Lamins

Question 1

lamin localisation

Answer 1

filament system in inner nuclear membrane
elastic enough to allow nucleus to distend
MT/actin would fall apart

Question 2

lamin building blocks basic

Answer 2

coiled coils come togehter
Ig folds stick out to side

mostly parallel coiled coil
but possible for it to take on ohter conformations

come together to produce 10nm filaments
severas strands of head-tail connected dimers

Question 3

lamin assembly

Answer 3

twisting together of ropes
that then themselves twist together

many lamins in cross section of eventual 10nm fibre

stick together well

heve hydrophobuc and polar/charged regions on in and outside
allows rearranging of structure
can disassemble (phosphorylation) and reassemble

Question 4

filament periodicity

Answer 4

at what point does the pattern repeat

originally seen as 25nm
but buffer conditions cause lamins to alter in their periodicity
could be due to flexibility of lamins

Question 5

CLMS

Answer 5

cross linking mass spectrometry

protein in solution
cross linking agents added
trypsin digest

mass spectrometry
unique mass and fragmentation spectra for diff proteins in trypsin digest - cross linking creates unique cross-linked structures in the digest that would not normally be there
can decode into a list of interacting/proximal residues of other proteins

can do in vitro and in vivo

Question 6

distinguishing intra vs inter-molecular interactions of lamins (using CLMS?)

Answer 6

use different isotopes in AAs to produce lamin As (gives heavy and light)

see if CLMS comes up with interactions between heavy and light
(will see heavy AND light isotopes in the cross linked species - ie will be species between the weight of the same heavy and light species)

can see if specific interaction between two lamin proteins is within the same dimer (will come up with only heavy or light X-linked species)
or if the interaction is between dimers (will also have mid-weigth X-linked species for that interaction)

Question 7

lamen disordered linkers

Answer 7

cross links concentrated disordered linkers indicated that electrostatic interactions fold individual coils over each other

this electrostatic compression of the lamins condenses the fibres

vimentins put them under tension to condense them in vivo
could explain why in vitro studies w just recombinant lamins didnt get right length

Question 8

lamin stretching

Answer 8

could alter the thickness
can see with GFP-Lamin A

GFP density higher at one end of fibre
stretched out - lower GFP density
could explain differences in thickness
10nm one part
3.5nm seen in another
could explain why diff studies see diff thicknesses

pulling on the edge of cells affects the nucleus

Question 9

diff lamin subtypes

Answer 9

multiple lamin genes
Lamin A - many splice isoforms (incl, lamin C)
LaminB - B1 and 2 diff splice isoforms

can give diff characteristics to nucleus w diff lamin compositions
different distinct organisations
can see by overexpressing diff lamin types

FRET can be used to observe diff lamin interactions (closer than 10nm)
lamin B1 and A show this (may not be direct interaction

Question 10

lamin dynamics

Answer 10

nuclear envelope disassembles in mitosis
increase in lamin phosphorylation seen
this drops again in G1
phosphorylation changes lamin properties allowing lamin polymer (hence NE) disassembly

in interphase
see many phosphoryalted groups on lamins
but at low frequency
can allow some dynanmicity in the lamina if needed
eg need to grow nucleus as DNA content is doubled in S-phase
allows breaking apart and reassembly of some NE areas to grow them?

Question 11

c-Fos TF interaction w lamin A

Answer 11

FRET interaction between c-Fos/Lamin A
photobleach c-Fos
CFP-lamin A signal goes up as c-FOS can no longer act as acceptor for that energy
exist v close in cell

overexpress lamin A
recruits c-Fos to lamin A
>c-FOS can no longer go to its target genes
>drop in activity of its target genes
>Lamin-A acts to sequester c-FOS TF

Question 12

lamins and disease

Answer 12

diff lamin subtypes
diff amounts of diff subtypes in diff cell types
have tissue specific binding partners

so can explain the variety of tissue specific diseases that come from lamins
even diff muscle groups affected preferentially by diff laminopathies

Less disease linked to lamin B1 and B2 as there are to Lamin A
because Lamin A not as important in early developmental steps
so can get by with mutant Lamin A better until alter on when disease can develop

Question 13

adjacent mutations in lamin and diff diseases

Answer 13

diff diseases can come from mutatiosn close by
shows that different disease function not so closely linked to structures within the lamin eg the coiled coil domain

Question 14

progerin processing

Answer 14

lamin A processed from Prelamin A (progerin)

in processing:
50 amino acid deletion
then farnesylation of C-terminus
AAX cleaved from C-term (leaves farnesylation as that is on C before AAX - C/AAX)
-methyltation of now terminal C
-this forms prelamin A (progerin)

in HGP - mutant progerin is not removed/processed to lamin A

causes genetic expression regulation changes

Question 15

lamin and cardiomyopathy

Answer 15

ERK signalling pathway lined with Lamin A

in lamin associated cardiomyopathy
can treat with ERK inhibitor - rescue WT phenotype
dropped signalling molecule levels (eg Beta-catenin) in cardiomyopathy mice

many other signalling pathways involved too

Question 16

IF proteins in industry

Answer 16

unique elastic properties in lamins

can take IFs from hagfish slime
dialyse and bring them together to give big sheets (kinda like paracrystals)

gives tough but flexible polymer (eg for armour)

stronger than silkworm/dragline silk
break strain gets higher when wet even