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 multicellular
• 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
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
Fluorescent tag
Antibody labeling
• Direct immunofluorescence
• Antibodies only bind if specific
antigen (protein) is present!
Antigen
binding
Cell
surface
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
Be able to define each of the following, and give an
example:
• Observation
•Necessity
• Hypothesis
•Sufficiency
• Prediction
•Necessary but not sufficient
• Experiment
•Sufficient but not necessary
• Correlation
•Necessary and sufficient
• Evidence
– Loss-of-function
– Gain-of-function