How Does Intracellular Molecular-Motor-Driven Transport Work?

Joseph Snider2, Frank Lin2, Neda Zahedi3, Vladimir Rodionov3 , Clare Yu2 and Steve Gross1,2

1Department of Developmental and Cell BiologyUniversity of California, Irvine;

2Department of PhysicsUniversity of California, Irvine;

and3King’s College, London

A Cell Is Like a City• Workers• Power Plant• Roads• Trucks• Factories• Library• Recycling center• Police• Post office• Communications

• Proteins• Mitochondria• Actin fibers, microtubules• Kinesin, dynein, myosin• Ribosomes• Genome• Lysosome• Chaperones• Golgi apparatus• Signaling networks

Intracellular Traffic

© Scientific American

How is intracellular transport regulated?

Microtubules (MT) are like freeways and actin filaments are like local surface streets.

Filaments

Actin filament• 10 nm diameter• 2.77 nm rise• 26 subunits/74 nm repeat

Microtubule 25 nm diameter 13 protofilaments

+ end- end

+ end

- end

Motor proteins

Myosin

Kinesin

Kinesin Myosin-V Dynein

Head (ATPase)

1

43

5

c

6

2

Head(ATPase)

Lever (?)

StalkPi

Pi

KAPP

KHC

KLC

KR2

KR3

Cargo

Ca2+

MR2

MR1

Cargo

KR1

Dynactinbinding

MT binding

Biochemistry, 4ht Ed, 1995

Motor proteins move cargo along filaments Molecular Biology of the Cell, 3rd Ed, 1994

Herpes Virus Transport in Neurons Along Microtubules

• Virus Movie: VirusMov.mov

How does the cell regulate the transport of vesicles?

Microtubules (MT) are like freeways and actin filaments are like local surface streets.

Model System:Melanophores (Pigment granule cells)

• Cells change color by Dispersing or Aggregating pigment granules• granules move bi-directionally along microtubules

(Kinesin-II and Dynein)

•Granules also move along actin filaments (Myosin-V)

Dispersing Pigment Granules

• Dispersion: Granules move out from nucleus:

Dispersion1.mov

Aggregating Pigment Granules

• Aggregation: Granules move in toward nucleus:

Aggregation1.mov

Dispersion: MTActin

Spread cargos throughout the cell

Aggregation:

ActinMT

Bring cargos back to nucleus

Experiment

• Pigment cells (melanophores) only have actin filaments (no microtubules)

• Pigment granules (melanosomes) are tracked

• Record position r vs. time t

• Plot average <r2> vs. time t

Experiment: Cargos traveling solely on actin filaments go farther during dispersion than during aggregation.

Do cargos go faster during dispersion? No, the velocity of the cargos is the same for aggregation and dispersion.

Why do cargos go farther during

dispersion than during aggregation?

• During dispersion cargos go “straight” to the end of the actin filament and do not turn at intersections with other actin filaments. This is good for spreading out pigment granules uniformly.

• During aggregation cargos have a 50-50 chance of switching to another actin filament at each intersection. So they don’t go as far. Frequent switching is a good way to find a nearby microtubule.

Two Types of Theoretical Modeling Confirm This Scenario

• Langevin solution interpolates between short time ballistic (straight-line) motion and long time diffusive motion.

• Computer simulations of cargos moving along actin filaments also confirms this picture.

Solution to Langevin Equation

• Langevin solution interpolates between short time straight line motion and long time diffusive motion.

• Fitting displacement data yields D and which can be used to obtain the mean free path ℓ (distance traveled before turning).

• The mean free path is given by

2 /( ) 1 tr t D t e

/ 2D

Fitting Parameters

• Fit to Langevin Solution

• Fit to power law

2 ( )r t At

2 /( ) 1 tr t D t e

Langevin Fits Yield Mean Free Path

• Dispersion: <ℓ> = 539 ± 9 nm • Aggregation: <ℓ> = 237 ± 12 nm

/ 2D

Compare Langevin ℓ with Electron Micrographs of Actin Filaments

Dispersion: Langevin ℓ L/2 where L≈1300 nm is a typical filament length implying cargos go to end of filamentAggregation: Langevin ℓ ≈ 1.5 d where d≈160 nm is the typical distance between filament intersections consistent with cargos switching with 50% probability at intersections

The actin filaments appear denser during aggregation which would encourage frequent switching from one filament to another. (Not enough EMs to confirm this.)

Electron Micrographs of Actin Filaments

Filament Density and Touching Number• To quantify the

density of filaments, randomly place circles on the EM.

• Circle diameter = 568 nm = cargo diameter

• Touching number = number of filaments in contact with circle

Touching Number nt

Aggregation Dispersion

< nt > = 7.8 < nt > = 4.2

Simulations of Cargos Moving on Actin Filaments

• Distribution of filament lengths taken from EMs (electron micrographs)

• Vary density of filaments (touching number)

• Vary switching probability at filament intersections

Simulations of Cargos Moving Along Actin Filament Networks

Trajectories more localized Trajectories more spread out

Switching Probability

Aggregation: nt = 7.8, <ℓ> = 237 nm, switch = 50%

Dispersion: nt =4.2, <ℓ> =539 nm, switch = 0% - 6 %

Result of Simulations of Cargos Moving on Actin Filaments

Using the density of filaments taken from EMs and the Langevin mean free path, we find:

• For aggregration, switching probability is 50% at filament intersections

• For dispersion, cargos go to end of filament and then attach to a new filament (switching probability is very small ~ 0%)

• Result: Average mean free paths agree with EMs and Langevin, confirming scenario

Simulations of Cargos Moving Along Actin Filament Networks

Trajectories more localized Trajectories more spread out

Cargo displacement after 30 sec from simulations

•Cargos are more localized during aggregation•Cargos are more evenly spread out during dispersion

SUMMARY:We have explained how and why

cargos go farther during dispersion than during aggregation

• During dispersion cargos go “straight” to the end of the filament and do not turn at intersections with other filaments. This is good for spreading out pigment granules uniformly.

• During aggregation cargos have a 50-50 chance of switching to another filament at each intersection. So they don’t go as far. Frequent switching is a good way to find a microtubule that leads to the nucleus.

Possible Way that the Switching Probability Is Regulated

• During aggregation, there are about 60 motors per cargo, but only one active motor pulls a cargo along a filament. Another motor can attach to a nearby filament and cause a switch to the new filament. Switch probability is 50%.

• During dispersion, there are about 90 motors per cargo, but only 2 active motors pull a cargo. Another motor may try to attach to another filament but it is not strong enough to cause a switch to a new filament. Switch probability is 0.

Actin Collaborators• Steven Gross (Cell and Dev. Biology, and Physics and

Astron., U.C. Irvine)

• Joseph Snider (Physics and Astron., U.C. Irvine) (Langevin and simulations)

• Francis Lin (Physics and Astron., UC Irvine) (data analysis)

• Neda Zahedi (King’s College London and U. Conn. Health Sci. Ctr.) (experiments)

• Vladimir Rodionov (U. Conn. Health Sci. Ctr.) (experiments)

• Snider et al., PNAS 101, 13204 (2004). • Website: http://bioweb.bio.uci.edu/sgross/

THE END

Fraction of cargos that have touched a MT 15 times by time t (from

simulations)

Collective Motion vs. Single Motor Properties

•Cargos go further in dispersion due to collective motion rather than the properties of individual molecular motors.

•Analogy: To understand traffic flow patterns in southern California, you don’t need to know how a car works. Learning about tires and internal combustion won’t tell you why there’s a traffic jam.

Actin Filament Length Distribution

Quantification of motion

• Particle tracking: 8nm resolution, 30 Hz

• Analysis: Displacement vs. time R (t) random motion