Exam 2 Flashcards
(144 cards)
1
Q
Basic difference between cell membranes of bacteria vs eukaryotic cells
A
In some bacteria, the plasma membrane is the only membrane
Eukaryotic cells also have internal membranes that enclose individual organelles
2
Q
Examples of internal membranes in eukaryotic cells
A
ER, vesicles, peroxisome, lysosome, endosome, golgi apparatus
Enclosed by 2 membranes: nucleus, mitochondria
3
Q
Structure of cell membrane
A
lipid bilayer with proteins embedded
Phospholipids: hydrophilic head + hydrophobic tails
4
Q
Where do kinks form in phospholipids?
A
In one of the hydrocarbon chains where there is a double bond between two carbon atoms
4
Q
Most abundant phospholipid in the cell membrane
A
phosphatidylcholine:
hydrophilic head - choline linked to a phosphate group
hydrophobic tails - two hydrocarbon chains w/ a carboxyl
A molecule of glycerol links the head to the tails
5
Q
Movement of lipid molecules in lipid bilayer
A
Membrane phospholipids move within the lipid bilayer.
Behaves as a two-dimensional fluid, in which the individual lipid molecules are able to move in their own monolayer. (lateral diffusion)
Note that lipid molecules do not move spontaneously from one monolayer to the other. (flip-flop)
6
Q
Role of cholesterol in fluidity
A
cholesterol stiffens membranes by filling in gaps between phospholipids, making the bilayer less flexible and less permeable
7
Q
Relation of kinks in phosopholipids with fluidity
A
The more unsaturated the hydrocarbon, the more kinks, the more fluid as it makes it harder for the tails to pack against one another
8
Q
do colder environment animal cells want more or less fluidity?
A
more fluidity so they dont freeze
9
Q
What is the role of flippases (what do they do and what is the result)
A
Flippases help to establish and maintain the asymmetric distribution of phospholipids
They selectively remove specific phospholipids (phosphatidylserine and phosphatidylethanolamine) from the side of the bilayer facing the exterior space + flip them into the monolayer that faces the cytosol
The resulting curvature of the membrane may help drive subsequent vesicle budding.
10
Q
Name the phospholipids and glycolipids that are distributed asymmetrically in the lipid bilayer and which side they lay on
A
Due to flippase: phosphatidylcholine and sphingomyelin concentrated in the noncytosolic monolayer.
Phosphatidylserine and phosphatidylethanolamine are found mainly on the cytosolic side.
Phosphatidylinositols are in the cytosolic monolayer (participate in cell signaling)
Glycolipids found exclusively in the noncytosolic monolayer of the membrane.
Cholesterol is distributed almost equally in both monolayers.
11
Q
What are extracellular vesicles and what is their role?
A
Extracellular vesicles are cell-derived membrane particles involved in signalling: exosomes, microvesicles, and apoptotic bodies.
Released under physiological conditions, but also upon cellular activation, senescence, and apoptosis.
Important role in intercellular communication.
May maintain cellular integrity by ridding the cell of damaging substances.
12
Q
What are the various types of plasma membrane proteins
+ examples
A
Transporters: e.g Na+ pump which actively pumps Na+ out of cells and K+ in
Ion channels: e.g K+ leak channel allows K+ ions to leave cells, influencing cell excitability
Anchors: e.g integrins which link intracellular actin filaments to extracellular matrix proteins
Receptors: binds extracellular molecule and generates intracellular signals
Enzymes: e.g adenylyl cyclase catalyzes production of intracellular cAMP in response to extracellular signals
13
Q
Role of the cell cortex: In blood cells
A
Cortex made largely of spectrin
Spectrin dimers are linked end-to-end to form longer tetramers.
Spectrin tetramers + actin molecules = a mesh
This network is attached to the plasma membrane by the binding of at least two types of attachment proteins to two kinds of transmembrane proteins
14
Q
How can a cell restrict the movement of its membrane proteins
A
Membrane proteins are restricted to particular domains of the plasma membrane of epithelial cells in the gut.
Proteins are prevented from entering other domains by tight junctions which separate the domains
(polarity is also achieved by this)
15
Q
What is the carbohydrate-rich layer coating the cell surface made of
A
Oligosaccharide side chains attached to membrane glycolipids and glycoproteins
And polysaccharide chains on the membrane of proteoglycans
Glycoproteins that have been secreted by the cell and then adsorbed back onto its surface can also contribute.
All the carbohydrate is on the external (noncytosolic) surface of the plasma membrane.
16
Q
Role of neutrophils in recognition
A
Recognition of cell-surface carbohydrates on neutrophils allows these immune cells to begin to migrate out of the blood and into infected tissues.
Specialized transmembrane proteins (called lectins) are made by the endothelial cells (lining the blood vessel) in response to chemical signals from a site of infection.
Lectins recognize particular sugar groups carried by glycolipids and glycoproteins on the surface of neutrophils circulating in the blood.
Neutrophils stick to the endothelial cells that line the blood vessel wall.
Neutrophil rolls along blood vessel wall.
Much stronger protein–protein interaction helps the neutrophil slip between the endothelial cells, so it can migrate out of the bloodstream and into the tissue at the site of infection
16
Q
How can one measure the rate of lateral diffusion of a membrane protein
A
Using photobleaching techniques such as FRAP
A specific type of protein can be labeled with a fluorescent antibody or tagged with a fluorescent protein, such as GFP.
A small area of the membrane containing these fluorescent protein molecules is then bleached using a laser beam.
As the bleached molecules diffuse away, and unbleached, fluorescent molecules diffuse into the area, the intensity of the fluorescence is recovered.
The diffusion coefficient is then calculated from a graph of the rate of fluorescence recovery: the greater the diffusion coefficient of the membrane protein, the faster the recovery.
17
Q
To which molecules is the membrane quite permeable to
A
small nonpolar molecules diffuse rapidly
small uncharged polar molecules diffuse readily if they are small enough
18
Q
To which molecules is the membrane hardly/not permeable to?
A
large uncharged polar molecules hardly cross
highly impermeable to ions
19
Q
Ion concentrations inside and outside mammilian cell
Na+
K+
Mg2+
Ca2+
H+
Cl-
A
Inside vs outside
5-15 : 145
140 : 5
0.5 : 1-2
10^-4 : 1-2
7x10^-5 : 4x10^-5
5-15 : 110
20
Q
How can inorganic ions and small, polar organic molecules cross a cell membrane ?
A
through either a transporter or a channel
A channel forms a pore across the bilayer through which specific inorganic ions or polar organic molecules can diffuse. (based on size and charge)
Ion channels can exist in either an open or a closed conformation
Channel opening/closing is usually controlled by an external stimulus or by conditions within the cell.
A transporter undergoes a series of conformational changes to transfer small solutes across the lipid bilayer. Transporters are very selective for the solutes that they bind, and they transfer them at a much slower rate than do channels.
21
Q
Outline the difference in passive vs active transport
A
Passive transport:
- allows solutes to move down their concentration gradients
- occurs spontaneously
Active transport
- against a concentration gradient
- requires an input of energy (ATP from hydrolysis, transmembrane ion gradient, or sunlight)
Only transporters can carry out active transport, and they are called pumps
22
Q
What are the main ways pumps carry out active transport
A
Transports solute against its electrochemical gradients
1) Gradient driven pumps: link the uphill transport of one solute across a membrane to the downhill transport of another (transmembrane ion gradient)
2) ATP-driven pump: uses the energy released by hydrolysis of ATP to drive uphill the transport of the solute
3) light-driven pump: use energy from sunlight to drive uphill transport of solute (found mainly in bacterial cells)
23
How does the Na+ pump work?
ATP-driven Na+ pump transports Na+ out of the cell (against its electrochemical gradient) and carries K+ into the cell
3 binding sites for Na+ and two for K+
Conformational changes occuring during:
1) The binding of cytosolic Na+
2) phosphate group removed from ATP and transferred to the cytosolic face of the pump
3) high-energy linkage of the phosphate to the protein induces conformational changes that transfer the Na+ across the membrane and release it outside the cell
4) Binding of K+ from the extracellular space
5) Dephosphorylation of pump
6) Protein returns to original conformation = transfers the K+ across the membrane and releases it into the cytosol
24
What inhibits the Na+ pump? and how?
Ouabain inhibits the pump by preventing K+ binding
25
What is the net result of the Na+ pump?
The net result of one cycle of the pump:
3 Na+ out and 2 K+ in
26
What happens in the Ca2+ pump in the sarcoplasmic reticulum?
When a muscle cell is stimulated, Ca2+ floods into the cytosol (which originally has a low Ca2+ conc) from the sarcoplasmic reticulum
Influx of Ca2+ stimulates the cell to contract
To recover from the contraction, Ca2+ must be pumped back into the sarcoplasmic reticulum by this Ca2+ pump.
The Ca2+ pump uses ATP to phosphorylate itself, inducing a series of conformational changes (similar to the ones of the Na+ pump); when the pump is open to the lumen of the sarcoplasmic reticulum, the Ca2+-binding sites are eliminated, ejecting the two Ca2+ ions into the organelle
Ca2+ pumps return to their original conformation w/o the requirement of a second ion
27
What are symports and antiports?
Gradient-driven pumps can act as symports or antiports.
symports: transfer solutes in the same direction
antiports: transfer solutes in the opposite directions
Uniports: only facilitate the movement of a solute down its concentration gradient. Because such movement does not require an additional energy source, uniports are NOT pumps but are still transporters.
28
Example of a symport
Glucose-Na+ symport: uses the electrochemical Na+ gradient to drive the active import of glucose
Fluctuates between outward-open (extracellular) and inward-open (cytosolic) states
Pump can only transition between the states when both Na and glucose are bound or neither are bound (so not when ONLY one is bound)
Because Na+ conc is high in extracellular, Na+ binding site is readily occupied in outward-open state and is waiting for a rare glucose molecule to bind > flips to occluded-occupied state (both molecules bound)
Now it can either flip back to outward-open state = solutes dissociate + nothing happens
OR
flips to inward-open state = Na+ dissociates and then glucose eventually dissociates > occluded-empty state > outward-open state
REPEAT
29
What maintains the steep Na+ gradient?
When Na+ is pumped into the cytosol it is also pumped back out of the cell
30
What are the diff types of glucose transporters that enable gut epithelial cells to transfer glucose across the epithelial lining of the gut?
Glucose-Na+ symport:
In apical domain of plasma membrane = faces gut lumen = import glucose into epithelial cell, creating high conc of sugar in cytosol
Na+ is pumped out by Na+ K+ pumps
Passive glucose uniports:
basal + lateral domains of plasma membrane
release glucose down its conc gradient to be used by other tissues
gut lumen (low glucose conc) > epithelial cell (high glucose conc) > extracellular fluid (low glucose conc)
31
What is the basis of electrical signalling in many cells?
changes in membrane potential
32
What plays a major role in generating the resting membrane potential across the plasma membrane?
K+ conc gradient and K+ leak channels
33
How does K+ conc gradient and K+ leak channels generate a resting membrane potential?
When K+ leak channels open, K+ will tend to leave the cell (down its conc gradient)
K+ will cross the membrane but -ve ions will be unable to flow = charge imbalance > a membrane potential that tends to drive K+ back into the cell
At equilibrium the effect of K+ conc gradient is exactly balanced by the effect of the membrane potential = no net movement of K+ across the membrane
34
How can we monitor ion channel activity ?
Patch-clamp recording
To expose the cytosolic face of the membrane, the patch of membrane held in the microelectrode can be torn from the cell. This technique makes it easy to alter the composition of the solution on either side of the membrane to test the effect of various solutes on channel activity.
35
Structure of a neuron + how it works?
a cell body, a single axon, + multiple dendrites
The axon conducts electrical signals away from the cell body toward its target cells, while the multiple dendrites receive signals from the axons of other neurons.
36
How does membrane potential influence voltage-gated Na+ channels
A voltage-gated Na+ channel can flip from one conformation to another, depending on the membrane potential.
When membrane at rest + highly polarized, +vely charged amino acids in the voltage sensors of the channel are oriented by the membrane potential in a way that keeps the channel in its closed conformation.
When membrane is depolarized > voltage sensors shift = change in the channel’s conformation so the channel has a high probability of opening (when threshold is reached Na+ channel opens = Na+ influx further depolarizing membrane)
But in the depolarized membrane, the inactivated conformation is more stable than the open conformation so after a while in the open conformation (when peak mV is reached),
Refractory period:
the channel becomes temporarily inactivated and cannot open. After the membrane has repolarized the channel returns to its original conformation
37
Voltage-gated Na+ channels: mV and conformation of the channel
AP is triggered by a brief pulse of electric current = partially depolarizes the membrane, reaches threshold = Na+ channel opens > mV reaches peak and starts to depolarise = inactivated > hyperpolarization > resting membrane potential > return to original closed confirmation
Even if restimulated, the plasma membrane cannot produce a second action potential until the Na+ channels have returned from the inactivated to the closed conformation. Until then, the membrane is resistant, or refractory, to stimulation.
38
How is a chemical signal converted into an electrical signal
Done by postsynaptic transmitter-gated ion channels at a synapse
Released neurotransmitter binds to + open transmitter-gated ion channels in the plasma membrane of the postsynaptic cell = resulting ion flows alter the membrane potential of the postsynaptic cell
39
give examples of water soluble molecules that diffuse freely across cell membranes
CO2 and O2
40
Thousands of ______ form on the cell body and dendrites of a motor neuron in the ________ synapses
synapses, spinal cord
41
What can control the activity of specific neurons in a living animal + study that discovered this
light-gated ion channels
Study of optogenetics (showed aggressive behaviour in rats was influenced by presence / absence of blue light as it activated rhodopsin channels to open
42
What is AE2
anion exchanger across plasma membrane
exchanging intracellular Cl⁻ with extracellular HCO₃⁻
Cl⁻ out of the cell
HCO₃⁻ is moved into the cell
43
What is the result of the absence of AE2 (from knockout study) + interpretations
results in more bone mass
AE2 is linked to osteoclast activity
Osteoclasts are responsible for bone resorption (breaking down bone).
Without AE2 (-/-), osteoclasts are inactive, and bone resorption does not occur as efficiently, leading to increased bone density.
44
Role of AE2 in osteoclast functioning?
anion exchange process is necessary for the proton pumps to work correctly
= help osteoclasts acidify the environment for bone breakdown
45
What are the general principles of cell signalling
Signals Can Act over a Long or Short Range
A Limited Set of Extracellular Signals Can Produce a Huge Variety of Cell Behaviors
A Cell’s Response to a Signal Can Be Fast or Slow
Cell-Surface Receptors Relay Extracellular Signals via Intracellular Signaling Pathways
Some Intracellular Signaling Proteins Act as Molecular Switches
Cell-Surface Receptors Fall into Three Main Classes
Ion-Channel-Coupled Receptors Convert Chemical Signals into Electrical Ones
46
What determines whether and how much gene is transcribed?
signals from outside, transcription factors, signal transduction, at DNA: repressor and enhancer sequences
47
What determines how much a functional protein is produced by the cell?
Efficiency of posttranslational modifications:
- cleavage of signaling peptides
- glycosylation
- phosphorylation
miRNAs
Ubiquitination
Repressors at the 3’UTR of mRNA
Stability of mRNA:
- CAP
- Poly A tail
48
The same signal molecule can induce different responses in different target cells. Give an e.g
acetylcholine reacts on pacemaker cells, salivary gland cells, skeletal muscle cells
48
Which type of receptors do extracellular signal molecules bind to?
cell-surface receptors (generate one or more intracellular signaling molecules in the target cell) or to intracellular receptors (pass through the target cell’s plasma membrane and bind to intracellular receptors—in the cytosol or in the nucleus—that then regulate gene transcription or other functions)
48
What are the 4 types of cell communication?
1) Endocrine: Hormones produced in endocrine glands are secreted into the bloodstream and are distributed widely throughout the body
2) Paracrine: paracrine signals are released by cells into the extracellular fluid in their neighborhood and act locally
3) Neuronal: Neuronal signals are transmitted electrically along a nerve cell axon. When this electrical signal reaches the nerve terminal, it causes the release of neurotransmitters onto adjacent target cells
4) Contact dependent: a cell-surface-bound signal molecule binds to a receptor protein on an adjacent cell.
49
Explain why certain extracellular signals act slowly or rapidly
Certain types of cell responses—such as cell differentiation or increased cell growth and division—involve changes in gene expression and the synthesis of new proteins; they therefore occur relatively slowly.
Other responses—such as changes in cell movement, secretion, or metabolism—need not involve changes in gene expression and therefore occur more quickly
50
Pathway of extracellular signals that alter the behavior of a target cell
extracellular molecule binds to receptor protein > intracellular signalling molecules > interact with specific effector proteins (e.g metabolic enzyme, cytoskeletal protein, transcription regulator) altering them > changes in target cell behavior (e.g gene expression, metabolism etc)
51
Components of intracellular signalling pathways perform one or more crucial functions. What are they?
1) they can RELAY the signal onward + help spread it through the cell
2) AMPLIFY the signal, making it stronger so that a few extracellular signal molecules are enough to evoke a large intracellular response
3) detect signals from more than one intracellular signalling pathway and INTEGRATE them before relaying a signal onward
4) DISTRIBUTE the signal to more than one effector protein
5) MODULATE the response to the signal by regulating the activity of components upstream in the signalling pathway (feedback regulation)
52
Explain further how feedback regulation within an intracellular signalling pathway works?
Positive feedback:
A downstream protein in a signalling pathway, protein Y, acts to increase the activity of the protein that activated it
Negative feedback:
protein Y inhibits the protein that activated it
53
What are 2 examples of an/off molecular switches
signaling by protein phosphorylation
signaling by GTP-binding proteins
54
what are molecular switches
some intracellular signaling proteins behave as molecular switches: receipt of a signal causes them to toggle from an inactive to an active state
Once activated, these proteins can stimulate (or in some cases suppress) other proteins in the signalling pathway
for every activation step along the pathway there exists an inactivation mechanism
Importance: e.g a signalling pathway that boosts heart rate, if it were to remain indefinitely active this would not be good
55
Explain how signaling by protein phosphorylation works
proteins are either activated or inactivated by phosphorylation
off to on: A protein kinase covalently attaches a phosphate group onto the switch protein (transfers phosphate group from ATP to signalling protein leaving you with ADP)
on to off: phosphate is removed by a protein phosphatase
56
explain how signalling by GTP-binding proteins works
off to on:
a GTP-binding protein is activated when it exchanges its bound GDP for GTP
on to off: the protein then switches itself off by hydrolyzing its bound GTP to GDP
57
What are the 3 types of cell-surface receptors
Ion-channel-coupled receptors:
opens in response to binding an extracellular signal molecule
G-protein-coupled receptors:
extracellular signal molecule binds to GPCR > activated receptor signals to a trimeric G protein on the cytosolic side of the plasma membrane = then turns on (or off) an enzyme (or an ion channel
enzyme-coupled receptors:
its extracellular signal molecule binds > an enzyme activity is switched on at the other end of the receptor, inside the cell. Many enzyme-coupled receptors have their own enzyme activity while others rely on an enzyme that becomes associated with the activated receptor
58
G proteins structure
composed of 3 protein subunits: alpha, beta, gamma
Unstimulated state: alpha subunit has GDP bound to do, G protein is idle
59
When an extracellular signal molecule binds to GPCR how does the altered receptor activate the G protein?
by causing the alpha subunit to decrease its affinity for GDP, which is then exchanged for a molecule of GTP
In some cases this activation breaks up the G-protein subunits > activated alpha subunit (clutching its GTP) detaches from the beta-gamma complex (which is also activated)
These two activated parts can then interact w/ target proteins in the membrane > relay signal to other destinations in the cell
60
The longer target proteins remain bound to an alpha or beta-gamma complex, the more prolonged.....
the relay signal will be
and thus how long a response lasts
60
How is the G protein returned to its original inactive conformation
the alpha subunit has intrinsic GTPase activity which eventually hydrolyzes its bound GTP to GDP = inactivated the alpha subunit which dissociated from its target protein and ressociates with a beta-gamma complex to reform an inactive G protein
61
When does the inositol phospholipid pathway occur?
Some GPCRs exert their effects through a G protein called Gq > activates enzyme phospholipase C (instead of adenylyl cyclase) > this enzyme propagates the signal by cleaving an inositol phospholipid = 2 second messenger molecules (IP3 and DAG) which play a crucial part in relaying the signal
62
What are the 2 signalling pathways that phospholipase C activates?
1) IP3
IP3 diffuses through the cytosol + triggers the release of Ca2+ from the ER into the cytosol by binding to and opening Ca2+ channels in the ER membrane
2) DAG
DAG remains in the plasma membrane + together w/ Ca2+ they activate the enzyme PKC (which translocated from the cytosol to the plasma membrane). PKC phosphorylates its own set of intracellular proteins = further propagating the signal
63
What are enzyme-coupled receptors
transmembrane proteins that display their ligand-binding domains on the outer surface of the plasma membrane
the cytoplasmic domain acts either as an enzyme itself or forms a complex with another protein that acts as an enzyme
respond to extracellular signal proteins that regulate growth, proliferation, differentiation, and survival of cells
64
what is the largest class of enzyme-coupled receptors?
receptor tyrosine kinases (RTKs)
their cytoplasmic domain functions as a tyrosine kinase which phosphorylates particular tyrosines on specific intracellular signalling proteins
have only one transmembrane segment (alpha helix)
65
How are RTKs activated?
Binding of a signal molecule to the extracellular domain of an RTK = two receptor molecules associate into a dimer.
If the signal molecule is itself a dimer = physically cross-links two receptor molecules
Other signal molecules induce a conformational change in the RTKs, causing the receptors to dimerize.
Dimer formation brings the kinase domain of each cytosolic receptor tail into contact with the other; this activates the kinases to phosphorylate the adjacent tail on several tyrosines.
Each phosphorylated tyrosine serves as a specific docking site for a different intracellular signaling protein, which then helps relay the signal to the cell’s interior
These proteins contain a specialized interaction domain that recognizes + binds to specific phosphorylated tyrosines on the cytosolic tail of an activated RTK or on another intracellular signaling protein.
66
important components (e.g proteins, receptors etc) in Fibrodysplasia ossificans progressiva
BMPs: bone morphogenetic proteins
ACVR1: is one of the receptors for BMPs, (mutated in FOP)
Activin A: Ligand for especially the mutated ACVR1.
Smad1/5/8: intermediate signal from BMP to nucleus
FKBP12: inhibitor, binds ACVR1, prevents BMP signaling through Smad1/5/8 – does not bind properly to mutated ACVR1.
RUNX2: critical transcription factor bone formation
67
Explain the process of BMP signalling in FOP
Binding of BMP to BMPRs dimer:
BMPs bind to a heterodimeric receptor complex consisting of Type I (ALK2, 3, or 6) and Type II receptors (BMPRII) on the cell surface.
Phosphorylation of Smad1/5/8:
Binding of BMP to BMPRs triggers the phosphorylation (activation) of intracellular proteins, particularly the R-Smad proteins: Smad1, Smad5, and Smad8.
Migration to the nucleus:
The phosphorylated Smads (1/5/8) form a complex with the Co-Smad (Smad4).
This complex then migrates from the cytoplasm to the nucleus.
Binding to other transcription activators:
Once inside the nucleus, the Smad1/5/8-Smad4 complex interacts with other transcription factors,
allowing the complex to influence gene expression
Activation of Runx2:
The Smad complex and co-factors activate Runx2, a critical transcription factor for bone formation.
Transcription of osteogenic genes:
Activation of Runx2 leads to the transcription of genes involved in osteogenesis (bone formation)
- Alkaline phosphatase (a marker of osteoblast differentiation).
- Collagen I (a major component of the bone matrix).
- etc
68
Structure of BMP receptors
ALK2= ACVR1
- Type I receptor
- Has a GS domain: Glycine and serine rich
69
Inhbition of BMP activity by FKBP12
FKBP12 binds to the GS domain of the Type I receptor, preventing its activation.
Upon BMP binding and activation of Type II receptor, FKBP12 is released.
Once released, Type I receptor phosphorylates Smad1/5/8 and other pathways
This is a Serine/Threonine kinase receptor, a type of enzyme-coupled receptor, facilitating BMP-mediated signaling pathways.
70
what does Activin-A do in FOP
Activin-A abnormally transduced BMP signaling in FOP-iMSCs
71
What do antibodies against activin-A do?
inhibit heterotopic ossification
72
Normally binding of activin .....
Binding of activin to the mutated receptor ...
inhibits smad 1/5/8 phosphorylation
activates the receptor and subsequently smad 1/5/8 phosphorylation
73
Only in which disease are there sig differences in gene expression based on the absence/presence of activin A?
FOP
so... Differential gene expression in FOP is due to Activin A
74
Pathways upregulated by Activin A in FOP
TGF-beta signalling
BMP receptor signalling
Signalling by activin
75
Membrane-enclosed compartments: numbers and % volume
Nucleus - 1 (6%)
Mitochondrion - 1700 (22%)
Golgi apparatus - 1 (3%)
ER - 1 (12%)
peroxisome - 400 (1%)
cytosol - 1 (54%)
lysosome - 300 (1%)
endosome - 200 (1%)
76
Membrane-enclosed compartments: functions
Nucleus - genetic information
Mitochondrion - energy
Golgi apparatus - Modification, sorting, packaging of proteins
ER - Lipid synthesis, packaging, delivery to organelles
cytosol - contains metabolic pathways, protein synthesis, cytoskeleton
peroxisome - Oxidative breakdown of toxic molecules
lysosome - Intracellular degradation
endosome - Sorting of endocytosed
material
76
What is the advantage of compartmentalization?
Compartmentalization separates processes
+ so they can occur in different environments e.g acidity
processes are concentrated due to this compartmentalization
77
How did compartmentalization arise?
pre-eukaryotic cells ingesting prokaryotic cells
78
3 ways of transport to organelles
proteins are made in the cytosol > organelles
To the nucleus:
1) through nuclear pores (selective gates + protein remains folded)
Into chloroplasts, mitochondria, peroxisomes, ER
2) across membranes (by protein translocators + protein must unfold)
Onward from ER > another compartment of the endomembrane system (e.g golgi apparatus)
3) by vesicles (pinch off membrane of one then fuse with membrane of the 2nd)
79
What do certain peptides determine?
certain peptides determine where a molecule is transported to (organelle wise)
signal sequences direct proteins to the correct compartment (usually removed from finished protein once it has been sorted)
80
Signal sequences for proteins destined for the ER vs those that remain in the cytosol
destined for ER:
N-terminal signal sequence directing them there
destined to remain in cytosol:
lack any such signal sequence
81
What techniques can be used to change the destination of proteins?
Recombinant DNA techniques in which the signal sequence is removed (from an ER protein for example) and attached to a (cytosolic protein for example). Both proteins are reassigned to the expected inappropriate location
82
What forms the nuclear envelope
Inner nuclear membrane + outer nuclear membrane which is continuous
Is perforated by nuclear pores
83
Nuclear pore complex of the nuclear envelope
forms a gate through which selected macromolecules and larger complexes enter or exit the nucleus
Protein fibrils protrude from both sides of the pore complex, on the nuclear side they converge to form a basketlike structure
- prevent the passage of large molecules but allow small water-soluble molecules to pass freely and non-selectively between the nucleus and cytosol
84
How does a protein move through the nuclear pore?
Proteins that migrate to the nucleus need a nuclear localization signal that is recognized by a nuclear import receptor which then directs the protein to a nuclear pore by interacting with the protein fibrils
85
Energy necessary for protein nuclear transport
Nuclear import receptor w/ prospective nuclear protein enters the nucleus
Encounters Ran-GTP which binds to the import receptor = releases nuclear protein
Receptor w/ ran-GTP is transported back through the pore to the cytosol where Ran hydrolyzes its bound GTP.
Ran-GDP falls off the import receptor and is carried back into the nucleus by its own unique import receptor
86
Unfolding of mitochondrial precursor proteins during import
Signal sequence on mitochondrial precursor protein is recognized by import receptor protein which is associated with a protein translocator
This complex diffuses laterally in the outer membrane until the signal sequence is recognized by a 2nd transolator in the inner membranes unfolding the protein in the process
signal sequence is finally cleaved off by a signal peptidase in the mitochondrial matrix
chaperone proteins help pull the protein across the membranes and help it to refold (energy from ATP hydrolysis helps chaperone proteins)
87
proteins enter the ER whilst...
they are being synthesized
88
free ribosome cycle vs membrane-bound ribosome cycle
free ribosome cycle: ribosomes translating proteins with no ER signal sequence remains free in the cytosol > polyribosome free in cytosol
membrane-bound: ribosomes translating proteins containing an ER signal sequence on the growing polypeptide chain
the ribosomes are attached to the cytosolic side of the ER membrane and are making proteins that are being translocated in to the ER
At the end of each round of protein synthesis, the ribosomal subunits are released and rejoin the common pool in the cytosol
89
How are soluble proteins made on the ER released into the ER lumen?
SRP (signal-recognition particle) binds to ribosome + ER signal sequence = slows protein synthesis until SRP engages with SRP receptor (on ER membrane) = SRP is released
Receptor passes the ribosome to a protein translocator in ER membrane + protein synthesis recommences
the protein translocator binds the (N-terminal) signal sequence + threads the rest of the polypeptide across the lipid bilayer as a loop
at some point the signal peptide is cleaved from the growing protein by a signal peptidase (this is ejected into the bilayer + degraded)
Once protein synthesis is complete, translocated polypeptide is released into ER
90
What determines the arrangement of a transmembrane protein in the ER lipid bilayer?
start and stop signals
91
transmembrane proteins with a single membrane-spanning segment in the ER
N-terminal ER signal sequence initiated the transfer (aforementioned process).
transfer process is halted by a second hydrophobic sequence which acts as a stop-transfer sequence
when this sequence enters the protein translocator, the growing polypeptide chain is discharged into the lipid bilayer
the N-terminal signal sequence is cleaved off = leaving the transmembrane protein anchored in the membrane
92
double-pass transmembrane protein in the ER
These have an internal signal sequence which starts the protein transfer and it is called a start-transfer sequence (+ its never removed from the polypeptide)
Internal sequence is recognized by an SRP which brings the ribosome to the ER membrane
When a stop-transfer sequence enters the protein translocator, the translocator discharges both sequences into the lipid bilayer
neither start nor stop sequence is cleaved off
93
What happens with proteins that span the ER membrane more than twice
they contain additional pairs of start and stop transfer sequences and the same process as the double-pass transmembrane protein is repeated for each pair
93
Explain the coated characteristic of vesicles
Vesicles that bud from membranes have a distinctive protein coat on their cytosolic surface that sheds off after budding from the parent organelle, allowing its membrane to interact directly with the membrane to which it will fuse
93
functions of the protein coat on vesicles
helps shape the membrane into a bud
captures molecules for onward transport
94
Transport of membrane components and soluble proteins between compartments of the endomembrane system
Endocytosis
Inward endocytic pathway that is responsible for ingestion + degradation of extracellular molecules
vesicles from plasma membrane delivered to early endosomes > late endosomes > lysosomes
Exocytosis
Outward secretory pathway
protein molecules transported from the ER, through the golgi, to plasma membrane or lysosomes
(movement through golgi occurs via vesicles that shuttle between individual cisternae)
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what is clathrin?
a protein that coats vesicles
clathrin molecules assemble into a basketlike network on the cytosolic surface of the membrane
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what is dynamin?
A GTP-binding protein that asssembles as a ring around the neck of each deeply invaginated clathrin-coated pit
dynamin causes the neck to construct, pinching off the vesicle from its parent membrane
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What is adaptin?
A class of coat proteins which secure the clathrin coat to the vesicle membrane and help select cargo molecules for transport
98
Outline the creation of a vesicle
cargo receptors with their bound cargo are captured by adaptins which also bind clathrin molecules to cytosolic surface of the budding vesicle
dynamin proteins assemble around the neck of the budding vesicles, then dynamin molecules hydrilyze their bound GTP + pinch off the vesicle
once budding is complete, coat proteins are removed + naked vesicle can fuse w/ its target membrane
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what are cargo receptors
they are transmembrane proteins which bind cargo molecules that have specific transport signals
99
receptors in vesicle docking
the target membrane has complementary receptors that recognize molecular markers displayed on the surface of each type of transport vesicle in the cell
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how does vesicle docking occur
tethering protein on a membrane binds to a Rab protein on the surface of a vesicle = allows the vesicle to dock
a v-SNARE on the vesicle then binds to a complementary t-SNARE on the target membrane = ensure that transport vesicles dock at their appropriate target membranes + catalyze the final fusion of the 2 membranes
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what can be used to tag a protein when a fusion of a protein wants to be tracked throughout the cell
green flourescent protein
102
What are the 2 pathways in secretory cells exocytosis ?
regulated pathway:
Extracellular stimulus (e.g hormone or neurotransmitter) is needed for release of cargo of the vesicles that are in the cytoplasm
golgi > secretory vesicle storing secretory proteins > signal transduction from extracellular signal > regulated exocytosis into extracellular space
constitutive pathway:
occurs w/o external signals
protein from golgi > transport vesicle > newly synthesized lipids and proteins exocytosed (unregulated) to extracellular space
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outline endocytosis
Pinocytosis: drinking
- Fluid containing smaller vesicles (< 150 nm)
Phagocytosis: eating
- Larger particles (> 250 nm)
- Bacteria
- Red blood cells/ defective cells
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receptor mediated endocytosis process
LDL (low density lipoproteins) binds to LDL receptors on the cell surface + it is internalized in clathrin-coated vesicles
vesicles lose their coat + fuse w/ endosomes (have acidiv env = LDL dissociates from its receptors)
LDL ends up in lysosomes where its degraded to release free cholesterol
LDL receptors are returned to the plasma membrane via transport vesicles
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what happens to receptor proteins after endocytosis
Receptors not specifically retrieved from early endosomes go to lysosomes where they are DEGRADED
or
retrieved receptors are returned to the same plasma membrane domain and RECYCLED
or
retrieved receptors are returned to a different plasma membrane domain and TRANSCYTOSED
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Degradation in lysosomes
Different pathways to lysosomes lead to the intracellular digestion of materials from different sources
early endosomes, phagosomes, and autophages can fuse with either lysosomes or late endosomes (both contain acid-dependent hydrolytic enzymes)
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what is autophagy
degrades obsolete parts of the cell, the cell eats itself
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when to use molecular cell bio techniques?
What is the molecular basis of differences between cells ?
How do cells respond to stimuli?
Which genes are important for the function of the cell?
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Principle of (Q)PCR
Specific amplification of DNA in a biological sample
Study amount of certain mRNA in cell
Isolate RNA from cell
Amount of RNA is too small to measure / visualize
Perform PCR for that specific gene
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what is cDNA and when is it used
To measure RNA levels, a DNA version of the RNA sequence is needed. This DNA copy is called complementary DNA (cDNA)
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role of reverse transcriptase in qPCR
Reverse Transcriptase is the enzyme responsible for converting RNA into cDNA, enabling DNA Polymerase (does not originally recognize or bind to RNA molecules) to subsequently amplify the sequence in qPCR
111
how is cDNA made ?
Total mRNA is extracted from a selected type of cell
Hybridize the mRNA with poly T primer
Make DNA copy with reverse transcriptase = mRNA/cDNA double helix
Partially degrade RNA with RNAse
RNA fragment that is left over and base-paired to the 3' end of the first DNA strand acts as the primer for DNA polymerase to synthesize the 2nd cDNA
Any remaining RNA is degraded during subsequent cloning steps
Left over w/ double stranded cDNA molecule
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Outline the 1st line of amplification in qPCR
1) double-stranded DNA is heated to separate the strands
2) DNA is exposed to a large excess of a pair of specific primers + the sample is cooled to allow the primers to hybridize to complementary sequences in the DNA strands
3) this mixture is incubated w/ DNA polymerase + 4 deoxyribonucleoside triphosphates so that DNA can be synthesized
process can then be repeated (only heating + cooling bc primers are there in excess and enzyme can be reused)
DNA is doubled at each amplification
113
use of flourescence in qPCR
Add fluorescent dye to PCR reaction mix (SYBR Green fluoresces when
bound to double stranded DNA)
During denaturation SYBR Green
is released and fluorescence reduces
During extension new double
stranded product is produced
SYBR Green binds to new product
resulting in increased fluorescence
Amount of fluorescence = amount of PCR product
Measure the amount of fluorescence after every cycle
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How to interpret a qPCR graph
when the line(s) cross the threshold value (horizontal line) these are the Ct values of each sample where each curve crosses the threshold, representing the cycle number at which detectable amplification occurs
lower Ct values = fewer cycles to reach the threshold = more of the target molecule was initially present
115
What suggests amplification is 100% efficient
double-ing per cycle
116
detecting only cDNA, not DNA
design primers that recognize two parts of adjacent exons, then you avoid that you amplify traces of DNA
So, only cDNA (the copy of mRNA) is amplified
117
The types of exon spanning primers
1) primers that hybridize sequences in 2 exons
2) primers that hybridize within different exons separated by a large intron
3) primers that hybridize within non-consecutive exons
118
Open ended vs. closed-ended approaches to molecular testing
Closed: PCR, gene sequencing: with known primers, you want to detect known sequences
Open ended: (Micro-array) or DNA or RNA seq. You do not know what to expect.
119
What is DNA sequencing
Process of determining the precise order of nucleotides (G, A, T, C) in a DNA molecule
120
Outline the components of Dideoxy sequencing (Sanger sequencing)
DNA Library: Collection of DNA fragments to be sequenced.
DNA Primer: Short DNA strand that initiates synthesis.
DNA Polymerase: Enzyme that synthesizes new DNA by adding nucleotides.
Dideoxy Ribonucleoside Triphosphates (ddNTPs): Modified nucleotides that terminate DNA synthesis when incorporated, creating DNA fragments of varying lengths.
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Outline the old and new ways of sanger sequencing
Old Way:
Gel-Based Separation: DNA fragments are separated by size on a gel (smallest DNA fragments migrate faster / further)
Manual Reading: Sequence is manually determined by reading the positions of each fragment on the gel.
New Way:
Fluorescent Labeling: Each nucleotide (G, A, T, C) is labeled with a distinct fluorescent color.
Automated Sequencing: Sequencing machines detect the colors and translate them into a nucleotide sequence, enabling faster and more accurate analysis
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what are ddNTPs structurally
derivatives of the normal deoxyribonucleoside triphosphates that lack the 3' hydroxyl group
they block further elongation
123
what happens when there is a surplus of normal nucleotides (DNA sequencing)
per chance you will get incorporation of the dideoxy form and the reaction will stop
124
illustrate the Sanger sequencing (dideoxy sequencing) process
Old way
A single-stranded DNA fragment is used as the template for sequencing.
DNA primer binds to the template DNA. (necessary for initiating DNA synthesis)
Add DNA Polymerase + normal deoxynucleotides (dNTPs)
Divide into four tubes, each containing a small amount of one type of chain-terminating dideoxy nucleotide (ddNTP): ddATP, ddTTP, ddCTP, or ddGTP.
Each reaction produces fragments that end at every occurrence of a specific base (A, T, C, or G) in the sequence.
The resulting DNA fragments of varying lengths are separated by size on a gel.
Shorter fragments (those closer to the primer) migrate further down the gel, while longer fragments stay closer to the top.
The gel is read from bottom to top to determine the sequence of the complementary strand (3' to 5').
By matching the sequence read from the gel with the primer sequence, the original DNA sequence can be determined.
125
illustrate the sequencing (dideoxy sequencing) process
New way
DNA is first hybridized with a short DNA primer.
The DNA is then mixed with DNA polymerase, an excess amount of normal dNTPs, and a mixture containing small amounts of all four chain-terminating ddNTPs, each of which is labeled with a fluorescent tag of a different color.
Each reaction produces a diverse set of DNA copies that terminate at different points in the sequence. The reaction products are loaded onto a long, thin capillary gel and separated by electrophoresis.
A camera reads the color of each band on the gel and feeds the data to a computer that assembles the sequence. The sequence read from the gel will be complementary to the sequence of the original DNA molecule.
A tiny part of the data from such an automated sequencing run. Each colored peak represents a nucleotide in the DNA sequence.
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which mutations/polymorphisms are visible in gene sequencing?
Visible:
- gene defect: protein is not formed or incomplete
Not visible:
- heterozygous (one of the 2 copies is affected)
- non-coding region of the DNA
- protein is still functional
127
What can mutations cause?
A mutation can give rise to a totally different amino acid sequence, resulting in a defective, non-functional protein
128
What causes juvenile periodontitis?
Mutation in Cathepsin C causes juvenile periodontitis
129
What role does cathepsin C play? + what process is interrupted?
Cathepsin C: necessary in granulocytes to kill bacteria
process interrupted:
1.Adhesion/recognition of a bacterium by a granulocyte
2. Uptake / phagocytosis
3. Degradation by the lysosome (proteases are needed here)
Normally granulocytes (neutrophils) resolve inflammation by killing bacteria. When inflammation around teeth can not be resolved and becomes chronic this can lead to periodontitis.
In periodontitis the alveolar (jaw) bone is broken down and ultimately teeth fall out.
130
What increases when marrying your cousin (represented by a double line)
chances increase that heterozygous, non- visible mutations become apparent
131
(bulk) RNA sequencing
- Open ended approach for differential gene expression
- High throughput sequencing
- Differences in expression between different conditions:
Control vs experimental condition
Healthy vs diseased sample
Wild type vs mutant sample
132
RNA sequencing methods
1) RNA isolation, make cDNA, add bar code (addition of a unique sequence per cDNA sample)
2) Sample and library preparation
3. Sequencing
4. Raw data analysis
133
Role of RNA sequencing in FOP
With RNA sequencing, you can analyse which pathways are altered
134
bulk sequencing VS single cell sequencing
Bulk sequencing is performed on all the (different) cells in a sample.
Single cell sequencing is performed on one single type of cell.
