Biochem-13-Structure of Tubulin and Microtubules- assembly/disassembly of microtubular structures

what are the 3 main cytoskeletal filaments and their composition- what cellular structures made of these?

1. microfilaments- actin- mitotic spindle/cytoplasmic networks/cilia/axons

2. microtubulin- tubulin

3. intermediate filaments- 70+ protein types

diameter of microtubules and who discovered them

George Palade- 24nm

composition and the structure of microtubules?

• hollow like structure- 13 protofilaments

• protofilaments- linear chain of polymerised alpha/beta tubular heterodimers

• alpha(55kDA) tubulin at the minus end, beta(53kDA) tubulin at the plus end

• both sides that GTP binding sites

how do microtubules assemble? what are the key interactions? what’s stronger?

• adding alpha/beta/alpha/beta sequentially- helical fashion- these make longer protofilaments

• protofilaments- linear chain of dimers. 13 of these make a hollow tube- microtubulin

• this doesn’t happen sequentially!!! all protofilaments grow TOGETHER at the same time

• top to bottom is stronger from + beta to - alpha end than side to side protofilament next to another protofilament

what conditions need to be met for experiments to look at microtubulin assembly?

• GTP present for tubular polymerisation

• low Ca2+- high calcium inhibits this

• high tubulin conc.

• normal 37. temperature

what assays can be done to measure assembly of microtubulin?

1. electron microscopy- time consuming and expensive

2. turbidity assay- the more polymers, its more turbid- measured at 340nm. shows the RATE of polymerisation. determine critical concentration of tubulin needed for growth

phases of assembly observed by turbidity assay? what is the critical concentration?

1. lag phase- where the 13 heterodimer protofilaments come together to form the hollow tube

2. exponential- where microtubules grow quickly

3. plateu- tubulin is limited

critical concentration is the concentration of tubulin needed for microtubulin growth

what is the microtubule organising centre? where is it located? key features

• MTOC: centrosome near the nucleus

• controls microtubule polymerisation throughout the cell

• has gamma tubulin ring complexes y-TuRC- rapidly nucleate microtubule assembly. microtubules grow from the PLUS end(beta)

how do microtubules grow after nucleation?

• grow rapidly after nucleation when alpha/beta heterodimers are available

• alpha/beta have GTP binding sites

• GTP on beta(+) is hydrolysed to GDP

• GTP Cap is added to the new growing end to stabilise it

GTP role in assembly and disassembly?

• + end Beta tubulin E-GTP is hydrolysed to GDP and is less stable and has lower affinity

• - end alpha tubulin N- GTP is still tightly bound and not hydrolysed

• GTP cap- formed by the newly added beta tubulin and is GTP bound-slows the shrinkage and added to the new beta dimers to stabilise the growing end

• when GTP cap is lost- protofilaments peel away and it shrinks

what are MAPs? what drugs are associated with this?

microtubule associated proteins

• bind polymerised microtubules to keep the, stable and filaments organised

• protects them from severing and crosslinks them to actin and membrane association

• controls the filament numbers and bundling

• Tau protein- when hyperphosphorylated the microtubules are disorganised and dissociate easier- neurodegeneration

Taxol- binds to Microtubule and blocks it from dissociation- a stabiliser.

colcemid/colchicine- blocks the assembly of microtubules to ctop cell division- in cancer

difference between a growing and shrinking microtubule?

growing: adding of alpha/beta GTP tubulin to the + end is faster than GTP hydrolysis. GTP cap on new beta tubulin stabilises longitudinal interactions. alpa tubulin - keeps GTP bound

shrinking- GTP hydrolysis on beta tubulin is faster than the addition of new dimers, GTP cap is lost and is less stable, begins to dissociated. released GDP can be recharged with GTP to grow again