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)

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

Movie About the movie: –Made in by John T. Bonner, Princeton Univ –Amoebae are um in size –“Slugs” are 1 mm, with about 100,000 cells

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

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

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

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

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

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?

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.

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

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

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

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

Correlation: SHOW IT Co - relation: –two events occur together (space or time) –Example: Fig (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)

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

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? Cells adhere via 24kD protein interactions: Add antibody: cells do not adhere

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

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?

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

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.

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