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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 secrets of a slime mold Dictyostelium discoideum, “Dicty” Protozoan: similarities to both plant and animals –cell wall with cellulose (like plant/fungus)

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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

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

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