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Cell Bio Mechanisms > MTs (incl. Cilia + Centromeres/Centrioles) > Flashcards

MTs (incl. Cilia + Centromeres/Centrioles) Flashcards

1
Q

gamma-TuRC

A

Gamma tubulin ring complex

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2
Q

MT filament destabilisation

A

GTP hydrolysis
leads to GDP tubulin
longitudinal interactions in GDP-Tub curve the protofilament
catastrophe

MAPs can temper the dynamic instability allowing rescue
so grow and shrink all the time

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3
Q

MT polarity

A

Beta tubulin exposed at plus end
+ and - ends give polarity to filament
motors can go diff directions

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4
Q

MT polarity use in diff cells

A

see radial MTs coming from centrosome

epithelial cells:
eg gut
apical toward lumen
basolateral to bloodstream
columns of MTs with - end at apical
trafficking from apical to basolateral

Neurons:
axons
minus to cell body
plus to dendrites

chromosome segregation
cell trafficking

need to organise MTs for this

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5
Q

MTOCs

A

MT organising centres
(or MT nucleation sites)
used to organise MT directionality

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6
Q

MT nucleation in cells

A

tubulin conc in cells at level that MTs dont spontaneously assemble
but have nucleation sites that can allow them to assemble below Critical Conc
localise this to one place in cell-specifically nucleates MTs where needed

CENTROSOME does this
(purified centrosomes can nucleate MTs below conventional CC)
centrosome = an MTOC

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7
Q

Gamma tubulin

A

3rd familiy of tubulin protein
localised to centrosome
in the Pericentriolar material around centrioles

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8
Q

Isolating Gamma TuRC

A

Density gradient separation
gamma tubulin sediments lower than a and b tub (heterodimer) (see in western blot of fractions)
exists in much larger complex

Pull out complex w IP or affinity purification to see other components of this complex
Complex promotes better MT nucleation at same conc of pure tubulin

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9
Q

Gamma TurC nucleation model

A

hypothetical
rings in complex structure with g-tub at top that interacts w a and b tubs

allows bringing together of AB heterodimers into filament better than just pure heterodimers
overcomes kinetic barrier for MT formation - so can start at lower than CC conc

Creates MTOCs by being localised to specific point in cell

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10
Q

Members of G-TuRC complex

A

IP of gamma tubulin
found GCP proteins - have gamma tubulin grip protein domain(s)

density gradient sedimentation + western blot
all co-sediment at same distance as g-tubulin

GCP2/3 form smaller complex
Gamma-TuSC (small complex)
cannot nucleate MTs on own
smaller Gamma TuSCs make up some of gamma-TuRC

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11
Q

Cryo-EM for finding structure

A

look at complex structure by EM
take class average to reduce noise in image of one complex

look at different average images of many complexes
taken at diff angles
can add these up to compute 3D shape

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12
Q

G-TuSC structure

A

V/Y shape
GCP2 one arm
GCP3 the other
gamma-tubulin at each lobe at arms’ ends

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13
Q

Gamma-TuRC structure

A

extended Flowers seen in vitro via recombinant proteins
helical symmetry - artifact of in vitro formation but allowed many angles to be seen to build image

GCP2/3 V shapes (Gamma-TuSCs) assembling together w G-Tub at ends
makes up the Gamma-TuRC

in metazoans:
other GCPs exist in TuRC too

GCP4 - made of GRIP motifs
similar structure to GCP2/3
GRIPs make up core-give structure
sirface residues may change but grip is conserved - so all GCPs likely have similar bent finger structure

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14
Q

different types of G-TuRCs in diff organisms

A

budding yeast = no GCP4,5,6 so cant ahce this
TuRCs just from GCP4,5,6

Fission yeast: has Mzt1 (essential) and GCP4,5,6

Metazoans:
-4 GCP2/3 TuSCs
-next pair GCP4/5
-next is GCP4/6
-then one more GCP2/3
makes up stalks 1-14 (7 v shaped GCP pairs)
makes one turn
this localises GCP4/5/6 to one side of ring, not radially distributed

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15
Q

Non GCP/tubulin proteins in the TuRC

A

single actin molecule in centre of cone

tiny Mozart (Mzt1) protein
essential for MT nucleation
sits at TuRC base

many other proteins other than GCPs involved in TuRC formation

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16
Q

Issues with this model of the TuRC

A

Cyo-EM not perfect at structure identification
electron densities can have ambiguity between proteins (cant assign to specific one)
possible to misalign things if not everything present there is known

Gamma-TuRC constructed here is not seen to be radially symmetrical
but MTs are
possibly some conformational change needed to put TuRC in right shape to form Rad-symmetric MT

some organisms lack some of these components
-B yeast - no Mzt1 or GCP4,5,6
-F yeast has both of these and Mzt1 is essential
-Drosophila- Mzt1 non-essential (just causes fertility issues as is expressed in testes)

these studies also looked at cytosolic Gamma-TuRC
could have conformational change when localised where it needs to be - harder to study

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17
Q

Pericentrin

A

exists in Pericentriolar material of centrosomes around the centriole
involved in recruitment of g-TuRC
eg as cells enter mitosis from interphase
-also involves CDK5RAP2

KO pericentrin - MTs not well organised at spindle pole anymore as g-TuRC localisation to pericentrolar area is not happening

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18
Q

human pericentin mutations

A

cause specific types of dwarfism
MOPD II/Seckel syndrome

primordial dwarfism-from beginning of development unlike pituitary growth hormone based dwarfism

short
small head
learning disability

due to patients having less cells
lack of increasing cell no. during development
organ development roughly in line but just less cells

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19
Q

pericentrin and disease view 1: improper spindle organisation

A

mutation in pericentrin
reduced recruitment of g-TuRC to centrosome MTOCs
chromosome segregation defects at higher frequency
daughters w incorrect complement apoptose
increased cell death
lower cell numbers

20
Q

competing DNA damage view of pericentrin and disease

A

seckel syndrome has defect in DNA damage detection
(could potentially apply to MOPD II too)

DNA damage
ATR activation
Chk1 activation
CDC25 inactivation

CDK1/CyclinB remains hyperphosphorylated

This DNA damage sensing mechanism is centrosome localised requiring pericentriolar material
if not correctly localised
improper DNA damage detection/checkpoint activation
cell cycle can progress w DNA damage present
increased cell death of daughters as incorrect DNA complement

21
Q

Centrosomes in human v fission yeast

A

humans have 1 centrosome in interphase
2 in mitosis

F yeast:
have mant and can nucleate from many cytosolic regions
in mitosis - have SPB spindle pole body- spindle nucleation from that
can use this system to study diff behaviours throughout cell cycle w/out diff cell types

22
Q

mto1 and 2 localiser

A

involced in gamma-TuRC regulation
Mto1/2 form large complex
goes to site
interacts w g-TuRC and recruits it to site

similar to pericentrin protein G-TuRC targeting

23
Q

Mto1/2 activator role

A

also acts as activator of g-TuRC (in fission yeast at least)

remove parts of Mto1/2 that allow it to localise to sites
drifts in cell
is still sufficient to activate nucleation of MTs (so this behaviour is independent to its localisation of nucleation at Mto1/2 sites)

add this truncated Mto1/2 to G-TuSCs, Mzt1, and tubulin in vitro
greatly increases nucleation

24
Q

CM1 domain

A

centrosomin motif 1
conserved region of Mto1 N-terminus

mutate it
kills ability of Mto1/2 to interact with G-TuRC

conserved in 2 human genes - linked to microcephaly conditions

25
CM1 domain genes and microcephaly
autosomal recessive primary microcephaly shows up in consanguineous populations no dwarfism just microcephaly from reduced brain development huge amount of brain devo in higher apes eg mutations in CDK5RAP2 in MOPD II dwarfism - cell division affected through whole body development these mutations just affect brain
26
CDK5RAP2 mutation effects
could be: -not enough Stem cell renewal - not enough made to give enough differentiated brain cells -premature SC differentiation - so dont have enough here either as not enough time to self renew enough -defects in specific neurogenic divisions Nuclei miugrate within neuronal stem cells when at apical surface -> mitosis+division is spindle is orthogonal ti apical surface - get two stem cell daughters if not orthogonal - get differentiated daughters -CDK5RAP2 involved in orienting mitotic spindle mutation give issues w this leads to too few neuronal stem cells -> microcephaly mice may not be best model as not as much cortical development as higher apes
27
new developments in g-TuRC stuff slide
conformational change of G-TuRC to radial shape could be from beggining to nucleate an MT actin molecule could be there in inhibitory fashion to keep complex in place - pops out when need to nucleate
28
Centrosome make-up
Centriole - + Pericentriolar material - where G-TuRC localises
29
Centriole structure
Cylinders of MTs Pinwheel shapes 9 MT triplets organised to make 2 cylinders orthogonal to each other 2 in centre duplicate in cell to give 4 then 2 in each daughter
30
Centriole purpose
form the base of Cilia no direct role in mitosis laser ablate - segregation occurs (checkpoint causes issues next cycle tho) basically hitchhike on segregation machinery by being in centrosomes KO sas-4 in drosophila centriole KO drosophila develops well compared to WT though has issues in cilia mediated processes (sperm cells, sensory neurons)
31
Cilia/flagella basics
only 2 out of 3 MTs in each triplet extend out duplets extend out of cell body w membrane surrounding can be used to move fluid and mucus around move oocytes move neuronal fluid Dyeinin proteins connect the MT doublets try to move MT its attached to to the MT next to it causes it to beat
32
Chlamydomonas model use for cilia
has 2 cilia simple genetics
33
Building cilia
No ribosomes present offsite synthesis + trafficking to cilium at full length steady state - there is still dynamically on/offloading of tubulin cut and it grows back subunits added at tip (not from base+pushed up) found by fusing normal strain w epitope tagged tubulin to a short cilia strain short cilia then grew labelled tubulin added at tips of shorter flagella
34
IFT
intraflagellar transport flagella paralysed w drug/mutation and so doesnt beat can see dense nodules under microscope moving towards tip then back to base Trains located between MT doublets and PM mutate kinesins known to affect flagellar formation - IFT also affected IFT involved in making flagellum?
35
Purification of IFT particles
sucrose density gradient many proteins coming down on relatively dense fraction sequence them (mass spec?) see the chlamydomonas IFT proteins see that they are homologous to proteins known to be linked to C. elegans sensing so IFT proteins also important in sensing (does he know)
36
IFTA and IFTB
IFTa particles move anterograde up to tip via heterotrimeric kinesin (Kinesin III) IFTb particles down to base via cytoplasmic dyenin 2 mess up either of these either cant bring stuff to end to add to cilium or cant bring stuff away to recycle it both affects ciliogenesis
37
Cilium gate
at base where centriole is gate impedes localisation to cilia Transition fibres -control what gain access to ciliary space requires specific sequence tags
38
Cilia in body patterning - Kartagener Syndrome
sinus infections ling problems infertility (sperm flagella, oocyte wafting) and Situs inversus left-right asymmetry generated by nodal cilia E8 - symmetry breaks certain genes expressed one side not other - left+right sides defined epithelial sheet - 1 cilium per cell (9+0-no central doublet) beat different in circular pattern causes fluid flow past the embryonic node form R to L (leftward flow)
39
How does leftward flow produce left+right asymmetry?
model 1: extra-embryonic fluid contains vesicle particles swept leftward burst signalling chemical released can be sensed preferentially on left side model 2: leftward flow causes cilia on left to bend opens up mechanosensing ion channels (Ca2+ methinks) letting in extra-embryonic fluid
40
Non-motile cilia as sensory organelles
like an antenna sensory organ put all sensor machinery in one place - improves sensitivity even if low whole-cell concentration (of sense machinery?)
41
sensory cilia examples
Rod cell photoreceptors: photoreceptors localised to basically a diverged cilium all photosensing done here iuses cilium as basic structure upon which sensory machinery is built Olfactory neuron: olfactory cilia at one end of neuron all olfactory sensing machinery here non-specialised cells: even if no cilium present before if starved for growth factors a Primary cilium will develop (9+0) -cells can grow a primary cilium when starved of serum (eg quiescence promoting conditions over cycling) -many sensory functions ascribed to them now
42
Polycystic kidney disease
autosomal dominant late onset underlying symptoms that worsen later on uncontrolled cell division in kidney tissue forming cysts can block tubules - issues autosomal recessive PKD in mouse model: IFT88 (human: polaris) mutant defect in intraflagellar transport prevented mouse from making primary cilia - stunted at a couple microns -in WT these are sensory organelles sense flow - bent - open mechanosensing ion channels downstream gene expression halts cell division autosomal dominant PKD in humans cause: -usually mutant in the ion channel -influx of Ca2+ not regulated as well by cilium? can sense Ca2+ flow with Fuo4 fluorescent Ca2+ reporter
43
Hedgehog pathway
invilved in developmental patterning (eg segmentation) mutants in drosophila hh signal pathway conserved Ci in Dros = Gli in humans can be either activator or repressor of gene expression (C-terminus cleaved = repressor, present = activator) Hh signal ->inhibits patch (smoothened inhibitor) ->so Smo activated ->Smo promotes activatory Gli (w/ C-term present) ->gene expression activation from Hh signalling
44
Hh signalling and IFT in vert development
Shh present inhibits repressive Gli form limb bud: lose Shh activity - fingers not made in right way in embryo so Hh important in limb devo eg ZPA - releases Shh - defines Posterior end of limb bud for organising digit order neural tube: Shh at ventral different cells along dorsoventral axis no Shh = more dorsal identity Shh = more ventral identiy removing Patch (Ptc) Dorsalises the Neural tube (IDK why this cause removing patch activates Smo->mimics activatory signalling by Shh? could use diff mechanism here)
45
Hedgehog signalling in cilia
grow epithelial cell layer tag smoothened Smo add Shh smothened becomes targeted to the primary cilia Patch removed from them Ci/Gli then exported as activator
46
Ciliary membrane formation
Still unknown how things pass the gate or how membrane does this membrane from around primary ciliun can have different origins centriole gets close to PM grows Doublet MT - extend membrane forwards OR can have internally forming cilia membrane comes from intracellular vesicle (may have diff properties in membrane) -doublet MTs grow and distort this membrane and fuses it with PM to extend cilium out of cell certain cell types prefer one of these mechanisms over the other may correlate to cilium function

Cell Bio Mechanisms (11 decks)

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