the secrets of a slime mold dictyostelium discoideum, “dicty” protozoan: similarities to both...
TRANSCRIPT
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 1940-41 by John T. Bonner, Princeton
Univ– Amoebae are 10-15 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
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
Antigenbinding
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. 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)
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