A role for Numb in Protein kinase M (PKM)‐mediated increase in surface AMPA receptors during facilitation in Aplysia

Abstract

There is considerable evidence from both vertebrates and invertebrates that persistently active protein kinases maintain changes in synaptic strength that underlie memory. In the hermaphrodite marine mollusk, Aplysia californica, truncated forms of protein kinase C (PKC) termed protein kinase Ms have been implicated in both intermediate‐ and long‐term facilitation, an increase in synaptic strength between sensory neurons and motor neurons thought to underlie behavioural sensitization in the animal. However, few substrates have been identified as candidates that could mediate this increase in synaptic strength. PKMs have been proposed to maintain synaptic strength through preventing endocytosis of AMPA receptors. Numb is a conserved regulator of endocytosis that is modulated by phosphorylation. We have identified and cloned Aplysia Numb (ApNumb). ApNumb contains three conserved PKC phosphorylation sites and PKMs generated from classical and atypical Aplysia PKCs can phosphorylate ApNumb in vitro and in cells. Over‐expression of ApNumb that lacks the conserved PKC phosphorylation sites blocks increases in surface levels of a pHluorin‐tagged Aplysia glutamate receptor measured using live imaging after intermediate‐ or long‐term facilitation. Over‐expression of this form of ApNumb did not block increases in synaptic strength seen during intermediate‐term facilitation, but did block increases in synaptic strength seen during long‐term facilitation. There was no effect of over‐expression of this form of ApNumb on other putative Numb targets as measured using increases in calcium downstream of neurotrophins or agonists of metabotropic glutamate receptors. These results suggest that in Aplysia neurons, Numb specifically regulates AMPA receptor trafficking and is an attractive candidate for a target of PKMs in long‐term maintenance of synaptic strength. Open Science Badges This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/

Excitatory postsynaptic calcium transients at Aplysia sensory-motor neuron synapses allow for quantal examination of synaptic strength over multiple days in culture

“…We limited GCaMP6s to membranes by fusing it to the Aplysia Src N-terminal that contains myristylation and palmitylation signals (src-GCamP6s), similar to previous membrane delimited versions of GCaMP (Shigetomi et al 2010; Akbergenova et al 2018). We believe this approach is better than using a transmembrane domain due to the lack of a requirement for passage through the secretory system, often leading to accumulation in the ER, as we have observed for other tagged transmembrane proteins expressed in Aplysia sensory neurons (Nagakura et al 2008; Farah et al 2019).…”

“…The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.10.434766 doi: bioRxiv preprint for other tagged transmembrane proteins expressed in Aplysia sensory neurons (Nagakura et al 2008;Farah et al 2019).…”

“…The generation of pNEXp-APsrc-gCAMP6s (Src-GCaMP6s) with the firsts 34 amino acids of Aplysia Src (NP_001191639.1) inserted in front of GCaMP6s was previously described (Farah et al 2019). The first 34 amino acids of Aplysia Src contains conserved myristoylation and palmitylation sites that lead to membrane insertion.…”

Excitatory postsynaptic calcium transients at Aplysia sensory–motor neuron synapses allow for quantal examination of synaptic strength over multiple days in culture

“…We limited GCaMP6s to membranes by fusing it to the Aplysia Src amino terminal that contains myristylation and palmitylation signals (src-GCamP6s), similar to previous membrane-delimited versions of GCaMP ( Shigetomi et al 2010 ; Akbergenova et al 2018 ). We believe this approach is better than using a transmembrane domain because of the lack of a requirement for passage through the secretory system, often leading to accumulation in the endoplasmic reticulum (ER), as we have observed for other tagged transmembrane proteins expressed in Aplysia sensory neurons ( Nagakura et al 2008 ; Farah et al 2019 ).…”

“…The generation of pNEXp-APsrc-gCAMP6s (Src-GCaMP6s) with the first 34 amino acids of Aplysia Src (NP_001191639.1) inserted in front of GCaMP6s was previously described ( Farah et al 2019 ). The first 34 amino acids of Aplysia Src contain conserved myristoylation and palmitylation sites that lead to membrane insertion.…”