BIOLOGY 2B03 Lecture Notes - Lecture 15: Atp Hydrolysis, Dynein, Kinesin
3 May 2018
School
Department
Course
Professor
Cytoskeleton*
The*cytoskeleton*provides*cell* shape*and*structure,*cell* shape*depends*
upon*the*unique*functions*of*different*filaments*
•
There*are*3*classes*of*filaments*in*eukaryotic*cells:*Actin*filaments,*
Microtubules*and*Intermediate*filaments;*they*can*be*visualized*using*
various*methods*such*as*GFP*antibodies*
•
Actin*filaments*are*the*thinnest*filament*and*are*comprised*of*actin*
subunits*
•
Microtubules*are*the*thickest*filaments*and*are*made*up*of*dimeric*
subunits*of*a-and*b-tubulin*
•
There*are*filament*specific*motor*proteins*that*track*along*actin*filaments*
and*microtubules,*they*are*Myosin*proteins*and*kinesin*&*dynein*
respectively*
•
The*head*domains*bind*to*the*cytoskeletal*fiber*and*the*tail*domain*
attaches*to*a*cargo,*with*ATP*hydrolysis*providing*the*energy*for*
movement
•
Actin
The*highest*density*of*actin*is*at*the*cell* periphery,*they*determine*the*
shape*and*movement*of*the*cell*surface
•
Actin*filaments*are*two-stranded*helical*polymers,*each*built*from*actin*
monomers*called*G-actin
•
G-actin*can*be*divided*into*four*structural*domains*with*a*large*cleft*
between*domains*2*and*4,*which*forms*an*ATP-nucleotide*binding*site*
•
Actin*filaments*are*polar,*the*plus*end*grows*more*quickly*through*the*
addition*of*more*actin*subunits*and*has*a*barbed*appearance*whereas*the*
minus*end*grows*more*slowly*and*may*shrink*and*has*a*pointed*
appearance
•
There*tends*to*be*more*growth*at*the*plus*end*and*more*shrinkage*at*the*
minus*end
•
ATP*regulates*the*growth*and*disassembly*of*the*actin*filaments*•
Actin*has*an*intrinsic*ATPase*activity*that*hydrolyzes*ATP,*so*most*of*the*
actin*filament*is*made*up*of*actin*ADP*(which*isn't*released*on*the*plus*
end,*but*is*so*on*the*minus*end)*
•
Critical*concentration -the*concentration*at*which*the*rate*of*actin*
monomer*addition*is*equal*to*the*rate*of*removal*
Factors*regulating*the*rate*of*these*reactions:*
Profilin*binds*to*actin-ATP,*promoting*ATP*binding*and*activating*the*
monomer
-
Thymosin*binds*to*actin*monomers*and*inhibits*polymerization*
-
Capping*proteins*on*the*ends*of*actin*filaments*can*inhibit*
polymerization*or**depolymerization*
-
•
Tread*milling -a*phenomenon*observed*when*there*is*no*net*increase*in*
the*length*of*the*filament,*but*the*filament*effectively*moves*forward
Myosin*
8*types*of*Myosin*have*been*identified,*with*Myosin*I,*II*&*V*being*present*
in*nearly*all*eukaryotic*cells*
•
They*all*share*a*characteristic*motor/head*domain*however,*tail*domains*
are*highly*divergent*in*order*to*carry*different*cargo*at*different*rates*with*
most*moving*towards*the*plus*end*of*actin*filaments*
•
Myosin*II*contains*two*heavy*chains*and*four*light*chains,*phosphorlyation*
of*these*light*chains*by*initiating*extension*of*the*myosin*tails*and*
activating*the*actin-binding*domains*on*the*motor*heads*
•
An*assembly*of*15-20*myosin*II*proteins*forms*a*bipolar*filament*called*
the*myosin*II*thick*filament*
•
The*motor*heads*of*the*myosin*II*thick*filament*are*exposed*for*
association*with*actin*filaments*
•
Sarcomere*
Myosin*II*thick*filaments*&*actin*filaments*make*a*structure*known*as*a*
sarcomere*
•
Components*of*the*sarcomere:*
Z*discs*fix*the*plus*ends*of*actin*filaments*to*the*sarcomere*
-
Tropomodulim*caps*the*minus*end*
-
CapZ*caps*the*plus*end*
-
Nebulin*binds*together*parallel*actin*filaments*
-
Titin*attaches*the*myosin*thick*filaments*to*the*Z-discs
-
•
Cycle:*
Myosin*is*attached*to*actin*
-
ATP*binding*to*myosin*releases*actin*
-
ATP*is*hydrolyzed*to*ADP*and*Pi,*which*changes*the*myosin*
conformation,*returning*it*to*the*relaxed* conformation*
-
The*release* of*Pi*increase*the*affinity*of*the*myosin*head*for*actin*
and*allows*binding*
-
The*release* of*ADP*changes*myosin*conformation*again,*putting*the*
cycle*back*at*step*1
-
•
Myosin*V*protein*powers*intracellular*trafficking*of*cargo*along*actin*
filaments*
•
Ex.*The*movement*of*melanosomes*in*skin*cells* called*melanocytes
A*loss*of*function*mutations*in*myosin*V*leads*to*phenotype*called*
the*dilute*phenotype*in*which*pigments*are*associated*with*fur*
color*not*distributed*to*the*fur*resulting*color*is*diluted*
-
The*rate*of*myosin*motor*protein*movement*varies*with*the*different*
myosin*proteins*and*depends*upon*the*cycle*of*nucleotide*binding*and*
hydrolysis*and*varies*with*the:*
The*rate*of*ATP*hydrolysis*and*ATPase*
-
The*proportion*of*time*myosin*is*bound*to*the*actin*filament*(a*
result*of*affinity)
-
•
Myosin's*step*size*depends*on*its*lever*arm*length*as*this*is*the*distance*
by*which*the*power*stroke*propels*the*myosin*forward
•
Module'7:'Lecture'1'
Tuesday,* March* 27,*2018
8:15*PM
Cytoskeleton*
The*cytoskeleton*provides*cell* shape*and*structure,*cell* shape*depends*
upon*the*unique*functions*of*different*filaments*
•
There*are*3*classes*of*filaments*in*eukaryotic*cells:*Actin*filaments,*
Microtubules*and*Intermediate*filaments;*they*can*be*visualized*using*
various*methods*such*as*GFP*antibodies*
•
Actin*filaments*are*the*thinnest*filament*and*are*comprised*of*actin*
subunits*
•
Microtubules*are*the*thickest*filaments*and*are*made*up*of*dimeric*
subunits*of*a-and*b-tubulin*
•
There*are*filament*specific*motor*proteins*that*track*along*actin*filaments*
and*microtubules,*they*are*Myosin*proteins*and*kinesin*&*dynein*
respectively*
•
The*head*domains*bind*to*the*cytoskeletal*fiber*and*the*tail*domain*
attaches*to*a*cargo,*with*ATP*hydrolysis*providing*the*energy*for*
movement
•
Actin
The*highest*density*of*actin*is*at*the*cell* periphery,*they*determine*the*
shape*and*movement*of*the*cell*surface
•
Actin*filaments*are*two-stranded*helical*polymers,*each*built*from*actin*
monomers*called*G-actin
•
G-actin*can*be*divided*into*four*structural*domains*with*a*large*cleft*
between*domains*2*and*4,*which*forms*an*ATP-nucleotide*binding*site*
•
Actin*filaments*are*polar,*the*plus*end*grows*more*quickly*through*the*
addition*of*more*actin*subunits*and*has*a*barbed*appearance*whereas*the*
minus*end*grows*more*slowly*and*may*shrink*and*has*a*pointed*
appearance
•
There*tends*to*be*more*growth*at*the*plus*end*and*more*shrinkage*at*the*
minus*end
•
ATP*regulates*the*growth*and*disassembly*of*the*actin*filaments*•
Actin*has*an*intrinsic*ATPase*activity*that*hydrolyzes*ATP,*so*most*of*the*
actin*filament*is*made*up*of*actin*ADP*(which*isn't*released*on*the*plus*
end,*but*is*so*on*the*minus*end)*
•
Critical*concentration -the*concentration*at*which*the*rate*of*actin*
monomer*addition*is*equal*to*the*rate*of*removal*
Factors*regulating*the*rate*of*these*reactions:*
Profilin*binds*to*actin-ATP,*promoting*ATP*binding*and*activating*the*
monomer
-
Thymosin*binds*to*actin*monomers*and*inhibits*polymerization*
-
Capping*proteins*on*the*ends*of*actin*filaments*can*inhibit*
polymerization*or**depolymerization*
-
•
Tread*milling -a*phenomenon*observed*when*there*is*no*net*increase*in*
the*length*of*the*filament,*but*the*filament*effectively*moves*forward
Myosin*
8*types*of*Myosin*have*been*identified,*with*Myosin*I,*II*&*V*being*present*
in*nearly*all*eukaryotic*cells*
•
They*all*share*a*characteristic*motor/head*domain*however,*tail*domains*
are*highly*divergent*in*order*to*carry*different*cargo*at*different*rates*with*
most*moving*towards*the*plus*end*of*actin*filaments*
•
Myosin*II*contains*two*heavy*chains*and*four*light*chains,*phosphorlyation*
of*these*light*chains*by*initiating*extension*of*the*myosin*tails*and*
activating*the*actin-binding*domains*on*the*motor*heads*
•
An*assembly*of*15-20*myosin*II*proteins*forms*a*bipolar*filament*called*
the*myosin*II*thick*filament*
•
The*motor*heads*of*the*myosin*II*thick*filament*are*exposed*for*
association*with*actin*filaments*
•
Sarcomere*
Myosin*II*thick*filaments*&*actin*filaments*make*a*structure*known*as*a*
sarcomere*
•
Components*of*the*sarcomere:*
Z*discs*fix*the*plus*ends*of*actin*filaments*to*the*sarcomere*
-
Tropomodulim*caps*the*minus*end*
-
CapZ*caps*the*plus*end*
-
Nebulin*binds*together*parallel*actin*filaments*
-
Titin*attaches*the*myosin*thick*filaments*to*the*Z-discs
-
•
Cycle:*
Myosin*is*attached*to*actin*
-
ATP*binding*to*myosin*releases*actin*
-
ATP*is*hydrolyzed*to*ADP*and*Pi,*which*changes*the*myosin*
conformation,*returning*it*to*the*relaxed* conformation*
-
The*release* of*Pi*increase*the*affinity*of*the*myosin*head*for*actin*
and*allows*binding*
-
The*release* of*ADP*changes*myosin*conformation*again,*putting*the*
cycle*back*at*step*1
-
•
Myosin*V*protein*powers*intracellular*trafficking*of*cargo*along*actin*
filaments*
•
Ex.*The*movement*of*melanosomes*in*skin*cells* called*melanocytes
A*loss*of*function*mutations*in*myosin*V*leads*to*phenotype*called*
the*dilute*phenotype*in*which*pigments*are*associated*with*fur*
color*not*distributed*to*the*fur*resulting*color*is*diluted*
-
The*rate*of*myosin*motor*protein*movement*varies*with*the*different*
myosin*proteins*and*depends*upon*the*cycle*of*nucleotide*binding*and*
hydrolysis*and*varies*with*the:*
The*rate*of*ATP*hydrolysis*and*ATPase*
-
The*proportion*of*time*myosin*is*bound*to*the*actin*filament*(a*
result*of*affinity)
-
•
Myosin's*step*size*depends*on*its*lever*arm*length*as*this*is*the*distance*
by*which*the*power*stroke*propels*the*myosin*forward
•
Module'7:'Lecture'1'
Tuesday,* March* 27,*2018 8:15*PM
Document Summary
The cytoskeleton provides cell shape and structure, cell shape depends upon the unique functions of different filaments. There are 3 classes of filaments in eukaryotic cells: actin filaments, Microtubules and intermediate filaments; they can be visualized using various methods such as gfp antibodies. Actin filaments are the thinnest filament and are comprised of actin subunits. Microtubules are the thickest filaments and are made up of dimeric subunits of a- and b-tubulin. There are filament specific motor proteins that track along actin filaments and microtubules, they are myosin proteins and kinesin & dynein respectively. The head domains bind to the cytoskeletal fiber and the tail domain. The head domains bind to the cytoskeletal fiber and the tail domain attaches to a cargo, with atp hydrolysis providing the energy for movement. The highest density of actin is at the cell periphery, they determine the shape and movement of the cell surface.
