Local Force Transduction Acts Globally Through Oncogenes and Cytoskeletal Transport of Signals

1
Local Force Transduction Acts Globally Through
Oncogenes and Cytoskeletal Transport of Signals
Pilus Retraction is Strong

• Michael P. Sheetz
• Biological Sciences
• Columbia University

2
Cell Biophysics Reverse Systems Engineering of
Cell Functions

• To understand functions in cells,we need to
  approach the problem as an engineer, trying to
  understand how an existing factory functions
• Physical factors are integrated with biochemical
  activities in most cellular functions

3
Critical Physical Aspects of Biological Systems

• Reversibility and Specificity by Spatial
  Dependence of Interactions
• 2 Mechanisms of Force Transduction in Cells
• Complex Integrated Functions Define Motile
  Processes
• Generation of High Force by Bacterial Pilus
  Retraction

4
Mechanical Sensing in Normal and Transformed Cells

• What differentiates a formed organism from a
  sphere of cells is the ability to apply and sense
  forces at specific places and times
• Rapid neuronal sensing of force is through ion
  channels. Turgor in tissues and long-term forces
  are sensed by cytoskeletal-based mechanisms.
• Transformation is described as the ability to
  grow on soft agar, i.e. in the absence of force.

5
Cell migration a cyclic process of force
generation
Membrane-Cyto. Adhesion Resists Extension a PIP2.
Sheetz, Nature Rev. MCB. 2392 (2001)
Extracellular Matrix Adhesion Linked to Force
Generation by Rearward Flow
How do cells locally trans-duce Matrix rigidity
into a signal used to direct cell edge extension?
6
FN Bead Binding Movement and Release
Displacement
Nishizaka et al., PNAS 97692 (1999)
7
How does a cell treat the ECM?
8
Phases of Cell Motility
2---
2-10
5---
Rate-limiting Step
Unpolarized spreading
Regional Activation Inactivation
Myosin-activated Clock
9
Structure of FN Monomer Trimer
Coussen et al. J. Cell Sci. 1152581-2590 (2002)
10
Fibronectin Trimer Binds Specifically to Edges,
Monomer Binds Uniformly
a
FN monomer
b
FN trimer
Jiang et al., (2003) Nature. 424334-7.
11
Breaking Events Are Common for Single FN Trimer
Beads
12
?v?3 is involved in the 2pN linkage mediated by
one FN-trimer
13
Spatial Distribution of Integrins is Important
for Talin Binding
FN trimer
?3
?v
?v
?3
?3
?v
membrane
talin1
N
C
2 pN Slip Bond
Jiang et al., (2003) Nature. 424334-7.
14
Attachment and Contraction
15
Force Causes GFP-Paxillin Accumulation in
RPTPa/ with FN
Without Force
FN Bead
Con A Bead
Von Wichert et al., 2003. J. Cell Biol.
161143-153
16
RPTPa Deletion Blocks But Transfection Restores
Binding
-/- Cells with Force
-/- Transfected wtRPTPa
Von Wichert et al., 2003. J. Cell Biol.
161143-153
17
Force on avb3 Activates RPTPa to Cause Fyn-Dep.
Assembly and c-Src Activation in Early Focal
Complex Formation
c-Src FAK
RPTPa Fyn
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Cytoskeleton Stretching Causes Focal Contact
Protein Binding
We tested the hypothesis that the cytoskeleton is
being altered directly by force on matrix
molecules using a stretchable silicon substrate
and detergent-treated cells.
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Stretching of Cytoskeletons
Subconfluent L929 Cells
Stretch
Triton
Collagen on Silicon Elastomer
Add biotin-Supernatant
Elute 1M NaCl
Wash
Analyze 2-D Gels Purify Sup. Prot.
Sawada and Sheetz, JCB 156609 (2002)
20
2-D Analysis of Bound Proteins
Stretched
Relaxed
Molecular Wt. kDa
21
Antibody Analysis of Bound Proteins
22
GFP-Paxillin Stretch-Dep. Binding
23
Titin Domains Unfold as Expected for a Force
Sensor
Li et al., (2002) Reverse Engineering of Titin.
Nature, 418998-1002
24
Total Internal Reflection Fluorescence of
Calcein-loaded Cell Spreading
25
(No Transcript)
26
Dubin-Thaler et al., Biophys. J, March, 2004
27
Anisotropic
Isotropic
28
Isotropic Spreading with GFP-a-actinin
29
Local periodic retractions of the cell edge
during lamellipodial extension
30 s
2 µm
Period 24 7 s
2 µm
fibronectin 10 µg/ml
Giannone et al., (2004) Cell. 116431-43.
30
Periodic contractions depend on a stiff substrate
30 s
5 µm
2 µm
Periodic contractions are used to stabilize the
propulsive lamellipodia
31
Periodic Retractions Are Periodic Contractions
of the Lamellipodia
32
Periodic Contractions Require MLCK
4 µm
30 s
2020 1000 ?m2
MLCK 790 440 ?m2
33
Periodic contractions induced the periodic
transport of a-actinin and MLCK from the leading
edge to the base of the lamellipodia
30 s
2 µm
5 µm
Cyclical transport of proteins from the front to
the back of the lamellipodia
34
Lamellipodial extension driven by actin assembly
Rac-GTPase
lamellipodia
lamella
Actin plolymerization (front) and
depolymarization(rear) define the lamellipodial
width
Forces generated by Myosins?
Cell spreading and migration require myosin
activity
35
Period Set by Width of Actin Meshwork
36
Model of signaling by cytoskeletal transport
37
Pilus-mediated Pathogenesis
Twitching motility Colonialization of host
cells
Pilus attachment to mammalian host cells
triggers downstream responses and invasion
Virulence
Biofilm formation Increases antibiotic
resistance
Horizontal gene transfer Antibiotic
resistance and virulence
38
Type IV pili mediate adhesion to host cells
WT
pilT
Bacterial colony
39

• The type IV pilus is a helical polymer
• homopolymer of pilin
• pitch of 4nm with 5 pilins per turn
• up to 5µm long
• 6nm in diameter

Pilin
Model system Neisseria gonorrhoeae
Forest et al Gene, 1997
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Organization of Pili in Bacteria
0.8 nm/pilin
PilT
ATP
5 pilins per 4 nm
Inner Membrane
Outer Membrane
41
Type IV pilus generate Motility and Force by
Extension and Retraction
N.gonorrhoeae moving on a glass surface
5µm

• We hypothesize that force is critical for
  virulence
• Evokes cellular response required for bacterial
  invasion
• Generates movement through viscous mucous layers

42
machine
What molecular machine powers pilus
retraction? What is the level of force
generated by a single pilus? How does force
generation affect cell function?
force
sensor
signal
response infection
43
How large is the force generated by pilus
retraction?
2µm
Time resolution 30ms Spacial resolution 10nm
Force range 1-150pN
Maier, B., Potter, L., Seifert, H.S., So, M.,
Sheetz, M.P. PNAS 2002
44
Maximum force of pilus retraction
Deflection d of the pilus-bound bead from the
centre of the laser trap
Distribution of stall forces
Average stall force ltFstallgt(110?30)pN
45
Pilus expression level of de-repressible pilin
mutant
Pilin concentration
Inducible pilin mutant at low expression level
Wild type
46
A single pilus pulls!
pilin mutant, 0.01mM IPTG pilin mutant, 0.1mM
IPTG pilin mutant, 10mM IPTG wild type
47
A Single Pilus Pulls!
Retraction Frequency correlates with number
of expressed pili.
Stall force independent of pilus expression
level.
Kinetics of pilus retraction is not modified by
pilus expression level.
pilin mutant, 0.01mM IPTG pilin mutant, 0.1mM
IPTG pilin mutant, 10mM IPTG wild type
48
What Molecular Machine Powers Pilus Retraction?
Genetic studies suggest
possible stepsizes one pilin subunit (0.8nm) or
one helical turn (4nm)
49
Twitching Motility at Low Levels of PilT
Wild type
Inducible pilT mutant
50
Kinetics of Pilus Retraction Single PilTs
pilT mutant, 0.01mM IPTG pilT mutant, 0.1mM
IPTG wild type
The mechanical translocation step is not
rate-limiting at forces Flt40pN.
51
A Single PilT Complex generates 110pN!
52
Reversibility of type IV pilus retraction
Skerker and Berg, PNAS 2001
53
Force-induced pilus extension
retraction
extension
Maier, B., Koomey, M. Sheetz , M.P. submitted
54
Force-induced pilus elongation
55
Reduced level of PilT is required for
force-induced pilus extension
reduced level of PilT
WT
56
Chemical Kinetics
Elongation
Retraction
A force-dependent switch reverses type IV pilus
retraction
57
PilT as a switch between retraction and elongation
Crowther et al, Mol Microbiol.2004
58
Conclusion

• A single PilT complex generates forces
• exceeding 100pN
• PilT, a force-dependent switch between
• pilus retraction and extension

Pilus dynamics in Neisseria gonorrhoeae
59
General Lessons for Robust Functions at Submicron
Level

• Compartmentation and Modular design
• Spatial Ordering of Multiple sites for
  Specificity
• Functional Activity Turns Off Automatically
• Multiple Activity Cycles for Important Functions
• Many Low Fidelity Steps for High Fidelity Process

60
Phases of Cell Motility
2---
2-10
5---
Rate-limiting Step
Unpolarized spreading
Regional Activation Inactivation
Myosin-activated Clock
Doebereiner et al., in preparation
61
Berenike Maier

• Department für Physik,
• Ludwig-Maximilians-Universität München, Germany
  and
• Center for NanoScience
• www.softmatter.physik.lmu.de

62
Lamellipodial Clock Set by Cytoskeletal Transport
of Contractile Signal
Grégory Giannone and Michael P. Sheetz
Department of Biological Sciences, Columbia
University, New York
63
Substrate Rigidity Can Control Direction of
Motility by Cytoskeletal Transport of Signals
Non-Rigid
Rigid
64
Horwitz, Scientific American
65
Acknowledgements
Sheetz Lab Drazen Raucher Jianwu Dai Catherine
Galbraith Goetz von Wichert Dan Felsenfeld Julia
Sable Daniel Choquet Guoying Jiang Ben
Dubin-Thaler Adam Meshel Yasuhiro Sawada Masako
Tamada Gregory Giannone Ana Kostic Hans-Guenther
Doebereiner
Duke University Dr. Harold Erickson Burnham
Inst. Gen-Sheng Fen NYU Medical Center Jan
Sap NIH Ken Yamada David Critchley Beatrice
Haimovitch Nelly Kieffer Anne Bresnick
66
Acknowledgments
Michael P. Sheetz Julia Sable Goetz Von
Wichert Masako Tamada Yasuhiro Sawada Anthony
Baer et les autres membres du laboratoire
Periodic contractions
Ravi Iyengar (New York) Kyle Miller Patricia
Gallagher (Indianapolis) Audrey Minden (New
York) Nelly Kieffer (Luxembourg) Anne Bresnick
(New York) Hans-Günther Döbereiner Benjamin
Dubin-Thaler