You are studying chemotaxis in two different strains of Dictyostelium discoidum.

Question

You are studying chemotaxis in two different strains of Dictyostelium discoidum. The first strain has a mutation in the solo gene (solo mutant), which encodes the G subunit of the heterotrimeric G-protein complex that is coupled to the cAMP receptor. The resulting protein product has normal GTPase activity, but it is unable to bind downstream targets. The second strain has a mutation in the clumpy gene (clumpy mutant), which encodes the G subunit of the same heterotrimeric G protein complex. However, the resulting mutation prevents G from interacting with G. This causes constitutively active G.

A) A single cell from each strain is placed on dish near a needle that flows cAMP. The cAMP that flows from the needle establishes a gradient with the highest concentration at the needle tip. Briefly describe how each mutant strain will respond to the cAMP gradient. Justify your answer in 1-2 sentences.

i) solo mutant strain:

ii) clumpy mutant strain:

B) A population of cells is evenly distributed onto a dish and starved. Under these conditions, wild-type D. discoidum will produce a short pulse of cAMP (via a pathway that is unaffected in either mutant strain). Wild- type ameoba will then aggregate to form a fruiting body. Predict how a population of cells from each strain will respond to starvation. Will a fruiting body form? Justify your answer in 1-2 sentences.

i) solo mutant strain:

ii) clumpy mutant strain:

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Explanation / Answer

A. The subunits of this G protein, once activated in response to cAMP binding, dissociate into and subunits that go off to activate yet more pathways. One result is an increase in the intracellular levels of cGMP. This cGMP appears to be necessary for eliciting a response from the cytoskeleton (actin & myosin, in this case), which eventually results in cellular movement.

i. The solo mutant strain which is mutated in binding the downstream signals will not respond to the cAMP signal as the movement of the amoeba is controlled by the downstream signals.

ii. The clumpy mutant strain will actively move towards the cAMP gradient as the GB is constitutively expressed assisting the movement of amoeba towards the chemoattractant.

B. Extracellular cAMP is the chemoattractant that attracts starving cells to one another to begin the developmental phase of the life cycle. As well as moving towards cAMP, cells that encounter cAMP also secrete cAMP, thereby amplifying the chemoattractant gradient. This relay system increases the distance over which starving cells can be recruited to an aggregate.

i. The solo mutant strain which is devoid of cellular movement will not aggregate to form fruiting bodies.

ii. The clumpy mutant strain which has constitutive BG activation will respond to starvation and form fruiting bodies.

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