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Lecture 10

Section 2 - Microtbules - Lecture 10 11 12.docx

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Biology 2382B
Robert Cumming

Section 2 - Microtubules: Tracks for Transport Microtbules : Tracks for transport Things within the cell are moving – they’re moving on what’s like tracks, not completely random Some things are moving one way and some moving other ways – at different speeds  Vesicle transport (Bi-directional)  Motor proteins – require energy (ATP)  People know that there are a lot of things going – we know that the microtubules are being used for transport Axonal Transport  Squid axons are a model system  Labeled proteins travel at different speeds in cells (not diffusion)  Amino acids are involved in transmission – making radioactive proteins  Those proteins that you ake using the radiolabelled amino acids will be transported down the axons  You take the little piece of axon with the radiolabelled amino acids incorporated and cut it up at different points  You isolate the tproteins rom those bits of axon You run this on a gel  Thesse are radioactive amino acnds that will give you bands – the bands won’t be colors! (just give you a sense of similarities/differences)  You repeat this with some more radiolabeled amino acids  You now find that the protein bands hat wre in segment 1 are in segment 2  You keep doing this at different time points ( inject again and wait a 3 hours now) and you see where these segments end up  Waiting different time intervals each time to see how far these amino acids are travelling  Overall – GROUPS OF PROTEINS ARE MOVING AT A CONSTANT RATE (BLUE PROTEINS ARE MOVING AT A CONSTANT RATE, RED ALL THESE DIFFERENT COLORS MOVING AT A CONSTANT RATE) Kinesin (Mts plus (+) end directed motor protein)  14 known classes of kinesin (45 genes in huamns) but 14 classes  2 heavy chains – head flexible neck and stalk o Head chain has ATPase activity and can bind to microtubules o Binds to the microtubule and uses ATp to move to the plus end (  2 light chains (variable) o 2 light chains are binding to the end of the tail at the stalk  Heavy chain heads have ATPase activity and MT binding ability  Light chains recognize cago o The light chains recognize different cargoes – o  MOST (+) directed Section 2 - Microtubules: Tracks for Transport Structures of Selected Kinesin members  Kinesin 5 – is bi polar – the tails of the kinesin5 bind to each other so you have 2 heads – if you have two micrtubles and the kinesin o The tail regions bind together – o Both Kinessins heads therefore will be trying to move to the + ends – the kinesin molecule will as a result be relatively still (since you have one trihng to move to the left and right) o As a result these microtubules will end up sliding o Organelle transport  Kinesin 1 –and Kinesin 2 – Kinesin 1 is the most abundant kind in the cell o Light chains will bind to cargo o Variable light chains allow to bind to multiple cargo o Microtuble sliding  Kinesin-13 o Made up of just the head domains – can bind to the ends of the microtubules o Use ATP to remove the dimers from the Microtuble ends Kinessins Continued:  Anterograe movement  ATP hydrolysis casues conformational changes in kinesin  ATP is ydrolysed as each head moves 16nm o The heads hydrolysize while we walk o We know it’s moving two dimers at a time (since 16nm) Cytoplasmic Dynein Kinesin Family member + end is always towards the outside of the cell – Dyneinin will move hthings towards the inside of the cell while kinesin will move it outside of the cell Cili and Flagella  Two versions of the same thing o Cilia 2-10 micro meters o Flagela 10-2000 micro meters  Flagella – Propel Cells o Used for propulsion (like sperm cells)  Cilia sweep material across tissue (many of these) o Collecting dust for example  Overall these two structures simply blend  How does it bend? Axoneme – The underlying structure of cilia and flagella  The structure of both is the same underneath Section 2 - Microtubules: Tracks for Transport  You have inside something called an axoneme  Over 250 proteins – you see a ring of microtubule doublets  There is an A ring that has 13 protofilaments and a B ring that has  You have singlets in the middle as well  The singlits inside are stable  You can get a variety of doublet and singlet combinations including a situation with no singlets in the middle – we have no idea what the singlets do  There are things that come from the dobulets that are aimed towards the middle o These are radial spoke heads that come into the center to stabilize the doublets  In between the doublets is a nexin molecule  Off the A ring of each double there are two dyneins that come off the A tubules from each doublet  The heads come off such that their heads are pointing towards the B tubules  The dynein is not typical dynein – it is reaching from one tubule to another tubule Axoneme and basal body  Axoneme continuous – attach to basal body in cell  The axoneme is continuous into the cell with a structure known as the basal body  The basal body is similar to a centriole  If an axonene has 9 doublets the basal body will have 9 triplits  The basal body will have the same number of triplets as the axonene has doublets o Triplts have A B and C tubules  Basal body may or may not have singlets in the middle –  Singlets between the basal body and the axonene don’t seem to have any relation  Basal bodys are similar to centrioles – will often see them in 90 degrees together  Microtbuules polymerize towards the cell surface Ciliary beating  Generated by sliding of mictorbultes against each other – powerd by dynein “A” tubule of one doublet “walks” along neighbor B  Result = MTs slide past each other – but linked to basal and nexin o Dyneine is trying to get to the minus end – will result in sliding if the two MTs are not attached to each other  In cilia and flagella however we don’t want sliding we want bending – this occurs because the microtubules are linked together and LINKED to the basal body Section 2 - Microtubules: Tracks for Transport  The dynein is tring to get to the minus end resulting in bending  Microtubles linked together by nexin and also niked to the basal body – as a result you get bending, if you leack etither nexin or atta chment to basal body you get sliding  For bending to occur you must have very well regulatedness – everything can’t slide at the same time, if you want bending only one segment can bend, you can’t get the sliding everywhere – has to be localized within the axoneme  The bending is restricted within the axonene – not all dynein are migrating at the same time Intraflagellar transport moves material up and down Movement is not related to bending May be related to stability and signaling events  Dynein found in axonene is bound permanently so can’t be transporting anything  When looking at celila or flagella there is something called intraflagellar transport – moving things towards or away from the tip  THIS DYNEIN IS DIFFERENT FROM THE ONE INSIE THE AXONEME  This allows the + end to be continually remodeled  The + end this way can also be used as a receptor o If you have a signal there you also want to carry the signal back Many interphase cells contain a non-motile primary cilium Mutations in these cilia or transport in them can have developmental consequences These cilia look a little bit like an axoneme but can’t bend These cilia work in signaling – they’re “out there” waiting for signals Without them you get abnormal developemtn In wildtype animals you see normal cilia – if you mutate them you get a mutated embryo Normal development requires the non-motile Cilia on the cells Karyokensis and cytokensis  Mitosis can be broken down in many ways – one way is to break down to karyokinesis and cytokenisis o Cytokensisi – dividing the cytoplasm, splitting the cell into two – this requires actin o Karyokensisi has to do with splitting up th
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