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Home , Ankyrin, Lamellipodium, Profilin, Spectrin

MEDICAL BIOLOGY

MICROFILAMENTS September 24, 2003

Thomas J. Schmidt, Ph.D. Department of Physiology and Biophysics 5-610 BSB, 335-7847

Reading Assignment: Molecular Biology of the Cell (4th ed..), 2001, by B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter; Chapter 16, pp. 907-925, 927-939, 943-981

Key Concepts:

1. The is a complex network of filaments ( filaments, intermediate filaments and ) that traverses the cell and performs many important and diverse cellular functions.

2. Thin actin filaments, which are present in all cells, are composed of two helically interwined chains of G-actin monomers.

3. A variety of including , , , thymosin, , and α- regulate the dynamic state of actin filaments

4. The spectrin membrane skeleton, which is composed primarily of actin filaments located at the cytoplasmic surface of the , is essential for maintaining cellular shape and elasticity as well as membrane stability.

5. Cell is mediated by actin-filaments organized into specific cellular projections referred to as lamellipodia and .

Medical 1 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003

Key Terms: cytoskeleton cytochalasins actin filaments (actin) phalloidins intermediate filaments (, spectrin membrane skeleton ) spectrin microtubules () actin microfilaments F-actin band 4.1 G-actin glycophorin II band 3.0 myosin I hereditary actin microfilaments hereditary elliptocytosis sickle cell anemia actin-binding proteins spectrin supergene family spectrin spectrin filamin α-actin fimbrin α-actinin microvilli gelsolin terminal web thymosin lamellipodium profilin filopodia villin stress fibers contractile bundles

Medical Cell Biology Microfilaments 2 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003

Clinical Correlation: situation

Judy, a 19 year-old African-American woman, was admitted to the hospital because of dyspnea, severe fatigue and a hemacrit of 6.4%. She had a history of anemia since childhood that was treated intermittently with iron supplements. There was no family history of sickle cell disease but her mother reported being told previously that she also was anemic. The patient took no medications and had no allergies. She worked as a cashier. A diagnosis of severe anemia of unknown cause was made.

Medical Cell Biology Microfilaments 3 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003 Lecture Outline:

I. Cytoskeleton

A. Definition - “a complex network of protein filaments that traverse the cell cytoplasm”

B. Functions

1. maintain or change cell shape

2. provide cells with mechanical strength and support fragile plasma membrane

3. contraction of muscle cells as well as non-muscle cells

4. transport of within the cell

5. cell division (segregation of chromosomes at mitosis)

6. and exocytosis

7. movement of cilia and flagella

8. provide binding sites for the specific localization of RNAs and proteins that were once thought to diffuse freely throughout cytoplasm

C. Principal types of protein filaments 1. actin filaments (actin) constitute microfilaments • 7-9 nm diameter

2. intermediate filaments (family of related fibrous proteins such as vimentin or lamin) • 10 nm diameter

3. microtubules (tubulin) • 24 nm diameter

D. Accessory or regulatory proteins are responsible for dynamic structures formed from cytoskeletal filaments

Medical Cell Biology Microfilaments 4 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003 II. Microfilaments

A. Actin-present in all eukaryotic cells

1. most abundant cytoskeletal protein (5-30% of total protein in nonmuscle cells)

2. well conserved protein (six different genes in human)

B. Actin (thin filaments) and myosin (think filaments) first isolated from skeletal muscle (Fig. 16-69) but known to be ubiquitous component of nonmuscle cells

1. actin/myosin ratio in nonmuscle cells is about 100:1 Medical Cell Biology Microfilaments 5 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003

2. although in nonmuscle cells the myosin and actin filaments don’t form highly structural array, they are responsible for contraction of these cells and cytokinesis (Fig. 18-34)

remaining overlap microtubules from central spindle

contractile ring of actin and myosin filaments in cleavage

Medical Cell Biology Microfilaments 6 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003 C. Structure of actin filament

1. thin (~8 nm wide) and flexible

2. each filamentous or F-actin is composed of two helically interwined chains of G-actin monomers

3. each G-actin monomer must have an ATP molecule bound to polymerize onto an actin filament (ATP is hydrolyzed to ADP during polymerization)

4. the actin filament is a polar structure with a relatively inert and slow-growing minus end and a faster-growing (dynamic) plus end (page 912, lower panel)

5. treadmilling refers to addition of G-actin to the plus end while it is being subtracted from minus end (page 913, center panel)

6. filaments usually exist as cross-linked aggregates and (networks) parallel bundles • stress fibers • terminal web

7. actin is in a dynamic state, undergoing polymerization and depolymerization as required

Medical Cell Biology Microfilaments 7 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003 D. Actin-binding proteins

1. a variety of binding proteins regulate the dynamic state of actin

2. examples of specific actin-binding proteins a. spectrin • terminal web (, cortical network)

Medical Cell Biology Microfilaments 8 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003 b. filamin • cross-links filaments into gel or networks (Figure 16-42)

c. gelsolin - Ca2+-sensitive actin severing protein; forms cap on the newly exposed plus end of filament, thus breaking up the cross- linked network of actin filaments (may help to loosen or liquefy cell cortex to allow membrane fusion events)

i. gelsolin-null mice have multiple defects in cell morphology and motility

ii. overexpression of gelsolin in some tumor cells (pulmonary carcinomas) may enhance their motility (more metastatic)

d. thymosin - blocks polymerization and maintains G- actin monomer pool

e. profilin - binds to actin monomers and catalyzes ATP-ADP exchange; when profilin is released from plasma membrane by external stimuli it may convert inactive ADP actin to active ATP actin to induce local formation of actin filaments

Medical Cell Biology Microfilaments 9 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003

f. fimbrin and α-actinin - bundle filaments (Fig. 16-40A)

3. drugs that interact with actin filaments and block cell movement

a. cytochalasins - block polymerization (bind to plus end) and by so doing inhibits cell motility

b. phalloidins - bind tightly along the side of actin filaments and stabilize them against depolymerization

Medical Cell Biology Microfilaments 10 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003 F. Spectrin membrane skeleton (Fig. 10-31A)

1. anchoring of actin filaments to the cytoplasmic surface of cell membrane

2. essential for maintaining cellular shape and membrane stability

3. first described and best understood in erythrocyte where it maintains biconcave shape, endows cell with properties of elasticity and flexibility, and stabilizes plasma membrane

4. major proteins are spectrin I, actin, protein 4.1, and ankyrin

a. spectrin tetramers cross-link actin filaments into a two-dimensional meshwork that covers cytoplasmic surface of plasma membrane

b. tail ends of four or five spectrin tetramers are linked together by binding to short actin filaments and other cytoskeletal proteins (including band 4.1) in a “junctional complex”

• protein 4.1 binds to a member of glycophorin family (transmembrane proteins) and thus serves as a link to bilayer

c. attachment of spectrin skeleton to bilayer

• ankyrin (peripheral protein) links spectrin to cytoplasmic domain of band 3 (a transmembrane protein)

Medical Cell Biology Microfilaments 11 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003 5. clinical correlations (anemias)

a. • ~75% of cases due to mutations in ankyrin • ~20% of cases due to band 3 mutations

b. hereditary elliptocytosis • majority of mutations in spectrin • some mutations in protein 4.1 c. sickle cell anemia • problems with posttranslational modifications of actin

G. Spectrin (exists in two isoforms, spectrin I, spectrin II) is also a ubiquitous component of nonerythroid cells

H. Spectrin supergene family of actin-binding proteins

1. spectrin I and II - in link actin filaments to microtubules, , organelles and synaptic vesicles.

2. α - actinin - actin bundling protein

3. dystrophin - flexible, elongated protein that may anchor actin filaments to plasma membrane in skeletal muscle

I. Microvilli (Fig. 16-41A)

Medical Cell Biology Microfilaments 12 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003

1. fingerlike projections that are located on the apical surface of epithelial cells and increase the surface area available for absorption of important nutrients

2. core consists of actin bundles that serve as scaffolding

3. fimbrin and villin are cross-linking proteins that bundle actin filaments

J. terminal web • a meshwork of actin filaments, actin-cross-linking protein (spectrin and myosin II) and intermediate filaments

Medical Cell Biology Microfilaments 13 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003 K. Role of actin-filaments in cell motility

1. lamellipodium - a thin sheetlike projection that forms on leading edge of motile cell

2. filopodia (Fig 16-38)

a. thin, stiff projections on a crawling cell that serve as feelers, helping direct cell

b. formed by rapidly polymerizing tight parallel bundles of actin filaments

c. rapid burst of actin polymerization (treadmilling) caused by dissociation of G-actin - profilin complex

Medical Cell Biology Microfilaments 14 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003 3. cell movement divided into distinct actin-dependent processes (Fig. 16-85)

a. protrusion - lamellipodium and filopodia extended from front of cell

b. attachment - actin cytoskeleton makes a connection with the substratum

d. traction - body of cell moves forward

4. monomeric G proteins-small

a. Rho- induces stress fibers and focal adhesion formation

b. Rac- induces larmellipodia

c. Cdc 42- induces formation of filopodia and activation of Rac

L. So why is the actin cytoskeleton and cell movement so important in medicine?

1. lymphocyte immune response

2. localization of neutrophils to site of infection

3. ability of fibroblasts to migrate and heal wounds

4. phagocytosis of pathogens by macrophages

5. muscle and cell contractions

Medical Cell Biology Microfilaments 15 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003

Medical Cell Biology Microfilaments 16 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003

Reprints on Reserve at Hardin Library:

1. Hassoun, H. and Palek, J. Hereditary spherocytosis: A review of the clinical and molecular aspects of the disease. Blood Rev. 10:129-147, 1996.

2. Carpenter, Christopher. Actin Cytoskeleton and Cell Signaling. Critical Care Medicine. 28(Suppl. 4):N94-N99, 2000

3. Thor, A.D., Edgerton, S.M., Liu, S., Moore, D.H. and Kwiatkowski, D.J. Gelsolin as a negative prognostic factor and effector of motility in erbB-2-positive epidermal growth factor receptor-positive breast cancers. Clin. Can. Res. 7:2415-2424, 2001.

Medical Cell Biology Microfilaments 17 Thomas J. Schmidt, Ph.D. Email: [email protected] September 24, 2003