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Adv Cell Bio > Midterm 3 > Flashcards

Midterm 3 Flashcards

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1
Q

Light microscope

A

●light source illuminate system
●condenser: below specimen, and gathers light
●objective lens magnify specimen
●tube lens collect parallel light from objective
●eyepiece

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2
Q

How light refraction work

A

●light absorbed by lenses slows down due to interaction between light and glass particles -> light is reflected or total internal reflection (TIR)

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3
Q

Fluorescent microscopy

A

●very powerful source of light (mercury/xenon lamp/LED)
●light passes through two filters: one before specimen and one after specimen
●one filter passes just the light to excite the fluorochrome, another filter to pass just the light emitted by the fluorochrome
●photobleaching: sample is exposed to lot of light or for very long time

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4
Q

Excitation and emission of fluorochromes

A

●proteins, organelles, mRNA
●excitation: absorption of photon
●emission: emission of photon at longer wavelength

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5
Q

Cell fluorescent signaling

A

●multiple dyes can be used at the same time to visualize diff molecules or cell elements
●fluorophore can’t overlap
●emitting in same light is bad (can’t differentiate)
●

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6
Q

Protein can be coupled with fluorescent dyes for visualization

A

●genetic manipulation: fusion protein
●antibodies (antibodies dyes-conjugated) (think ELISA)

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7
Q

Immunofluorescence: antibodies dyes-conjugated are used

A

●very sensitive
●primary antibody recognized by many secondary antibodies -> amplify signal
●secondary antibody is conjugated with fluorescent dye or other probes (electron microscopy)

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7
Q

Protein fusion

A

●individual proteins can be tagged with fluorescence proteins
●GFP is excited by blue light
●shows localization of protein

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8
Q

Why are fluorescent from corals?

A

●can absorb wavelength
●emit fluorescent

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9
Q

Reporter gene expression system

A

●promoter activates reporter

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10
Q

Cell communication

A

●chemical signal and physical signal
●generate response

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11
Q

Unicellular communication

A

●bacteria can coordinate, balance and influence their behavior collectively: quorum sensing
●yeast communicating for mating, pheromones (no food, want to reproduce)
●cells communicate and influence one another’s behavior

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12
Q

Cell communication generalized

A

●extracellular signals mediate cell communication
●extracellular signal binds to receptor
●binding activates an intracellular signaling cascade or intracellular signaling molecules
●targets of signaling cascade are effector proteins

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13
Q

Examples of cell responses

A

●TF
●phosphorylation
●methylation

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14
Q

purpose of cytoskeleton in signaling

A

●related to cell apoptosis

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15
Q

Four types of cell communication (receptor dependent)

A

●Contact dependent (membrane-bound signal molecules)
●paracrine (local mediator- signal from another cell)
●synaptic (neurons)
●endocrine (signal through bloodstream)

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16
Q

Receptor specifics

A

●highly specific
●signaling molecules have small concentration in extracellular environment
●very specific functions, produce specific responses
●specific -> conformation is specific to one ligand
●Ka dissociation constant very low -> high affinity
●transmembrane proteins (some inside cell: nuclear membrane)
●hydrophobic

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17
Q

4 common elements of signaling

A

●Receptor and ligand
●secondar messenger
●signal cascade
●target

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18
Q

Cell can respond to many signals

A

●key challenge is understanding how cell integrates all those signals to generate answer

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19
Q

Three types of receptors

A

●Ion-channel-coupled receptors: ligand bind to receptor for ion release
●GTPase-coupled receptors: target 30% of FDA approved drugs, receptor coupled to heterotrimeric G protein (B, a, y), Ga bound to GTP/GDP
●Enzyme-coupled receptors: RTKs receptor-tyrosine kinase

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20
Q

Secondary messengers

A

●made in large amount in response to receptor activation
●signal is spread in cell
●Ca2+ or cAMP or diacylglycerol
●higher conc of secondary messengers inside cell, activate quickly, amplify signal

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21
Q

Molecular Switches: GTPase

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A

●switch from active to inactive
●GTP binding proteins
●based on GTP or GDP bound
●Large heterotrimeric protein, G protein, or small monomeric GTPase
●GAP (GTP->GDP) and GEF (GDP->GTP)

22
Q

Molecular Switches: Protein kinases and phosphatases

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A

●kinase= phosphorylation of target substrate
●phosphatases= dephosphorylation on target
●present in multicellular and unicellular
●30 to 50% of human proteins are phosphorylated
●human genome encodes for 520 protein kinase and 150 protein phosphatase
●phosphate usually from ATP

23
Q

Two main protein kinases

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A

●serine/theronine kinase
●tyrosine kinase
●phosphate added onto OH group
●catalytic death by mutating amino acid

24
Protein Kinases
●target substrate can be protein or TF or kinase protein ●kinases cascades ●sequence of phosphorylation
25
Scaffold proteins
●used to bring together signaling proteins that interact together/work in same signaling pathway ●large proteins ●some are part of receptors ●activated at high local concentrations, efficiency, rapidity and no cross-talking ●puts certain molecules together
26
G-Protein-Coupled Receptor (GPCR)
●binds to proteins, small peptides, neurotransmitters, hormones ●7 transmembrane domains ●in olfactory receptors, light receptor
27
Activation of G protein
●signal binds to receptor ●GPCR receptor changes conformation and it activates G-protein (a,B,y) ●GDP is released from a subunit and exchanged with GTP ●a subunit-GTP dissociates from B/y subunit and receptor -> G subunits interact with other proteins (signal transmission start) ●when GTP is hydrolyzed the trimeric G-protein reforms (temporary) ●GEF is receptor because G protein changes when interacting with receptor ●alpha subunit has hydrolytic activity (GTPase)
28
Inactivation of G proteins
●activation of G protein is transient because a subunit has GTP hydrolysis activity ●When GTP is hydrolyzed the heterotrimeric complex forms again ●GDP replaced with GTP ●AH domain keeps GDP in place ●Ras domain (hydrolysis of GTP), small GTPase
29
cyclic-AMP (cAMP)
●some G proteins regulate production of cAMP ●cAMP activate PKA (protein kinase A)
30
Activated PKA
●activated by cAMP ●enter nucleus (w/ NLS) and phosphorylates CREB ●CRE binding protein -> stimulates expression of target genes
31
Activation curve of target molecules
●response to signals vary ●hyperbolic: smooth response, fine-tuning of metabolic processes (hormones) ●sigmoidal: threshold to overcome to have sharp response -> can go to all or none ●can change to all or none when conc. of signal molecule increase faster
32
PKA activation graphs
●four molecules of cAMP activates the PKA ●phosphorylation to activate target ●higher # is more like all or none
33
RTK: Receptor Tyrosine Kinase
●growth factors and insulin bind to receptors ●ECM and surface proteins bind to receptors ●60 human RTKs ●20 diff families in mammals ●each one dedicated to family of ligands ●has ligand binding domain in extracellular which activates the kinase domain in the intracellular environment
34
RTKs activation mechanism
●1) ligand binding ●2) dimerization or cross-linked dimers, trans-autophosphorylation activation ●3) autophosphorylation, cross-phosphorylation ●4) docking platform for other proteins involved in signal transduction ●probably follows all or nothing because activated when all sites are phosphorylated
35
RTKs activation specific parts
●proteins have SH2 domain that recognize phosphate ●find candidate by looking at domain sequence ●SH3 domain to bind other domains of protein ●some binding proteins turn off signal ●catalytic activity is phosphorylation
36
RTK Ras (GTPase)
●activated RTK receptor recruits GRB2 ●GRB2 recruits Ras protein to receptor (with SH2 domain) ●SOS is Ras-GEF promoting exchange of GDP to GTP ●Ras-GTP activates (by phosphorylation on Ser residue) the first MAP kinase (MAPKKK) -> signaling cascade activation
37
Mitogen Activated Protein (MAP) Kinase Cascade activation
●well conserved ●phosphorylate by ATP ●each kinase in cascade is activated by previous one by phosphorylation ●signal is amplified in each step ●last kinase in module phosphorylates multiple targets ●growth factors and pheromones use this pathway
38
MAPK
●three serine-threonine kinases ●Ras protein, small G protein, activates pathway ●Not all MAPK cascade activated by RTK
39
How to stop cascade
●inactivate Ras ●remove ligand ●internal receptors ●phosphatase
40
Insulin-like growth factors-dependent signaling
●RTK activation recruits and activates PI 3-kinase ●PI 3-kinase phosphorylates PI(4,5)P2 inositol to PI(3,4,5)P3 ●PI(3,4,5)P3 interacts with PDK1 and Akt (PKB) at PH domain ●Akt or PKB is phosphorylated by mTORC2 (mTOR in complex2) ●phosphorylation of Akt by mTOR causes change in conformation in Akt that promotes PDK1 phosphorylation ●upon phosphorylation Akt dissociates from membrane
41
Insulin-like affect on Bad
●Akt phosphorylates targets such as Bad protein ●14-3-3 protein sequester bad ●inhibition of apoptosis by Bcl2 activation -> growth and survival
42
TOR
●kinase named as Target of Rapamycin, bacterial toxin used as immunosuppressant and cancer drug ●eukaryotes mTOR conserved ●mTORC1 contains Raptor ●mTORC2 contains Rictor
43
mTOR
●essential for cell physiology and regulates cell growth ●two major activators of mTORC1 are growth factors and amino acids ●mTOR and cancer: TSC1/2 is GAP of Rheb GTPase that activates mTORC1 ●when mTORC1 is active, promotes cell growth ●TSC1/2 is negative regulator of mTORC1
44
How does signal amplification work in activating by G-protein receptor and by RTK-coupled receptor?
●cAMP and phosphorylation
45
Cross-talking between MAPK cascades
●pheromones, starvation osmotic shock are all signals transmitted by MAPK cascade in yeast ●pheromones bind to G-coupled receptor which activates (B,y) the MAPK cascade ●osmolytes binds to the sensor which activates the signaling pathway, called HOG pathway ●both pathways shared same kinase (Kinase A) ●cross-talking avoids by presence of two different cascades. same in higher eukaryotes
46
What is cross-talking?
●when signaling molecules activate multiple signaling pathways
47
Rho, GTPase family
●activated by RTK receptor ●Rho family proteins regulate actin and microtubules in cell -> shape, polarity, motility, movement, adhesion ●Rho family proteins regulate cell-cycle progression, gene transcription and membrane transport ●inactive when bound to GDI
48
Members of Rho family
●Rho ●Rac ●Cdc42
49
Neural Development
●cell shape is fundamental ●very hard to shape to obtain ●neuron must connect with other neurons ●can't increase number of neurons so you increase neuron connections
50
Rho activated by RTK receptor extension
●development of neuron is highly regulated ●neuronal extensions are guided by set of cues: neuronal guidance ●Ephexin stably bound to EphA-receptor and activates Rhos proteins -> stimulate the activity of cytoskeleton -> generate of forward movement ●Ephexin is GEF
51
Rho Retraction
●Ephrin A bound to receptor EphA (dimerization) -> Cdc42 and Rac1 are inactivated -> depolarization of cytoskeleton -> inhibition of filopodia and lamellipodia ●contractility active in retraction and extension ●filopodia and lamellipodia generate force force to elongate
52
PLC Pathway
●activated by G-coupled receptor ●alpha subunit activates phospholipase C (PLC) ●PLC splits PIP3 in two parts : DAG and PIP3 ●PIP3 binds to Ca2+ channels in SER -> muscle contraction ●DAG activates PKC (Protein Kinase C) ●PKC phosphorylates targets are important for muscle contractions
53
What is a GTPase?
●protein that can bind to and hydrolyze GTP ●act as molecular switch

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