S elf-organization of moving nanoscale objects leading to order over distances >> the objects and times >>

1. Self-organization of moving nanoscale objects leading to order over distances >> the objects and times >> that of individual steps A motor protein that regulates microtubule length Motor-driven, density-dependent alignment and aggregation of linear polymers

2. Review of main concepts from last week Some linear polymers use chemical energy to assemble/disassemble in response to binding/ hydrolysis of GTP (“dynamic instability”) Non-linear response to [GTP] Push and pull associating objects Form tracks along which other molecules move Examples: tubulin-microtubles-kinesin motors Actin-filaments, “comets”-myosin motors

3. Protein molecular motors move along polymer tracks via sequential shape changes associated with binding/ hydrolysis of smaller molecules like ATP 2 views of motor mechanism (not clear if either is right): sequential shape changes convert molecular asymmetry in motor to uni-directional motion e.g. trailing head hydrolyzes ATP-> dissociates from track->neck pushes trailing head forward, it binds ATP and track, etc., like macroscale motor Brownian ratchet – asymmetry of binding along repeating track unit moves motor “within” unit; conformational change “flattens” energy landscape -> unbiased diffusion; commensurately timed rebinding -> directionally biased movement

4. Today – some higher order interactions Howard – a kinesin motor that promotes track disassembly from + end ~14 protofilaments/tube circumference ~8nm/tubulindimer

5. 0:1 60:1 ?1500:1 Unlabeled:labeledkinesin What is red? Green? Why do they use TIRFM? How “wide” is microtubule in microscope image? What is shown in photomicrograph? How fast does kinesin move (mm/min, dimers/s)? About how long does kinesin stay at + end? Why are green diagonals discontinuous? Why are there red “stripes” horizontally?

6. Histogram of times Kip3p kinesin stayed at ends of tubules (when present at low concentration) What does this suggest about number of events required to get it off the end? What do you estimate for the rate of leaving?

7. 0:1 60:1 ?1500:1 Unlabeled:labeledkinesin B. Is kinesin speed affected by unlabelled kinesin? How many kinesin molecules come to end/min? If each took a tubulin with it, how fast would tube shorten? Why is residence time at end reduced commpared to A?

8. 0:1 60:1 ?1500:1 Unlabeled:labeledkinesin C. How fast is tube shrinking (mm/min, dimers/s)? Caption says 150:1 unlabeled to labeled kinesin; why do I think this is a misprint? Check out movie 1 with raw data. How does figure above compare to movie in terms of conveying results of experiment?

9. Here only the Kip3p motor is labeled Why might the rate of shrinkage be slower in the beginning (phase I)? How long would you expect phase I to last? If motors bind tubule at constant rate, why does the rate of shrinkage slow?

10. Note how apparently “inefficient” this is in terms of ATPs consumed/tubulindimer dissociated Each Kip3p kinesin uses 1000’s of ATPs to walk to end of tubule, to dissociate one dimer; Kip3p have no other known role in terms of moving cargo What do authors’ propose as explanation? Control of microtubule length so important biologically that cells devote large fraction of energy resources to its regulation; “walking” as part of “antenna” model is one way nm-size objects can regulate phenomena on mm-scale

11. Relationship to Reif DNA tubes (class 2) Could similar methods be used to observe growth/ disassembly of Reif tubes? E.g. label one “U”-oligo with red fluor, another w/biotin; make tubes; attach to avidin-coated glass slide; add more “U”-oligos, now with green fluor and no biotin. Will red tubes grow green extensions visible in TIRFM? Add complements of U-oligos. Will DNA tubes disassemble from green ends? At what rate? Can rates be regulated?

12. Gliding assay: fix (green-labeled) motors to glass, watch them slide red microtubules along Why might tubes rotate when bound by a single kinesin? Should they rotate if bound by more than 1 kinesin? See movie 2

13. Why might you expect a pause when a Kip3p motor reaches the end of a tubule? If you add excess, unlabeled motors, what should happen to rotation? to rate of gliding? Look at movie 3

14. Schaller et al, Polar patterns of driven filaments Immobilize myosin (similar to kinesin) motors, watch fluorescent actin filaments (like m-tubules) glide

15. At low density of unlabled filaments, spatial distribution and motion of labeled filaments is random

16. As (unlabeled) filament density is increased above a threshold,distribution and motion of labeled filaments becomes ordered Movies are dramatic!

17. Order can be characterized by correlation functions – e.g. average value of cosq between filaments separated by distance s, or same filament at time t and t+s, or velocity vectors of filaments separated by distance s… 1 s <cosq> 0 p s q conceptually related to persistence length

18. Why does order emerge? Intuitively, filaments bumping into each other may push each other into alignment, or slow down, allowing other fragments to “catch up” to form denser aggregates. Interactions are highly non-linear as function of density. Hard to explain this quantitatively – a problem in search of good mathematicians! Similar phenomena seen in simulations – but is this a satisfying “explanation”? Does it provide insight, e.g. why diff. thresholdsfor diff. forms of order?

19. Other examples of self-organization associated with energy consumption and far-from-equilibrium systems (potentially good topics for class presentations): Actin “comets” – swirling aggregates of polymerizing actin filaments, can push beads around chaotically in vitro, induced by various infectious agents inside cells, apparently involved in cell-to-cell transmission of infection “Reaction-diffusion” processes, -> moving “waves” of reactants in various inorganic and biochemical reactions, organized electrical activity in the heart