Food poisoning and the cytoskeleton

1. Food poisoning and the cytoskeleton German Research Center for Biotechnology

2. Listeriosis • Symptoms • Influenza-like early on • The infection quickly progresses to cause… • septicemia • meningitis and encephalitis • intrauterine or cervical infections in pregnant women, which may result in spontaneous abortion • Prevalence • 2500 cases of listeriosis in the US each year, with about 500 deaths

3. Transmission • Normally orally through contaminated food • Many animals carry the bacterium without symptoms • L. monocytogenes has been associated with raw foods • raw milk • soft-ripened cheeses • raw meats • raw and smoked fish • It can grow in cans and at temperatures as low as 3°C • It is resistant to dehydration and temperature extremes

4. Listeria lifecycle: an intracellular pathogen • Phagocytosis by monocytes • Lysis of phagosome • Proliferation in the cytoplasm • Locomotion • Cell-cell spread through filopodia • What survival advantage does this life cycle afford Listeria? http://www.med.ufl.edu/biochem/DLPURICH/Listeria.html http://www.med.sc.edu:85/ghaffar/zoonoses.htm

5. Listeria life history http://cmgm.stanford.edu/theriot/movies.htm

6. Cytoskeleton • The cell interior is highly ordered as a result of the cytoskeleton. • The cytoskeleton is a filamentous network of protiens that pervades the cytoplasm • The protein filaments consist of protein monomers that self-assemble into the final structure, and can dissociate to destroy that structure. • In general, these filaments are highly dynamic - they are not permanent or static structures.

7. Three filament types • Intermediate filaments • Microtubules • Microfilaments (actin filaments)

8. Intermediate filaments • 8-12 nm in diameter (intermediate between microtubules and actin) • The most stable and least dynamic of the filaments • Filaments are not polarized • Six different related classes • These include • Nuclear lamins • Keratins, which make up the tonofilaments of epithelia • Thought to serve a strengthening and stabilizing role in cells

9. Microtubules • Tubes made of α- and β-tubulin • Exist in vivo as heterodimers • Form protofilaments, which circularize in groups of 13 (usually) • One end is all alpha (+ end), the other all beta (- end)… that is, they are polarized • In cells, growth occurs preferentially at the + end • Mechanically analogous to rigid rods

11. Polymerization of microtubules from tubulin dimers

12. Critical concentration, Cc, is the monomer concentration at which the rate of monomer addition equals the rate of monomer loss from one end of the filament. Chemistry of polymerization

13. …but in a cell… • Microtubules are nucleated by microtubule organizingcenters (MTOCs) • The centrosome is an example of a MTOC • MTOCs contain microtubule structuresthat form stable (-) endsfor growing microtubules • Contain γ-tubulinwhich may stabilize these nascent microtubules

14. …and microtubules are GTPases • Tubulin dimers bind GTP • Tubulin-GTP adds to the (+) end of the filament with a lower Cc than [tubulin]. • Once in the microtubule, the GTP is almost immediately hydrolyzed, leaving a GTP cap on the (+) end. • If the cap is lost, Cc for the (+) end is suddenly higher than [tubulin], and the microtubule catastrophically disassembles.

15. Handy poisons • Many chemotherapeutic drugs are antimitotics (shut down mitosis) • vinblastine causes tubulin aggregation • nocodazole caps microtubules so that they can’t grow • taxol stabilizes microtubules, blocking complete celldivision

16. “Super Pot” • Made using colchicine • from the Autumn Crocus (a lavender) • causes disassembly of microtubules • Sometimes used to enhance the potency of marijuana • Causes some chromosomes to not always be evenly divided between daughter cells • Causes some cells to become polyploid • induces overexpression of genes. FYI: The “cannabinoid receptor” in the brain normally binds an arachidonic acid derivative, anandamide.

17. Microtubule-based motors • Two major kinds: • Kinesins (most are plus-end directed) • Dyneins (minus-end directed) • Both have motor domains (heads) and cargo-binding domains (tails) • Dyneins are of two kinds • Cytoplasmic – cargo transport • Axonemal – ciliary and flagellar motion

18. Why do you need transporters anyway? • Otherwise you would rely on diffusion • The distance traveled by diffusion is not linearly related to time!

19. How long does it take to diffuse? • For a sphere… • “Stokes-Einstein equation” • kB = Boltzmann constant (1.38x10-23 J Kº) • T = Absolute temperature • η = viscosity of the medium (0.001 for water in SI units) • r = radius of the sphere • n= “dimensionality” (1D, 2D or 3D) • Example: How long would it take for a ribosome to diffuse down the length of a 1m axon?

22. Actin (microfilaments) • Over 5% of the intracellular protein • The most ubiquitous of all intracellular proteins • Functions • Cell motility and muscle contraction • Cell shape (like microvilli) • Vesicle transport near cell periphery

23. Actin filaments • Actin monomers stack to form a double-helical filament, 7 nm in width • While microtubules are GTPases, actin filaments are ATPases

24. Actin polymerization • The + end grows faster than the – end • Like microtubules, the + end has a lower critical concentration than the – end, and… • This difference is accentuated when monomers have ATP bound, as opposed to ADP. • Once in the filament, the rate of ATP hydrolysis increases • As a result, the + end has predominently ATP-monomers, and the – end ADP-monomers + end - end Actin monomers

25. What will happen to filament length if… • The concentration of monomers is greater than the CCs at both ends of the filament? • The concentration of monomers is lower than the CCs at both ends of the filament? • The concentration of monomers is greater than CC at the + end but lower than CC at the – end?

26. Treadmilling + end - end Actin-ADP Actin-ATP

27. Not-so-mellow mushrooms • Phallotoxin (phalloidin) • an actin filament stabilizer • the poison in some mushroom genera • It kills by stabilizing actin filaments (inhibiting disassembly) • Immediate cause of death is liver failure • Cytochalasin • an actin filament de-stabilizer • also derived from mushrooms Death Cup mushroom

28. Myosins: actin-based motors • With few exceptions, plus-end directed • Myosins have heads that serve the motor-function, and tails that either: • Form filaments, as in muscle • Bind cargo, like kinesins and dynein

29. Controlling filament growth Thymosin is a monomer buffer Profilin is a shuttle, and promotes nucleotide exchange

30. Cell crawling • Lamellipodia and pseudopodia are pushed forward by actin polymerization and other forces • How does a cancer cell or leukocyte crawl “in the right direction,” like towards a cytokine?

31. Molecular basis of cell crawling

32. Nucleation and branching: Arp2/3 and WASP • Arp2/3 is a very important for making branch points on actin filaments, and nucleating new filament growth • WASP is a key activator of Arp2/3 that is itself activated by cell-surface receptors.

33. Molecular basis of cell crawling

34. Listeria lifecycle: an intracellular pathogen • Phagocytosis • Lysis of phagosome • Proliferation in the cytoplasm • Locomotion • Cell-cell spread through filopodia • How does Listeria accomplish cell-cell spread? http://www.med.ufl.edu/biochem/DLPURICH/Listeria.html

35. Stealing the machinery • Listeria has on its surface the protein ActA • ActArecruitsArp2/3 from the cytoplasm and activates it (basically substituting for WASP) • Promotes actin filament nucleation and growth Listeria

36. Visualizing the actin http://cmgm.stanford.edu/theriot/movies.htm