MTs (incl. Cilia + Centromeres/Centrioles)

Question 1

gamma-TuRC

Answer 1

Gamma tubulin ring complex

Question 2

MT filament destabilisation

Answer 2

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

Question 3

MT polarity

Answer 3

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

Question 4

MT polarity use in diff cells

Answer 4

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

Question 5

MTOCs

Answer 5

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

Question 6

MT nucleation in cells

Answer 6

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

Question 7

Gamma tubulin

Answer 7

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

Question 8

Isolating Gamma TuRC

Answer 8

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

Question 9

Gamma TurC nucleation model

Answer 9

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

Question 10

Members of G-TuRC complex

Answer 10

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

Question 11

Cryo-EM for finding structure

Answer 11

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

Question 12

G-TuSC structure

Answer 12

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

Question 13

Gamma-TuRC structure

Answer 13

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

Question 14

different types of G-TuRCs in diff organisms

Answer 14

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

Question 15

Non GCP/tubulin proteins in the TuRC

Answer 15

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

Question 16

Issues with this model of the TuRC

Answer 16

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

Question 17

Pericentrin

Answer 17

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

Question 18

human pericentin mutations

Answer 18

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

Question 19

pericentrin and disease view 1: improper spindle organisation

Answer 19

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

Question 20

competing DNA damage view of pericentrin and disease

Answer 20

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

Question 21

Centrosomes in human v fission yeast

Answer 21

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

Question 22

mto1 and 2 localiser

Answer 22

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

Question 23

Mto1/2 activator role

Answer 23

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

Question 24

CM1 domain

Answer 24

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

Question 25

CM1 domain genes and microcephaly

Answer 25

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

Question 26

CDK5RAP2 mutation effects

Answer 26

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

Question 27

new developments in g-TuRC stuff slide

Answer 27

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

Question 28

Centrosome make-up

Answer 28

Centriole -
+
Pericentriolar material - where G-TuRC localises

Question 29

Centriole structure

Answer 29

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

Question 30

Centriole purpose

Answer 30

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)

Question 31

Cilia/flagella basics

Answer 31

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

Question 32

Chlamydomonas model use for cilia

Answer 32

has 2 cilia
simple genetics

Question 33

Building cilia

Answer 33

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

Question 34

IFT

Answer 34

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?

Question 35

Purification of IFT particles

Answer 35

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)

Question 36

IFTA and IFTB

Answer 36

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

Question 37

Cilium gate

Answer 37

at base where centriole is
gate impedes localisation to cilia

Transition fibres -control what gain access to ciliary space
requires specific sequence tags

Question 38

Cilia in body patterning - Kartagener Syndrome

Answer 38

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)

Question 39

How does leftward flow produce left+right asymmetry?

Answer 39

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

Question 40

Non-motile cilia as sensory organelles

Answer 40

like an antenna sensory organ
put all sensor machinery in one place - improves sensitivity even if low whole-cell concentration (of sense machinery?)

Question 41

sensory cilia examples

Answer 41

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

Question 42

Polycystic kidney disease

Answer 42

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

Question 43

Hedgehog pathway

Answer 43

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

Question 44

Hh signalling and IFT in vert development

Answer 44

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)

Question 45

Hedgehog signalling in cilia

Answer 45

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

Question 46

Ciliary membrane formation

Answer 46

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