How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda...
-
Upload
brenda-lloyd -
Category
Documents
-
view
216 -
download
0
Transcript of How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda...
![Page 1: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/1.jpg)
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
![Page 2: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/2.jpg)
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
![Page 3: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/3.jpg)
Intracellular Traffic
© Scientific American
How is intracellular transport regulated?
![Page 4: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/4.jpg)
Microtubules (MT) are like freeways and actin filaments are like local surface streets.
![Page 5: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/5.jpg)
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
![Page 6: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/6.jpg)
Motor proteins
Myosin
Kinesin
![Page 7: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/7.jpg)
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
![Page 8: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/8.jpg)
Biochemistry, 4ht Ed, 1995
Motor proteins move cargo along filaments Molecular Biology of the Cell, 3rd Ed, 1994
![Page 9: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/9.jpg)
Herpes Virus Transport in Neurons Along Microtubules
• Virus Movie: VirusMov.mov
![Page 10: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/10.jpg)
How does the cell regulate the transport of vesicles?
Microtubules (MT) are like freeways and actin filaments are like local surface streets.
![Page 11: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/11.jpg)
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)
![Page 12: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/12.jpg)
Dispersing Pigment Granules
• Dispersion: Granules move out from nucleus:
Dispersion1.mov
![Page 13: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/13.jpg)
Aggregating Pigment Granules
• Aggregation: Granules move in toward nucleus:
Aggregation1.mov
![Page 14: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/14.jpg)
Dispersion: MTActin
Spread cargos throughout the cell
Aggregation:
ActinMT
Bring cargos back to nucleus
![Page 15: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/15.jpg)
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
![Page 16: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/16.jpg)
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.
![Page 17: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/17.jpg)
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.
![Page 18: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/18.jpg)
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.
![Page 19: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/19.jpg)
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
![Page 20: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/20.jpg)
Fitting Parameters
• Fit to Langevin Solution
• Fit to power law
2 ( )r t At
2 /( ) 1 tr t D t e
![Page 21: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/21.jpg)
Langevin Fits Yield Mean Free Path
• Dispersion: <ℓ> = 539 ± 9 nm • Aggregation: <ℓ> = 237 ± 12 nm
/ 2D
![Page 22: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/22.jpg)
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
![Page 23: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/23.jpg)
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
![Page 24: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/24.jpg)
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
![Page 25: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/25.jpg)
Touching Number nt
Aggregation Dispersion
< nt > = 7.8 < nt > = 4.2
![Page 26: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/26.jpg)
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
![Page 27: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/27.jpg)
Simulations of Cargos Moving Along Actin Filament Networks
Trajectories more localized Trajectories more spread out
![Page 28: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/28.jpg)
Switching Probability
Aggregation: nt = 7.8, <ℓ> = 237 nm, switch = 50%
Dispersion: nt =4.2, <ℓ> =539 nm, switch = 0% - 6 %
![Page 29: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/29.jpg)
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
![Page 30: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/30.jpg)
Simulations of Cargos Moving Along Actin Filament Networks
Trajectories more localized Trajectories more spread out
![Page 31: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/31.jpg)
Cargo displacement after 30 sec from simulations
•Cargos are more localized during aggregation•Cargos are more evenly spread out during dispersion
![Page 32: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/32.jpg)
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.
![Page 33: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/33.jpg)
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.
![Page 34: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/34.jpg)
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/
![Page 35: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/35.jpg)
THE END
![Page 36: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/36.jpg)
Fraction of cargos that have touched a MT 15 times by time t (from
simulations)
![Page 37: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/37.jpg)
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.
![Page 38: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/38.jpg)
Actin Filament Length Distribution
![Page 39: How Does Intracellular Molecular- Motor-Driven Transport Work? Joseph Snider 2, Frank Lin 2, Neda Zahedi 3, Vladimir Rodionov 3, Clare Yu 2 and Steve Gross.](https://reader036.fdocuments.in/reader036/viewer/2022062409/56649ccc5503460f949960b2/html5/thumbnails/39.jpg)
Quantification of motion
• Particle tracking: 8nm resolution, 30 Hz
• Analysis: Displacement vs. time R (t) random motion