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Advanced Cell Biology > Final Exam Study guide - experiments and pathways > Flashcards

Final Exam Study guide - experiments and pathways Flashcards

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1
Q

Dorsal pathway in Drosophila D/V patterning

A
  1. Follicle cells will secrete proteins into the space between the egg shell and egg membrane, one of these being Spaetzle and certain proteases (Easter)
  2. Spaetzle is locally activated on the ventral side by cleavage by the Easter protease (i.e. easter protease is only secreted on the ventral side whereas the Toll receptor and Spaetzle will be everywhere0
  3. Activated Spaetzle ligand binds to Toll receptor, activating it
  4. Toll activation leads to Myd88 adaptor protein binding
  5. Myd88 recruits and activates the IRAK kinase complex
  6. IRAK phosphorylates and activates IKK
  7. IKK phosphorylates Cactus (IkB), targeting it for proteasomal-mediated degradation
  8. Dorsal (NFkB) is no longer sequestered by Cactus, enters the nucleus and promote transcription of Twist in ventral cells
  9. Twist TF promotes EMT
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2
Q

NFkB signaling in the inflammatory response

A
  1. Cells of the innate immune system release cytokines, including TNFα at the site of inflammation
  2. TNFα, a trimer, binds to the TNFα receptor on the surface of a macrophage, activating it
  3. Activated TNFα receptor recruits Myd88 adaptor protein
  4. Myd88 recruits and activates the IRAK complex
  5. Activated IRAK phosphorylates and activates the IKK complex
  6. Activated IKK phosphorylates IkB leading to its ubiquitination and proteasome-mediated degradation
  7. Relieved of its inhibition, NFkB enters the nucleus and promotes the transcription of cytokines and other target genes
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3
Q

Toll pathway in Drosophila innate immunity

A
  1. A secreted protease is activated in response to bacterial/fungal proteins
  2. The activated protease cleaves Spaetzle, activating the ligand
  3. Spaetzle binds to Toll, activating it
  4. Activated Toll recruits Myd88, the adaptor protein
  5. Myd88 recruits and activates IRAK
  6. Activated IRAK phosphorylates and activates IKK
  7. IKK phosphorylates IkB leading to proteasome-mediated degradation
  8. NFkB is liberated and enters the nucleus to promote transcription of genes such as Drosomycin and other anti-bacterial/fungal genes
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4
Q

Toll pathway in vertebrate innate immunity

A
  1. Toll-like receptors, a type of PRR, binds to PAMPs from microbial pathogens
  2. Binding of PAMPs to the toll-like receptor leads to its activation
  3. Toll-like receptor activation leads to signaling via NFkB which enters the nucleus and promotes transcription of cytokines and other innate stimulatory proteins
  4. Cytokines activate innate immune cells
    - pathway will be similar to the other NFkB ones
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5
Q

Antigen presentation to TH-cells (i.e from dendritic cell binding to the T-helper cell to the activation of something by the T-cell)

A
  1. Dendritic cell contacts TH-cell through a heterophilic interaction between the Ig-superfamily molecule on the dendritic cell and the LFA-Integrin on the T-cell
  2. If the TCR recognizes the MHC-II + peptide, it is activated
  3. Activated TH-cell stimulates the humoral immunity
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6
Q

MHC-II presentation by an APC

A
  1. Endocytosis of antigenic peptide/phagocytosis of bacterial pathogen and delivery to the endosome (early –> late)
  2. Partial proteolysis of bacterial peptides in the late endosome (partial acid hydrolase activity)
  3. Peptides are diverted to specialized secretory vesicles where they bind to an MHC-II receptor
  4. MHC-II with bound antigenic peptide are delivered to the cell surface to be recognize by a TH-cell with a TCR specific to that antigen
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7
Q

Sequential activation of the immune response

A
  1. Bacterial infection
  2. Recognition by cells of the innate immune system (macrophages, neutrophils and dendritic cells)
  3. Dendritic cell display peptides derived from bacterial proteins on their MHC-II platform, to T helper cells
  4. TH-cells with matching TCR is activated
  5. Activated TH-cell stimulates humoral response
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8
Q

Axon guidance – growth cone collapse mediated by ephrin

A
  1. Leading edge of the growth cone from a motor neuron comes into contact with a non-target cell, expressing an ephrin on its membrane
  2. Ephrhin binds to the ephrin receptor (Eph4A), an RTK, on the surface of the growth cone
  3. Ligand (Ephrin) binding to the RTK (Eph4A) activates it leading to dimerization and autophosphorylation  fully active
  4. Activated Eph4A recruits a tyrosine kinase that phosphorylates and activates Ephexin, the Rho-GEF
  5. Ephexin promotes the GTP-bound state of Rho, thereby promoting its activity
  6. Rho activity at the leading edge leads to retreat of the growth cone
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9
Q

Chemotaxis of a neutrophil towards a bacterium

A
  1. Bacteria releases peptides that are N-formylated
  2. N-formyl peptides from bacteria bind to GPCR on cell surface of neutrophil, activating it
  3. Activated GPCR activates two different G-proteins: Gi and G12/13
  4. Gi at the leading edge activates PI3K
  5. PI3K phosphorylates a membrane-bound PIP to make PIP3
  6. PIP3 serves as a docking site for Rac-GEF and recruits Rho-GAP
  7. Rac-GEF promotes local activation of Rac and local inhibition of Rho at the leading edge
  8. Rac-GTP activates PI3K (+ve feedback loop)  PI phosphatases ensure transient activation
    a. Rac-GTP activates WASp  activates Arp2/3  promotes actin polymerization/branching  actin recruits integrins and lamellipodia protrusion in target direction
    b. Rac-GTP activates PAK:
    i. PAK stimulates filamin  stabilizes overlapping actin filaments
    ii. PAK inhibits MLCK  leads to decreased myosin activity  decreased stress fiber formation
  9. The polymerized actin filament undergoes treadmilling at the leading edge and actin will be ADP-bound due to hydrolysis of ATP near the minus end
  10. Cofilin binds to actin-ADP and promotes actin disassembly here
  11. Behind the leading edge, G12/13 activates Rho-GEF (by default)
  12. Rho-GEF promotes Rho-GTP, active state
  13. Rho-GTP promotes formin activity  recruits actin monomers to + end  actin bundle growth
    a. Rho-GTP activates ROCK
    i. ROCK phosphorylates MLC  increased myosin activity
    ii. ROCK phosphorylates LIM kinase and activates it  LIM kinase phosphorylates cofilin, inhibiting it
    iii. Rho-GTP promotes stress fiber formation  integrin clustering and focal adhesion formation  promote adhesion to substratum
  14. At the trailing edge  focal adhesion disassembly, actin/myosin contraction around cell cortex
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10
Q

Actin treadmilling in lamellipodia extension

A
  1. WASp activation of Arp2/3 at leading edge nucleates new actin polymers at sides of newly formed polymers.
  2. (Actin plus ends become capped)
  3. Actin hydrolyses its ATP as filaments age
  4. Cofilin binds ADP-actin
  5. Cofilin mediates disassembly of filaments behind the leading edge.
  6. filaments that persist will initiate focal adhesions
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11
Q

Focal adhesions mediate integrin signaling

A
  1. (Integrin head domain binds to RGD domain on fibronectin activating it)
  2. Active integrin recruits Talin
  3. Talin recruits Fak (Focal adhesion kinase)
  4. Fak binding to Talin causes it to be active, autophosphorylate itself and recruit Src kinase
  5. Src kinase phosphorylates Fak
  6. Phosphorylated Fak recruits Grb2  Grb2 recruits Sos (Ras-GEF)  Sos promotes GTP-bound state of Ras (membrane associated)  active Ras activates Raf (P)  Mek (P) MAPK  MAPK enters nucleus and phosphorylates/activates TF  promote transcription of genes for cell division
    a. Fak/Src inhibit recycling of E-cadherin to the cell surface upon internalization  this leads to reduced E-cadherin  reduced cell-cell adhesion
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12
Q

Experiment to show that Talin is a tension sensor

A
  1. Genetically engineered talin with tag (fusion protein) that can adhere to glass slide (N-terminus) and with its c-terminus conjugated to a magnetic bead
  2. Apply magnet to c-terminal end to make the protein stretch out
  3. Add fluorescently labelled vinculin
  4. Wash
  5. See fluorescence emanating from slide
    • If you do this without the magnet, get no fluorescence
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13
Q

Inside-out activation

A
  1. Integrin intracellular tail domain (beta) binds to Talin which leads to unfolding of extracellular head domain
  2. Unfolding of the extracellular head domain leads to increased affinity for the extracellular ligand (fibronectin)  binds strongly to RGD motifs
  3. Tension from actin/myosin contraction leads to further activation  focal adhesions
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14
Q

Outside-in activation:

A
  1. Head domain of Integrin binds to RGD motifs on extracellular ligand/substratum in the ECM (such as fibronectin)
  2. Binding of integrin to fibronectin leads to integrin activation
  3. Outside activated integrin has its intracellular domains separate  α and β subunits separate
  4. β domain binds talin strongly
  5. Talin binds to and recruits actin; also binds vinculin which binds actin
  6. α-actinin promotes formation of anti-parallel contractile actin bundles
  7. Myosin recruited to actin leading to contraction
  8. Contraction of actin by myosin II motor and binding of integrin to fibronectin leads to tension
  9. Tension on talin exposes its vinculin binding sites  increases vinculin binding
  10. Vinculin promotes further actin polymerization
  11. Further actin polymerization leads to greater recruitment of integrins (sort of positive feedback pathway)  focal adhesions
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15
Q

Planar polarity signaling – Fat and DS

A
  1. Fat atypical cadherin binds to DS in neighbouring cell
  2. Fat in one cell leads to inhibition of DS at that site in the same cell; DS inhibits Fat at the opposite site  mutually exclusive domains of localization
  3. Fat and DS promote localization of each other (i.e. fat  fat and dsds) in the cells
  4. Once planar polarity is established, Fat cadherin inhibits Dachs
  5. Dachs generally binds to and promotes the closed conformation of Warts
  6. When Warts is in the closed conformation  unable to function as a kinase even if phosphorylated by Hippo
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16
Q

Hippo pathway – apical/basal polarity

A
  1. Establishment of proper apical/basal polarity leads to activation of Hippo
  2. Activated hippo kinase phosphorylates and activates Warts kinase
  3. Activated Warts kinase phosphorylates and inhibits Yorkie (or Yap the mammalian equivalent)
  4. Inhibited yorkie cannot enter the nucleus and promote transcription of DIAP (drosophila inhibitor of apoptosis; or IAP mammalian equivalent) and cyclin E
     Disruption of apical/basal polarity (loss of crumbs, par proteins, aPKC, Lgl (parts of crumbs), scribble, E-cadherin) leads to yorkie activation
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17
Q

Making mutant clones in the drosophila eye – white gene marker

A
  1. Start with chromosome that has FRT sites on both arms and is w(-)/- on one arm and w+/+ on the other (i.e. mutation is heterozygous) – wt cell; normal colour
  2. Induce mitotic recombination using flp recombinase  produce two cells:
    a. w-/w-; -/- and w+/w+;+/+
  3. If the patches of cells are roughly the same size (identify mutant since white colour in ommatidia), than the mutation is not in a tumour suppressor
    a. If the patch of cells corresponding to the mutant is much larger than the wt one, then the mutation is in a tumour suppressor gene
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18
Q

EMT and B-catenin

A
  1. B-catenin has dual roles in the cell, generally degraded by inactive wnt pathway or stably associated at the adherens junction as an anchor for actin/cadherin – cell not responsive to Wnt signal
  2. Loss of adherens junction (Cell loses association with neighbouring cell, accident or LOF mutation in E-cadherin or [overexpression of twist  would not causes this])
  3. B-catenin free from adherens junction and enters nucleus
  4. B-catenin induces transcription of myc
  5. Myc promotes cell division and cell growth
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19
Q

Organization of junction complex – specifically from an unpolarized cell

A
  1. Par3-Par6-aPKC complex localized to apical site on cell and recruit:
    a. Recruits Rac
    b. Recruits tight junction components (claudin and occludin)
    c. Organizes basal Scribble complex and apical crumbs complex
  2. Rac organizes polarized actin cytoskeleton at its site  Actin further recruits Rac
    a. Scribble complex recruits adherens junction proteins  cadherins and B-catenin
  3. Cadherins in one cell recruit cadherins in a neighbouring cell
    a. Claudins in one cell recruit claudins in another cell
  4. Cadherins/claudins in neighbouring cell recruit Par3-Par6-aPKC complex
  5. Par3-Par6-aPKC complex recruits rac and organizes basal scribble complex and apical crumbs complex
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20
Q

Compaction

A
  1. In mammalian embryogenesis, at the 8 cell stage, cells start to express E-cadherin and as a result being to tightly adhere together
  2. At the 16-cell stage, inner cells determined to be embryo
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21
Q

Identification of cadherins

A
  1. Take tissue from animal and add trypsin (non-specific protease + EGTA (chelates cations such as Ca2+)  get individual cells
    a. If calcium is added back, cells will re-aggregate
  2. Take cells and raise antibodies against cell surface proteins (by injecting into a rabbit for example)
  3. Test antibody ability to prevent aggregation of cells even after Ca2+ is added
    a. If antibody blocks aggregation, may be due to binding and blocking of cadherin activity
  4. Purify antibody and use it to affinity purify cadherin
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22
Q

mRNA transport in Drosophila oocytes

A
  1. Egl binds to specific sites on the 3’UTR of bcd mRNA
  2. Egl with Bcd binds to dynein light chain
  3. BicD binds to dynactin and BicD-CTD binds to the first 79 amino acids of Egl, stabilizing its interaction with dynein (BicD-CTD can also bind to Rab6-GTP)
  4. Dynein migrates along the microtubules to the anterior (minus end)
  5. Localized Bcd mRNA is anchored by association with actin
    • BicD mutant phenotype is the same as Egl mutant phenotype; both necessary
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23
Q

Yeast 2-hybrid interaction assay

A
  1. Make two fusion proteins; Protein A + TF activating domain (prey) and Protein B + DNA binding domain (bait)
  2. Introduce transgenes into yeast
  3. If the proteins are binding partners (or at the very least interact) get a functional TF against a certain reporter gene in the genetically modified yeast
    a. Reporter gene can be LacZ or gene required for viability
  4. Presence of blue colonies when stained with X-gal or just presence of viable colonies indicates binding
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24
Q

Yeast two-hybrid results using BicD-CTD as prey and Rab6/BicD/Egl (1-79) as bait.

A

• Made fusion protein of BicD-CTD + AD and did the assay with fusion proteins including BicD, Rab6 and Egl 1-79
o BicD-CTD can bind itself  dimer
o BicD-CTD can bind Egl 1-79
o BicD-CTD can bind to Rab6
 Thus, Rab6 and Egl 1-79 both bind to BicD CTD, probably compete

25
Q

Egl binds to 3’UTR sequences

A
  1. Add in-vitro translated 35S-labeled Egl protein to beads that have the 3’UTR of minus end localized mRNAs conjugated to them
  2. Incubate
  3. Wash
  4. Measure radioactivity by radiography
26
Q

BicD and Egl proteins interact

A
  1. Incubate BicD-CTD-GST fusion protein with glutathione conjugated to agarose beads
  2. Add in-vitro translated 35S Egl
  3. Wash
  4. Elute
  5. Run out on SDS-PAGE
  6. Autoradiography
  7. See that BicD-CTD can interact with first 79 amino acids of Egl
27
Q

Egl interacts with Dynein light chain (DLC)

A
  1. IP using α-Egl  DLC co-immunoprecipitates
    a. Wash, run on SDS-PAGE, transfer to membrane
  2. Do western on membrane with α-DLC  get band
    • Western on supernatant lane  lots of DLC that didn’t bind
    • Western with control IP (random antibody for IP)  no bands
    • IP against mutant Egl  no band
28
Q

Bcd and osk mRNA transport by kinesin and dynein

A
  1. Wt oocyte  bcd and osk mRNA localize properly
  2. Dynactin mutant  bcd fails to localize
  3. KHC mutant  osk fails to localize
    • Seemed to have done enhancer trap for this
29
Q

Fusion of head domains of MT motors to B-gal reveal MT orientation

A
  1. Fuse head domain of Nod (minus-end directed KLP) with B-gal  localizes to anterior end
  2. Fuse head domain of kinesin (plus-end directed MT motor) with B-gal  localizes to posterior end
30
Q

MTs and Actin are necessary for mRNA localization in the Drosophila oocyte

A
  1. Incubate ovaries in colchicine (MT depolymerizing drug)  Bcd and Osk mRNA fail to localize
  2. Incubate ovaries in Latrunculin B (Actin depolymerizing drug)  Bcd and Osk mRNA fail to localize
31
Q

Ash1 mRNA localization in budding yeast

A
  1. Ash1 mRNA 3’UTR bound by She2
  2. She3 linker protein binds to Myosin V
  3. She2/Ash1 bind to She3/Myosin V
  4. Complex migrates along actin, that is anchored by formin, towards plus end
32
Q

Major motor proteins – Substrate direction, tightly bound when, powerstroke, features

A
  • Kinesin  MT plus end directed  tightly bound to MTs when ATP bound  powerstroke occurs during ADP to ATP exchange  highly processive
  • Dynein  MT minus end directed  tightly bound to MTs when ADP bound  powerstroke when ADP + Pi are released  highly processive (partly due to dynactin association)
  • Myosin  actin plus end directed  Myosin II has low processivity but fast; Myosin V has high processivity but slow
33
Q

Muscle contraction

A
  1. Motor neurons stimulate action potential in muscle membrane
  2. T-tubules propagate action potential rapidly and cause opening of voltage-gated Ca2+ channels
  3. Ca2+ flows into the cell and binds to Ca2+-gated calcium channels in the SR causing release of calcium into the cytosol
  4. Ca2+ binds to SR channels  positive feedback
  5. Ca2+ binds to troponin which causes it to release tropomyosin
  6. Tropomyosin slides off of the actin binding sites, exposing them to myosin
  7. Myosin binds to actin in the sarcomere
  8. Myosin-II motors of each thick filament rapidly pull actin towards the midline of each sarcomere  contraction
  9. Ca2+ rapidly returns to the SR via Ca2+ ATPase
34
Q

Organization of a sarcomere

A
  • Myosin in bipolar arrangement associated tail-tail
  • Titin – extends from Z-cap to M-line
  • Z-cap found at the plus ends, protects it
  • 4 thin filaments associated with each thick filament
  • Multiple sarcomeres are aligned within a myofibril
35
Q

Regulation of myosin II by MLCK

A
  1. Myosin Light chain kinase phosphorylates myosin regulatory light chain
  2. Phosphorylation causes release of tail domain from the head domain thereby exposing actin binding sites and promotes the straightened configuration of the myosin tail
  3. Spontaneous self-assembly of myosin into a bipolar filament
36
Q

Different regulatory proteins for actin

A

• Arp2/3: heterodimer that nucleates actin from the minus end and can promote branching of actin to form networks
• WASp: activates Arp2/3
• Formins: dimers that associate with actin plus ends and will attract free actin to the growing filament.
o Translocates after addition to attract another free actin
o Also typically associated with the plasma membrane
• Profilin: binds to actin monomers and promotes their addition to actin plus ends; then falls off after addition
o Profilin activity can be regulated by phosphorylation, association with specific phospholipids and works with formins
• α-actinin: promotes anti-parallel actin bundles which are important for contractility
• Filamin: cross-links overlapping actin at right angles  promotes actin network
• Cofilin: promotes actin disassembly
• Fimbrin: stabilizes overlapping actin; important in fillipodia

37
Q

Treadmilling – own equivalent of dynamic instability

A
  1. Actin subunits are ATP-bound in the free state and are added to the plus end in the ATP-bound state
  2. Inherent ATPase activity of the subunits leads to ADP state over time
  3. ADP-bound actin comes off of the minus end
  4. Length of the microfilament stays constant
38
Q

Dynein motor activity

A
  1. ATP-bound dynein is not associated with MTs
  2. ATP hydrolysis leads to dynein being ADP-bound and tightly associates with MTs (only associated through 1 AAA of the 6 through stalk protein)
    a. Tail domain associated with dynactin and cargo protein
  3. Loss of ADP + Pi leads to the power stroke  rotation of the 6 AAA domains of the dynein heavy chain causing movement 8nm in the (-) direction
  4. Binds ATP again, causing release from MT
39
Q

Kinesin movement on microtubules

A
  1. ATP-bound lagging head tightly associates with MTs while the ADP-bound leading head associates weakly
  2. ATP hydrolysis and Pi release on the lagging head converts it to weak binding, readying it for translocation
  3. ADP exchange for ATP on the leading head causes strong binding with MTs and causes its linker region to adopt a forward pointing conformation
  4. This causes the powerstroke which pulls the lagging head forward 8nm in the (+) direction to the next available MT binding site
40
Q

Identification of a cytoplasmic motor protein

A
  1. Take whole cell extracts from squid axoplasm (Contains motor protein)
  2. Incubate with purified MTs + AMP-PNP (motor protein binds to MTs)
  3. Centrifuge  microtubule motor co-sediments with MTs
  4. Wash
  5. Add ATP to elute motor protein (and other contaminants)
  6. Fractionate + in-vitro assay to isolate pure protein
  7. Run out on SDS-PAGE, excise band and sequence  110 kDa kinesin
41
Q

• In vitro assay for motor activity

A
  1. Take total cell extract from squid axoplasm and pour it onto coverslip
  2. Wait and let the proteins there (contains motor protein) adhere to the slide
  3. Add purified MTs and add ATP  movement of MTs relative to slide
42
Q

• SDS-PAGE

A

o SDS page of in-vitro synthesized MTs  1 band (alpha and beta tubulin are the same size)
o SDS page of total cell extracts from squid axoplasm  smear

43
Q

• In-vitro MTs

A
  1. Take tubulin dimers purified from cells + ATP and assemble them in-vitro  pure MTs
44
Q

• House-keeping part

A
  1. Add inert beads to cell body and they will move towards the axon terminal (anterograde)
  2. Add non-hydrolyzable ATP-analogue  inhibits movement and stabilizes bead/MT interactions
    • Thus, since we know MT run the length of the axon and from the above information we know that ATP hydrolysis is involved, must be an ATP-dependent motor protein that moves along MTs to transport the bead
45
Q

Ciliopathy – Kartagener’s

A
  1. Individual has a mutation in any of the genes encoding the axonemal dynein subunits
  2. Cilia of the midline do not beat correctly, if at all so the Nodal pathway is not activated
  3. Leads to situs invertus (and other symptoms)
46
Q

Kartagener’s syndrome and Nodal pathway

A
  1. Cells of the node (midline) contain cilia that beat rhythmically in the counter-clockwise which pushes ECF towards the left
  2. Something in the ECF induces cells on the left side to express Nodal (a TGFβ homologue)
  3. Secreted Nodal binds to and activates the TGFβ receptor of nearby cells (and perhaps of the secreting cell as well)  activation of Smads (effector proteins)
  4. Activated smads induce the transcription of Pitx2 TF and Nodal  positive feedback
  5. Pitx2 promotes heart fate
47
Q

Axonemal dynein

A
  1. Axoneme is a specialized bundle of MTs and associated proteins including axonemal dynein
  2. Axonemal dynein has a head domain associating with one MT doublet and the tail associates with the other
  3. Dynein is minus end directed but, linker proteins limit the sliding of MTs relative to each other
  4. This combined action leads to a stirring motion
48
Q

processivity

A

The ability of an enzyme to repetitively continue its catalytic function without dissociating from its substrate.

49
Q

Centrosomes and centrioles  primary cilium

A
  1. Centrosome within the cell has a single centriole pair
  2. Centrioles replicate in S-phase of the cell cycle
  3. Replicated centrioles separate from each other in prophase of mitosis, centrosomes split into two
  4. Centrosomes organize the mitotic spindle within mitosis
  5. Each daughter cell inherits a single centrosome
  6. Centrosomes disassemble in interphase
  7. In some cells, centrioles initiate the formation of the primary cilium
  8. Centrioles migrate to the cell surface and organize the primary cilium; (one of the two nucleates the MTs)
50
Q

Nucleation of MTs

A
  1. γ-tubulin dimers + associated proteins form the γ-tubulin small complex (SC)
  2. 7 γ-tubulinSC assemble into a corkscrew-like structure – last γ-tubulin sits on top of the first  13 γ-tubulins. With the associated proteins  γ-tubulinRC
  3. γ-tubulinRC nucleate 13 microtubule protofilaments
51
Q

Microtubule dynamic instability in-vitro

A
  1. B-tubulin is GTP-bound in free tubulin
  2. Growth and shrinkage at the minus end is slow
  3. Free tubulin is added to the plus end; growing filament will have a GTP-cap
  4. GTP hydrolysis on B-tubulin can occur stochastically anywhere on the MT
  5. Exposed GDP-B-tubulin at the plus end heads to rapid disassembly  catastrophe
    a. GDP-B-tubulin at the plus end promotes a curved protofilament – loss of lateral contacts
  6. Addition of a new GTP-B-tubulin dimer at the plus end leads to stability and growth  rescue
    a. GTP-B-tubulin promotes straight conformation – filaments interact
52
Q

Signaling in plants – Auxin

A
  1. Membrane-bound Auxin influx transport brings Auxin into the cell
  2. Auxin binds to Auxin receptor (TIR1)
  3. Aux-TIR1 bring Aux/IAA to the SCF ubiquitin ligase
  4. Aux/IAA is ubiquitinated and degraded by the proteasome
  5. Arf inhibition by Aux/IAA is relieved and Arf is now able to promote transcription of Auxin target genes
53
Q

Signaling via membrane soluble molecules and intracellular receptors

A
  1. Steroid hormones are transported in the blood by specific carriers since they are water insoluble
  2. Steroid hormones diffuse directly through the membrane
  3. Steroid hormone binds to the ligand binding domain of its receptor (generally located in the cytoplasm) resulting in dissociation of the inhibitory domain and, typically, binding to a co-activator
  4. Ligand binding may lead to nuclear entry
  5. Receptor-ligand binding typically leads to activation of transcription at specific genes  primary response
    a. May also lead to transcriptional repression
  6. Many of the primary response genes encode transcription factors that promote the transcription of other genes  secondary response
54
Q

GPCR desensitization

A
  1. Extracellular ligand binds to and activates a GPCR
  2. The activated GPCR stimulates GRK (GPCR kinase) to phosphorylate it at multiple sites
  3. This phosphorylation allows arrestin-family proteins to bind to the GPCR and block further G-protein activation
    • GPCR desensitization can also occur by receptor-internalization (receptor-mediated endocytosis)
55
Q

Photoreceptors and GPCR

In the absence of light

A
  1. Rhodospin (GPCR) located on the internal membrane – rhabdomere – is inactive in the absence of light
  2. cGMP levels are high in the cell and bind to cGMP-gated sodium channels  leading to influx of sodium and membrane depolarization
  3. Inhibitory sensory neuron fires and inhibits its post-synaptic partner; indicates the absence of light
56
Q

Photoreceptors and GPCR In the presence of light

A
  1. Photon of light strikes the chromophore associated with rhodopsin causing a light-dependent isomerization of the chromophore (cis  trans), activating rhodopsin (GPCR)
  2. Activated Rhodopsin activates its G-protein (transducin)
  3. Activated transducin activates cGMP phosphodiesterase which converts the intracellular cGMP to GMP
  4. cGMP levels go down and no longer bind to cGMP-gated Na+ channels leading to repolarization of the membrane
  5. The repolarized sensory neuron no longer inhibits its post-synaptic partner which now signals to the brain the presence of light
  6. After the GPCR is activated, it gets phosphorylated by GRK which allows arrestin-family protein binding, turning off the signaling pathway
    a. Mutations in arrestin or GRK leads to night-vision problems
57
Q

PLC/Ca2+ signaling

A
  1. Extracellular signaling molecule binds to extracellular domain of GPCR, activating it
  2. Activated GPCR activates its associated G-protein causing a conformational change, leading to GTP binding (exchange of guanine nucleotide) and separation of the α-subunit from the β and γ subunits
  3. Activated G-protein activates phospholipase C (PLC)
  4. Phospholipase C cleaves membrane bound PIP (PIP2) to form membrane associated DAG and free IP3
  5. IP3 binds to IP3-gated Ca2+ channels on the ER  rapid increase in cytosolic Ca2+ (which is normally kept low due to calcium binding proteins and Ca2+ pumps in the ER and plasma membrane)
  6. Ca2+ binds to calmodulin and activates it
  7. Activated calmodulin binds to and activates CaM kinase (II) by binding to its inhibitory domain thereby relieving inhibition of the CaM kinase catalytic domain
    a. Subsequently, activated calmodulin binds to and activates the Ca2+ ATPase that pumps Ca2+ out of the cell or into the ER  transient calcium signaling
  8. Activated CaM kinase phosphorylates substrates, including itself  autophosphorylation
  9. Calcium levels decrease and calmodulin no longer binds to CaM kinase; phosphorylated CaM kinase is still partially active
  10. (Phosphatase removes phosphate from CaM kinase, thereby turning it off)
58
Q

Olfactory system GPCR pathway

A
  1. Odorant molecule binds to GPCR on olfactory epithelium
  2. Activates G-protein (Golf) (conformational change allowing exchange for GTP and separation of α subunit from the β and γ subunits.
  3. Activated G-protein α-subunit activates adenylyl cyclase
  4. Adenylyl cyclase converts ATP to cAMP
  5. cAMP binds to cAMP-gated sodium channels  opening of channels sodium influx into the cell
  6. Membrane depolarization leads to firing of action potential from sensory neuron that is relayed to the brain
59
Q

GHRH pathway

A
  1. Neurosecretory cells produce GHRH
  2. GHRH binds to a GPCR found on pituitiary gland cells, causing it to undergo a conformational change and activating it
  3. Activated GPCR causes a conformational change in its associated G-protein (Gs) leading to separation of the α-subunit from the β and γ subunits, and binding to GTP  activates G protein
  4. Active G-protein activates membrane bound adenylyl cyclase
  5. Adenylyl cyclase converts ATP to cAMP
  6. cAMP binds to regulatory subunits of PKA  causes release of catalytic subunit
  7. PKA catalytic subunits enter the nucleus and phosphorylates CREB
  8. Activated CREB binds with co-factor CBP which act as a TF
  9. CBP+ CREB go to the promoter of growth hormone gene and induce its transcription

Advanced Cell Biology (15 decks)

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