week 6 Flashcards
what do microtubules do
- they act as tracks for transport
what do you see when you look at microtubules
- see things moving in. both directions on microtubules at different s[eeds
- can be seen when looking at in vitro experiments using axons of giant squids.
squid axons
- model system
radio active amino acids assay
- injecting radioactive amino acids into the cell body of a large axon.
- dividing axon into segments, and then collect different bits of the axon at different distances from the injection site.
- these isolated proteins are then run on a gel
- the transport of proteins that are made from the amino acids is not random (not just simple diffusion)
what does the gel protein assay teach us
which proteins are travelling together (i.e. they remain together at the different time frames)
Kinesin
- motor protein
- there many types, 14 known classes coded by 45 genes in humans
in what direction does kinesis move
moves along microtubules towards plus end
composition of kinesin
- 2 heavy chains
- 2 light chains
what are the kinesis heavy chains composed of
- head, neck/linker, stalk/tail
kinesin head
microtubule binding domain that has ATPase activity (is able to hydrolyze ATP while moving towards plus end)
kinesin neck/linker region
- flexible linker region
kinesin stalk/tail
stalk region that goes into the tail leads to light chains.
kinesin light chains
- variable light chains
- there are lots of different types present that bind to different types of cargo
- light chains are located at the end of tail regio
what would happen if u ran the heavy and light chain regions
you would get three bands (1 thick band ofr the heavy regions because same size and therefore occupy large molecular weight)
- and then each of the two light chains would be different sizes and have a small molecular weight (hard to indeitify how heavy they are)-
kinesin 1
- most important
- conventional kinesin, found all over cytoplasm
- head domains bind to microtubules
- does most of the work and is made of two heavy chains and two variable light chains
- light chains are variable and depend on cargo
kinesin 2
- heterotrimeric
- has two different heavy chains (not identical)
- head domains bind to the microtubules
- has different kinesin family member that is sort of like a light chain
- made up of three different molecules
- three different banding patterns on SDS page (i.e. you would see the three bands travelling together on SDS page)
roles of kinesin 1 and 2
organelle transport
kinesin 5
- bipolar (both sides are the same)
- does not bind cargo
- head domains bind to microtubules
- binds to stalk domain of two other heavy chains
- there are four heavy chains that come together in this bipolar chain
- head domains on both sides, both ends of the kinesin can bind to the microtubule
- causes microtubule sliding
kinesin 13
- does not bind cargo (not for transport)
- head domains bind to microtubules
- uses atp hydrolysis to remove dimers off of the ends of microtubules (uses ATP to cause depolymerization)
- no stalk or tail domain
- primarily works at the + end because - end is usually capped by gamma tubulin ring complex
- however, it is possible to have depolyermization at either end
kinesin 13 function
depolyermizataion
kinesin 5
microtubule sliding
how does cargo bind to light chain of kinesin
- cargo needs the right receptor that can be recognized by a specific light chain
kinesin movement
- usually anterograde
(uses atp hydrolysis to move head towards + end)
how is kinesin 1 regulated
- inactive when folding (no ATPase activity) but will be active once bound to receptor
how far does kinesin 1 go
- when hydrolyzed, the head moves 16 nm
- before that, the kinesin heads are 8 nm apart when kinesin is not mobing
- the behind head steps out in front of the other head (one head remains stationary and bound)
- in total, moves 16 nm