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