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Cell Signalling and Neurochemistry > New Deck > Flashcards

New Deck Flashcards

1
Q

what is cortical capture and its mechanism?

A

cortical capture of astral MTs

  • centrosome-mediated astral MT
  • one side tethered by cortical cap

mechansim:

  • dynein tether to cell cortex, movement creates a pulling motion (-end directed)
  • attached factors move by MT depolymerisation–> produces force
  • cell surface not static, myosin/actin motor contract cell together.
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2
Q

what are the 3 DAergic neuronal pathways?

A
  • nigrostiatal
  • mesolimib
  • tuberoinfundibular
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3
Q

how is mitochondrial dysfunction monitored?

A
  • transfer SH-SY5Y (neuroblastoma cell) with plasmid that directs expression of mito-GFP
  • confocal microscopt to monit the mitochoondrial morphology
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4
Q

how do ROS cause dysfunction of mitochondria?

A
  • CoQ is the major site of ROS production
  • Ubiquinone: the unpaired e- can be transderred directly from ubisemiquinone –> O2 to generate super oxide
  • mitochondrial can generate ROS; system overwhelmed
  • DNA in mito not histone bound therefore prone to damage
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5
Q

How is α-synuclein implicated in PD?

A
  • present in high concentrations in Lewy bodies
  • always phosphorylated at Ser129 in Lewy bodies by Polo-like kinase II
  • binds to membrane and inhibits its fusion, localised to outer memnrane
  • overexp –> fragmentation
  • high propensity for ampipathic binding (mem binding)
    • mutations occur in membrane BD
    • A30P mutation suppresses mem-binding (abolished physiological func)
  • all mutations increase rate of oligomer formation - protofibrils and B-sheet (fibrils)
    • mature insoluble fibril = formation of Lewy
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6
Q

what are the outstanding questions concerning PD?

A

what are the substrates of PINK1?

what are the causes of dysfunctional mitochondira in sporadic PD?

whyare the neurons of SN more susceptible to death then other DAergic pathways?

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7
Q

why is PINK1/Parkin enhanced mitophagy neuroprotective?

A
  • catastophic rupture of dysfunctional mitochondria can release pro-apoptotic proteins e.g. CytC –> neuronal death
  • eliminating these dysfunc mito prevents this process and in turn maintain neuronal survival
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8
Q

what is the spindle assembly checkpoint in metaphase-anaphase transition?

A
  • amphitelic attachment of MT to kinetichore –> mitotic checkpoint complex activation –> APC/C activation –> separase activation
  • separase: separates sister chromatin
  • metaphase: anaphase transition
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9
Q

describe the process of lipidation

A

Pro-LC3 –> LC3-1 –> LC3-II

  • proteolysis of Pro-LC3 to expose Gly at C-terminus
  • this is necessary for lipidation
  • covalent attachment of PS or PE to LC3-1 to form LC3-II
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10
Q

describe the MT motors in spindle formaiton.

A
  • motor domains utilise energy from ATP hydrolysis to catalyse stepwise motion in a directed mammer
  • cargo transport in retrograde/anterograde fashion also MT tracks
  • kinesins: +end directed motors (anterograde)
  • Dynein: -end directed motors (retrograde)
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11
Q

what are the current treatments for PD and their MAO?

A

Levodopa and DA receptor agonists

  • Tyr –> L-DOPA (by tyrosine hydroxylase)
  • L-DOPA –> DA (by Dopa decarboxylase)
  • L-DOPA: some can diffuse to parts of SN where DA is missing
  • side effects due to conversion to L-Dopa –> DA in periphery
    • solution - blockers of DDC in periphery

Surgical therapy

  1. pallidotomy - lesion of GPi, alleviates symptoms
  2. DBS of sub-thalamic nuc and GPi
  3. thalamotomy - stops tremour only
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12
Q

how do neuroprogenitor cells divide?

A

asymmetric division

axis and polarity of cells important in asymmetric division.

axis of spindle generates different daughter cells

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13
Q

what are the functions and features of LRRK2?

A
  • autosomal dominant, therefore gain of function –> PD i.e. toxic molecule

functions

  • regulates neurite outgrowth
  • in PD: activation of catalytic activity causes neuronal death
  • exp of LRRK2 with PD-associated mutation induced nuronal cell death in culture - dep on kinase activity
  • some mutations activate kinase domain
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14
Q

what are the problems with the “search and capture” model of bipolar spindle formation?

A
  • mathematically and kinetically inefficient
    • would take 20 times as long to serach for chromosomes
  • centrosomes are not essential for spindle formation e.g. plant spindles
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15
Q

what is the evidence that mitochondrial dysfunction and oxidative stress involved in PD?

A
  • mitochondrial overwhelmed by ROS production
  • more oxidised proteins and phospholipids were found in mito in brain
  • MPTP produces PD-like sympt’s
    • complex 1 inhibitor (in ETC)
    • taken up by DAergeic neurons
  • can be alleviated with L-DOPA
  • loss of neurons in DA-rich neurons in SN
  • presence of Lewy bodies in SN
  • immunostain reveals presence of α-synuclein and ubiquitins
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16
Q

what is the structure of centrioles?

A
  • MT-based structure
  • 9-blades of MT bundles - formed with triplets
  • amorphous cores not completely understood - cartwheel stuct?
    • proteins at core
  • distal and subdistal apprendages mark older mother centriole
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17
Q

describe the dynamic gradient II: the RanGTP gradient in bipolar spindle formation.

A
  • RanGDP/GTP switch is key in driving nuclear import through importins
  • RCC1 –> chromatin assocated RanGEF (GTE exchange factor)
  • RanGTP promotes MT stabilisation, as RanGTP binds importin b
  • in interphase Ran in nucleus so not MT stablising
  • RanGTP levels are high around chromosomes due to RCC1 which binds chromatin
  • RanGDP in distal cytoplasm - generates RanGTP gradient - concentrated around DNA
    • high FRET = RanGTP-IMP (centre)
    • low FRET = RanGDP, FRET molecules separated
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18
Q

describe centrosome maturation and pericentriolar matrix expansion

A
  • kinase signalling cascade recruits γTuRC and other PCM component proteins required for PCM expansion and support astral MT nucleation
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19
Q

describe the MT sliding model.

A
  • in mitosis, MTs are the cargo and transported along MT tracks creating a sliding motion in anti-parallel fashion
  • Eg5 (bipolar kinesins) organises adjacent MT into antiparallel arrach, focuses -ends together
  • dynein anchored on free MTs focuses spindle poles
  • chromatic motors captures MT to move -end away from chromosomes
    • move in +direction, sliding MT in opposite direction
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20
Q

describe the structure of PINK1 and what mutations can lead to pathogenesis.

A

structure:

mitochondrial targeting sequence - Hb domain - PK domain

  • most mutations occur in kinase dom - loss of kinase activity
  • point mutations G386A and G409V –> loss of PINK1 activity
  • mito-targeting sequence: needs to be imported into mito to perform physiological role, cleaved off after and processed to form PINK1 (2 sizes)
21
Q

where do PD-associated mutations occur in LRRK2?

A

point mutations:

  • D2019S
  • I2020T
  • both mapped to activation loop and –> activation of LRRK2
  • very common familal link

mutations scattered all over domains , so affects different funcitons –> cytotoxicity

how abnormal activation causes cytotoxicty is unknown, but clear that the kinase activity is essential for neurotoxicity

R144C/G mutation in Roc induces impaired DA neurotransmission

22
Q

what is the structure of LRRK2?

A
  • many repeat motifs - all rep domains implcted in protein-protein interactions therefore binds many cellular proteins
  • Roc-Cor domain: intrinsic GTPase activity
    • very low, implies GTP-activator (eg. GEF) necessary
  • 2 catalytic domains - Roc and KD
23
Q

what have we learnt about environmental factors and pathological brain examiniations?

A
  • oxidative stress and mito dysfunction are implicated as causes of DAergic death –> PD
  • loss of neurons, especially in DA rich neurons of SN
  • presence of Lewy bodies : abnormal protein aggregates
  • presence of α-synuclein and ubiquitins as major protein components
24
Q

what are some structural features of DJ-1?

A
  • E126, C106, E18 form supposed catalytic triad for protease activity
  • C106 can readily undergo oxidation
    • very susceptible to oxidative damage
25
Q

what is PD ?

A
  • hypokinetic movement disorder
  • premature death of DAergic neurons in substanti nigra
    *
26
Q

how do you detect and quanitate mitophagy?

A
  • biochemical marker = LC3-II
  • Use LC3-GFP: monitor co-localisation of LC3-GFP and mito-tracker red
  • LC3-GFP is recruited to autophagosome when it is lipidated
27
Q

what are the different subunits of tubulin in MT and the structure of it ?

A
  • 3 α-tubulins (1, 2 and 4)
  • 5 β-tubulins (I, II, III, IVa and IVb)
    • β-tubulin is GTP bound
  • γ-tubulin – nucletating tubulin
  • δ,ε-tubulin
  • form from polymerisation of α/β tubulin heterodimers
  • GTP ‘cap’ at + end is more stable than GDP tubulin
  • GTP hydrolysis regulates MT growth
  • equalibrium between dimers and polymer tend to s
28
Q

draw a flow chart of the PD-associated factors leading to neuronal death.

A
29
Q

describe the effect of CCCP on PINK1

A
  • CCCP used to dissipate the mitochondrial MP –> dysfunction
  • PINK1 is targeted to dysfunctional membrane
  • monitoring GFP-PINK1 shows accumulation on mitochondria with CCCP treatment
  • CCCP blocks PINK1 degradation
30
Q

describe centrosome replication

A
  • G1: mother-daughter centroles ready to duplicate
  • S: procentriole formation (coincides with Cdk2 activity)
    • spike of Cdk2 activity –> duplication begins
  • S: procentriole formed in perpendicular manner - once procentriole attached, it elongates until centrocome formed
  • G2: newly formed centrioles are “engaged: and unable to deuplicate further
    • procentriole site occupied
    • 1st daughter cell matures and takes on appendages
    • separase req to disengage centrioles
  • M phase: cell division segregates duplicated centrosomes into daughter cells
  • Go: mother-daughter centrioles “disengaged” - license to duplicate
31
Q

what signalling is occuring in the BG?

A

inhibition and dis-inhibition

  • input inhibition (-) –> less inhibitory output (-)–> more stimulatory output (+)
  • no input inhib, more inhibitory output (-)–>less stimulatory output (+)
  • substantia nigra sens out DA signals
32
Q

what are the contributors to mitochondrial dysfunction and what leads to neuronal death?

A
  • mitochondrial dsyfunction –>ROS prod –> oxidative damage
  • mito dys –> release of pro-apoptotic proteins e.g. CytC
33
Q

what are the general features of the DJ-1 gene?

A
  • unsure how it is regulated, so unable to active it and therefore physiological role is still unknown
  • encodes a redox sensor - sensors ROS production as it oxidises things
    • DJ-1 = redox activated chaperon, assits misfolding
  • DJ-1 -/- mice more susceptible to MPTP-induced toxicity
    • neurons more susceptible to oxidative stress
  • over-exp –> decrease in α-synuclein mito frag (morphology role)
  • selective protease?
34
Q

what do PINK1 knockout mice and flies exhibit? (7)

A
  1. mitochondrial dysfunction
  2. DAergic neuronal death
  3. susceptible to oxidative stress-induced neuronal death
  4. reduction in long term survival
  5. enhanced autophagy and mito fragmentation
    1. enhanced co-loc of LC3-II-GFP with mito
    2. i.e. autophag with mito
  6. deficiency of RINK1 can be rescured by overexp of Parkin
    1. Parkin is downstream of PINK1
  7. PINK1 over-exp suppresses α-synuclein-induced fragmentation - so related to morphology
35
Q

describe PINK1 regulation in normal and damaged mitochondria.

A
  • PINK1 resides on outer membrane of mito, with kinase dom facing cytosol

nomal mito: protease cleaves PINK1 on outer mem and then Ub –> degradation

damaged:

  • depolarised membrane (damaged) - inactives protease action
  • so PINK1 accumulates on outer membrane
  • leads to recruitment/activation of Parkin
  • Parkin and then Ub a substrate (mito proteins) –> degradation of damaged mito
36
Q

discuss the centrosome mediated “search and capture” bipolar spindle formation model.

A
  • centrosomes nucleate astral MTs
  • MT captures and stabilzes DNA at kinetichores on chromosomes
  • amphitelic attachment “pulls” chromosomes to metaphase plate
37
Q

what are the general features of α-synuclein (physiological function, etc)

A
  • involved in MT binding
  • concentrated at nerve terminal and involved in vesicle transport
  • implicated in learning, synpatic plasticity
  • exact physiological function unknown
38
Q

describe the motile cilium .

A
  • MT-based cell structures
  • centrosomes anchor them
  • MT motors crosslink MT bundles within cilia core and generates motion - dynein motors
  • non-motile, absent –> diseases (ciliopathies)
39
Q

describe Parkin and UCH-1 and their role in PD.

A
  • both involved in ubiquitination
  • mutations –> protein misfolding and accumulation of insoluble oligomers in PD pathogenesis
  • α-synuclein = potential substrate for Parkin
    • Ub –> proteasome
    • stop it from fragmenting mito - neuroprotective
    • PINK1 essential for Parkin interaction with mito
  • Parkin activity is altered by nitrosylation in oxidative stress
40
Q

what major questions/discoveries remain in PD?

A
  • need biomarker so high-risk patients can be treated byfore symptoms appear with neuroprotectors to stop disease
  • are Lewy bodies toxic or are they just a product of the disease? toxic precursor?
41
Q

what are the general feactures of centrosomes?

A
  • involved in nucleating, anchoring and organising MTs, also known as MTOC
  • centrosomes are platforms for coordinating cell signalling
  • tethered centriole pair embedded in amorphous “cloud” of proteins known as pericentrntiolar matrix (PCM)
  • PCM enriched in MT nucleating and anchoring complexes
42
Q

what is the “block and license” and what are the 2 “rules” in centriole replication?

A
  • rule 1: centroles duplicated once and only once
  • rule 2: mother centrioles make only one copy
  • duplication occurs at same time as DNA duplication during S-phase
  • G1 can/G2 can’t duplicate
  • block - centriole engagment prvents further copying
  • license - separase protease activity disengages mother-daughter centrioles
43
Q

describe how MT motors behind spindle self-organise.

A
  • motors act as dynamic cross-links between filaments
    • motors organised them into anti-parallel arrays
    • -end focussed at poles
  • slidling mechanism generates anti-parallel bipolar spindle
  • bipolar kinesins (Eg5) on overlapping MTs most abundant at spindle midline
  • dynein function at spindle poles
  • chromokinesines push -ends to away from chromosomes
44
Q

what can occur when there are defects in asymmetric division?

A
  • can —> cancer
  • centrosome loss/dysfunction –> uncontrolled proliferation and metastasis
  • due to wrong positioning in cell, do not divide properly but retain some stem-like features
45
Q

describe the dynamic gradient I: phosphorylation in bipolar spindle formation.

A
  • FRET assay reveals stathmin-tubulin interation gradient
    • high FRET - stathmin is elongated, inactive (centre)
    • low FRET - tubulin-stathmin interaction (periphery_
  • mitotic kinases localised to chromosomes, inactivates stathmin near chromatin
    • phosphorylated stath = steric hinderance, so tub binding
  • MT destabilised at cell periphery constraining length of spindle
  • this is how spindle lengths are determined
46
Q

describe the regulation of MT dynamics

A

stabilizing factors

  • MT associated proteins (MAPs): bind MT to keep stbale in polymer structure
    • end tip proteins (co-polymerizing factors)
  • MT bundling
  • NT nucleation

destabilizing factors

  • tubulin sequestering proteins = stathmin
    • sequestering protein binds heterodimer, has cap which prevents more than 2 heterodimers binding
    • do not degrade tubulin - can switch to polymerisation quickly
  • MT severing - cut then GDP bound so unravels
  • GTP hydrolysis - GTPases actively move tubulin away
  • MT depolymerase
47
Q

what are the steps of autophagy? (4)

A
  1. activating enzymes assemble at PAS which acts as a scaffold for protein recruitment to form autophagosomes
  2. phagophore must be populated with genes and starts engulfing damaged organelles etc
    1. recruitment of Atg8 (=LC3) to phagophore and lipidation of LC3-1–> LC3-II
    2. after activating enzymes assemble at PAS, lipidation occurs
  3. fusion with lysosome
  4. degradation
48
Q

what is the “inside out” mechanism for bipolar spindle formation?

A
  • MT formation around chromatic and chromosomes
  • this self-organises into bipolar anti-parallel array (from unfavourable conditions) with broad poles
  • MT cross-linking mechanism focus spindle poles
49
Q

describe the infiltration of Lewy bodies overtime in PD.

A
  • –> selective loss of midbrain DAergic neurons
  • deposition of brainstem Lewy bodies
  • clinical - on going degeneration of DAergic neurons
    • when disease progresses Lewy bodies infiltrate midbrain –> cortex

Cell Signalling and Neurochemistry (3 decks)

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