Centrosomes + Kinetochores

Question 1

MT dynamicity

Answer 1

Grow and shrink by addition and removal of tubulin heterodimers

+ end more dynamic

MT constantly switch between growth and disassembly states
catastrophe and rescue

Question 2

KT interaction w MT

Answer 2

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

Question 3

kinetochore interaction with dynamic MT plus ends

Answer 3

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

Question 4

different MT attachments

Answer 4

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

Question 5

Sensing Bi-oriented attachment

Answer 5

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

Question 6

Microneedle experiments for sister KT tension

Answer 6

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

Question 7

Yeast genetic KT tension experiments

Answer 7

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

Question 8

Error correction mechanism for incorrect kinetochore pole attachments

Answer 8

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

Question 9

centromere specific histones

Answer 9

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

Question 10

Budding yeast kinetochore model

Answer 10

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

Question 11

Spindle assembly checkpoint and KTs

Answer 11

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

Question 12

Epigenetic determination of KT positions

Answer 12

chromosome should have 1 KT
dicentric chromosomes can cause mis-segregation and breaks

Question 13

Different types of centromere

Answer 13

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

Question 14

Holocentric chromosome spindle binding

Answer 14

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

Question 15

Metapolycentric chromosomes

Answer 15

eg in Pisum (pea)
multiple foci of KT
multiple foci clustered in one place rather than scattered

Question 16

different attachment no.s on diff centromeres

Answer 16

point centromere
b yeast
125bp
1 MT binds

regional centromere
f yeast, human, trypanosomes, most
several kb-Mb

Holocentric
c elegans, butterflies

Question 17

how/when did KT evolve

Answer 17

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

Question 18

KT study model organism issues

Answer 18

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

Question 19

Kinetoplastids

Answer 19

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

Question 20

Kinetoplastid mitosis

Answer 20

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

Question 21

Kinetoplastid KT-like structure

Answer 21

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)

Question 22

Finding the kinetoplastid KT proteins

Answer 22

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

Question 23

kinetoplastid KT proteins

Answer 23

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)

Question 24

KKT4

Answer 24

has an MT binding domain
(different to canonical KT MT binding domains)

Question 25

KKT protein evolutionary origin

Answer 25

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

Question 26

why kinetoplastids use unique KT

Answer 26

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

Question 27

driver of chromosome segregation in prokaryotes

Answer 27

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

Question 28

Canonical KT evolution

Answer 28

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

Question 29

kinetoplastid KT evolution

Answer 29

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

Question 30

Membrane bound KT

Answer 30

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

Question 31

membrane bound KT evolution

Answer 31

2 independent evolution events
(lost in Oxyrrhinales)

Question 32

as yet unknown KT types

Answer 32

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

Question 33

Diplonemids as models

Answer 33

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