions; oftentimes spliced out.
PolyA signals
o Mature mRNA transcripts leave the nucleus and are brought to the ribosome, which subsequently assembles (small and large ribosomal subunits)
mRNA tRNA primary protein structure (amino acid sequence)
Simple Gene
a simple transcription unit without the need for splicing; the gene is transcribed by RNA polymerase II and does not need to be spliced.
Complex Gene
What most of our cells are
have multiple exons (regions that code for subunits or bits of the final protein) and introns are between them (to be spliced out; splicing = internal processing).
A typical complex gene has multiple exons and different splicing patterns provide variant mRNA transcripts that encode different proteins known as PROTEIN ISOFORMS
• Ex: muscular proteins, such as fibroblasts and striated muscles that are all products of differential splicing ( and subsequent cleavage and polyadenylation) of ONE complex gene, alpha-tropomyosin.
o Different gene structures different functions
Solitary Genes and Tandem Arrays
• Solitary Genes: genes that exist as ONLY 1 COPY 25-50% of all genes are single copy
o You only need a certain amount of that gene to be expressed; one gene is sufficient for the desired amount of protein produced/ proper functioning.
• Tandem Arrays: A series of copies of a gene arranged in tandem along a chromosome; exist for genes that code for products such as rRNA, for large quantities of rRNA are necessary for biological function.
o DUPLICATED GENE
o Tandem arrays are series of METABOLICALLY ACTIVE genes; still contain their own promoters, enhancers, etc.
o Spacers separate the genes within a cluster to insulate them and prevent “read through” by polymerase enzymes (if there were no insulators, then there would be no separation between the gene copies and there could be more transcription than desired).
Gene Families
• Gene Familes: multiple copies of genes that are similar, but not identical; have >1 member. Reside on the same chromosome.
o Similar to TANDEM ARRAYS you still have duplication/crossovers of one gene, but evolution results in modified genes from that same ancestral line
o Modified genes will have different functions.
o There can also be non-functional family members within a gene family PSEUDOGENES
Pseudogenes are nonfunctional genes that have been mutated over time; originated from functional genes but eventually usually nothing at all. “Ghosts of previous genes.”
o Beta-globin: a part of the HEMOGLOBIN molecule; an example of GENE DUPLICATION to create a GENE FAMILY.
Hemoglobin: binds oxygen; embryonic and fetal forms of hemoglobin exist in fetal blood, more oxygen must be bound more tightly.
In development, you first express embryonic form of hemoglobin, then a fetal form of hemoglobin, and FINALLY, a post-natal (adult) form of hemoglobin; earliest forms of hemoglobin are most avidly bound to oxygen.
• The genes start out as identical (gene family) but diverge into variants that perform different tasks.
Subtypes of RNA
o hnRNA = heterogenous nuclear RNA: RNA before processing (polyA tails, methyl caps, splicing)
o mRNA = RNA after processing; used for translation in to proteins
o tRNA = transfer RNA; bring amino acids to ribosomes
o rRNA = ribosomal RNA; part of the ribosome
o snRNA = small nuclear RNA; a variety of RNA that are part of ribonuclear protein complexes that perform function such as RNA splicing and telomere preservation.
o tRNA, rRNA, and snRNA are all from non-protein coding genes
Composition of a Genome
o ***1.5% of all DNA in body are EXONS (coding for proteins)
o Introns are much more prevalent; fill in large gaps between exons
o RNA products (tRNAs, rRNAs) are a small portion of the genome
o ***98.5% of the genome is non-coding (but not necessarily NONFUNCTIONING)
o Simple sequences; microsatellites and satellite sequences are small repetitive sequences in the centromere.
o ***Approximately 50% of the human genome is comprised of unique sequences; the other 50% is of repeating sequences
o ***45% of the repeated sequences are made of TRANSPOSONS (jumping genes) that can move around the genome. ALL are coded on DNA sequence.
DNA Transposons: a DNA sequence that can change its position within the genome; at low frequency, sequence is cut out of DNA and can be inserted somewhere else on the genome. The same number of bases/sequence quantity is the same. • "CUT and PASTE" = 1 copy, only
RNA Transposons (retrotransposons): transcribed into RNA, make a copy of that, and then go back to form a new DNA sequence that can be inserted into genome at a different location from the original sequence. • Retrotransposons require reverse transcriptase. 8% of our DNA is viral in origin (presence of endogenous retroviruses) • "COPY and PASTE " = 2 copies result • LTR: Long terminal repeats = sequences that are duplicated at each end of the retroransposon.
o Capable of making RNA and often also making proteins that are not useful to the cell; potentially harmful
o DNA methylation at endogenous retrovisues keep them “off”
Difference in the size of genomes of different species are due to the number of transposons that influence the quantity of DUPLICATED GENES, not that some species have more genes than others.
Mobile DNA: Transposable Elements = 45% of human DNA
o Do not make useful proteins; are essentially parasitic DNA and their origin is retroviral
o Transposition = moving around to different parts of the genome; relatively low frequency of transposition but they have an effect on evolution of organisms.
o When transposition occurs in GERM LINE CELLS, the sequence change is HERETIBLE.
o Some chromosomal transpositions/translations, etc cause various diseases, such as hemophilia and cancer, upon mutation.
o The genome works to keep the transposable elements transcriptionally silent by means of DNA and histone methylation = represses transcription.
** Overall takeaway: **
Transposable DNA can inactivate genes or their products by disrupting them; they can activate genes or their products by inserting near them; some can make RNA and proteins that are disruptive.
Hemophilia: caused by insertion into clotting factor
IX
Cancer: activation of oncogenes by insertational
mutagenesis
Transposable elements can evolve = duplicate genes and their products, modifying existing ones.
NON-RETROVIRUS-LIKE RETROTRANSPOSONS
Different from Conventional Retroviruses in that they
o Lack the structure of retroviruses
o Most abundant mobile DNA elements in
mammals (21% of human DNA)
DNA Fingerprinting/PCR
identify individuals based on their unique pattern of satellite or microsatellite DNA; variable number tandem repeats.
o Simple sequence repeats:
• Microsatelite DNA = 1-13 bp repeats; some occur within coding regions of disease and can thus cause mutations (Trinucleotide Repeat Disorders)
• Satellite DNA = mostly repeats of 14-500 bp; often at telomeres and centromeres
Gene
Region of DNA that controls a hereditary characteristic of an organism
Humans = 25,000 genes
Bacterium = 500 genes
BASIC GENETICS TERMS
Locus: specific position or location of a gene on a chromosome
Ploidy: number of chromosomes present in cell nucleus
Diploid: cell or organism containing 2 sets of chromosomes and hence 2 copies of each gene
Haploid: cell or organism containing 1 set of chromosomes
Genotype: Set of genes carried /inherited by an individual cell or organism
Phenotype: The observable characteristics of an organism
Genome: The total genetic information carried by a cell or an organism
Alleles
Allele: an alternative form of the same gene
Homozygous: An organism with identical alleles for a given gene
Heterozygous: An organism with different alleles for a given gene
Hemizygous: An organism with a single copy of a gene. Usually applies to X-linked genes in males
Dominant and Recessive Genes
Alleles that mask or hide other alleles, are said to be dominant.
An allele that is masked, or covered up, whenever the dominant allele is present is said to be a recessive allele.
Symbol: Dominant alleles – upper case letters
Recessive alleles - lower case letters
Complete (Classic) Dominance: when the heterozygous is completely indistinguishable from the dominant homozygous
Homozygous dominant
Heterozygous dominant
Homozygous recessive
Incomplete (Partial) Dominance
When one allele is NOT completely dominant over another (they blend – genetic blending)
Example: In flowers, the color red (R) is incompletely dominant over white (W). The hybrid color is pink
Co-dominance
when the contributions of both alleles are visible in the phenotype
example: In humans, blood type is determined by 3 alleles – A, B, and O
A and B are expressed equally i.e. co-dominant blood group alleles
Dominant – A and B (co-dominance)
Recessive – O
Allele Frequency
is the frequency (proportion) of alleles in a population
Allele frequency = (times an allele is observed in a population) / (total number of alleles in the population)
»_space; expressed as a percentage, decimal or fraction
Reflection of genetic diversity in a population changes in allele frequencies over time and can indicate that genetic drift is occurring or that new mutations have been introduced into the population
Genetic drift = random variation in allele frequency from generation to generation
Remains relatively constant unless there is a selective advantage (high frequency vs low frequency) – natural selection
Example:
high allele frequency when mutations in genes that promote survival or fertility will be favored by selection because it’s more likely that these mutants will survive and pass the mutations to their offspring
OR
low allele frequency when affected individuals die and their affected alleles are, over time, eliminated from the gene pool
