Bio 215 exam 4

Define ‘signal transduction’.

means of a cell sensing a signal from its environment

1. reception: cell senses a signal through receptor proteins

2. transduction: relaying the ‘message’

3. response

Explain how a signal molecule which does not enter a cell can alter cell activities.

ligand (or signal molecule) binds to receptor outside the cell, triggering a conformational change and a subsequent transduction/ response scheme

extracellular ligand

secondary messengers; small, non-protein, water-soluble molecules or ions that spread throughout a cell by diffusion

* function to propagate signal transduction pathways

cytoplasmic ligand

• found in the cell, tend to be hydrophobic and small since they cross across PM

• can activate intracellular receptors found inside the cell

• enter PM→ bind to receptor protein to create hormone receptor complex→ HRC enters nucleus and binds to DNA to create mRNA and new protein

• ex: estrogen, thyroid hormone, steroids

Describe the receptor-ligand interactions. Compare this interaction to previous intermolecular interactions discussed in class (e.g. enzyme-substrate, solute-transporter)

• R-L interaction is very specific;

  • ligand fits into ligand binding domain on receptor (similar to ES and Sol-Trans)

• temporary interaction

• receptors can be saturated (finite number of receptors)

Local regulators

-messenger molecules that travel only short distances

• cell junctions

• cell-cell contact

• secretion and diffusion of signals

• paracrine signaling (local regulator diffuses thru EC fluid)

• synaptic signaling (electrical signal along nerve cell triggers release of neurotransmitter which diffuses across synapse and targets cell)

Long-distance signaling

endocrine hormones

* synthesized in a particular location→ secreted into blood→ travels long distances from site of production→ induces a response in a target cell/tissue located a long distance from the OG source

What determines if a cell can respond to a particular signal?

depends on whether or not it has a receptor specific to that signal

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Paracrine vs endocrine hormone

• paracrine- which local regulator diffuses through extracellular fluid

• endocrine- made n certain place and travels through b stream where it will induce response from OG cell

Explain signal amplification and its significance in cell signaling.

small quantities of ligand are often sufficient to elicit a response from the target cell

• at each step in the resulting cascade

  • signaling intermediate stimulates the production of many molecules needed for the next step and repeats

• this produces more secondary messengers

  • ex: Epineph= Gplr in 1:1 ratio

  • GPLR= G protein in 1:100 ratio

  • glycogen phosphorylase= G1P in

    10^6 :10^8 ratio

Signal Amplification

multiplication of the effect of the original signal

GPCRs

• G protein-coupled receptors

• multipass integral mem protein

• the largest fam of cell-surface receptors

• ligand binding causes a change in receptor conformation that then activates a particular G proteins

• GCPR structure

  • 7 trans mem spanning domains

  • ligand binding domain

  • G-protein interaction domain

G-protein

• Guanine-nucleotide binding proteins

• G protein acts as an on/off switch

  • bound to GDP= inactive G protein

  • GTP bund= active

two classes of G proteins

1. heterotrimeric

2. monomeric (cytoplasmic, no link to receptors)

Heterotrimeric G proteins

• mediate signal transduction cascades via interactions with GCPRs

• 3 subunits

  • alpha subunit

  • beta/gamma subunits (permanently bound to each other)

G alpha

• binding domain for GDP to GTP

• also has GTPase activity

• when Ga binds to GTP= Ga detaches from the G b/g subunits

G protein Activation/Inactivation Cycle

1. Ga bound to GDP (inactive) - resting state

2. ligand binds receptor

3. receptor binds G protein; Ga releases GDP and acquires GTP (active)

4. Ga and Gby subunits separate

5. G protein subunits activate or deactivate target proteins; initiate sig trans

6. Ga subunit hydrolyzes its bound GTP to GDP, becoming inactive

7. Ga and Gby recombine to form inactive G protein

What is cAMP? Outline the general signal transduction pathway involving cAMP.

• cyclic adenosine monophosphate

• in response to signals, an enzyme called adenylyl cyclase converts ATP into cAMP, removing 2 phosphates and linking the remaining phosphate to the sugar

• once generates cAMP can active protein kinase A (PKA) enabling it to phosphorylate its target and pass along the signal

• PKA is found in a variety of cells and it has diff target proteins in each

• this allows the same cAMP to produce diff responses in diff context

Explain how cAMP levels are regulated by adenylyl cyclase and phosphodiesterase.

• adenylyl cyclase- enzyme to catalyze the conversion of ATP into cAMP

• phosphodiesterase- enzyme that cleaves remaining cAMP into AMP and deactivates cAMP

• once G protein is inactive→ AC stops making cAMP

• remaining cAMP continues to propagate signal until phos degrades it

Kinase

catalyze addition of phosphate group form ATP to substrate

Phosphorylase

catalyzes addition of phosphate group from an inorganic phosphate to a substrate

Phosphatase

type of hydrolase that removes a phosphate group

Recall the pathways involved in glycogen metabolism. 

• glycogenolysis- phosphorolysis of glycogen to release G1P (G1P→G6P)

• Glycogenesis: G6P→ G1P

• glycolysis: glucose→ G6P→F6P→ F1,6P → (DHAP and G3P)→ 2 pyruvate

Explain the mechanisms involved in regulating the glycogen phosphorylase enzyme.

• glycogen phosphorylase b: inactive can become active using phosphorylase kinase and ATP→ADP

• glycogen phosphorylase a: active can become inactive using phosphorylase phosphatase and h2o → inorganic phosphate

• cAMP regulates PKA which then regulates phosphorylase kinase

Outline the signaling cascade involved in the "fight or flight" response, as discussed in class.

* threat→ hypothalamus→ pituitary → adrenal gland (top of kidney, endocrine gland)→ epinephrine (hormone released from adrenal gland, then travels through circulatory system)→ epinephrine molecules on the heart and liver cells (molecules contain receptors that are the target cells) → if on heart cell then increases heart rate; if on liver cell then breakdown of liver glycogen

Flight or fight response- Students should specifically be able to describe the epinephrine-triggered increase in blood-glucose levels as part of this response. 

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1. cAMP binds to and activates protein kinase A

2. Protein kinase A phosphorylates phosphorylase kinase, activating it

3. active phosphorylase kinase phosphorylates phosphorylase b, converting it to phosphorylase a, the active form of the enzyme

4. phosphorylase a catalyzes cleavage of a terminal glucose form glycogen as glucose-1-phosphate, which increases glucose levels in the blood

PKA

also phosphorylates glycogen synthase; which inactivates it for regulation

Describe in detail the IP3/DAG signaling pathway.

1. receptor is activated by ligand binding (recall how GTP and alpha subunits react when ligand binds)

2. subunits split up

3. GTP-Ga complex then binds to phospholipase C (P), activating it and causing cleavage of PIP2 into IP3 and DAG

4. IP3 is released into the cytosol where it triggers calcium release

5. DAG remains in the membrane, where it activates protein kinase C

Recall the basal cytoplasmic \[Ca2+\]. Compare that to \[Ca2+\] in the ER and/or the extracellular space.

• most Ca is stored in the smooth ER

• resting [Ca] in cyto= 10^-4

• [Ca2+] in EC= 1mM (bc stimulated→ levels increase→ higher conc in cytoplasm)

• [Ca 2+] in ER= 0.2-0.3 mM

Describe the structure of the calmodulin protein.

• small dumbbell-shaped protein composed of 2 globular domains connected together by a flexible linker

• each end bonds to 2 Ca ions (alpha helix structure)

Explain the regulation of calmodulin involving calcium levels.

when Ca in the cytoplasm is greater than 10^-4→ calmodulin complex binds 4 calcium ions, calmodulin changes conformation resulting in an active complex, and the 2 globulars “hands” of the complex wrap around a binding site on a target protein

List and describe the different signaling components involved in calcium-mediated signaling.

• calmodulin- protein that changes conformation after binding ca ions

• calcium-calmodulin complex- active complex that results from changes conformation

• target protein- contains calmodulin binding site that calmodulin protein binds to for signaling

• exerts allosteric regulatory effects

Describe the general tissue structure of a blood vessel.

• contains the endothelial cell and smooth muscle cells

• endothelial lines the inside of the vessel and smooth muscle cells line the outside

Outline in detail the steps involved in calcium-mediated vasodilation.

Ca induces Nitric oxide production leads to vasodilation

1. acetylcholine binds to G protein-linked receptor on endo cell surface and IP3 enters cell

2. IP3 travels to ER

3. Ca2+ activates calmodulin→ activates nitric oxide synthase → moves to smooth muscle cell

4. activates guanylyl cyclase which used GTP to produce cGMP

5. cGMP activates protein kinase G

6. muscle relaxation dilation of blood vessels

Describe the effects of nitroglycerin. How do they influence the pathway? At which specific step are they involved?

• catabolized to release NO, therefore involved in the activation of guanylyl cyclase

• stimulates vasodilation

Describe the effects of Viagra on the vasodilation pathway. How do they influence the pathway? At which specific step are they involved?

• inhibits cGMP-phosphodiesterase (cleaves cGMP to GMP→ DEACTIVATES cGMP)

• competitive inhibitor of cGMP PDE (therefore inhibits the breakdown of cGMP)

• prolongs vasodilation

• stops vasodilation sig trans

What is cGMP-phosphodiesterase?  How is it involved in the vasodilation pathway?

• enzyme that cleaves cGMP to GMP

• deactivates cGMP

• stops the vasodilation sig trans cascade

Describe an enzyme-coupled receptor.

• transmembrane proteins (subunits consist of single pass polypeptides)

• cytosolic domain - some ECRs have intrinsic enzymatic activity while others directly associate w/ an enzyme

• 6 diff classes

• ECRs and GPLRs can activate same signaling pathways- safety mech if mutation causing deficit in one of them

Describe in detail the structure of ECR

* structure: monomer; receptor binding site outside of cell, goes through plasma membrane , tyrosine kinase in cytosol, attached to cytosolic tail

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function of receptor tyrosine kinases (RTKs). 

• ligand binding induces dimerization→ activation and autophosphorylation

• autophosphorylation- phosphorylate tyrosine residues on themselves (and other transduction components)

What is a growth factor?

• common ligands for RTKs

• small molecules capable of simulating cell growth cell division and/or cell differentiation

• proper cell behavior often requires a specific combination of growth factors

Outline the events involved in a growth-factor/RTK-mediated pathway.

1. ligand binds to receptor

2. autophosphorylation of tyrosine

3. binding of cytosolic proteins w/ SH2 domains

4. activated PLCy stimulates IP3-DAG pathway

Purpose of IP3 -DAG pathway

• DAG and IP3 are important secondary pathways

• DAG activates protein kinase C

• IP3 stimulates release of Ca release from ER

Levels of blood glucose

normal (homeostasis): 70-110 mg glucose/100 ml blood

hyperglycemia: high blood glucose, stimulates insulin

hypoglycemia low blood glucose. stimulates glucagon

Hyperglycemia- process

stimulus: rising blood glucose (after eating high carb)

1. beta cells of pancreas stimulated to release insulin into the blood

2. liver takes up insulin and stores it as glycogen

3. OR body cells take up more glucose

4. blood glucose level declines to set point; stimulates for insulin release diminishes

5. normal glucose level

hypoglycemia- process

stimulus: removal of excess glucose form blood; low blood glucose level

1. alpha cells of pancreas stimulates to release glucagon into the blood

2. glucagon goes to liver; breaks down glycogen and release glucose to the blood

3. glucose levels rise to a set point and stimulus for glucagon release diminishes

In the context of regulating blood glucose, are glucagon and insulin functioning as endocrine or paracrine hormones?

peptide hormones: insulin and glucagon; regulates blood glucose levels

* endocrine

Systematize the signal-transduction pathway involved in insulin-mediated uptake of glucose and  insulin-mediated glycogenesis.

• involves RTK signaling

• Insulin receptor binds insulin→ activates IRS1

• IRS-1 activates PI3 kinase, which catalyzes addition of phosphate group to membrane lipid PIP2 and converts to PIP3

• PIP3 recruits kinase to the inner surface of PM, leading to phosphorylation and activation of a protein kinase called Akt

• Akt catalyzes the phosphorylation of key proteins, which leads to an increase in glycogen synthase activity and recruitment of GLUT 4 transporter to membrane

Glycogen synthase

• catalyzes glycogenesis

• activated by insulin sig trans (bc too much glucose and needs to be converted to glycogen)

Define diabetes.

* chronically high blood glucose levels

Explain how defects in the insulin signal transduction pathway may lead to diabetes. 

kidneys respond by trying to remove excess glucose in urine→ frequent urination→ loss of water→ water mvmt from tissues→ thirst, blurred vision (bc no fluid in eyes)→ fatigue→ insulin resistance (bc promotes conversion of glucose to glycogen and body needs energy)→glucose is not getting to cells and insulin sig trans blocked

Type 1 diabetes

• juvenile onset

• autoimmune disease in which pancreatic beta cells are destroyed

• lack of insulin production

• leads to failure of somatic cells to take up glucose from the blood

Type 2 diabetes

• adult onset

• results from insulin resistance

• primarily caused by obesity/lifestyle

Gestational Diabetes

• high blood sugar when pregnant in a mother w/out previous diabetes diagnosis

• usually dissipates after birth but higher risk of type 2 later

List and describe the treatments available to treat diabetes.

• type 1: insulin injections, human cadaveric islet implants, stem cells

• type 2: reducing activity of liver (therefore reduction of glycogenolysis); meds that increase insulin production by pancreas; meds that increase sensitivity of target organs to insulin; decreasing glucose absorption from GI tract or glucose release; reduce hepatic glycogenolysis

• gestational: special meal plans, physical activity, insulin injections

cytoskeleton.

• found in cytoplasm and nucleus

• network of protein fibers

List the functions of the cytoskeleton.

• organization- spatial org of cell comps

• cell shape- provides mechanical support to the cell and nucleus

• motility-mvmt of cell, intracellular mvmt of structure/mlcl

• cell division- manages chromosomes; cytokinesis

• regulation- transmits mech signals form environ; intracellular mvmt of mclc involved in signaling/metabolism

• ROMCC

Main function of Microtubules

• spatial organization

• intracellular transport

Main function of Microfilaments

• cell shape

• cell locomotion

• cytoplasmic MTs

• Axonemal MTs

Main function of Intermediate Filaments

• mechanical strength

• nucleus shape

Cytoplasmic MTs

• cell organization

• intracellular movement

  • vesicles, organlles, chromosomes

Axonemal MTs

* components of motility structures
* flagella
* cilia

Describe in detail the structure of MTs.

• hollow tube of protofilaments

• protofilaments comprised of tubulin heterodimers (a and b monomer subunits held together by non-covalent interaction)

• each subunit is bound by GTP

• B has GTPase activity (hydrolyze GTP to form GDP and vice versa)

• subunits in each protofilament all point in the same direction

• structural polarity to MT: + and - ends

Define 'critical concentration' as related to MTs. (Cc)

\[free tubulin\] cyt at which rate of subunit addition is \~ equal to rate of subunit loss

Explain the effects of the critical concentration on MT assembly. Explain how critical concentration is related to the polarized assembly of MTs.

• polarity of growth rates seen at +/- ends is due to diff Cc requirements

  • • end: lower Cc

  • • end: higher Cc

• as MT growth occurs at both ends, conc will decrease → growth will first stop at - end

• once Cc is reached at - end, depolymerization will occur and growth may continue at + end, if cont, then cont will until Cc reached→ depolymerization of + end

Describe the process of 'dynamic instability' and the role played by GTP in this process. 

• model to explain MT behavior

• at any time, MT are growing or shrinking (polymerize or depolymerize)

• poly may continue for some undefined period

• MT may suddenly shrink rapidly or shrink partially and recommence growing or completely stop

• all determined by Cc

What is the "GTP cap" and how does it influence MT stability?

• dynamic stability is regulated by GTP cap

• tubulin dimers bind GTP

• GTP-tubulin dimer added to end of MT during polymerization

• GTP in dimer is slowly hydrolyzed to GDP (in b subunit only)

• GTP hydrolysis destabilizes MT structure

• if GTP hydro rate> rate of MT growth→ depolymerization of MT end occurs

  • means stop growing

Explain the function of MTOCs.

• microtubule organization center

• nucleation site for MTs= site where MT assembly is initiated

• serves to anchor and organize MTs

• contain y-tubulin ring complexes (yturc); serves as nucleating sites within MTOC, - ends of MT subunits bind to y turcs; + ends of MT grows outward from y turc

Describe the structure of the centrosome.

\-MTOC in al animal cells = centrosomes

* contain:
* centrioles: 2 symm barrel-shaped structures; unclear fun
* pericentriolar material: gel-like proteinaceous matrix; includes yturcs

Explain how MTOCs are involved in cell shape/differentiation in polarized cells, e.g. neuron differentiation.

• polarized cells; ends of the cell are structurally and/or functionally diff

• underlying MT arrangement determines cell polarity

• Axonemal MTs controlled by MTOC (+ driving axonal growth)

• dendritic MTs are mixed polarity

Describe the role of Tau 

• defn: MAP found in axons of nerve cells

• binds to MTs in axons

  • stabilizes MTs

  • promotes outgrowth of axons by promoting MTpolymerization

how is Tau correlated with some neurodegenerative diseases

• tauopathies: neurodegenerative disorders involving Tau aggregation

• tau=soluble protein so it normally does not aggregate

• aggregation→ paired helical filaments→ neurofibrillary tangle (NFTs)

• disorders: dementia, Alzheimers

• kinase destabilizes

Describe the structure and functions of microfilaments.

• 7nm diameter polymers

• comprised of 2 strands of polymerized Actin protein

  • G-active (globular)= free form

  • F-actin (filamentous)= polymerized form

  • all G subunits have the same orientation within the microfilamnet layer

MF assembly 

G actin bound to ATP→ ATP hydrolyzed to ADP in MF polymer → hydrolysis of ATP into ADP destabilizes MF→ eventual depolymerization

Describe the similarities between microtubules and microfilaments. How are they different? 

* both of +/- ends; all active monomers are oriented in the same direction; MFs have structural polarity; + ends grow faster than - end

Describe the structure/function of microvilli.  What types of cells have a lot of cillia?

• microvilli= cell surface projection

• formed from ordered arrays of MF bundles

• prominent features in cells w/absorption function

• 1000 per cell

• increase SA

• apical cells of intestinal epithelial cells have a lot of cilia

Describe the structure and functions of intermediate filaments (IFs).  Be able to distinguish the cytoplasmic IFs from those that form the nuclear lamina.

• function: structural support (tension bearing; very stable) and nuclear lamina scaffolding (determine nuclear shape)

• structure: 8-12 nm diameter; variety of protein components (protein identity depends on cell tissue type; proteins that make up nuclear lamina don’t vary)

Compare the diameter sizes of the three cytoskeletal elements. Why is there variation in the diameter of cytoplasmic IFs?

• MF: 7nm

• IF: 8-12 nm (depends on proteins components)

• MT: 25 nm

Explain how and why cytoplasmic IFs can be used in tissue/cell-typing and why nuclear IFs or other cytoskeletal elements (e.g. MTs or MFs) cannot.  Why would nuclear IFs not be used for IF typing?

• IF typing: tech used to identify cell type

• used for tumor diagnosis bc IFs are specific to certain cells bc tumor cells retain the IF protein of the cell type from which they originated

• cytoplasmic IFs are cell/tissue specific while nuclear IFs are not

Three types of cell movement

1. intracellular movement (mvmt contents within the cell)

2. movement of the cell itself

3. movement of environment past/through the cell

example of intracellular movement

motor proteins transporting cargo along MTs

examples of movement of cell

swimming cell/ crawling cell

ex of movement of environment past/through cell

ciliated epithelial cells in the trachea moving mucus over the apical surface

Recall the types of microtubule-associated motor proteins and describe their general overall structural/functional domains.

• kinesin and dynien

• structure of both: 2 globular ATP binding heads and a tail ; heads interact with MTs; ATPase activity and tails interact with cargo

Recall the direction of movement associated with each MT motor protein.

• kinesin- towards POSITIVE end

• dynein- towards NEGATIVE end; moves cargo towards the end of MT linked to gamma tubulin

Describe how vesicle-based movement occurs in the cell. Specifically describe how vesicles might be transported between the components of the endomembrane system using the different MT motor proteins. e.g. which motor protein would carry a vesicle to the PM? which motor protein would transport cargo from the ER to the Golgi?

• dynein carry cargo:

  • from PM during endocytosis

  • from ER to Golgi

• kinesins carry cargo:

  • from Goldi to PM

  • retrograde from golgi to ER

explain why motor proteins are necessary

• because diffusion would be too slow to transport things long distance

• diffusion alone could not sustain life

• has a CONSISTENT rate (not dependent on conc)

Recall the rates of transport associated motor proteins, and with axonemal dyneins.

• rate of transport associated motor proteins: 2 um/sec

• rate with axonemal dyneins: 14 um/sec

Describe the motor proteins associated with microfilaments. What similarities do they share with microtubule-based motor proteins?

• myosin: converts chem e in form of ATP to mech E, generating force and movement

• move MF not MT

• forms cytoskeleton and contractile filaments of muscle cells

• active transport of molecules

Explain why motor proteins are only associated with MTs and MFs, but not IFs. i.e. what is different about IF structure compared to the other cytoskeletal elements?

• both MTs and MFs have structural polarity (+/-) ends

• this structural polarity is what allows the motor proteins to have directionality

• IF do NOT have structural polaruty→ no motor proteins

  • ends of IF are identical

Compare and contrast the structure and function of cilia and flagella.

• contained in Axonemal MTs

• both: motile appendages of eukaryotic cells; moth made of MTs

• diff: # per cell and size (length)

  • cilia→ many per cell (2-10 um)

  • flagella → one to few per cell (10-200 um)

List and describe the three types of cilia found in eukaryotic cells. Provide an example of each to support your description.

1. motility→ movement of cells (C and F); cilia involved in the movement of environ past/through cell

   1. transmission of egg cells through the fallopian tube (CF), epithelial cells lining the trachea (C)

2. sensory (cilia ONLY)- function as cellular antennae, involved in detecting external cells, present in specialized cells

   1. chemoreceptors and mechanoreceptors

3. nodal (cilia ONLY)- cilia on the surface of embryo, movement generates a unidirectional flow (right to left)-?therefore only left side cascade is activated

Describe the signal transduction pathway involved in odor detection. 

• inhalation of odorant molecules into nasal cavity→ nasal cavity is lined by olfactory epithelium→ odorant molecules cross over surface of OE→ detecting scents required specialized cells present in OE (Olfactory sensory neurons)

• signal transduction inside OSNs is transmitted to neurons in olfactory bulb→ then to your brain

• dendrites of OSNs have cilia with odorant receptors

• odorant receptors in sensory cilia bind odorant molecules

• requires GPCR receptor

• Ca coming into cell to activate PDE doenst require direct energy

What is the difference between cillia and microvilli?

• cilia: MT based, involved in cell movement

• microvilli: MF- based, function to increase SA

Describe the ultrastructural details of cilia and flagella.

• 9+2 array of MTs (nine doublets arranged in a ring; surround pair of single MTs)

• MTs associated with many accessory proteins → bind at regular points along length of MT

  • some serve to x-link bundles

  • some generate forces to cause bending

Explain how the axonemal components interact to generate movement in cilia and flagella.

• axonemal dyneins: specialized motor proteins in axoneme structures

• dynein attached by its tail to one MT while the other end interacts with adjacent MT

• causes MT to slide against one another

  • bending deformations propagate along length of axoneme

• movement of cilia/flagella produced by bending of core

Describe the *situs inversus* disorder. Explain the underlying cause and the connection to the various issues that arise in an individual suffering from this disorder. 

• reversal of organs in thoracic and abdominal cavities

• defect in ciliary dynein (proper symmetry is not established during embryogenesis)

• can lead to infertility issues or respiratory problems

Define cell locomotion (aka: cell crawling). How is this different from a 'swimming' cell?

• actin based cell motility (MF)

• crawling cells

• swimming cells use help of cilia/flagella

Create a detailed model outlining the subcellular events involved in cell locomotion. 

1. PROTRUSION: leading edge extends via the polymerization of actin at its tip

2. ATTACHMENT: new adhesions anchored by actin, form on the undersurface of the lamellipodium

3. CONTRACTION and DETACHMENT: trailing edge of the cell detaches and is drawn forward by contraction of the cell body

Specifically explain the components involved in cell locomotion and how the microfilament arrangements contribute to this process.

• Trailing edge: a contractile bundle of stress fiber to push forward

• Lamellipodium: gel, cell cortex for support

• Leading edge: parallel bundle, filopodium