Summary of single-molecule experiments

Motor proteins: Are uni-directional, and move along straight filaments Exert 1-6 pN force Typically go ~ 1m before detaching Kinesin motors take 8 nm steps, Dynein takes a variety of step

sizes, Myosins take 36 nm steps Move between 0.1 and 2 m/s

Is this how transport functions inside cells?

How do we go from single-molecule

characterization to in vivo function?

Herpes virus in cultured neuron

Why do cargos need multiple motors?

Many intercellular distances are longer

than 1 micron

Motion in cells is different from what might be expected based on single-molecule propertiesCargos can move long

distances Maybe multiple motors?

Bead moved by multiple kinesin motors

So, multiple motors can move a cargo long distances.

Now, lets look more carefully…

Start to build complexity in a

controlled environment, i.e. in vitro, and understand

how motors work together

Poisson statistics: Getting down to the single molecule limit…

For single motor, use Binding/moving fraction ≤ 0.3

• Catch Dynein- or kinesin-coated beads, bring in contact with MT

• Find probability for Binding/motion (Bind fraction)

• Repeat at different motor:Bead ratios

• Plot the Bind fraction Vs motor:Bead ratio

• Stay where probability for “doubles” is negligible

Motor - polystyrene bead assays

Kinesin I: single motor30% or less of beads bind to MTs

Run Length (Processivity)

Decay constant ± SEM :

1.46±0.16 µm

Peak center ± SEM :

4.8±0.06 pN

Force production

Poisson statistics: Getting down to the single molecule limit…and then back to

multiple motors

• Catch motor-coated beads, bring in contact with MT

• Find probability for Binding/motion (Bind fraction)

• Repeat at different motor:Bead ratios

• Plot the Bind fraction Vs motor:Bead ratio

• Now, use concentration where probability for “doubles” is high: mixed population

Mixed bead population--> How do we know how many motors are moving a specific bead?

What we think is going on

Increasing Kinesins per bead

Bf~0.3 Bf~0.7 Bf~1.0Bf~1.0

Evolution of force production with

increasing kinesins per bead

Single motor

(Bf ~0.3)

1-2 motor

Mostly single motor

(Bf ~1)

Conclusion: for multiple-motor driven transport, binding

fraction cannot tell you how many motors

engaged.

Stalling forces are additive at low motor number; use this as a

readout of the number of instantaneously engaged

motors

Motor - polystyrene bead assays

Kinesin I: ~two motors driving polystyrene bead

Run Length Force production

Summary for ~2 engaged Kinesins:

* Velocities unchanged (not shown) * Stall forces ~ additive* Cargo travel lengths very long, but this is not really correct (see next)

>> Similar results for cytoplasmic dynein (see Mallik et al, Curr. Bio, 2005)More: see website bioweb.bio.uci.edu/sgross

Conclusion: motion in cells is different from what might be expected based on single-molecule properties

Cargos can move long distances Cargos can reverse course, move bi-directionallyCargo transport can be regulated

We have three ‘systems’ level questions to understand:

What single-molecule properties are particularly important for how multiple motors function together?

Cartoon of processive motion of a cargo moved by two motors

From cartoon…

On-rate

Off-rate

Overall number of motors

Analytic Mean-field theory of average cargo travel carried by two motors

Klumpp and Lipowsky, PNAS, 2005Xu et al, Traffic, 2012

Velocity: crucial initial condition: binding rate (1/s): unbinding rate (1/s)

d=v*(1/v/

Experiment: established for single-motor study

Valentine et al., Nat. Cell Bio., 2006

Experiment: established for single-motor study

Valentine et al., Nat. Cell Bio., 2006

Experiment: difficult to interpret for more motors

?

Experiment: difficult to interpret for more motors

?

Experiment: difficult to interpret for more motors

?

?

Experiment: difficult to interpret for more motors

Experiment: modify surface chemistry for two-motor

Experiment: modify surface chemistry for two-motor

Experiment: force to further require two-motorP

osit

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Time

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Experiment: clean one- vs. two-motor system!

Goal: Test

Strategy: reduce ATP to slow down motor

Dd

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Experiment: two-motor travel D

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Rogers et al., Phys. Chem. Chem. Phys, 2009

D=1.7d

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Velocity tunes travel distance for two-motor system

d D

Motors work in small ensemble in cells

We establish velocity as a control for ensemble travel

May be particularly important, as beautiful work by Joanny (Campas, et al, Biophys. J. , 2008) suggests a limited number of motors (~ 9 max) can be active.

Hw #2 : Model two kinesin motors functioning together, and then investigate velocity effects.

In Hw #1 you developed a simulation for 1 motor. Here, stick two such motors together. Assume initially that the motors here have the same properties as in the previous hw.

The main goal here is to get the simulation working, and compare its results for a few different choices of ‘on’ rates and ‘off’ rates to the order-of-magnitude theory developed in class. How similar are the two sets of predictions?

For a single motor with processivity of 1.2 microns, what is your prediction for the mean travel of a cargo with two such motors, assuming an ‘on’ rate of 2/sec or 5/sec. Do this assuming a velocity of 800 nm/sec, and a velocity of 100 nm/sec.

Velocity: the link between temporal and spatial

of individual motor unbinding from microtubule

Slower velocity buys more time for additional motor to bind before the current bound one detaches.

(see Xu et al, Traffic, 2012)