D103 Intermediate Filaments (ALS 6, Video 11)

migrating cell contains 2 sets of MTs

golgi apparatus and centrosome

golgi apparatus

• additional site of MT nucleation

• MTs are directed to the leading edge

• function: enhancing vesicular transport to the leading edge

centrosome (MTOC)

• primary site of mt nucleation

• radial array

• function: organelle positioning, vesicular transport routes, mitosis (chromosome segregation)

• - end is fixed = no treadmilling 

how would you test if golgi-nucleated MTs are more stable than centrosome-nucleated MTs in interphase 

tag alpha-tubulin with GFP and perform a FRAP experiment of each MT population  

• frap could indicate movement and stability

which of these graphs describes the most stable population of microtubules (cells express GFP-tagged alpha-tubulin)?

no recovery

• you cannot have recovery if very stable because it cannot freely move

• dependent on depol with repol with new mix of gfp tag

researchers treated cultured human cancer cells with different concentrations of taxol and measured 2 parameters:

1. percentage of cells in mitosis

2. average rate of microtubule depol

based on the data, which conclusion best explains how taxol inhibits cancer cell division?

taxol arrests cells in mitosis because MTs are stabilized

what happens to the nuclear lamina during mitosis 

disassembles 

• look for depolymerization and lining of nuc envelope 

• MTs provide structural support 

which of the following conclusions is best supported by this experiment

in mutant cells, the nuc lamina does not break down

you now focus on lamin a and its role in nuclear envelope breakdown. the mutation prevents lamin a phosphorylation. which of the following statements best explains your result

lamin a phosphorylation is necessary for NEB

• no lam A = no NEB

which is NOT true for primary cilia 

they function in moving liquids over a surface 

f-actin overview

size: 6 nm

energy for assembly: ATP

energy for disassembly: none

building block: g-actin

polarity in filament: yes

typical features: treadmilling

molecular motor: myosins

one prominent funct: migration

MT overview

size: 25 nm

energy for assembly: GTP

energy for disassembly: none

building block: a/ B-tubulin

polarity in filament: yes

typical features: dynamic instability

molecular motor: kinesins/ dynein

one prominent funct: chromosome seg

intermediate filament overview

size: 10 nm

energy for assembly: no

energy for disassembly: yes, ex - phosphorylation

building block: 70 different kinds of monomers

polarity in filament: monomer and dimer, not others

typical features: sturdy structure

molecular motor: no

one prominent funct: support for epithelial cell layer

functions of intermediate filaments

70 members with diverse cellular functions

• provide structural support

• plectin, desmoplakin, filaggrin

plectin 

links IFs to other cytoskeletal structures 

desmoplakin

desmosomes/ cell junction

filaggrin

bundles keratin filaments

intermediate filament monomers 

polar

intermediate filament dimer (homo or hetero)

polar because of head-to-head assembly

intermediate filament of staggered tetramer of 2 coiled-coil dimers

anti-parallel = apolar

intermediate filament of 2 tetramers packed together end-to-end 

apolar 

are intermediate filaments dynamic

yes, must assemble and disassemble to incorporated (dynamic)

• assembly occurs independent of energy input

keratin function

provide structural support to epithelial cells

epidermolysis bullosa

keratin mutations 

• blistering in response to minor injury and inside the skin 

• caused by mutations in keratin 5 (type II) and keratin 14 (type I) 

nuclear lamina function

provides structural support to the nucleus

• nuclear lamins form a 2-dimensional meshwork

• lamins are nuclear protein and contain nuclear localization signal

• the nuclear lamina has at least 4 different roles

• dynamic and changes as the cell goes thru the cell cycle

lamin phosphorylation and nuclear envelope breakdown

phosphorylation induces nuclear lamina disassembly, which is necessary for nuclear envelope breakdown

• MT and F-actin require energy for assembly, but none for disassembly

hutchinson-gilford progeria syndrome

mutatiosn in lamin a gene → point mutation prevents heterodimer formation with lamin C → support for nucleus is lost 

• accelerated aging 

intermediate filament overview

tripartite structure

monomer and dimer = polar; higher order = non-polar

dynamic → assembly occurs spontaneously, but energy is required for disassembly (generally thru phosphorylation)

provide structural support to the cell and the nucleus

keratins overview

assembly of keratins is essential for epithelial cell stability, mutations cause severe human diseases

nuclear lamina overview

intermediate filament that supports the nuclear envelope: 

• disassembles during mitosis 

• reorganization occurs during the cell cycle 

• reorganization is induced by lamin phosphorylation 

• mutations: progeria and muscular dystrophies