4/17 - lec 22:1. Using the components discussed in class,
propose a signal transduction pathway that would result in the
release of cortical granules during fertilization. From this
pathway, identify three potential causes of CG-related
infertility.
Cortical granules: releasing components that form fertilization
envelope
Signal Transduction Pathway Leading up to CG release: 1. Ligand
binds to GPLR receptor on egg cell and activates it2. L-R complex
triggers conformational change, which is transduced to G-protein 3.
G-protein exchange GDP for GTP and becomes activated a. It also
separates into G-alpha and G-beta,gamma 4. G-alpha (bound to GTP)
travels along membrane until it encounters Phospholipase C5. Once
Phospholipase C is activated, it catalyzes the hydrolysis of PIP2
(phospholipid in the egg cell membrane) into DAG and IP3 a. DAG
will remain in the membrane, while IP3 is soluble in the
cytoplasm6. IP3 will diffuse into the cytoplasm and will bind to
calcium channels on membrane of Smooth ER, opening them and
releasing calcium into the cytoplasm by facilitated diffusion7.
Calcium levels in the cytoplasm will go above the basal level (10-4
mM) 8. Calcium will then bind to Calmodulin, activating it 9.
Calmodulin will then bind to Calmodulin-induced Kinase , 10.
Calmodulin-induced kinase will the phosphorylate (using ATP)
myosina. Myosin : cytoskeletal motor proteins11. Activated myosins
will carry cortical granules being formed up to the plasma membrane
for release. a. Cortical granules made in the endomembrane system
(being secreted outside the cell) 12. Myosins move CG up to the PM,
where their internal contents will be released and form the
fertilization envelope outside the fertilized egg
Causes of Infertility: Lack or abnormal CG formation Misfolded
G-protein Initial receptor is defective or absent G-protein cannot
exchange nucleotides Calcium levels do not get high enough to
activate calmodulin
2. Describe an enzyme-coupled receptor. Detail in detail the
structure and function of receptor tyrosine kinases (RTKs).
Enzyme-Coupled Receptors: Transmembrane proteins Subunits
consist of singlepass polypeptides Cytoplasmic domains Either have
intrinsic enzymatic activity Or associated with an enzyme Receptor
Tyrosine Kinases: class of ECRs Phosphorylate hydroxyl group on
tyrosine residues Involved in autophosphorylation Will
phosphorylate serine residues on themselves Changes properties of
molecules (adding polar, negative group) 3. Describe growth factor
characteristics and the roles of played by growth factors in cells.
Growth Factors: Common ligands for RTKs Small molecules capable of
stimulating cell growth, division, or differentiation Some can act
Broadly Affect many classes of cells Some can have Narrow targets
Affect one/few cell types (specific) EX: Erythropoietin Proper cell
behavior requires a specific combination of growth factors
Epidermal Growth Factor: broad range, stimulates proliferation of
many cell types and acts as an inductive signal during embryonic
development
4. Describe a monomeric G protein (e.g. Ras).
Ras Monomeric G-Protein Signal transduction component and
important regulator of cell growth Identified in rat sarcomas
Similar in function to heterotrimeric G-protein Associated with
cytoplasmic face of PM (monotopic membrane protein) Alternatively
binds to GDP and GTP Inherent GTPase activity can convert GTP back
to GDP by hydrolyzing phosphate group Only associate with RTKs not
GPLRs
5. Outline the events involved in a growth-factor/RTK-mediated
pathway. Using the example outlined in class, explain why
expression of cyclin/CDK is an appropriate outcome for such a
pathway.
Signal Transduction Growth Signaling Pathway via Ras: 1.
Epidermal Growth Factor acts as ligand or primary messenger and
binds to receptor, RTK 2. Binding of growth factors to individual
RTK polypeptides cause them to dimerize and autophosphorylating the
OH group on the tyrosine residues on eachother, activating the
complex 3. Activated RTKs transduce cascade to adaptor molecules
(or secondary messenger) a. Message transduced by a series of
conformational changes
4. Secondary messenger interacts with Ras (G-protein) 5. Ras
switches out GDP for GTP and becomes activated a. Ras changes
conformation when it binds with secondary messenger, thereby
changing affinity for nucleotides (GDP for GTP) 6. Phosphorylation
cascade occurs where is message is transduced down the rest of the
cascade by phosphorylation of one molecules triggering the
phosphorylation of another molecule 7. A molecules activated by
phosphorylation and enters the nucleus , where it causes changes in
gene expression 8. The products of the pathway are Cyclin and CDK
a. The intended message of this pathway is mitosis or cell
division/growth b. Cyclin and CDK regulate the progression of the
cell through the cell cycle c. The primary messenger was a growth
factor, so cell would eventually be triggered to go through
mitosis
6. Explain how cytosolic calcium levels can be regulated by
either a GPLR-mediated pathway or a RTK-mediated pathway.IP3
mediated release of calcium can involve: GPLR pathway RTK pathway
Involves activation of phospholipase C Triggers IP3 release, which
triggers calcium release in the cytoplasm EGF
Example of signal integration in which different receptors
activate the same pathway Could be a redundancy issue to ensure the
pathway gets activated If one pathway is faulty or doesnt get
activated, there is another pathway there to ensure other pathway
gets activated
7. Diagram the insulin/glucagon system used in our bodies to
maintain blood glucose homeostasis.
Normal Fasting Blood Glucose Level: 70-110 mg of glucose per 100
mL of blood
Insulin: peptide hormone, involved in uptake of glucose (to be
stored as glycogen) Glucagon : peptide (very small) hormone
involved in triggering the breakdown of glycogen and release of
free glucose into the blood
In Hyperglycemic State: Glucose levels above normal range Body
detects increased glucose levels Signal sent to Beta Cells in
Pancreas to become activated and produce and secrete insulin
Insulin released into bloodstream and binds to receptors on
different cells, causing them to take up glucose Also binds to
receptors on liver, causing it to take up glucose and store it as
glycogen Glucose is eventually cleared out of the bloodstream,
bringing blood glucose levels down to homeostatic level
In Hypoglycemic State: Glucose levels below normal range Signals
sent to pancreas and Alpha Cells, which are stimulated and produce
and secrete glucagon Glucagon binds to cells on liver, and triggers
breakdown of glycogen and release of it into the bloodstream
Continues until blood glucose levels rise back to homeostatic
level
8. Systematize the signal-transduction pathway involved
ininsulin-mediated uptake of glucose and glycogenesis. Explain how
defects in this pathway may lead to diabetes.
Insulin-mediated regulation of Blood Glucose Levels: 1. Insulin
acts as a ligand and binds to RTK receptora. Binding of insulin to
each polypeptide of RTK causes them to dimerize and
autophosphorylate 2. Binding of R-L recruits in molecule of IRS
(Insulin Receptor Substrate) a. IRS is a target of the RTK, which
will phosphorylate the IRSb. IRS also involved in transducing cell
division pathway involving Ras 3. IRS activates PI-3 Kinase4. PI-3
Kinase targets PIP2 (lipid in PM) and phosphorylates it, forming
PIP3 5. PIP3 recruits protein kinases, which leads to
phosphorylation and activation of Akt6. PIP3 activates Akt 7. Akt
causes production and activation of GLUT (Glucose Transporter) 8.
GLUT gets put into the membrane of the hepatocyte and facilitates
entry of glucose into the cell (and out of the bloodstream) 9. Akt
also activates Glycogen Synthase (by phosphorylation) a. Glycogen
synthase synthesizes glycogen
9. Define diabetes. What are the long-term effects of diabetes.
Differentiate between Type I and Type II diabetes.
Diabetes: a result of high blood glucose levels 2 Types: Type 1
Diabetes : Insulin Dependent (Insufficient Insulin) Results from
defect in beta cells of pancreas Beta cells being destroyed by the
body (autoimmune disorder), cells attacking beta cells, and
therefore, no insulin is being produced There is no insulin ligand
present to bind to receptor to trigger uptake of glucose Glucose
levels remain chronically high
Type 2 Diabetes : Insulin Independent (Insulin Resistance) Body
is producing enough insulin to begin with, but signal transduction
cascade is not responding properly Defect in cells ability to
recognize and process insulin message Blood Glucose Levels remain
chronically high
Long term effects of Diabetes: Can lead to heart disease, kidney
failure, and blindness
4/22 - lec 23:10. Describe in detail the steps taken by insulin
from gene expression to secretion from beta cells of the pancreas,
all the way to the binding of insulin to its receptor on target
cells. Explain why not all cells respond to insulin.
Insulin goes through the Co-Translational Import Pathway It is a
protein secreted from the beta cells of the pancreas It is a
vesicle that goes through the Endomembrane System Inside the Golgi,
it goes through post-translational processing Signaling sequence
gets cleaved off and becomes proinsulin Different chains held
together by disulfide bridges fully functional insulin
Once it leaves the beta cells, it travels to its target cells,
like the live, and binds to receptors on these cells, causing them
to take up glucose and store them as glycogen
11. List the functions of the cytoskeleton.
1. Organization:a. Spatial organization of cellular contents b.
Like cytoplasmic and nuclear (genetic) materials 2. Cell Shapea.
Provides mechanical support to the cell and nucleusb. Scaffolding
that helps generate maintain and maintain cell shape3. Motility: a.
Movement of the cell itself (cell crawling) b. Intracellular
movement of structures4. Cell Division: a. Manages chromosomes
(Microtubules bind to and help separate them) b. Cytokinesis
(Microfilaments actually divide the cell itself) 5. Regulation: a.
Transmits mechanical signals from environment i. Like adhesion
molecules, either directly or through adaptor molecules b.
Intracellular movement of molecules in signaling
12. Recall the three different elements that make up the
cytoskeleton and describe their main functions.
1. Microtubules: a. Spatial organization in cytoplasmb.
Intracellular transport (motor proteins) 2. Microfilaments: a.
Forming and maintaining cell shape b. Cell locomotion (specific to
crawling cells) 3. Intermediate Filaments: a. Mechanical strength
b. Nucleus shape ( and organizing chromosomes)
13. Describe the roles of cytoskeletal accessory proteins.
The cytoskeletal accessory proteins: Link cytoskeletal elements
to each other or to other cellular components Regulate
assembly/disassembly of elements 14. List the functions specific to
microtubules (MTs).Differentiate between cytoplasmic MTs and
axonemal MTs.
Microtubules: Cytoplasmic MTs : dynamic MTs Cell organization
(contents of cytoplasm) Intracellular movement (tracks for motor
proteins) Vesicles and organelles Chromosomes Axonemal MTs : static
MTs axoneme is a motility structure Components of motility
structures Flagella, Cilia
15. Describe in detail the structure of MTs.
Microtubule Structure: a single MT is a hollowed out tube made
out of protofilaments each MT is 25 nm in diameter (largest of the
elements) protofilaments made up of dimers of the protein tubulin
beta tubulin and alpha tubulin form tubulin heterodimers tubulin
heterodimers come together to form protofilaments, which come
together to form hallow tube oriented in a specific polarity beta
subunits face plus(+) end alpha subunits face minus (-) end Tubulin
Heterodimer : alpha and beta tubulin both bind to nucleotide GTP
only beta tubulin can hydrolyze GTP to form GDP (GTPase activity)
(and can exchange GDP for GTP) alpha tubulin is permanently bound
to GTP alpha and beta tubulin held together by non-covalent
interactions structural polarity to MT because subunits in each
protofilament all point in the same direction
Cytoplasmic MTs singlet arrangement of protofilaments hollow
ring comprised of 13 parallel protofilaments Axonemal MTs Doublet
arrangement Singlet ring with partial ring of 10 protofilaments
Triplet arrangement Singlet ring with 2 partial ring (10
protofilaments each)
16. Explain the model of MT assembly.
MT Assembly : Start off with population of tubulin dimers (alpha
and beta) Link together to form short fragments : oligomers Oligos
come together to form longer strands : protofilament Protofilaments
line up laterally to form sheet of protofilaments Protofilament
sheet folds in on itself to form hollowed out tube Hollowed out
tube grows from either by the addition of free tubulin dimers in
the cytoplasm to form fully functional Microtubule Growth of MT
starts out really slow with free dimers in the cytoplasm Growth
gets rapid once the dimers start linking to form protofilaments 17.
Define 'critical concentration' as related to MTs. Explain the
effects of the critical concentration on MT assembly. Explain how
critical concentration is related to the polarized assembly of
MTs
Critical Concentration : The concentration of free tubulin in
the cytoplasm at which rate of tubulin subunit addition is
approximately equal to the rate of subunit loss MTs tend to grow
when tubulin concentration exceeds Cc and depolymerize when tubulin
concentration falls below Cc Overall length change is constant
Reached during the plateau phase Point where growth slows down
dramatically and subunits and coming off and being added Cc differs
for plus end and minus end of MT, which is why is there is a
difference in rates of growth and disassembly Plus end grows much
faster than minus end ( elongates faster) Growth Rates reflect
differences in Cc requirements Plus (+) end: lower Cc Minus (-) end
: higher Cc Minus end will reach Cc earlier than plus end, as it
does so at a higher concentration of free tubulin Treadmilling:
simultaneous polymerizing (at plus end) and depolymerizing (at
minus end) within one MT Once Cc is reached at plus end (at a lower
concentration of free tublin), catastrophe event can occur Rapid
depolymerization at + end Growth/Shrinkage determined by critical
concentration
18. Describe the process of 'dynamic instability' and the role
played by GTP in this process.
MTs are continually changing and are very dynamic, at any given
time, some MTs are growing while others are shrinking
Dynamic Instability: Model to explain constantly changing MT
behavior In a given population of MTs: Some polymerize and grow
while others are depolymerizing and shrinking Polymerization may
continue for some undefined period of time MT may suddenly shrink
rapidly (catastrophe) Can also shrink partially and recommence
growing (rescue) Can also completely depolymerize and growth does
not resume Cc extremely sensitive to environment immediately
surrounding that end of MTRegulated by GTP Cap: Free tubulin is in
the GTP form Tublin dimers (bound to GTP) added to the of MT during
polymerization Eventually GTP within tubulin dimer is hydrolyzed to
GDP Beta subunit hydrolyzes GTP when it is a part of MT GTP
hydrolysis destabilizes MT structure Events occur at both ends of
MT (more activity at the plus end) At High Tubulin Concentration:
GTP-tubulin subunits get added to MT If Growth Rate > Rate of
GTP Hydrolysis As subunits get incorporated into MT structure, more
subunits will get added Beta tubulin will hydrolyze GTP to GDP, but
rate of growth greater than GTP hydrolysis GTP Cap on the end of
the MT Stabilizes MT and promotes further growth As growth
continues, rate of growth slows down because amount of free tubulin
in the cytoplasm decreases as growth occurs
At Low Tubulin Concentration: Growth Rate