Meiotoc Spindle Flashcards
difference between spindles in mitotis and meiosis
centrosomes drive spindle formation in mitosis
appear to:
>define where it forms
>define its biopolarity
BUT
in meiosis
female meiosis in animals takes place without centrosomes
why do female ooctytes not have centrosomes
lack of centrosomes likely to prevent them from dividing and developing without first being fertilised
>unfertilised egg has everything it needs to divide
>EXCEPT centrosomes
>sperm brings centrosomes along with paternal DNA
this prevents the oocyte from mitosing before fertilisation
this couples division with fertilisation
evidence for lack of centrosomes being to couple fertilisation + division
inject centrosomes w/out fertilisation
egg begins to divide
creates haploid tadpole
sperm bringing in centrioles kickstarts division ONLY once fertilisation has occurred to prevent this
are centrosomes required for mitosis really?
centrosomes are not necessary for spindle assembly in mitosis either
destroy both centrosomes via laser ablation
flies w/out centrosomes more or less notmal
genetically remove centrosomes in drosophila by mutating gene necessary for their duplication
are able to form adult relatively similar to WT
lack cilia though which is what makes it sorta different (remember centrioles are cilium base)
RNAi culture cells
KO centrosome duplication gene
a spindle without a centrosome was found (i guess via tubulin-GFP)
in vitro spindle assembly (w/ and w/out centrosomes)
can add demembraned sperm to m-phase extract from xenopus eggs
forms spindle in this extract with centrosomes (from sperm)
instead can add bead coated in phage DNA to the extract
bioilar spindle forms without centrosomes
phage DNA is able to recruit cellular machinery and stimulate spindle assembly
SO:
ANY DNA can recruit cellular factors and assemble MTs into bipolar spindle
NEITHER centrosomes or kinetochores are required for spindle bipolarity
Ran assembly of spindle
Ran-GDP/Ran-GTP gradient
Ran GAP -> GDP form favoured
Ran GEF: RCC1 -> GTP form favoured
important in nuclear transport (you know this)
also regulates chromatin mediated spindle assembly
>Rcc1 chromatin bound -> Ran-GTP near chromatin
>Ran GAP - cytoplasmic -> Ran-GDP in cytoplasm away from chromatin
SAFs (spindle assembly factors) bound to importin - inactivated by this
Ran-GTP localised near chromatin binds importin and releases SAFs
localises spindle assembly to chromosomes
visualising Ran-GTP/GDP gradient
with FRET
YFP and CFP on FRET probe
probe binds target -> straightens out and fluorescent proteins no longer together
no fluorescence resonance energy transfer - just cyan fluorescence
2 diff setups possible:
probe binds Ran-GTP:
>FRET signal away from chromosomes where Ran-GDP
>no FRET signal close to chromosomes (probe bound)
probe binds importin unbound by Ran-GTP:
>binds importin away from chromosomes
>unbound near chromosomes (FRET signal here)
Ran-GTP requirement for spindle assembly experimenr
add excess of Ran-GAP
or inhibit Rcc1
>Ran-GTP low throughout celll
>no spindle formation near chromosomes
add excess of Rcc1
or excess of Ran mutant that cannot hydrolyse its GTP - stays Ran-GTP wherever
>always high Ran-GTP throughout cell
>Ran-GTP high far from DNA
>so MTs assembled every where as SAFs are released from importin throughout cell
testing what the SAFs that importin binds is/are
use xenopus extract
>add excess importin
>then remove importin from the cell
>the excess caused the Importin binding proteins to become importin bound in cell
>removing importin depletes it of IBPs
>incl. SAFs
>this extract can no longer form mitotic spindle in presence of DNA coated beads
> add fractionated HeLa nuclear extract
one of the fractions should contain SAFs
this rescues spindle formation
can fractionate this fraction
repeat until can isolate the protein that activates MT formation
here purified TPX2
TPX2 interactor assay
xenopus extract
add anti-TPX2 Ab
and protein A coupled beads (protein A binds Fc portion)
spin down will pull down TPX2 and its interactors
immunoprecipitation
western blot shows importin coming down too
in presence of Ran mutant stuck in GTP form - no importin seen as is only binding Ran-GTP in extract
TPX2 Ab could no longer pull down importin
shows that TPX2 binds importin and is released by Ran-GTP
TPX2 depletion
precents formation in m-phase extract around DNA beads
indicates its importance in spindle assembly
BUT depletion could also be removing a TPX2 binding protein that itself is required instead
so add back recombinant TPX2 to depleted extract
rescued spindle formation
so TPX2 itself is necessary for spindle formation
TPX2 basics
binds importin
released by Ran-GTP
required for spindle assembly from DNA beads
activates other proteins important for spindle assembly:
>NuMA (spindle pole associated)
>NuSAP (Spindle MT associated)
>HURP (KT associated)
in cell spindle formation dependent on Ran-GTP gradient
FRET shows this gradient is present in oocytes
inject an excess of Ran mutants that are uniformaly high in Ran-GTP or Ran-GDP
both of these cases abolish the gradient
spindle still manages to assemble localised only around the chromosoems
so must be an alternative MT assembly patheay activated by chromosomes
Chromosome passenger complex
CPC
4 subunit kinase complex
Aurora B (S/T kinase)
activated by chromosomes in a Ran-independent pathway
Incenp deletion:
aurora B cant interact w chromatin
meiotic spindle is delayed compared to WT
so CPC important for spindle assembly
CPC essential:
KO Aurora B = CPC depleted = spindle MTs fail to assemble
CPC targets
aurora B in CPC can phosphorylate kinesin-13 (MT depolymerase)
inhibits Kinesin-13’s depolymerisation activity
phosphorylates and activates Kinesin-14
an MT cross linker
allows formation of rigid MT filaments
CPC localises this essential spindle formation activity around the chromosomes
so get this spindle formation only near chromosomes
