Functional analysis of Drosophila Kekkon receptors at the neuromuscular junction of Drosophila

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Hasenauer, Jacob ORCID: https://orcid.org/0000-0002-1716-5309 (2024). Functional analysis of Drosophila Kekkon receptors at the neuromuscular junction of Drosophila. University of Birmingham. Ph.D.

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Abstract

Neurotrophin ligands signalling via Trk receptors are key regulators of activity-dependent structural plasticity and neuronal function in vertebrates. In Drosophila, conserved neurotrophins (DNTs) are encoded by the spaetzle genes. The six Kekkon receptors
(Keks) are the fly homologs of the Trk family. DNT signalling can also be mediated by Toll receptors. kek6 can regulate synaptic plasticity in Drosophila by increasing Ca2+ levels and activating CaMKII. However, it was unknown if the other five Keks function similarly. Here,
I investigated the Keks by asking: where are they expressed in the nervous system? Can they all regulate calcium? And what their impact is in activity-dependent structural plasticity?

Using RT-PCR I showed that all keks were expressed in the central nervous system (CNS), with only kek1 and kek5 being expressed in the periphery. I visualised the distribution of overexpressed Kek-RFP protein fusions and endogenous CRISPR/Cas9 knock-ins showing that Keks had overlapping subcellular localisations, with Kek2 being dendritic, Kek5 and -6 being presynaptic, and Kek3 and -4 localising throughout the neuron. Using Western blot I demonstrated that kek overexpression altered the abundance of CaMKII, with kek3 differentially regulating alternate CaMKII isoforms. Using live Ca2+ imaging, I demonstrated that overexpression of all keks, except kek5, sustained Ca2+ levels following stimulation, with kek2 and -4 also lowering the amplitude of Ca2+ influx. Focusing on kek4 and 6, I show with endogenously tagged protein fusions that Kek6 colocalised with the voltage-gated Ca2+ channel Cacophony, and that kek4 and 6 overexpression led to the clustering of Cacophony channels. Using optogenetics to induce activity-dependent plasticity, I demonstrated that kek4 and 6 were required for the activity-dependent synaptic growth following activity, with immunofluorescent stainings of synaptic markers Bruchpilot(Brp) and Discs-large (Dlg) at neuromuscular junction (NMJ) 6/7.

Altogether, these data have revealed that Keks have distinct but overlapping spatial distributions and functional properties in the nervous system. kek4 and -6 can alter Cacophony distribution and may physically associate with active zones, indicating that Keks may act via conserved Ca2+-dependent pathways to regulate activity-dependent synaptic plasticity. This work highlights the Kek receptor family as an emerging class of
regulators of synaptic plasticity and neuronal function in Drosophila.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):