Lecture 1 Notes
1. Signal transduction cascade
a. A receptor and subsequent elements of the response together
b. binding of the signal molecule causes a chemical or structural change to the receptor protein > causes a series of
intracellular signaling molecules > modified intracellular signaling molecules interact with effector proteins
(metabolic enzymes, cytoskeleton proteins, or transcription regulators) altering them to change the behavior of cells
in various ways
c. Examples
i. Cortisol > adrenal gland > steroid (derivative of cholesterol) > affects metabolism of proteins, carbs, and
lipids in most tissues
ii. Insulin > beta cells of pancreas > protein > stimulates glucose uptake, protein synthesis, and lipid synthesis
in various cell types
iii. Nitric oxide (NO) > nerve cells, endothelial cells lining blood vessels > dissolved gas > causes smooth
muscle to relax, regulates nerve-cell activity
iv. Acetylcholine > nerve terminals > derivative of choline > excitatory neurotransmitter at many nerve-muscle
synapses & in central nervous system
2. Four types of extracellular signaling:
a. Contact-dependent > Short distance, signal molecule is membrane-bound and binds to a receptor protein on a
neighboring cell
b. Paracrine > a cell releases a signal in their “neighborhood” and act locally
i. Examples > NO > gas molecule found in the endothelial cells in the blood vessels > can pass into cells and
activate enzymes directly
1. Endothelial cells release acetylcholine when increased shear stress is generated by the blood flow
(indication of limited blood flow) > acetylcholine binds to membrane receptor which activates the
NO synthase (produces NO)
2. NO diffuses to nearby smooth muscle cells in blood vessel walls > binds to guanylyl cyclase
(regulatory enzyme) that produces a signaling molecule cyclic GMP from GTP
3. Production of cyclic GMP causes rapid relaxation of smooth muscle cells > blood flow increases
4. Nitroglycerine (drug) can quickly be converted to NO in emergency situations > triggers relaxation
of the coronary blood vessels to relieve angina (chest pain when heart doesn’t get enough oxygenrich blood) usually a symptom of coronary heart disease
c.
Synaptic (Neuronal) > transmitted electrically along a nerve cell axon, when electrical signal reaches nerve terminal
a neurotransmitter is released onto a target cell
d. Endocrine signaling > hormones produced in endocrine glands are secreted into the bloodstream & are distributed
throughout the body
i. Examples > Small hydrophobic signaling molecules (can diffuse across plasma membrane and bind to
intracellular receptors) & response is slow because it involves making new proteins (activates transcription)
1. Sex hormones > estradiol & testosterone
2. Metabolic hormones > cortisol & thyroxine (physical development)
a. Hormone is released in bloodstream, it diffuses into the plasma membrane > it causes a
conformational change in a receptor protein activating it > the activated receptor-cortisol
complex moves into the nucleus > the complex binds to regulatory region of the target
gene and activates transcription
3. Insulin > Hydrophilic and bind to cell-surface receptors
3. How does insulin secretion lead to the lowering of blood glucose level?
a. High blood glucose level > islet cells secrete insulin (beta cell clusters) {regulated secretion} > insulin binds to
insulin receptors on cell surface > Insulin receptors activate (cross phosphorylation on tyrosine residues) > Many
intracellular signaling molecules activate > Vesicles containing glucose transporters fuse with the plasma
membrane (an example of regulated secretion) > increase in passive glucose transporters on the cell surface >
increase in glucose uptake > decrease in blood glucose level
4. What is the difference between type I and type II diabetes?
a. Type I > autoimmune disease, the immune cells kill beta cells within the pancreas
i. The immune cells kill almost all the beta cells shrinking islets, remaining beta cells produce insufficient
insulin
ii. The body cannot regulate blood glucose levels > high glucose levels can damage tissues
b. Type II > Caused by a high-carb diet, dysfunction of insulin cells ( eyelets stop secreting insulin), cells fail to
respond to insulin
5. What type of practice/treatments might help lowering the blood glucose level in type II diabetic patients?
Practice/Treatment
Type I
Type II
Metformin > Drug that increases glucose uptake in peripheral tissues (i.e. muscle cells)
✔
Metformin > Drug that decreases hepatic glucose export (liver cells convert glycogen to
glucose and export glucose to the bloodstream to increase blood sugar level)
✔
Control carbohydrate intake in the diet
✔
✔
✔
Glipizide > Drug that stimulates eyelet cells to secrete more insulin
insulin injection
✔
✔
Exercise
needs careful
monitoring
✔
6. Is the insulin receptor a GPCR or a RTK?
a. RTK > Receptor tyrosine kinases
i. Class of enzyme-coupled cell-surface receptors
7. RTK intracellular pathway
a. When the RTK is activated by signal molecule and phosphorylation occurs, an adaptor protein docks on a
phosphotyrosine (each phosphorylated tyrosine serves as a docking site for a different intracellular signaling
protein) > Ras guanine nucleotide exchange is recruited (Ras GEF) to adaptor protein, stimulates Ras which is
anchored to the plasma membrane by a covalently bonded lipid group to exchange its bound GDP for GTP > Ras
protein activates 3-kinase signaling module > the final kinase MAP kinase phosphorylates various downstream
signaling or effector proteins (changes in protein activity or gene expression)
8. Why is ATP needed for the activation of RTKs?
a. It provides the phosphate group necessary for the phosphorylation of tyrosine residues on the RTKs, a crucial step
in their activation and initiation of downstream signaling pathways.
9. RTK info
a.
b.
c.
d.
Transmembrane protein
Enzyme activity >
The signal molecule is in the form of a dimer so it can physically cross-link 2 receptor molecules
Ligand-binding on the plasma membrane & the cytosolic domain has either intrinsic enzyme activity or directly
associated with the enzyme
e. Intracellular change - When dimerization occurs > kinase domains on cytosolic tail are in contact > kinases
activated to phosphorylate several tyrosines on the adjacent receptor cell > this serves as a specific docking site for
a different intracellular signaling protein > this helps relay the signal to the cell’s interior
f. What will happen if the RTK is stuck in the active state and cannot be turned off?
i. It could lead to uncontrolled cell signaling, promoting aberrant cellular responses such as excessive growth
and survival. This dysregulation may contribute to cancer development, metabolic disorders,
developmental abnormalities, and cellular stress responses.
10. Ras Info
a. Belongs to a large superfamily of monomeric GTPase (like Ran- protein import into the nucleus and Rab - vesicular
targeting)
i. Ras is inactive when its bound to GDP (base, sugar, diphosphate) and is active when bound to GTP (base,
sugar, triphosphate)
ii. Made of conserved protein loops that cluster at one end of the protein
iii. Switch 1 & 2 change in conformational states when activated
iv. Ras hydrolyzed GTP to switch off > requires RasGap (Ras GTP activating protein)
1. RasGap binds tightly, burying the bound GTP & inserts an arginine side chain into the active site >
arginine, gln (in Ras), and thr (in Ras) promote the hydrolysis reaction
b. What will happen if Ras is stuck in the GTP-bound state?
i. Ras Is an oncogene. It is often highly expressed or overactive in cancer cells which results in continuous
cell proliferation
Lecture 2 Notes
1. GPCR Info
a. Largest family of cell-surface receptors
b. All have similar structure > polypeptide chain traverses the membrane as 7 alpha helices
i.
Cytoplasmic portion of receptor bind to a G protein inside the cell
c. Essential for vision, smell & taste
i.
Dysfunction results in numerous human disorders
ii.
~400 GPCRs in humans for the sense of smell alone
2. G-protein (Alpha-subunit of trimeric) Info
a. G-protein is made of 3 parts alpha & gamma (both covalently attached to the membrane) & beta
b. G-protein binds only after the GPCR is activated by ligand protein
c. The alpha-subunit is inactive when GDP is bound and active when GTP is bound (occurs when G-protein binds to
receptor)
i.
Then the G-protein activated a target protein in the membrane to send the signal downstream in the
signaling cascade
ii.
Eventually the alpha subunit hydrolyzes the GTP to GDP inactivating the subunit
1. Often accelerated by the binding of RGS (regulator of G-protein signaling > bound to the
membrane by covalent bonds)
iii.
Once re-inactivated the alpha subunit now reforms a G-protein with the gamma/beta complex
d. The activated G protein may activate an important functional protein directly, such as triggering an ion channel to
open, but commonly G proteins activate specific target enzymes that produce many additional small signaling
molecules, such as cAMP, cGMP and IP3.
3. How might ligand-binding be transduced into an intracellular signal?
a. When the protein ligand binds to the receptor it is activated,undergoing a conformational change that is transmitted
to the cytosolic region recruiting & activating a G-protein (trimeric GTP binding protein) > the activated receptor
causes a conformational change in the G-protein releasing the GDP > GTP binds to activate the protein > G-protein
dissociates into alpha and beta/gamma complexes > the activated complexes activate target proteins that relay the
signal downstream > eventually alpha subunit hydrolyzes GTP to GDP (often accelerated by RGS [regulator of Gprotein signaling] another protein) > inactive subunit reforms complex with beta and gamma subunits > turns of
other downstream events
b. As long as the receptor is activated by a ligand protein it will continue to activate G-proteins
i.
After prolonged stimulation the receptor eventually inactivates even if the ligand remains bound
1. A receptor kinase phosphorylates the cytosolic portions of the activated receptor & then binds to an
arrestin protein inactivating the receptor by preventing the binding of G-proteins > Arrestin acts as
an adaptor protein recruiting the receptor to clathrin coated pits > Receptors are endocytosed &
sent to lysosomes to be digested or sent to activate new signaling pathways
4. What is the signaling protein that a GPCR acts on directly?
a. G-protein or trimeric GTP binding protein
5. How is that protein activated?
a. A protein ligand binds to the ligand domain on the receptor & undergoes a conformational change that is
transmitted to the cytosolic region that activates a trimeric GTP binding protein (G-protein) by causing it to
dissociate from the GDP and replace it with GTP
6. The G-protein alpha-subunit activates adenylyl cyclase as one primary target.
What does this enzyme do?
a. Adenylyl cyclase converts ATP to cAMP which acts as the 2nd messenger that relays the signal from the G protein
coupled receptor to other components in the cell
7. What is the small molecule that needs to bind to PKA in order for it to be activated? How is this small molecule
produced (i.e. from what molecule? by what enzyme?)?
a. cAMP binds to PKA (cAMP protein kinase) on the regulatory subunit portion > the complex undergoes
conformational changes that free the catalytic subunits (now active) & phosphorylate specific target proteins
i.
Inactive PKA is made of 4 subunits > 2 regulatory & 2 catalytic
ii.
cAMP mediates signaling transduction in a wide range of tissues
b. Rise in intracellular cAMP concentration alters gene transcription:
i.
In some cases the activated catalytic subunits enter the nucleus where they phosphorylate a transcription
factor CREB > CREB recruits a CREB binding protein > the complex activates transcription by binding to a
regulatory region in promoters of target genes
8. How do cells respond to cAMP?
Epinephrine
Heart
Increase in heart rate and force of contraction
Epinephrine
Skeletal muscle
Glycogen break down
Epinephrine, glucagon
Fat
Fat breakdown
Adrenocorticotropic hormone (ACTH)
Adrenal gland
Cortisol secretion
9. Pathway of G-proteins & adenylyl cyclase
a. Activated G-protein activates a target protein adenylyl cyclase which converts ATP to cAMP > cAMP bind to PKA
on 2 regulatory subunit portions > the complex undergoes conformational changes that free the 2 catalytic
subunits (now active) & phosphorylate specific target proteins > activated catalytic subunits enter the nucleus
where they phosphorylate a transcription factor CREB > CREB recruits a CREB binding protein > the complex
activates transcription by binding to a regulatory region in promoters of target genes
10. Response of a rod photoreceptor cell in the dark
a. The rod cell is resting state without light the rhodopsins are inactive & the cGMP concentration in the cell is high
b. The cGMP gated cation channels are therefore kept open to maintain our constant influx of sodium and calcium
ions
c. The plasma membrane is kept at a depolarized state > Stimulates the release of neurotransmitter from the synaptic
region of the rod cell that signals to neurons in a retina
d. When the rod cells are in the dark and rhodopsin molecules are inactive
> the rate of neurotransmitter release is high
i.
Neurotransmitters are inhibitory > the bindings of these
neurotransmitters to the receptors in the downstream neuron
inhibits the activity of the neurons
11. Response of a rod photoreceptor cell in the light
a. Rhodopsin molecules in the disks decrease the cGMP concentration >
causes the closing of the cGMP gated cation channels
b. The plasma membrane is hyperpolarized (decrease in membrane
potential)
c. This inhibits this synaptic signaling by decreasing the rate of transmitter
release (in the dark neurotransmitter release from the rod photoreceptor
inhibits the postsynaptic retina neurons) > light activation serves to free
the neurons from inhibition exciting them
d. Activation is achieved by the removal of an inhibition (downstream neuron
activated)
12. What are the steps at which the signal is amplified? Activation
a. 1 rhodopsin molecule absorbs 1 photon > activates GPCR (rhodopsin)
b. 500 G-protein (transducin) molecules are activated by GPCR molecules
c. 500 cyclic GMP phosphodiesterase molecules are activated
d. 105 cyclic GMP molecules are hydrolyzed by cGMP phosphodiesterase > degradation of cGMP
e. 250 cation channels in the plasma membrane close blocking Na+ ions from entering
f. 106 - 107 Na+ ions per second are prevented from entering the cell for a period of ~1 second
g. Membrane potential is altered by 1 mV > membrane hyperpolarization due to Na+ ions not being able to enter the
cell
h. Signal is relayed to the brain
13. How is the rod cell restored to the resting state when the light exposure is removed? Inactivation
Uses multiple negative feedback loops to allow cells to revert quickly to an inactive state after light exposure
a. 1 rhodopsin molecule is inactivated by inhibitors prevent the rhodopsin molecule from activating more G-proteins
i.
Rhodopsin kinase partially inhibits the rhodopsin molecule by phosphorylation
ii.
Arrestin binds to the phosphorylated rhodopsin molecule
b. 500 G-protein (transducin) molecules are inactivated by RGS inhibitor protein
i.
Turns the G-protein to its inactive GDP binding state
c. 500 cyclic GMP phosphodiesterase molecules are activated
d. 105 cyclic GMP molecules are hydrolyzed > guanylyl cyclase activated to produce more cGMP
i.
This restores the cGMP concentration
e. 250 cation channels in the plasma membrane are re-opened
i.
Due to the presence of cGMP restoring the membrane potential to its previous state before activation
f. Influx of Na+ ions restores the cell’s membrane potential
14. Why is this important?
a. Maintaining visual sensitivity, adapting to changing light conditions, and ensuring continuous visual perception
15. What will happen to one's vision if this pathway fails to activate? Stays inactive.
a. If the pathway to restore the rod cell to its resting state fails to activate, it can lead to reduced dark adaptation,
decreased visual sensitivity in low light, impaired visual perception, and increased sensitivity to light.
16. What will happen if this pathway fails to deactivate? Stays active.
a. If this pathway fails to deactivate, the rod cell may remain in an overly sensitized state, resulting in prolonged light
sensitivity, difficulty adapting to changes in light levels, and potential visual disturbances such as glare or
discomfort in bright environments. This could also lead to difficulties in discerning fine details in well-lit conditions
and may contribute to visual fatigue or strain over time.
17. cAMP and cGMP are small molecule mediators used in many GPCR controlled pathways. How are they produced
and destroyed?
a. cAMP > concentration is dependent on relative activities of the cyclase (regulated by G-proteins) and the
phosphodiesterase
i.
Produced by adenylyl cyclase (enzyme) when it is activated by G-proteins & converts ATP to cAMP
ii.
Destroyed by cAMP phosphodiesterase (enzyme) & converts it to AMP
b. cGMP
i.
Produced by guanylyl cyclase
ii.
Destroyed by cGMP phosphodiesterase
18. Calcium release pathway
a. Signal molecule activated GPCR > G-protein is activated by GTP then activates target protein phospholipase C to
produce IP3 & diacylglycerol (membrane bound) > IP3 opens the calcium channel ( ligand-gated channel on the ER
membrane) to release calcium from ER lumen to cytoplasm > diacylglycerol & calcium attaches to PKC (protein
kinase C) > pathway is continued
b. Calcium functions as a intracellular mediator found in other areas > muscle contraction, regulated secretion
(secretion of neurotransmitters & proteins that change the surface structure of an egg upon fertilization)
19. Right after fertilization, how does regulative exocytosis help to prevent polyspermy?
a. Fertilization by sperm triggers an increase in cytoplasmic Ca2+ in the egg from the point of fusion > this triggers
vesicles in the plasma membrane that store hydrolytic enzymes that are released > swelling occurs that elevates
the fertilization envelope (also rising from the fusion point) to prevent the fertilization by more than one sperm
through exocytosis
20. What is the range of calcium concentration in the cytoplasm at the resting state (i.e. without stimulation)? What
about in the extracellular space? (Slide 19-Lecture 4: Refresh your knowledge about the calcium concentration
gradient across the plasma membrane).
a. Cytoplasmic Ca2+ free ion concentration at the resting state > 10-4 mM
i.
There is a total of 1-2 mM within cells that is bound to other molecules or stored in organelles
ii.
Free Ca2+ is tightly controlled because it is an important signaling molecule a low concentration must be
maintained
b. Extracellular Ca2+ concentration > 1-2 mM
21. Which organelle is the main calcium store for signaling?
a. Endoplasmic reticulum lumen
22. What are the three locations of the calcium pumps that help a cell to maintain a very low concentration of free
calcium in the cytoplasm at the resting state?
a. The plasma membrane
i.
Na+ driven Ca2+ exchanger & an Ca2+ pump powered by ATP
b. The ER
i.
Ca2+ pump powered by ATP
c. The mitochondria
i.
Active Ca2+ import using H+
d. Within the the cytoplasm
i.
Ca2+ binding molecules
23. Calmodulins
a. Cytoplasmic signaling proteins that sense the change in cytoplasmic [Ca2+] and relay to downstream factors (such
as calcium/calmodulin dependent kinases) by modifying their activities
b. Extremely abundant in eukaryotic cells and can constitute as much as 1% of the total protein mass
c. Serves as a multi-purpose extracellular calcium receptor > governed many calcium regulated processes
d. Consist of a highly conserved single polypeptide chain with a N & C terminal globular domain at each end
separated by an extended central helix with a total of 4 high-affinity calcium binding sites (2 in each domain)
i.
When activated by calcium binding > a conformational change occurs causing major allosteric changes the
two globular domains rotate relative to each other
ii.
Calmodulin then binds to, changes, and regulates the activity of target proteins
1. Wraps tightly around the target proteins > central helix breaks into 2 flexible helices creating a loop
24. What are the main ways for a cell to adjust its sensitivity to a signal?
a. Adaptation allows cells to respond to changes in the concentration of an extracellular signal molecule
(e.g.chemotaxis) rather than to an absolute concentration
i.
Reduction in the # of receptor proteins(storing or degradation), inactivation of receptors from cytoplasmic
side, & inactivation of intracellular signaling proteins by inhibitory proteins
b. Examples > Hormonal regulation, neuronal signaling & immune response
c. Why does a cell need to adjust the sensitivity?
i.
Cells need to adjust sensitivity for homeostasis, adaptation, signal integration, efficiency, and disease
prevention.
ii.
If a cell is exposed too long to the same signal it will desensitize or reduce its response even when the
concentration of the signal molecule remains the same
iii.
Failure to regulate the activity of signaling molecules properly can result in disease (e.g.retinitis
pigmentosa, cancer)
25. What determines the speed of a cellular response to an extracellular
signal?
a. Depends on the mechanism of signal delivery but also on the nature
of the target cells response
i.
Using existing proteins = faster response (seconds to
minutes)
1. Ex: neuronal signaling & insulin-induced glucose
uptake
ii.
New proteins as a result of altered gene expression = slower response (many minutes to hours)
1. Ex: cell growth & division
iii.
The speed of a response to an extracellular signal depends not only on the mechanism of signal delivery
but also on the nature of the target cells response
26. Signaling cascades depend on relay chains of intracellular signaling proteins and small intracellular mediators
A relay chain usually greatly amplifies the initial signal
a. Signal cascades are like computing devices
i.
They relay the signal from the side of reception to the site of response within the cell
ii.
They transform the signal from its original form to a form that can stimulate the response
iii.
They usually amplify the signal from a few molecules originally to a large cellular response
b. Different pathways might share downstream signaling molecules that
allow integration and distribution of the signal & also allows for
stimulating diverse responses by multiple cellular components
c. Many intracellular signaling proteins function as molecular switches
activated by phosphorylation or GTP binding
i.
Signaling by phosphorylation
1. Human genome encodes ~ 520 protein kinases and
150 phosphatases
2. Protein kinases & phosphatases
ii.
Signaling by GTP binding
1. Trimeric G-proteins & small monomeric GTP-ases
27. The response to a certain signal depends on the cell type, concentration of the signal molecule and the sensitivity
of the cell
i.
Different types of cells can respond differently to
the same extracellular signal
1. Acetylcholine receptor is a GPCR
a. Heart pacemaker cell > activated
receptor > Decrease firing rate
b. Salivary gland cell > activated
receptor > Increase in secretion
c. Endothelial cell > activated
receptor > Production of nitric oxide (NO)
d. Skeletal muscle cell > activated receptor > Acetylcholine acts as a neurotransmitter binding
to an ion channel > Leads to action potential in the muscle cell leading to contraction
ii.
Animal cells depend on multiple extracellular signals
1. Every cell type displays a set of receptor proteins that enables it to respond to a specific set of
extracellular signal molecules produced by other cells. These signal molecules work in
combinations to regulate the behavior of the cell.
a. May require multiple signals ( blue arrows) to survive, additional signals (red arrows) to
grow and divide, and still other signals (green arrows) to differentiate.
b. If deprived of the necessary survival signals, most cells undergo a form of cell suicide
known as apoptosis
Lecture 2 Notes
1. The cytoskeletal polymers form the structural framework for the cell (cell shape)
a. Examples
i.
Red blood cells
1. Cell shape allows the cell to undergo mechanical stress
2. Mutations in cytoskeletal components will alter the shape of the cell and weaken the cortex >
typically anemic
ii.
Epithelial cells
1. Have microvilli at the apical surface to increase surface area for material uptake > Microvilli are
supported by bundles of actin cytoskeleton
iii.
Muscle cells
iv.
Neurons
2. Three major kinds of biopolymers forming the cytoskeleton
a. Microtubules, Intermediate filament
b. Acton filament or microfilaments
3. Intermediate filament info
a. Properties - Only found in vertebrates and soft-bodied animals
i.
Non-polar
ii.
Form thick bundles in its distributed throughout the cytoplasm
iii.
Diameter of 10 nm
iv.
Made of rope-like fibrous intermediate filament proteins
v.
One type forms a meshwork called the nuclear lamina just beneath the inner nuclear membrane
vi.
Other types extend across the cytoplasm
1. Gives cells mechanical strength and distributing the mechanical stresses in an epithelial tissue by
spanning the cytoplasm from one cell-cell junction (desmosomes) to another
vii.
Very flexible and have great tensile strength, they deform under stress but do not rupture
b. Function : reinforcement of the cell membrane between cells and extracellular matrix through cell junctions to hold
cells together
i.
Cell shape, adhesions, movement & division
c. Assembly: Polymers composed of alpha-helical monomers more flexible
than actin and microfilaments & rope-like > formed by lateral bundling and
twisting of coiled coils
i.
2 monomers coil to form a dimer with asymmetric head and tail
(48nm) > 2 dimers stack together in an antiparallel, staggered
conformation, making them symmetric a tetramer > 8 tetramers
form the filament
ii.
Intermediate filaments are non-polar > The structures of both ends
of the intermediate filament are indistinguishable
4.
5.
6.
7.
8.
9.
10.
d. Intermediate filament types
i.
Keratin > protect epithelial
ii.
Keratin > found in hair, nails, & scales
iii.
Vimentin > strengthen cells & secure cells > connective-tissue cells, muscle cells, & glial cells
1. Type 3 is the most abundant & universally expressed among cell types
iv.
Neurofilaments > strengthen axons & regulate nerve impulse speed, change nerve axon diameter
v.
Laminin > nuclear lamina (in all animal cells)
vi.
Nestin > nerve cells, regulate diameter
How do cytoplasmic intermediate filaments, such as keratin, help to hold epithelial
cells together in a cell sheet?
a. Keratin filaments > mechanical stability in epithelial cells
b. Using these connections, intermediate filaments connect the entire cell sheet
forming a network > able to withstand mechanical stress
i.
Desmosomes interact with adjacent cells using transmembrane proteins
1. On the cytosolic side transmembrane proteins interact with
intermediate filaments using adaptor proteins
ii.
Hemidesmosomes > at the base of the epithelial cells & anchor the
intermediate filaments to the extracellular matrix
Keratin mutations (intermediate filaments)
a. Can cause skin blisters due to loss of tensile resistance in the basal cell of the
epidermis (cells in basal layer of epidermis detach from the connective tissue)
i.
Truncated keratin proteins are too short to form a complete network >
cells in the basal layer of epidermis are no longer firmly attached to the
basal lamina or to each other
b. Keratin mutations have been correlated with various defects in tissues of
epithelial origin (skin, hair, nails)
Do the subunits of intermediate filaments associate with any nucleotide?
a. Unlike tubulin and actin, the subunits of the intermediate filaments do not bind a nucleotide
What are the nuclear intermediate filaments in animal cells?
a. Form the nuclear lamina and provide structural support for the nuclear envelope
i.
During mitosis the lamins are phosphorylated > disassembly occurs & fragments the nuclear envelope >
allows the release of mitotic chromosomes to the cytosol
What type of human diseases could result from mutations in nuclear intermediate filaments?
a. Progeria > Defects in the nuclear lamina causes premature aging
i.
Children with progeria begin to show features of advanced aging and early life
Microtubule Info
a. Properties
i.
Polarized polymer
ii.
Tube-like polymers formed from alpha-beta protein tubulin heterodimers
iii.
Tubulin subunits are globulin proteins that bind to GTP
1. In the �-tubulin subunit the GTP is so tightly bound that it is considered to be an integral part of the
structure that it is ignored
2. In the �-tubulin subunit the GTP is more loosely bound and can be hydrolyzed to GDP > plays
important role in assembly regulation
3. �/� subunits are aligned into columns called protofilaments
4. Each protofilament in the microfilament has the same orientation causing polarization so each
hollow cylinder has structurally distinct ends
a. �-tubulin subunit forms the minus end > probability of depolymerization is higher
b. �-tubulin subunit forms the plus end > grows rapidly
iv.
Long, straight, hollow & typically have one in attached to a single microtubule organizing center called a
centrosome
v.
Outer diameter is 25nm
vi.
More rigid than actin or intermediate filaments & rupture when stretched
What is the role of GTP hydrolysis in regulating microtubule polymerization and depolymerization?
a. Growing microtubules
i.
Free tubulin GTP bound dimers add to the growing end of microtubule > addition happens faster than GTP
hydrolysis by the dimers
11.
12.
13.
14.
15.
16.
b. Shrinking microtubules
i.
Free tubulin GTP bound dimers add to the growing end of microtubule > GTP hydrolysis is faster than
addition of new GTP-tubulin dimers > GTP cap is lost > protofilaments containing GDP-tubulin peel away
from the microtubule wall > GDP-tubulin is released to the cytosol
c. Probability of growth or shrinkage is dependent on the rate of the addition of GTP subunit with respect to the rate of
GTP hydrolysis
Does the minus end tend to have a string of GDP-tubulin or GTP-tubulin? What about the plus end?
a. The minus end tends to have a GDP cap & the plus end tends to have a GTP cap
What is "treadmilling"?
a. Caused by adding microtubule subunits at the plus end and losing subunits at the minus end & at certain
concentration ranges of free tubulin subunits > GTP-tubulin adds to the plus end of a microtubule at the same time
as GDP-tubulin is lost from the minus end
b. Energy input from GTP hydrolysis by tubulin subunit can drive directional translocation of microtubules (active
transport)
What is the effect of taxol on microtubule polymerization?
a. Taxol (paclitaxel) binds to filaments and prevents depolymerization
i.
Stabilizes microtubules by forcing the tubulin subunit into the straight conformation that favors continuous
growth
ii.
Naturally produced by a fungus living in the pacific yew trees and some other fungi
b. Colchicine or colcemid forms a complex with tubulin dimers that binds to the ends of a microtubule preventing
further polymerization
c. Nocodazole binds tubulin and prevents their polymerization
Why can microtubule drugs be used as anti-cancer drugs?
a. Can be used to treat aggressive cancers (lung, breast, head & neck)
b. Microtubule drugs disrupt cancer cell division, induce cell death, and inhibit blood vessel formation crucial for tumor
growth. They're selective for fast-dividing cancer cells, sparing normal cells to some extent. Often used in
combination therapies, they're effective against various cancers, although side effects need management.
What is the core component of the microtubule nucleation complex in a cell? Which end of the microtubule does
this complex associate with?
a. �-tubulin ring complex nucleates and caps the minus end of a microtubule
Why does polymerization happen faster at the microtubule organizing centers compared with other parts of the
cell?
a. The �-tubulin ring complexes In the centrosome facilitates the nucleation of microtubules skipping the lag phase so
microtubules preferentially grow from the centrosome
How does this explain why often most microtubules in an animal cell radiate from the centrosome?
b. The �-tubulin ring complexes are part of the centrosome matrix & There is a high concentration of �-tubulin ring
complexes here compared to other parts of the cell
i.
The centrosome is also called the microtubule organizing center (MTOC)
17. Time course of polymerization in vitro
a. The assembly of a protein into a long helical polymer such as cytoskeletal filament or a bacterial flagellum
b. Lag phase > rate limiting step, time taken for nucleation, seeding structures (nucleus) made of several subunits
formed (contains a ring of 13 or more tubulin molecules) probability is low
c. Growth phase > occurs as monomers add to the exposed ends of the growing filament elongation
d. Equilibrium phase or steady state > reached when the growth of the polymer due to monomer addition precisely
balances the shrinkage of the polymer to disassembly back to monomers
18. Use of microtubules for transport
a. Not only individual microtubules themselves but the entire microtubule array also needs to have a distinct polarity
b. Microtubule-based motors are important vesicle transporters.
c. 2 globular heads bind & walk on the microtubule
d. The tail motor protein bind to the cargo (vesicle or another microtubule) directly
or indirectly through adaptor proteins
i.
Different motor proteins transport different types of cargo along
microtubules
1. Kinesin > can use different types of kinesin motors sometimes
binding directly to the cargo
a. Moves to the plus end
b. Transports vesicles, organelles, or molecules
2. Cytoplasmic dynein > always uses adaptor proteins to interact
with its selected cargo
a. Moves to the minus end
b. Cytoplasmic dynein > always uses adaptor proteins to
interact with its selected cargo
19. What is the energy source that drives directional movement?
a. ATP hydrolysis by the globular heads provides the energy source for directional movement
b. ATP hydrolysis loosens attachment of one head to the microtubule > ADP release & ATP binding change
conformation of the second head which pulls the first head forward
20. Mitotic spindle
a. Dynein pull the centrosome towards the plasma membrane by walking towards the minus end on astral
microtubules
b. Kinesin-4, 10 transport chromosomes to the plus end
c. Kinesin 5 is a tetrameric motor that pushes the centrosomes apart by walking to the plus ends of the antiparallel
microtubules
d. Spindle length is determined by the balance of all these different forces
21. How do microtubules work together with dyneins to generate the beating motion of a flagellum? What is
the energy source that drives this motion?
a. 9 outer microtubules (each are a paired structure) carry 2 rows of dynein molecules
b. The heads of each dynein molecule reach out toward the adjacent doublet microtubule
c. Periodically the dynein heads make contact with the adjacent doublet microtubule & move along it
producing the force
22. How is the direction of the flagellum bending related to the direction of dynein movement along the
microtubule?
a. Dynein movement towards the minus end of the microtubule on one side causing bending
23. What will happen if nexin (the linking protein) is removed from the flagellum/cilium?
a. Without linking proteins the microtubule bundles can slide apart when the dynein motors walk (the tail
end moves towards the minus end and the other to the plus end)
24. Why do people who have dysfunctional ciliary dynein often have issues with chronic lung infection?
a. Cilia also sweep inhaled particles of dust smoke as well as bacteria up and out of the respiratory tract
b. If the ciliary dynein is dysfunctional their lungs cannot clear particles
25. Actin Filament Info
a. Polarized polymer
b. Thin flexible helical polymers made of protein actin
i.
Thinnest of the 3 types
c. 7nm diameter
d. Organized in linear bundles, 2D networks, & 3D gels
e. Highly concentrated in the cortex just beneath the plasma membrane
i.
Form transverse bundles closer to the front edge of the cell
f. Actin monomers exist in ATP and ADP binding form > mainly ATP binding in cytosol
g. Plus end is the barbed end (where monomers are added) & likely to have a string of ATP actin
h. Minus end is the pointed end (where monomers are disassembled)
26. What do actin and actin binding proteins do?
a. Involved in cell division, movement, endocytosis, muscle contraction, and determining cell shape
i.
All use one or both of the basic properties of actin polymers > actin polymerization & actin-myosin
contraction
b. Nucleating proteins (ARP & formin), monomer sequestering protein, severing protein, cross-linking protein (in cell
cortex), capping protein (plus-end blocking), side-binding protein (tropomyosin), myosin motor protein, bundling
protein (in filopodia)
27. The ARP Complex or Actin-Related Protein
a. Specifically the ARP2/3 complex, nucleate actin filaments from existing
filament at a 70 degree angle, which are essential for various cellular
processes such as cell motility, endocytosis, and cell shape
maintenance.
b. The ARP2/3 complex has a high concentration at the cell front > activity
for actin polymerization is higher in this region
c. The branched actin mesh work nucleated by ARP complex help to
generate membrane protrusion (polymerization) at the cell front >
directional cell movement
i.
The cell adheres to a surface through focal contacts > when the
new protrusion is generated it is attached by a new focal contact
ii.
Contraction occurs at the back end of the cell > breaking of focal
contacts using actin myosin driven contraction from the
attachment
iii.
The combination of actin polymerization at the front of the cell
and the actin-myosin contraction at the back of the cell, the cell can move directionally
What kind of higher order actin structure does it organize?
d. The higher-order actin structure organized by the ARP2/3 complex is known as the dendritic actin
network > actin filaments branch off from pre-existing filaments, forming a complex and interconnected
network resembling the branches of a tree.
28. How can the binding of a signal molecule to its surface receptor result in localized actin polymerization
to change cell shape and direction of movement?
a. A signal (like a nutrient source) appears and is detected by the cell > Disassembly of filaments and rapid
diffusion of subunits occurs > Reassembly of filaments at the new site takes place > Enables cell to
move towards the source
b. Cooperation between signaling and structural proteins occurs to accomplish localized polymerization
i.
Links extracellular signals to the response needed from the cell
29. Structure of myosin II.
a. The coiled-coil tails of myosin II molecules associate with one another to form a bipolar myosin filament
(the heads project outward from the middle in opposite directions) The bare region in the middle of the
filament consists of tails only.
b. In muscle cells, Myosin filaments are also called “thick filaments”
c. Myosins > motors that bind to actin filaments > uses ATP to walk towards the plus end
d. Myosin II molecules can associate with one another to form myosin filaments
30. Myosin power cycle (how the myosin walks on the actin filament)
a. An empty myosin head binds to the actin filament > rigor state > rigor mortis (when ATP isn’t available)
b. Muscle myosin is a dimer with 2 identical motor heads that act independently > each one has a catalytic
core and attached lever arm w/ a coiled-coil rod tying the 2 together tethering them to the thick filament
i.
They myosins heads interact with the actin filament
c. The head have ADP & P & have a weak affinity for actin > when one of the heads docks on the actin > P
is released strengthening the bond between the head and actin > triggers the force generating power
stroke that moves the actin filament
d. ADP dissociated from the docked head > ATP bind to the empty nucleotide binding site > causes the
head to detach from the actin > ATP is hydrolyzed and recocks the lever arm back to the pre-stroke state
e. The arm stores the energy released by ATP hydrolysis > cycle is repeated
f. Actin does not slide back because other myosin molecules are attached holding the tension
31. How is the signal transmitted from a motor neuron to a muscle cell? What happens right after the action
potential reaches the nerve terminal of the motor neuron?
a. An upstream motor neuron send an excitatory signal onto junctions between the neurons and muscles
b. The electrical signals have to get through the synapse before you get electric signals on the muscle
c. At the nerve end, calcium channels open up as a result of the electrical signal > calcium enters and
vesicles fuse to the membrane and release content (neurotransmitter: acetylcholine) that binds to the
receptors on the muscle side [receptors are ion channels that open up] > Sodium enters the muscle &
triggers electrical signal to the muscle which depolarizes the membrane
32. How does the cone shell toxin, prialt, cause paralysis? Or block activation of the muscle cell?
a. Prialt plugs up the calcium channels on the nerve > electrical signal still arrives at nerve end but no Ca+
enters the nerve stopping the cycle
i.
No release of neurotransmitters, opening of the receptors on the muscle end > signal is stopped
33. How does the propagation of action potential on muscle cell membrane result in the rapid increase in
calcium concentration in the entire muscle cell? Focus on the distribution of the two types of Ca2+
channels and the structural features of the SR and the plasma membrane of the muscle cell.
a. When cell is stimulated, depolarization occurs > the action potential travels along the t-tubules
(invaginations of the plasma membrane)
b. Voltage-sensitive proteins opens a calcium channel releasing Ca2+ into the cytosol from adjacent
sarcoplasmic reticulum (specialized ER in muscle cells)
i.
The t-tubules and SR are only about ~35nm from each other > they work together to regulate
cytoplasmic calcium concentration in response to action potentials
c. Within the myofibril (contractile bundle of a muscle cell) Ca2+ interacts with protein filaments to trigger
contraction
i.
Myofibrils are ~2 microns in diameter
ii.
Muscle cells are ~50 microns in diameter
d. In each sarcomere (contracting unit) thin actin and thick myosin filaments are juxtaposed
i.
Sarcomeres contain: a dark band (more proteins), 1 half of a light band (fewer proteins) on each
end, and ends with a Z disc
1. Dark band > thick filaments or myosin II filaments
2. Light band > thin filaments or actin
ii.
Cannot interact without calcium
1. Myosin binding sites on the filaments are all covered by tropomyosin (rod-shaped protein),
at the end of each tropomyosin molecule is troponin (calcium sensitive complex)
2. When calcium enters > troponin binds to it moving tropomyosin off the myosin binding
sites > when the binding site is opened on the actin filaments, myosin motors can crawl
along the actin > muscle fiber contracts > calcium then returns to the sarcoplasmic
reticulum by calcium pump
3. Without calcium myosin releases actin > filaments slide back to original positions
34. Orientation of the actin filaments (i.e. the positions of the plus and minus end)
a. The Z discs are at the plus ends of the actin filaments & minus ends are near the myosin filaments
b. Myosin filaments > thick filaments
c. Actin filaments > thin filaments
d. Myosin motor heads walk to the plus end on actin filaments shortening sarcomeres causing muscle
contraction
35. Tropomyosin and Troponin complex
a. Actin-associated protein complex binds to calcium in the muscle cell > Critical for regulating muscle
contraction in response to change in cytoplasmic calcium concentration
b. Blocks the myosin binding site on the actin filament when calcium concentrations are low
c. Calcium will release the troponin from the actin allowing the myosin to bind > affecting the interaction
between the actin filaments and myosin motors
36. Restoring the system: relaxation
a. After each contraction Ca is pumped back by an ATP-dependent pump on the SR
i.
Muscle contraction consumes large amounts of ATP > to power the myosin motors for contraction
and to power the calcium pumps for relaxation
ii.
2 calciums bind to pump and are transported from cytosol to the lumen of the SR
37. Actin-myosin contraction is also present in non-muscle cells
a. Drives cell movement and cytokinesis [similar mechanisms > different cellular context and architecture of
contractile apparatus]
i.
When adherent cells move there is a contractile force at the back of the cell to break focal
contacts
1. Generated by actin-myosin structures
ii.
During cytokinesis actin & myosin filaments form the contractile ring that divide cells
1. Rings is essentially a series of mini sarcomeres
2. As ring contracts it pulls the plasma membrane inward