Characterization of neuronal synaptopodin reveals a myosin V-dependent mechanism of synaptopodin clustering at the post-synaptic sites

Abstract

STATEMENTHere we demonstrate that the spine apparatus is assembled locally in dendrites. This process, which relies on the presence of synaptopodin and F-actin, is disrupted by interfering with myosin-V activity. ABSTRACTThe spine apparatus (SA) is an endoplasmic reticulum-related organelle which is present in a subset of dendritic spines in cortical and pyramidal neurons. The synaptopodin protein localizes between the stacks of the spine apparatus and is essential for the formation of this unique organelle. Although several studies have demonstrated the significance of the SA and synaptopodin in calcium homeostasis and plasticity of dendritic spines, it is still unclear what factors contribute to its stability at the synapse and whether the SA is locally formed or it is actively delivered to the spines. In this study we show that synaptopodin clusters are stable at their locations. We found no evidence of active microtubule-based transport for synaptopodin. Instead new clusters were emerging in the spines, which we interpret as the SA being assembled on-site.Furthermore, using super-resolution microscopy we show a tight association of synaptopodin with actin filaments. We identify the actin-based motor proteins myosin V and VI as novel interaction partners of synaptopodin and demonstrate that myosin V is important for the formation and/or maintenance of the SA.

F‐actin patches associated with glutamatergic synapses control positioning of dendritic lysosomes

“…To test this hypothesis, we employed a dominant negative approach to inhibit myosins V and VI. Overexpression of a dimerized tail construct of myosin Va (myoV DN), containing the cargo‐binding domain, is frequently used to impair the function of endogenous myosin Va by competition (Correia et al , ; Balasanyan & Arnold, ; preprint: González‐Gallego et al , ). Live imaging experiments of DIV16‐17 neurons overexpressing mCerulean‐labeled myoV DN showed visible associations between the myoV DN tail and LAMP1‐positive vesicles, suggesting that lysosomes associate with myosin V motors in living cells (Fig D).…”

“…Next, we asked whether myosin VI might also be involved in the stalling of lysosomes in dendrites. To test this, we used a dominant negative construct, analogous to myoV DN, consisting of the C‐terminal cargo‐binding domain of myosin VI (myoVI DN) fused to GFP (Aschenbrenner et al , ; preprint: González‐Gallego et al , ). Kymograph analysis of lysosomal motility in myoVI DN expressing neurons showed only a minimal change in pausing time distribution (Fig L) while all other analyzed parameters were unaffected (Fig I–K; Appendix Fig S3C and D).…”

Endoplasmic reticulum visits highly active spines and prevents runaway potentiation of synapses

“…To generate MyoV DN, a sequence encoding the GTD of mouse MYO5A (starting at residue 1415, numbering according to brain-spliced isoform) was inserted in frame at the 3′-end of the leucine zipper of mCer-LZ. Plasmid mCer-LZ was generated by inserting a sequence encoding the leucine zipper of GCN4 (MKQLEDKVEELLSK NYHLENEVARLKKLVGE) in frame at the 3′-end of the mCerulean coding sequence 53 . When assessing the effect of MyoV DN, mCer-LZ was used in control cells in substitution of MyoV DN to maintain an identical DNA concentration in electroporation mixes and for post-hoc identification.…”

F-actin patches associated with glutamatergic synapses control positioning of dendritic lysosomes

“…Next, we asked whether myosin VI might also be involved in the stalling of lysosomes in dendrites. To test this, we used a dominant negative construct, analogous to myoV DN, consisting of the C-terminal cargo binding domain of myosin VI (myoVI DN) fused to GFP (González-Gallego et al, 2019). Kymograph analysis of lysosomal motility in myoVI DN expressing neurons showed only a minimal change in pausing time distribution while all other analyzed parameters were unaffected ( Figure 7I-K; S3C, D).…”