To study the function of the β‑spectrin N-terminus, the researchers engineered versions of β‑spectrin without the N-terminus and called this βspec^ΔN-WT. The researchers used this construct as well as full-length, or wild-type, β‑spectrin (βspec^WT) to generate genetically engineered fruit flies. When they compared flies that express βspec^WT and flies without the N-terminus (βspec^ΔN-WT), they noticed that βspec^ΔN-WT flies had lower survival rates. This proved that the N-terminus is not redundant, but important to the normal function of β‑spectrin and the survival of fruit flies.

To understand the effect of SCA5 mutations on β‑spectrin function, the researchers generated flies that expressed full-length β‑spectrin containing SCA5 mutations (βspec^SCA5) and flies that expressed β‑spectrin containing SCA5 mutations without the N-terminal (βspec^ΔN-SCA5). Since β‑spectrin is important for maintaining cell shape, the researchers next looked at the shapes of neurons for the different versions of β‑spectrin. In flies that had the SCA5 mutation (βspec^SCA5), neurons had fewer branches, and these branches were shorter. This means that the neurons were not formed properly. This could indicate that they make fewer connections with other neurons and therefore have a lower ability to receive signals from others. However, when the N-terminus was removed (βspec^ΔN-SCA5), the neurons appeared normal despite having the SCA5 mutations. This showed that the N-terminus is required for the SCA5 mutation to cause alternations in neuron branching: and thus makes the N-terminus an accomplice.

Next, the researchers explored why removing the N-terminus restored normal cell shape. They first checked if neurons are producing different amounts of the β‑spectrin protein. Indeed, compared to normal flies (βspec^WT), SCA5 flies (βspec^SCA5) expressed three times more β‑spectrin protein. This indicates that in SCA5, an abnormal amount of β‑spectrin accumulates in neurons. However, if the N-terminus is removed (βspec^ΔN-SCA5), the β‑spectrin protein level is similar to wild-type flies (βspec^WT). This means that the N-terminus is involved in increasing the levels of the mutated β‑spectrin protein: again, the N-terminus is an accomplice in SCA5 toxicity.

An important function of β‑spectrin is to bind to actin, another structural protein in cells. The researchers hypothesized that the N-terminus participates in this interaction. To test this, they compared wild-type flies (βspec^WT) and SCA5 flies (βspec^SCA5) and found that βspec^SCA5 showed abnormally strong binding to actin. This was abolished in SCA5 flies without the N-terminus (βspec^ΔN-SCA5). This suggests that the N-terminus is involved in binding actin and is necessary for the abnormal actin binding in SCA5.