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PIPs, Pattern Formation, and the Regulation of the Cytoskeleton William Foster, PhD, MD Department of Physics University of Houston
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PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Jan 02, 2016

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PIPs, Pattern Formation, and the Regulation of the Cytoskeleton. William Foster, PhD, MD Department of Physics University of Houston. How Cells Crawl. Some reasons why this problem is medically important. - PowerPoint PPT Presentation
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Page 1: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

William Foster, PhD, MD

Department of Physics

University of Houston

Page 2: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

How Cells Crawl

Page 3: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Some reasons why this problem is medically important

human polymorphonuclear leukocyte (neutrophil) on a blood film, crawling among red blood cells and "chasing" Staphylococcus aureus microorganisms

C. Cunningham, MD

Page 4: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Listeria

An INTRAcellular organism.Triggers actin polymerization at its trailing edge

Page 5: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Melanoma

Usually grows slowly but kills patients because it can spread to the liver, brainand other parts of the body.

Page 6: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Human plateletsChanges in cell shape are due to changes in

the cytoskeleton

Page 7: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

What is the cytoskeleton?

• The “skeleton” of a cell

• Made up of actin– a protein– Simplified in this

talk

Page 8: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Regulation of the cytoskeleton

• Actin monomers “G-actin” are added to the growing end of an actin filament “F-actin”

• Monomers fall off of the other end

• This process is driven by ATP

J. Käs, PhD

Page 9: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

What is happening at the membrane?

• Proteins (gelsolin and profilin) cap actin filaments and prevent further grown

• Removal of these proteins allows elongation of filaments

Page 10: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

• These proteins are regulated by CLUMPS of highly charged lipids

What is happening at the membrane?

Page 11: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

How is actin regulated?• The cell membrane plays a critical role both in the

regulation of the actin cytoskeleton as well as many other processes

• The actin polymers elongate at the membrane– Wouldn’t want them to elongate in the middle of the nucleus or

near the DNA

T Stossel, MD

Page 12: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Not part of this talk!

Page 13: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

What do we know about how the cytoskeleton works?

F-actin (polymerized actin) in the lamellipodia is (fluorescence labeled) GFP-Actin.

Page 14: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Intermediate Summary

• Actin is a protein that polymerizes to form much of the cytoskeleton.

• Some proteins that regulate the cytoskeleton are regulated in part by lipids (PIP) in the cell membrane.

• We want to understand the effect of these highly charged lipids (PIP) on cell membranes.

• We will now talk about some soft condensed matter concepts and techniques that will allow us to study this problem.

Page 15: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Lipids

PCPI PI(4,5)P2

Page 16: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Why PIP

• Usually a few % of total lipid in biological membranes

• In vitro, several proteins require 10 mole%

• Specialized regions of the plasma membrane (caveoli or lipid rafts) may be highly enriched (>20%) in PI’s

• This clumping of PIs may be important

Page 17: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Imaging MembranesLens

1 mm

Inject solublefactors intoaqueous phase

PC Texas Red - PE NBD-PIP2

PA Janmey, PhD

Page 18: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

What is the effect of these highly charged lipids (PIP) on cell

membranes?• Start with a lipid film

– contains varying quanties of PIP– Contains 1% fluorescent probe

• Image the lipid film

Page 19: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Line Tension

• There can be domains of different phases– say, differently ordered

phases

• Domains have line tension– The 2 dimensional analog of

surface tension

• Domains in a film are round because that lowers the energy of the system

• The bar is 100 microns

Page 20: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Changes in line tension with PIP concentration (0%, 10%, 50%, 90%)

Page 21: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Addition of NaCl to screen electrostatic interactions

Page 22: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

The labeled lipid in the prior slides also contains PIP

Page 23: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Line tension no longer dominates the shape of lipid domains

The free energy of the system is:

'

'

2'

'

2

2

3

2

rr

dAdA

rr

dAdAdrF

Where:=the line tension is the dipole density is the charge density DJ Keller, JP Korb, HM McConnell, J

Phys Chem 91 (1987).DJ Keller, HM McConnell, VT Moy, J Phys Chem 90 (1986).HM McConnell, VT Moy, J Phys Chem 92 (1988).

Page 24: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Consider evaluating this equation on an ellipse

• Using Green’s theorem:

'

'

2'

'

2)(

222

rr

dAdA

rr

drdrdrAF

Where:= the electrostatic energy/unit area= the line tension = the dipole density = the charge density

Page 25: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Evaluating the energy in terms of the complete elliptical integrals

)(43

4

'

'

2)()(

222 kaKab

rr

drdrkEAF

where 12

a

bk

K(k) is the complete ellipital integral of the first kindE(k) is the complete ellipital integral of the second kind

Page 26: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Expand in powers of =ln(b/a)• F=e0+e22+e44+…• e4>0, otherwise the system is unstable.• If e2>0, the minimum is at =0• If e2<0, the minima are at +e2/2e4

• The system undergoes a second order phase transition from a phase where round domains minimize the energy to a phase where distorted domains are favored.

de

abababe

3/10

2/12

22

2/1

2

)(4ln2

2

)()(3

16

)(20

Page 27: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Summary

• The cytoskeleton is a rich topic for biological physics research.

• Powerful techniques have been developed to study this system.

• We can quantatively understand the effect of different constituents to the structure of cell membranes

Page 28: PIPs, Pattern Formation, and the Regulation of the Cytoskeleton

Thank you

• Paul Janmey, PhD (U. Penn)

• Josef Käs, PhD (U. Leipzig)