Dynamic DissonantsCell and Developmental Biology

• New York UniversityDavid ScicchitanoMark SiegalKris Gunsalus

• University of HawaiiSteve RobinowAthula Wikramanayake

• University of Wisconsin, MadisonBrian Manske

• Part I: structure, assembly and dynamics of microfilaments

• Part II: the cytoskeleton organizes the cytosol and supports the function of other cellular organelles and processes

• Part III:cytoskeletal filaments form higher-order structures that perform diverse functions

Actin Cytoskeleton Teachable Unit

• Chemical principles govern the behavior of biological macromolecules.

• Cellular phenomena reflect the collective action of populations of molecules.

• The same molecule can have multiple, diverse functions within cells.

Teaching Challenges

Diseases of the cytoskeleton• Muscular Dystrophy Dystrophin

– muscular degeneration

• Kartagener Syndrome

Axonemal dynein intermediate chain– Infertility due to defective sperm motility – Situs inversus

• Usher Syndrome myosin VII– deafness

• Epidermolysis bullosa and bullosa pemphigoid keratin mutation or autoimmune disorder

– skin blistering

Actin

Actin

Microtubules

Intermediate filaments

Are microfilaments the same as F-actin?

A. YesB. No

What polymerizes to form microfilaments?

A. alpha-tubulin

B. intermediate filaments

C. beta-tubulin

D. G-actin

The cytoskeleton is composed of:

A. microtubules

B. microfilaments

C. intermediate filaments

D. A and B

E. A and C

F. B and C

G. A, B, and C

Are microfilaments the same as F-actin?A. YesB. No

What polymerizes to form microfilaments?

A. alpha-tubulin

B. intermediate filaments

C. beta-tubulin

D. G-actin

The cytoskeleton is composed of:

A. microtubules

B. microfilaments

C. intermediate filaments

D. A and BE. A and CF. B and CG. A, B, and C

Are microfilaments the same as F-actin?A. YesB. No

What polymerizes to form microfilaments?

A. alpha-tubulin

B. intermediate filaments

C. beta-tubulin

D. G-actin

The cytoskeleton is composed of:

A. microtubules

B. microfilaments

C. intermediate filaments

D. A and BE. A and CF. B and CG. A, B, and C

synonymous to

Learning Goals

• To understand the structure, assembly and dynamics of microfilaments

• Apply chemical principles to enhance understanding of actin polymerization

• Predict and understand a biological model of actin polymerization

• Predict and interpret experimental results

G-actin: globular monomerF-actin: filamentous polymer

G-actin F-actin

Experiment

G-Actin

+

ATP

G-Actin

I

Results

F-actin is present in I only (at steady state)

II

Additional Experiment

G-Actin

+

ATP*

G-Actin

+

ADP

A. No F-actin in eitherB. F-actin in bothC. F-actin in ID. F-actin in II

ATP* cannot be hydrolyzed to ADP

I II

Actual Results

G-Actin

+

ATP

G-Actin

G-Actin

+

ATP*

G-Actin

+

ADP

Amount of F-actin at steady state+++ - +++ ++

Conclusion

For G-actin to form F-actin:

A. ATP hydrolysis is necessary.

B. ATP is necessary.

C. ADP is necessary.

D. Either ATP or ADP is necessary.

E. Neither ATP nor ADP is needed.

APPP

APP

F-actin

G-actin

Fast Slow

Summary

1.G-Actin has four lobes and an ATP binding site

2.In a test tube, add magnesium, sodium, potassium and either ATP or ADP:

G-actin F-actin

3. ATP binding to G-actin provides the best conformation to promote F-actin assembly

4. F-actin has directionality (the two ends are not the same)

G-actin ↔ F-actin ATP

Actin Dynamics

ATP

G-actin ↔ F-actin ATP

Summary

• G-actin and F-actin will reach a steady state.

• The nucleation step is slow.

Acknowledgements

Thanks to our facilitators!

• Randy Phillis

• Lauren Gollahon