The secrets of a slime mold

1. The secrets of a slime mold • Dictyostelium discoideum, “Dicty” • Protozoan: similarities to both plant and animals • cell wall with cellulose (like plant/fungus) • motile cells (like animal) • cell movements in morphogenesis (like animal) • forms spores (like plant/fungus)

2. The Dicty slime mold life cycle • Switch from uni- to multi- cellular • Cells cooperate to form fruiting body with spores • 1-4 days for cycle

3. Movie • About the movie: • Made in 1940-41 by John T. Bonner, Princeton Univ • Amoebae are 10-15 um in size • “Slugs” are 1 mm, with about 100,000 cells

4. What are we seeing? • What behaviors can we directly observe in the movie? • What questions are raised?

5. Aggregation in Dictyostelium • Single cells: crawl around and engulf bacteria • Eventually: starvation • All local food is consumed • Program initiated: • cells cooperate as a large group to migrate, and sporulate in new locale

6. Observations: • EATING phase: cells grow and divide mitotically • free movement, no cell-cell adhesion • STARVATION phase: cells change behavior • migrate into streams • ADHERE • aggregation to form “multicellular” slug • long distance migration to new food source

7. Cell adhesion • How cells stick together, • How (and why) cell biologists study cell adhesion

8. Two examples of cell adhesion • 1. Compaction of early mammalian embryo • 2. Muscle precursor cells adhere and fuse

9. Making observations • “Just the facts, ma’am.” • Observations: • Cells stay separate when food is present • begin to stick together when food is depleted • How can these observations be explained?

10. Formulating a hypothesis • Hypothesis= how can observations be explained? • Needs to be consistent with all observations • Car break-down analogy • Often more than one hypothesis: “competing hypotheses” • Testing hypotheses: first make predictions • IF... hypothesis is true, THEN… this experiment is predicted to give result.

11. Another Dicty observation: • fluorescently-labeled antibodies against a 24 kD glycoprotein bind to the surface of starved cells • Antibodies: tools to observe (and manipulate) molecules in cells

12. How are antibodies made? • Isolate material from cells or embryo • isolate membranes from starved cells, • or purify a specific protein • Inject into animal, immune system reacts by producing large amounts of antibody that binds to antigen • 1-2 months later, collect blood and isolate serum • Purify antibodies specific for antigen • Example: specific 24 kD membrane protein

13. Fluorescent tag Antibody labeling • Direct immunofluorescence • Antibodies only bind if specific antigen (protein) is present! Antigen binding Cell surface

14. Three types of evidence • Correlation: SHOW IT • Loss-of-function: BLOCK IT • Gain-of-function: MOVE IT

15. Correlation: SHOW IT • Co - relation: • two events occur together (space or time) • Example: Fig. 1.25 (handout) • 24 kD antibody does not label dividing, non-adhering cells • 24 kD antibody does label starved, adhering cells • So, presence of 24kD protein correlates with adhering cells • adhering cells have protein • non-adhering cells do not have protein • (for later discussion: control experiments are key to be able to obtain a clear result)

16. Correlation: SHOW IT • Correlation is not Cause • suggests one event causes the other • leads to Hypothesis: • 24 kD protein causes cell adhesion • Careful! weakest type of evidence • Very useful for suggesting hypotheses • Other possible hypotheses? • Shoot down the first hypothesis… • Need a more definitive set of experiments

17. Loss-of-function: BLOCK IT • Experiments to provide loss-of-function evidence • block, interfere, prevent, remove, knockout, ablate • Example: antibody that binds 24 kD protein could block cell adhesion • Design an experiment using Dicty? Add antibody: cells do not adhere Cells adhere via 24kD protein interactions:

18. Loss-of-function: BLOCK IT • Stronger than correlation, but still limited power • How could the antibody blocking experiment give mis-leading results?

19. Necessity and sufficiency • Necessity = Requirement • Event or molecule is necessary for event to occur • But: • just because something is necessary doesn’t mean that it is sufficient • Sufficient = Enough to do the job alone • How can Sufficiency be demonstrated?

20. Gain-of-function: MOVE IT • Demonstration of sufficiency: • Force event or molecule at new time or place • move, transplant, over-express, activate, induce • Example: • 80 kD protein with similar adhesive function • Isolate gene and transfect into feeding cells • cause to be expressed at all times, even before starvation • Predicted result: feeding cells adhere! • Conclusion: 80 kD protein is sufficient to cause cell adhesion

21. Necessary or Sufficient? • Invent examples of : • Necessary but not Sufficient • “BLOCK IT” worked, but “MOVE IT” did not. • Sufficient but not Necessary • “BLOCK IT” did not work, but “MOVE IT” did. • Necessary and Sufficient • “BLOCK IT” and “MOVE IT” worked.

22. Analyzing experiment and results Be able to define each of the following, and give an example: • Observation • Hypothesis • Prediction • Experiment • Correlation • Evidence • Loss-of-function • Gain-of-function • Necessity • Sufficiency • Necessary but not sufficient • Sufficient but not necessary • Necessary and sufficient