microtubules Flashcards
what are the three types of cytoskeletal filaments?
- actin microfilaments
- aB tubular microtubules
- intermediate filaments
what are the properties of the cytoskeleton?
- dynamic but organised structure
- formed from the polymerisation of small subunit proteins
- provides cellular structure and organisation
- each type of cytoskeletal filament has unique properties that allow it to perform specific functions for the cell
what are the functions of intermediate filaments?
cell strength and mechanical support
what are the functions of actin microfilaments?
- cell structure and shape
- cell migration
what are the functions of microtubules?
- organisation of intracellular structures and organelles
- intracellular vesicle trafficking
- chromosome segregation during mitosis
what is the structure of microtubules?
- dimer of alpha and beta tubulin in a 13 protofilament hollow tube
- a tubulin has a GTP binding site which is constantly GTP bound
- B tubulin has an exposed GTP binding site
- microtubules have a polarity
what is the ‘minus end’ of a microtubule?
the end with a-tubulin at the end
what is the ‘plus end’ of the microtubule?
the end with B-tubulin at the end
which end of the microtubule are new subunits added and removed from?
added- plus end
removed - minus end
what is the significance of the B-tubulin at the ‘plus end’ of a microtubule?
the exposed GTP/GDP binding site permits hydrolysis, allowing it to regulate dynamics
what is the role of the centrosome?
it is the major microtubule organising centre of the cell
what is the structure of the centrosome?
consists of two pairs of centrioles and is surrounded by a protein network that is capable of nucleating microtubules
what end of the microtubule is nucleated at the centrosome?
the minus end
how is microtubule dynamics regulated by GTP hydrolysis?
- dimer with GTP bound is added to the plus end, over time, this can be hydrolysed
- this causes a bend in the protofilament, de-stabilising the microtubule
- this causes GDP bound subunits to dissociate
- if GTP is bound to the plus end, this is a stabilised microtubule
what is meant by dynamic instability of the microtubule?
there is a cap of GTP bound a/B tubulin, this stabilises the plus end
- in this state, the microtubules are able to grow
- however, it can switch to an unstable state rapidly when GTP is hydrolysed, causing curvature of the filament, de-stabilisation and rapid de-polymerisation of the microtubule
what is the function of dynamic instability?
it allows rapid re-organisation of microtubules in response to appropriate cell stimuli
what are the functions of microtubule associated proteins?
regulate microtubule dynamics by:
- stabilising filaments
- destabilising filaments
- linking filaments to other molecules or structures
how do MAPs control microtubule dynamics?
controlling when microtubules:
remain stable
depolymerise
grow
how does Tau regulate microtubules?
binds to the side of the microtubule, keeping it in rigid conformation
this allows them to form parallel bundles
what is kinesis-13 and how does it regulate microtubule dynamics?
- end binding protein that promotes catastrophe during mitosis
- causes curvature of the protofilaments, destabilising them
- they are motor proteins that use ATP
what feature of the microtubule allows motor proteins to travel in opposing directions down the microtubule?
polarity
which direction do kinesins move cargo?
towards the plus end, at the periphery of the cell
which direction do dyneins move cargo?
towards the minus end, at the centrosome
how do kinesins move?
a conformational change upon ATP hydrolysis allows one subunit to be moved in front of the other
what are the major functions of microtubules?
- determinant of intracellular organisation and movement
- positioning of organelles
- intracellular trafficking
- cell polarity
- formation of the mitotic spindle
- formation of cilia and flagella
give an example of when microtubules might contribute to cell polarity
in epithelial cells
the minus end of the microtubule is aligned to the apical domain and the plus end is aligned to the basolateral domain
which techniques might you use to investigate microtubule function?
- use of an inhibitor of the protein
- expression of a mutant that impairs or abolishes protein function
- knocking down protein expression by RNAi
what is nocodazole?
a drug used to depolymerise microtubules. binds to free a/B tubulin, preventing it from incorporation into growing microtubules
how can we demonstrate which motor protein is responsible for a function?
- dynein inhibition
- kinesin inhibition
how can we inhibit dynein?
- use inhibitor such as EHNA
- use RNA interferences to knock down expression of one of the dynein subunits
what happens when you overexpress a kinesin dominant headless mutant?
- binds to cargo by the tail domain
- outcompetes endogenous protein but cannot bind to the microtubule
- prevents cargo from being transported to its destination
