Cytoskeletal Dynamics David Stephans L1-2

Question 1

alpha and beta tubulin

Answer 1

capable of binding guanine NTs

Question 2

Microtubules

Answer 2

Predominantly involved in separating the chromosomes

Contact cortex of the cell where actin is

Question 3

Actin

Answer 3

Generate cleavage furrow for separating 2 daughter cells

Question 4

Dynamic instability of MTs
Reversible process
Filaments grow and shrink in an energy dependent manner

The property of dynamic instability means that the cell can respond to its environment very rapidly

Answer 4

GTP capped end promotes growth of MT
Promotes association of another a/b dimer
Accidental loss of GTP cap-CATASTROPHE, subunits are no longer added DEPOLYMERISATION
Rapid shrinkage
Regain of GTP cap, RESCUE-factors promote more association of tubulin to the ends of MTs (stabilisation) promotes association of GTP
Rapid growth of GTP-capped end

Question 5

Microtubules-GTP binding and conformational strain

Answer 5

GTP promotes a conf change of the whole protofilament that is straight
GTP hydrolysis changes subunit conformation and weakens bond in the polymer-curved protofilament
Depolymerisation
Protofilaments don’t stick together well in GDP bound form as forces weaken
Decreases tensile strength within a MT

Question 6

Microtubule binding proteins MAPs

Answer 6

Lots exist in cells
MT associated proteins-any protein that binds to the MT
eg all the motor proteins

MAPs bind to GTP cap(+end) and bridge the subunit-stabilisation, scaffolds the straight ends
RESULT:longer less dynamic microtubules

Catastrophe factor (KINESIN 13) eg motors-binds to the GTP cap and uses its energy from ATP hydrolysis to pull apart the protofilaments (even if GTP is bound)
Promotes GTP hydrolysis and disassembly of the filaments
RESULT:shorter, more dynamic MTs
Important in mitosis when dynamic reorganisation is required

Question 7

Gamma tubulin ring complex

Answer 7

Promotes nucleation of MTs
The centrosome (-end of MTs) contains gamma ring tubulin complexes
GCP proteins
gamma tubulin-encoded by separate gene-distinct in structure
Sits at base of MT onto of accessory proteins

Question 8

CENTROSOME=

Answer 8

Big balls of protein (matrix) that assemble around a pair of centrioles-these are always 90degrees to each other in a normal cell
Made up of several 100s of proteins that associate with the centrioles in a very ordered manner
PERICENTRIN-major proteins in centesimal matrix
Centrioles themselves are made up of short specialised MTs
Made up of TRIPLETS of protofilaments
2 Centrioles are different
The ability to find the middle of the cell is an intrinsic property of the centrisome
Approx 0.5micro-m across
Quite a big structure within a cell
Mother (more elaborate centriole with all the functions) and daughter centrioles-this difference is important in asymmetrical cell division

The centriole matrix itself has capacity to regulate signalling NEK2-kinase PP1-protein phosphatase

Question 9

Mother centriole

Answer 9

Distal appendages
Subdistal appendages-specialist factors associate here -used to detect only mother centriole

Intercentriolar link -connects mother and daughter centrioles

Primary cilia formation-axonemal MTs extending from the mother centriole

Question 10

Nucleation

Answer 10

a/b dimer more likely to bind adjacent to one that is already there-more interactions
A template increases the numbers of interactions
A nucleator gets rid of the lag phase and promotes process from the outset

Question 11

What proteins are in the gamma TuSC complex (Tubulin small complex) and how is it formed?

Answer 11

GCP2/3 with 2 gamma subunits on top
7 copies form a helical structure for the nucleator/template in a “lock washer” spiral
gamma tubulins define the protofilaments in your MTs
Adjacent complexes overlap by about 1/2 -lose one from your 14
13 fold symmetry of MTs-beta subunits overlay directly onto the gamma-tubulins

Question 12

Microtubule “seam”

Answer 12

Intergral for how MTs depolymerise, the helical arrangement zippers it up well yet since the subunits are so offset from each other you end up with a structural defect along it
The MT is discontinuous

Question 13

Proteins and structure of gamma-TuRC

Answer 13

GCP 456 and gamma-TRSC
The GCP attachment factors can also change the behaviour of the complex to make it more or less likely to polymerise its MT
It can also modulate behaviour since it can localise the centrosome
These factors can also specify binding of the gamma-TuRC to the side of an existing spindle MT. Increases density of meshwork
Attachment factors can alter MT stability, anchoring and even direct branching (mitosis spindle)

Question 14

What protein dictates the NINE FOLD SYMMETRY of the centriole hub?

Answer 14

Sas-6 assembly
Sas-6 dimer sticks out 9 times.
MT triplets are interlinked by a hub
hub defines the spatial organisation of the rest of it
hub is made up of Sas-6 DIMER-forms a base and a rod which sticks out

Question 15

Non-cycling cells

Cant send off cilium in a cycling cell

Answer 15

In non-cycling cells(means cell cycle) (FORMATION OF PRIMARY CILIA), the mother centriole matures to FORM the basal body

The basal body directs the formation of primary cilia
Centrosome function underpins both the cell cycle and cilia function
Mutations in genes encoding centrosomal proteins are linked to many developmental diseases and cancer

Non cycling-forms primary cilia
Cycling-need both centrioles to duplicate so that the 2 daughter cells each have a mother and daughter centriole pair

Question 16

Centrosome during cell polarisation

Answer 16

1) moves to the front of a migrating fibroblast- MTs concentrated at front of cell
2)T-target cell interaction, centrosome migrates to the contact point on the p.memb and delivers the granules for the active cytotoxic killing
3)axon specification-centrosome specifies the fates of one of the neurites in an immature neutron to become an axon-localises to neurite.
Stabilsing MTs within a neurite makes it become an axon

Position of centrosome acts to determine the fate of other parts of the cell

Question 17

TAXOL

Answer 17

Drug that stabilises MTs-used in treatment of breast cancer to stop the continual assembly and disassembly of MTs in mitosis which promotes cell division/proliferation

Question 18

Wounds and cell migration

Answer 18

Centrosome nucleus and golgi all polarise.
Polarisation of the centrosome and regrowth of the MTs towards the direction you want to migrate it
MTs interact with the p.memb (+ end)

Question 19

Plus-end complexes capture MTs at the cell cortex

Answer 19

GTPase effector
GTP-active
GDP-inactive and MT detaches from the membrane
More GTP more tight interactions with cell cortex

+ end tracking proteins that selectively bind to the + end of MTs e.g. EB1 (end binding protein 1acts as a scaffold for GTPase effectors)

Question 20

Does the centrosome move?

Answer 20

NO
The centrosome is maintained yet the nucleus moves to the “back” of the cell
MTs maintain the position of the centrosome in the middle of the cell since they pull from all directions
The dynein is not pulling on the centrosome
Dynein maintains the position of the centrosome

Myosin II drives the nucleus to the back of the cell.

Question 21

Dynactin

Dynein/dynactin mediates VTC movement
Vesicular tubular cluster
ER….COPII….VTCs….Golgi

Answer 21

Links dynein to cargo
Increases PROCESSIVITY of dynein
Dynactin has its own MT binding domain and gives dyne a third leg-there are always 2 attachment sites to the MT where-ever it is-therefore its more likely to stay associated with its MT

Question 22

Which motors are regulated by unfolding?

Answer 22

Myosin V
Kinesin I
They unfold upon cargo binding
Induced by binding of activators(KINESIN1)/adaptors(BOTH)

Question 23

Which motors are regulated by dimerisation?

Answer 23

Myosin VI
Exists in inactive monomeric state
Binding of cargo or cargo adapter triggers dimerisation and membrane association
This dimeric form is the active motor form

Question 24

Melanocytes-pigmented cells

Model for BIDIRECTIONAL motility

Answer 24

Kinesin-2
Dynein (drives things into middle of cell, -end directed)
Myosin Va
ALL ASSOCIATE WITH MELANOSOMES (pigment granules)
Kinesin 2 and dynein are both coupled to melanosomes by dynactin (control by same activator)
Movement regulated by hormones and neuronal activity
Dispersion vs aggregation controlled by signalling
Positioning of melanosomes are defined by crosstalk b/w actin and MT networks

Actin filaments distribute randomly through the cytoplasm

Question 25

Kinesin 2

Answer 25

Causes complete dispersion
When dynein is suppressed, plus end motility dominates
Melanosomes disperse
ONLY WHEN ACTIN BASED MOVEMENT IS INHIBITED
melanosomes accumulate in the periphery

Question 26

Balance of myosin Va, dynein, kinesin-2 leads to uniform distribution

Answer 26

If actin-based motility is intact, melanosomes transfer frequently between actin and MT networks and become spread throughout the cytoplasm

MT motors move fast
Actin motors move slow (10th of the speed)

Question 27

Griscelli syndrome (GS)
What are the symptoms?
3 types

Answer 27

Autosomal RECESSIVE hereditary disease
Rare-develops early in life
GS1 and GS2 are primarily characterised by immunological defects and immunodeficiency (GS2) or neurological dysfunctions and impairments (GS1)
GS3 is restricted to hypopigmentation disorder

GS1 - mutation in myosin Va (drives actin based motility)
Melanosomes aggregated
You can no longer control the tethering of the melanosomes to the ACTIN network because of this mutation

GS2 - mutation in Rab27a (small GTPase) provides a specific membrane anchor
GS3 - mutation in melanophilin (sticks to melanocytes and accumulate around nucleus - aggregates) acts as a cargo adapter - like spectrin

If you mutate any of these 3 components with COMPLETE loss of function you lose ability to couple melanosomes to the actin filaments