Centrosomes + Kinetochores Flashcards
MT dynamicity
Grow and shrink by addition and removal of tubulin heterodimers
+ end more dynamic
MT constantly switch between growth and disassembly states
catastrophe and rescue
KT interaction w MT
enters mitosis
spindle forms from centromeres
lateral side of MT interacts w KT
KT moves along until plus end connected to KT
Tubulin subunits incorporate onto and dissociate off the plus end
so need to maintain interaction w KT through this
proper KT-MT attachment needed for accurate segregation
kinetochore interaction with dynamic MT plus ends
search and capture
spindle forms
lateral side of MT attaches to KT (as have more surface so more likely)
kinetochore transported to plus end
astral/centrosomal MTs - before attached to KT
kinetochore MTs: attached to KT
eventually achieve bi-orientation as the sister’s kinetochore attaches MT from another pole
when an MT binds a kinetochore on one sister
then it orients it so that the other kinetochore is oriented away
makes it more likely to be bound by MT from other pole
however multiple MTs can still attach same pole so not perfect
different MT attachments
Bi-oriented (amphitelic?)
sister attached to opposite poles
syntelic mono-oriented
both sister KTs attached to same pole - both go to one daughter
monotelic mono-oriented
only one KT bound to MT - other not bound
both may end up in same daughter
Merotelic
KT on one sister binds to MTs from both poles
this one sister can get stuck in middle
need to detect and fix incorrect attachments
Sensing Bi-oriented attachment
one difference when chromosomes are bi-oriented
mitotic pulling forces create tension at the sister KTs
doesnt properly occur in incorrect attachments
tension - allow segregation
no tension - need to wait/fix - mediated by aurora B
Microneedle experiments for sister KT tension
use glass microneedle to apply tension to mitotic chromosomes
take mono-oriented chormosome - no tension
apply tension
causes less phosphorylation of the KT proteins on the KT under tension by microneedle (aurora B stuff)
v low picoNewtons of force produced
but just above background level enough to move entities in cell
Yeast genetic KT tension experiments
circular chromosome with centromeric sequence and replication origin
upon chromosome replication - these centromere sequences are duplicated
produce tension
can produce minichromosome with no replication sequence (i guess can have chromosome from earlier one with head to tail SSR sites around it)
so no tension can be produced
have another site with an unactivated centromere
so can turn on in experiement to give same chromosome 2 centromeres
have tet operator that can be bound by TetR-GFP
only one centromere
becomes mono-oriented to one pole
activate 2nd centromere
can bi-orient
nw found in middle of poles in metaphase
tension is sufficient to allow bi-orientation
Error correction mechanism for incorrect kinetochore pole attachments
absence of tension
Aurora B kinase phosphorylates kinetochores
destabilises inappropriate attachments
allows another try for correct ones
once tension successfully produced
PP1 phosphatase removes phosphorylations from KT to stabilise correct orientation
aurora B activation = chromosomes get stuck at incorrect attachment
centromere specific histones
CENP-A
Cse4 in B yeast (conserved between these two)
has homology to core histone H3
co-purifies with core histones so is part of a nucleosome
Budding yeast kinetochore model
centromere specific nucleosome containing Cse4 (CENP-A) found at base
40 unique proteins - structural and regulatory
many components and structure conserved in humans
inner KT important for DNA binding at centromere
outer KT important for binding MT - incl. Dam1
-aurora B phosphorylation of Dam1 causes MTs to bind less stably - can come off attachment - hopefully reattach correctly
Spindle assembly checkpoint and KTs
SAC inhibits APC/C when KT not bound by MT
prevent anaphase entry
SAC activated by unattached KT
inhibits APC/C
KT attaches to MT
SAC silenced from that kinetochore
when all KTs attached
no more SAC signalling
(but if tension lacking then Aurora B can still de attach MTs until correct orientation -cause can have incorrect orientation w/out unattached KT)
All KT attached
+ correct tension
=progression to anaphase
Epigenetic determination of KT positions
chromosome should have 1 KT
dicentric chromosomes can cause mis-segregation and breaks
Different types of centromere
monocentric: function localised to one region - seen in F yeast, humans, trypanosomes
holocentric - holocentromeres: assemble KT all along the chromosomes
seen in C. elegans, silkworms, butterflies
Holocentric chromosome spindle binding
one theory for an advantage conferred by this
in a monocentric chromosome
if there is a dsBreak and chromosome is cleaved - small piece of chromosome w no centromere/KT can be lost easily
may lead to cell death/cancer
in holocentric organism
this fragment can still be bound by MT and inherited in mitosis
salvage it to avoid the genetic instability
potential advantage
but holocentrics pretty rare
so maybe disadvantages to this too
Metapolycentric chromosomes
eg in Pisum (pea)
multiple foci of KT
multiple foci clustered in one place rather than scattered
different attachment no.s on diff centromeres
point centromere
b yeast
125bp
1 MT binds
regional centromere
f yeast, human, trypanosomes, most
several kb-Mb
Holocentric
c elegans, butterflies
how/when did KT evolve
eukaryote evolved from archaea that acquired prokaryote endosymbiont
prokaryotes (archaea, bacteria) have no kinetochores
eukaryotes do
so evolved in Eukaryogenesis?
some in eukaryote group thought to have branched off before mitochondrial acquisition
but found to be wrong as have Mt remnants
KT study model organism issues
popular eukaryotes in KT research (yeast, human…) in the Opisthokont group (1/10 groups) so less info on others
issue as many pathogens come from other groups so could be helpful to better understand them (eg differences to target)
eg Kinetoplastid group
Kinetoplastids
many genome sequences available for many eukaryotes
can find canonical KT proteins in many
but not so much in kinetoplastids
KTs may look different in them
have several conserved components of mitosis
but hard to identify conserved KT components
Kinetoplastid mitosis
eg in T. brucei
kinetoplast organelles contain mtDNA clusters
basal body for flagellla
nuclear DNA
all 1 copy organelles
need to be duplicated and segregated into both daughters
diamond shape spindle in metaphase
elongates in anaphase
see a mitosis specific kinetochore like structure
Kinetoplastid KT-like structure
MTs bind it
sit back to back (as opposed to canonical sister KT that have ~1micron distance)
so where do aurora B and cohesin accumulate (this inner centromere region is important for this, none in kinetoplastid)
Finding the kinetoplastid KT proteins
localisation based screen
take random uncharacterised gene whose expression upregulated in S/M-phase
try find KT related protein in (mutation?) screens
localisation screen - to look at where these genes localise - see if they localise to KT positions
kinetoplastid KT proteins
KKT1 gene
not much in G1
more in s/G2
lines up on metaphase plate
through IPMS found more KT proteins with similar localisation
can then IP/MS these to find more
found KKT-2-25
so KKT1-25
no similarity to canonical KT
KT have diff structure
instead of conserved
this difference between these and canonical KTs allow for specific targeting of pathogens by drugs (eg small molecule CLK1 protein kinase inhibitor)
KKT4
has an MT binding domain
(different to canonical KT MT binding domains)
KKT protein evolutionary origin
found domains similar to other proteins in some KKT proteins
esp SYCP2-like domains
KKT17/18 form complex
have SYCP-2 like domains
usually only expressed in meiosis
SYCP2 and 3 are meiotic proteins
part of synaptonemal complex SC formed between homologous chromosomes
hypothesus that kinetoplastid KTs evolved from meiotic synapsis/recombination machinery
speculative
why kinetoplastids use unique KT
if KT are essential for cell proliferation
why have different from canonical KT
hypotheses:
possibility 1:
derived condition
derived this new one and lost canonical KTs
problem - why abandon an already working system for a new one - would need to find specific advantage
Possibility 2:
ancestral
never had canonical KTs
KKTs arose as alternative system
but if never ahd canonical ones - how did they segregate chromosomes before the new unique ones
answer could be different depending on which eukaryotes branched off first
Kinetoplastids couldve been one of earliest branching before canonical KT-havers
-support: rest of eukaryotes have canonical, Kinetoplastids dont
-can explain this by kinetoplastids branching first - so have completely diff KT proteins
bacteria and archaea dont have KTs
driver of chromosome segregation in prokaryotes
Entropy
2 long chains (2 DNA molecules) in same space
can separate from each other in order to increase the entropy increases
-spontaneously separate as entropy increases/to increase entropy
Canonical KT evolution
from which cell process did they evolve?
repurposes machinery from many cell functions
one interpretation:
early eukaryotes did not need KT
then later proteins from different pathways were repurposed into canonical KT
kinetoplastid KT evolution
archaea have HR/cell fusion machinery (SYCP2) and no KT, eukaryote LUCA had these too
so Kinetoplastids branched off earlier than canonical-KT eukaryotes
so had not canonical KT
evolved their unique set of KKT proteins using recombination machinery
this would suggest that meiosis evolved before mitosis (eukaryotic style mitosis i guess this refers to?) - not proven tho
Membrane bound KT
in dinoflagellates, parabasalids
KT embedded in nuclear envelope
mitotic spindles assemble outside nucleus
chromosomes inside (attached to KT so are at envelope inner side)
so the KTs need to embed into the envelope to contact the spindle MTs
how do they do this and still have bi-orientation?
they have canonical KT proteins
but no membrane localisation signal found so unsure how they do this
membrane bound KT evolution
2 independent evolution events
(lost in Oxyrrhinales)
as yet unknown KT types
no identifiable KT proteins found in diplonemid genomes via sequencing
could identify them in similar way to kinetoplstids
ie by screening unknown funciton genes upredulated in S/M phase
Diplonemids as models
werent thought to be v abundant at first
as rRNA used often to get sequence info for this purpose
but the primer used for rRNA seq was too divergent to recognise diplonemid rRNA (present in sample but not recognised)
found to be one of most abindant/diverse eukaryotes in oceas when took environmental DNA samples
often is difficult to study species that cant be cultured in lab
but diplonemid pretty tractable model
culturable in seawater
genome/transcriptome sequenced
transformation possible
could be pretty tractable models
KT proteins unknown
