PFOS-014, 015, 011, 012

1. Foundation Studies PFOS-014, 015, 011, 012 Cytoskeleton and cell movement Cellular Organelles Cell membranes Membrane Transport Prof. K.M. Chan, Rm 513B, BMSB Dept. of Biochemistry Chinese University Email: kingchan@cuhk.edu.hk Tel: 3163-4420

2. Objectives: Be able to understand • cellular system for life processes • defects of the cellular system with clinical manifestations • cells coup with environmental changes or external stimuli with membrane proteins and transporters • basic structure and function of different cellular components, including membranes, cytoskeletons and organelles • Malfunctions of and drug actions on cellular components

3. PFOS-014 THE CYTOSKELETON & CELL MOVEMENT • Proteins are dynamic and movable • Interact to make up a cell and maintain cell shapes; they organize the cytoplasm, move organelles, etc • They form major machinery for cell movement in response to environmental changes.

4. The cytoskeleton composed of three systems: (1)actin, (2) microtubules, and (3) intermediate filaments; Extra-cellular Matrix will be discussed in other sections. (4) Summary and clinical correlations.

5. Extra-cellular Matrix and threetypes ofprotein filaments that form the cytoskeleton [4] Extra-cellular matrix, may restrict cell movement Cell membrane Nucleus [1] Actin Filaments (microfilaments, 7 nm) are helical polymers of actins [2] Microtubules with centrosome Using tubulin to form hollow cylinders of 25 nm diameter to move “cargoes” around [3] IntermediateFilaments: rope like fibers with 10 nm size.

6. 1. ACTIN FILAMENTS • Form micro-villi and contractile bundles • Form sheet-like or fingerlike protrusions (pseudopodia) from the leading edge of a moving cell • Many proteins (e.g myosin) bind to actin to modify its properties to form contractile structures in non-muscle cells and myofibril in muscle cells

7. 1.1 Actin filaments contain two chains of polymerised actin monomers coiled together. Thread-like structure Other proteins attach to actin filaments, e.g. myosin 7 nm in diameter Actin Filaments F-actin Twisted chain of polymerized globular actin molecules Polymerized actin Actin monomers G-actin monomers

8. 1.2 Migrating cells use actin filament to create podia (= foots) • Lamelli-podium (thin sheet) and filo-podium (thin, point) are supported by actin filaments made underneath the cell membrane. • The podia are for cell crawling. Podia

9. 1.3 Skeletal Muscle Cells • Muscle fiber cells are elongated • A few cm long, with a diameter of only 50 µm • Contain numerous myofibrils with actin and myosin filaments forming a highly ordered structure Myosin filaments Z disc Z disc Actin filaments By moving the mysoin closer to the Z disc alongside the actin filaments, muscle cells become contracted

10. Myosin molecule walks along actin filament using ATP energy Myosin head attached to actin filament Myosin filament ATP Actin filament ATP hydrolysis releases myosin head from actin Binding of myosin head to a new site ADP Pi

11. 1.4 The Erythrocyte Cytoskeleton • In red blood cells (erythrocytes), spectrins bind with actin to form spectrin membrane skeleton and maintain the biconcave (donut) shape of erythrocytes; keeping the elasticity as well as flexibility of the erythrocytes. • The actin filament is short and contains only 14 monomer. It is stabilized by tropomyosin, and each binds 6 spectrin tetramers of 2α2β heterodimer.

12. To hold up the red blood cell, intracellular protein network holds up the fluid like layer of plasma membrane with peripheral proteins. Erythrocyte Membrane structure Spectrin αβ Actin Tropomyosin Band 4.1 Ankyrin Band 3 Transmembrane protein

13. Hereditary spherocytosis : • A genetic disease characterized by anemia, jaundice and splenomegaly (enlargement of the spleen). • spherical and fragile red cells due to reduced spectrin content; the spectrin cannot bind with 4.1. protein to form the actin-4.1-spectrin complex. • In hereditary elliptocytosis, spectrins form dimer instead of tetramer.

14. 2. MICROTUBULES • Long, hollow cylinders made of tubulin proteins • Outer diameter could reach 25 nm • More rigid than actin or intermediate filaments • Usually attached to centrosome which is the microtubule-organizing centre • From free tubulin to a polymerized microtubule, the formation requires GTP binding • During cell division, the microtubule framework forms mitotic spindle to guide chromosomes to move and segregate

15. 2.1 Microtubules are stiff hollow tubes of protein monomers of tubulin polymerized together as proto-filament. 25 nm Tubulin is heterodimer of αand βsubunits Lumen at centre Protofilament αtubulin βtubulin GTP ( ) is needed to add on tubulin to the protofilament as a growing microtubule.

16. 2.2 Properties and function of microtubules • Dynamic instability: tubulin hydrolyzes GTP to GDP building different conformations, growing or shrinking, to make up the interior of the cell. • Proto-filaments with GDP are unstable and can peel away from the microtubule wall. • The cell’s polarity is also created by microtubules which move different organelles to different ends of the cell. Microtubule has its polarity. • Motor proteins like kinesins and dyneins are involved in controlling the two ends (+ and -) of microtubules. • Organelles move along the microtubules with the help of kinesins to the plus end, and dyneins to the minus end.

17. Dyneins and kinesins transport “cargoes” (organelles or macromolecules) along microtubules. Dynein 14 nm microtubules Kinesin 8 nm “Colchicine” makes microtubules disassemble and the organelles change locations and go all over the cell.

18. 2.3 Cancer Drugs kill cells by blocking the cytoskeleton • Colchicine blocks tubulin to form mitotic bundle and hence breaks tubulin; cell division stopped. • Taxol has an opposite action to hold microtubules and arrests dividing cells in mitosis, thus less side effects are found. • Taxol and colchicine are effective anti-cancer drugs.

19. 3. INTERMEDIATE FILAMENTS • Have great tensile strength to withstand mechanical stress. • Known as intermediate because its diameter is around 10 nm, in between actin (7 nm) and microtubules (25 nm). • They are tough and durable filaments to form a network in the cytoplasm or anchored to the plasma membrane for cell-cell interaction.

20. 3.1 Rope-like fiber of intermediate filaments form “web” like structure to hold up cell morphology, whereas actin and tubulin are globular proteins. C N N C C N N C C N C N C C C N N Protofilaments are tetramer of two coiled-coil dimers.

21. 3.2 Intermediate filaments can go through cell-cell junction via desmosome to make cell linked together, very common in epidermal cells. Intermediate filaments Desmosome

22. 3.3 Intermediate Filaments in various cell types • Three classes in different cell types: • Keratin filaments in epithelial cells; • Vimentin and vimentin-related filaments in connective tissues; • Neurofilaments in neurons: NFL, NFM, NFH.

23. Form nuclear lamina in the nucleus to strengthen nuclear envelope. • When phosphorylated, they fall apart to facilitate disassembly of the nucleus for cell division. • Chromatin in nucleus is also associated with nuclear lamina. • The proto-filament proteins are called lamins.

24. 4.1 Clinical correlations (1) • After a person has died, ATP synthesis stopped and thus myosin firmly attached to actin. This is why in the corpse, the muscle is stiff and rigid, a condition called rigor mortis. • Integrin connects ECM and cytoskeleton through plasma membrane, tumor cells start to migrate (metatasize) must first disrupt integrin, collagen and other ECM proteins with digestive enzymes (matrix metalloproteinases, MMPs). MMP inhibitors may be useful in stopping metatasis of cancer cells.

25. Clinical correlations (2) • Linker molecules in ECM or actin-anchored proteins can act as chemotactic receptors. • Neutrophils, e.g. respond to N-formylated peptides derived from bacteria by reorganizing the actin network and formmicrospikes that propel the cell toward the bacterium.

26. 4.2 The connective tissues control collagen secreted in a organized way by attachment to actin filament via fibronectin and integrin. Collagen Fibronectin Integrin going through the membrane Cell membrane Adaptor proteins Actin filament Collagen from ECM attached to cytoskeleton

27. Clinical correlations (3) • Muscular dystrophy: dystrophin is a cytoskeletal protein joining the membrane of muscle cells that mediates the interactions of extracellular matrix. • It is a progressive myodegenerative disease when dystrophin is mutated, and some may die by 20 with heart and lung failure.

28. 4.2 Summary • Cytoskeletons are macromolecules of protein with tertiary structures in a highly ordered organization. • ATP energy is required to change the conformation of myosin head for actin affiliation, leading to movement of cytoskeletons or muscle fibers. • GTP is required to assemble tubulin monomers into filaments of microtubules. • Anti-cancer drugs control cell division by attacking tubulins, e.g. Taxol (avoid GTP detachment) and Colchicines (avoid GTP binding for formation of filament). • Mutations of genes causing defects in proteins and malfunction of the specialized functions, e.g. muscle contraction, anemia or hemolysis.