MTs (incl. Cilia + Centromeres/Centrioles) Flashcards
gamma-TuRC
Gamma tubulin ring complex
MT filament destabilisation
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
MT polarity
Beta tubulin exposed at plus end
+ and - ends give polarity to filament
motors can go diff directions
MT polarity use in diff cells
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
MTOCs
MT organising centres
(or MT nucleation sites)
used to organise MT directionality
MT nucleation in cells
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
Gamma tubulin
3rd familiy of tubulin protein
localised to centrosome
in the Pericentriolar material around centrioles
Isolating Gamma TuRC
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
Gamma TurC nucleation model
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
Members of G-TuRC complex
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
Cryo-EM for finding structure
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
G-TuSC structure
V/Y shape
GCP2 one arm
GCP3 the other
gamma-tubulin at each lobe at arms’ ends
Gamma-TuRC structure
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
different types of G-TuRCs in diff organisms
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
Non GCP/tubulin proteins in the TuRC
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
Issues with this model of the TuRC
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
Pericentrin
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
human pericentin mutations
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
pericentrin and disease view 1: improper spindle organisation
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
competing DNA damage view of pericentrin and disease
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
Centrosomes in human v fission yeast
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
mto1 and 2 localiser
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
Mto1/2 activator role
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
CM1 domain
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
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
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
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
Centrosome make-up
Centriole -
+
Pericentriolar material - where G-TuRC localises
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
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)
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
Chlamydomonas model use for cilia
has 2 cilia
simple genetics
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
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?
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)
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
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
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)
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
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?)
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
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
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
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)
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
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
