Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
78 Cards in this Set
- Front
- Back
|
What is the big role of microtubules in transportation? |
- vesicle transport (bi-directional) -motor proteins require energy (ATP) **without ATP there is no movement :( |
|
|
What is a good model to study microtubule transportation? |
-axonal transport -squid axons are a model system -labelled proteins travel at different speeds in cells (not diffusion) |
|
|
How do we study axonal transport in squid axons? |
How are proteins moving from cell body to nerve? 1. inject radiolabeled amino acid 2. cut the nerve out in different sections (sciatic nerve) 3. isolate proteins from different sections, lyse them and run on SDS page 4. see different banding patterns across different times |
|
|
What do we learn from axonal transport experiment? |
-proteins move at set speeds -they travel together -can now take out bands and sequence aa. **thats how we found kinesin! |
|
|
What is kinesin? |
Microtubles + end directed motor protein |
|
|
How many types of kinesin are there? |
14 known classes 45 genes in humans |
|
|
What is the structure of kinesins? |
2 heavy chains: head, flexible neck and stalk 2 light chains (variable) |
|
|
What are the properties of the heavy chain heads of kinesins? |
ATPase activity MT binding ability |
|
|
What is the role of light chains of kinesins? |
recognize cargo is found at the tail of the heavy chain at the (+) end |
|
|
where do we find the head of the heavy chain in kinesins? |
they go the (-) end |
|
|
What are the kinesins that we have to remember? |
Kinesin 1 Kinesin 5 Kinesin 13 |
|
|
What is the role of kinesin 13? |
it uses ATP to rip out the dimers -only has heavy chain -end disassembly -no involvement in transport |
|
|
What is the role of kinesin 5? |
it is bipolar - both sides are the same (end in heavy chains) 4 heavy chains -both ends bind to microtubules -involved in microtubule sliding! -can move microtubules |
|
|
What is the role of kinesin 1? |
conventional 2 heavy chains, 2 light chains heavy chains: head domain (linker -bends), long tail called stalk. moves toward the + end |
|
|
How does kinesin move doe? |
-Anterograde movement -uses motor head -ATP hydrolysis causes conformational changes in kinesin -ATP hydrolysed as each head moves 16nm |
|
|
What are the steps in kinesin anterograde movement? |
1. leading head binds ATP 2. binding of ATP induces a conformational change causing the neck linker to swing forward and dock into head -> swings the former trailing head to become the leading head 3.now leading head finds a biding site on the microtubule 16 nm ahead of its previous site 4. leading head releases ADP and coordinately the trailing head hydrolyzes ATP to ADP+Pi released Pi, linker becomes undocked |
|
|
What is dynein? |
a MT (-) end directed motor protein |
|
|
What is dynein involved in? |
retrograde transport |
|
|
What are the differecens between kinesin and dynein? |
Kinesin = exocytosis -> anterograde transport Dynein = endocytosis -> retrograde transport |
|
|
What is dynein's structure? |
"Heavy chain" heads are bound to microtubles through stalk -have ATPase activity and move to the - end -linker attaches head to stem which in turn intearacts with dynactin hetero complex (recognizes and binds to cargo). -one end of the heavy chain binds to the microbules through the stalk -> microtubule binding domain -other end of the head interacts with the dynactin hetero complex through the stem and dynein intermediate light chain-> dynactin-binding region |
|
|
What roles does ATP hydrolysis have in dynein activity? |
ATP hydrolysis causes linker shape changes that drives movement |
|
|
How does dynein interact with dynactin ? |
Dynein has intermediate and light chains at the opposite end of the microtuble -binding domain and those form a hereocomplex with dynactin-binding region. the dynactin-biding region on the end of the stem of dynein binds to the dynein binding region on the stalk of dynactin |
|
|
How does dynactin bind cargo? |
through the dynactin hetero-complex which is made up of different proteins (different combinations of proteins recognize different cargo) -this is on the opposite side of the microtubule binding part of dynactin. -even though dynactin is bound to microtubles it doesnt use ATP |
|
|
When do we use dynein? |
when we want to move something from the cell surface to the inside of the cell -> toward the (-) end we use Dynein |
|
|
When do we use kinesin? |
when we want to move something from the inside of the cell to the cell surface or close to it -> toward the (+) end we use kinesin |
|
|
What is the relationship between cilia and flagella? |
they are two versions of the same thing. cilia move small things flagella move big things, propel cells -both work by bending |
|
|
What do flagella do? |
flagella 10-2000 micrometers -propel cells (few) |
|
|
What do cilia do? |
cilia 2-10 micrometers -sweep material across tissue (many) |
|
|
What is the underlying structure of cilia and flagella? |
the axoneme |
|
|
What is the axoneme made up of? |
-over 250 proteins
- 9+2 array of microtubules typical (others exist) - outer doublets consist of A and B tubules - Axonemal dynein |
|
|
What is the structure of the axoneme? |
has doublet (A and B) microtubules linked by nexin in a circle. -we think that axoneme might have 2 singlet microtubules (central pair of singlet microtubules) inside the ring structure |
|
|
What is the role of nexin in axoneme structure? |
in keeps the dimers together not present everywhere, only in a ladder effect. |
|
|
What is the role of axonemal dynein? |
in binds to the dimers and is different from normal dynein b/c it is always bound to the doublets (dynein arms - outer and inner). is bound permanently to A, and tries to reach to B. |
|
|
What is the MTOC (microtubule organizing center) for cilia and flagella? |
basal body |
|
|
What is the structure of cilia and flagella? |
Axoneme is bound to Basal Body basal body -> transition zone -> axoneme |
|
|
The basal body is made up of ? |
similar to centriole 9 basal body triplet MTs (A and B which pass through transition zone, C does not) |
|
|
What happens in the transition zone of cilia and flagella? |
in the basal body polymerization happens directly (unlike kinesin where u use other proteins e.g. MTs are not attached to MTOC), as u go through transition zone it goes from being a triplet to a doublet (losses C) now stable and do not polymerize or depolymerize |
|
|
How is bending generated in axoneme and basal body? |
Microtubule sliding =>>> Cilia beating |
|
|
How does Cilia beating work? |
generated by sliding of microtubules against each other - powered by dynein "A" tubule of one doublet "walks" along the neighbour "B" =>> MTs slide past each other- but linked to basal and nexin |
|
|
How do nexin, axonemal dyenin, A-tubule, B-tubule work to result in ciliary beating? |
there are two microtubules Nexin-links A and B microtubules Nexin link (-) ---------------------------------- (+) B-tubule ======================== A-tubule (-) ---------------------------------- (+) Axonemal dynein Axonemal dynein is permanently bound to A micortubules and it will try to bind to the B tubule of the next doublet |
|
|
What happens in ciliary beating when you move toward the (-) end? |
-activation of dynein bends microtubule doublets -motor walks toward (-) end, constrained by nexin links -lose nexin by protease -MTs slide pass each other nothing holds them together |
|
|
What does intraflagellar transport do? |
moves material up and down -movement is not related to bending -may be related to stability and signalling events (antenne sticking out of the cell) |
|
|
How does intraflagellar transport work? |
1. base-directed movement powered by cytoplasmic dynein 2. tip directed movement powered by kinesin-2 (complexes made up of Kinesin2-IFT particles-Cytoplasmic dynein) |
|
|
What is the structure of intraflagella ? |
flagellum tip flagellum base central microtubule outer doublet microtubule |
|
|
What other roles can cilia have? |
-non-motile signalling roles |
|
|
What is the non-motile primary cilium? |
many interphase cells contain a non-motile primary cilium used in signalling - in development mutations in cilia structure (or transport mutations) can have embryonic consequences mutating dynein or kinesin can impair signalling |
|
|
what is karyotokinesis? |
Dividing up the chromosomes |
|
|
What is cytokinesis? |
dividing up the cytoplasm -happens on the actin cytoskeleton |
|
|
What happens to the centrosome during interphase? |
centrosome duplication |
|
|
what happens in prophase (related to microtubules)? |
breakdown of interphase microtubules
kinetochore assembly |
|
|
What happens after you get rid of the interphase microtubules in prophase? |
you end up with spindle poles -make new different microtubules than interphase ones -these ones can capture chromosomes and align them |
|
|
What is a centromere? |
one on each chromsome it joins sister chromatids together during metaphase cohesins are located at the centromere so sister chromatids are only connected there direct the formation of the kinetochore which attaches to the microtubules in the mitotic spindle |
|
|
What are the new mitotic MT dynamics? |
interphase MT half-life of about 5 mins half-life drops to about 15 seconds during mitosis -very dynamic |
|
|
What is the contractile ring that has the role to divide the cell in two made up of? |
actin and myosin filaments |
|
|
Why do microtubule dynamics increase in mitosis? |
due to loss of a stabilizing MAP (XMAP215) -inactivation of XMAP215 by phosphorylation causes instability |
|
|
What is the major role of XMAP215? |
-to suppress catastrophes induced by kinesin-13 "keeps it in line" |
|
|
What are two things that happen in mitosis in terms of dynamics and MTs? |
-mitosis is much less stable than interphase -in mitosis MTOCs have an increased ability to nucleate microtubules |
|
|
Why does half-life of MT in mitosis drop to about 15 seconds ? |
because XMAP215 which has the role to stabilize kinesin-13 acitivity and tubulin, is inactivated by phosphorylation |
|
|
What is the mitotic apparatus made up of? |
-polar & kinetochore MTs (make Spindle) -Aster (astral) MTs |
|
|
What is the zone of interdigitation in the mitotic apparatus? |
it is where the interpolar (polar) MT's overlap |
|
|
What is the centromere? |
attachement site for microtubules (Lecture) |
|
|
What is the role of Kinetochore proteins? |
they mediate attachment of chromosomes to MTs -made of thousands of proteins -histologically can see two layers: inner and outer kinetochore -capture and polymerize microtubules |
|
|
How does the capturing of the MT on the outer kinetochore work? |
-they are caught on the (+) end by fingerlike projections that are coming out of the kinetochore so that the (+) end is still free -(+) end is not capped, can polymerize and depolymerize -the shrinking and growing of MTs can technically move the chromsome |
|
|
What does spindle formation imply? |
-all chromosomes must be captured -all must be aligned during metaphase -requires motor and MT dynamics |
|
|
How do you get the chromosomes to go to the spindle pole? |
-capture them from both sides -if when captured but not in the middle it must be moved in the middle of the metaphase plate -cell uses depolymerization and polymerization to move the chromosome right or left. -one one side we have force from dynein and MT depolymerization by kinesin 13, -4 -the other side we have growth a kinesin 7, MT assembly |
|
|
So how does chromsome movemnt happen on the metaphase plate? |
1. polymerization and depolymerization of MTs 2. shrinkage by dynein, kinesin 13 3. growth by kinesin 7 |
|
|
What is bi-orientation? |
that the chromosome (sister chromatids) are attached to both spindle poles -assured by tension |
|
|
How does tension assure bi-orientation? |
Ndc80 proteins (complex) holds on to the MTs (between outer and inner kinetochore) -it needs tension from the other sister chromatid being attached to a microtubule. -if there is no tension after some time Ndc80 is phosphorylated. |
|
|
How does Ndc80 ensure that the chromsome is attached to both spindle poles? |
under no tension phosphorylation of Ndc80 proteins at the kinetochore results in weak microtubule interactions with the kinetochore |
|
|
What phosphorylates Ndc80 (kinetochore protein) |
CPC |
|
|
What happens when Anaphase starts? |
1. cohesins separate Anaphase A & B |
|
|
Main role of Anaphase A? |
-requires MT shortening |
|
|
Main role of Anaphase B? |
-pole separation, requires motors -sliding by kinesin 5 -pulling by anchored dynein |
|
|
What happens in Anaphase A? |
-shrinkage of the kinetochore microtubules that bring the chromosome closer to the spindle pole -depolymerization of kinetochore microtubules at + end -kinesin and dynein can facilitate process but are not necessary |
|
|
What happens in Anaphase B? |
-while the chromsomes are moving towards the poles, the poles move futher apart so the cell is being elongated. -elongation is driven by kinesin 5 a + directed motor, has + on both ends, makes the MT's move toward their (-) ends relative to each (further away) |
|
|
What else contributes to the anaphase processes except MT shortening and pole separation? |
-the poles are being pulled toward the plasma membrane by astral kinetochores -at the plasma membrane there is dynein which is bound to astral MTs, since it's a (-) end directed motor protein it will pull toward the plasma membrane -> toward (+) -as dynein is pulling toward the plasma membrane the + end gets depolymerized so that it doesnt poke through the membrane |
|
|
What is chormosomal movement in Anaphase A driven by? |
Microtubule disassembly at the (+) end attached of MTs attached to kinetochore How do we know? bleach experiment |
|
|
What is chromosomal movement in Anapahse B driven by? |
Driven by motor proteins 1. overlap of polar microtubules, kinesin 5 is in between them and as you slide them there is less overlap and the poles are pushed apart 2. newly polymerized tubulin after initial pushing at the + end so that you can push more 3. dynien on the astral microtubules on the + end wants to move to the - end, so it pushes the micortubules to the plasma membrane |
