While the use of embryonic mixed primary cerebellar cultures has proven valuable for dissecting structure: function relationships in Purkinje Neurons (PNs), this technique is technically challenging and often yields few cells. Recently, mouse embryonic stem cells (mESCs) have been successfully differentiated into PNs, although the available methods are very challenging as well. The focus of this study was to simplify the differentiation of mESCs into PNs. Using specific extrinsic factors, we successfully differentiated mESCs into PNs without the use of a postnatal feeder-layer. The morphology of mESC-derived PNs is indistinguishable from PNs grown in primary culture in terms of gross morphology, spine length and spine density. Furthermore, mESC-derived PNs express Calbindin D28K, IP3R1, PLCb4 and GRID2, all of which are PN-specific markers. Finally, using collagen, poly-l-lysine and gelatin as the extra cellular matrix allowed us to grow mESC-derived PNs in monolayers, which is crucial for live cell imaging. Current efforts are focused on expressing exogenous DNAs specifically in mESC-derived PNs using the PCP2/L7 PN-specific promoter. If this is successful, we will then attempt gene editing in stem cells using CRISPR, followed by complementation using exogenous DNA. Together, this technology would provide a scalable, high-throughput method for dissecting specific molecular mechanisms in PNs, especially when a KO mouse is not available. Myosin Va, a class V processive motor, transports tubules of smooth endoplasmic reticulum (SER) into the dendritic spines of cerebellar Purkinje neurons (PNs). These SER tubules provide the source of calcium downstream of mGluR1 activation that drives synaptic plasticity at parallel fiber: PN synapses. Calcium release from SER tubules is mediated by the IP3 receptor (type 1 inositol 1,4,5-trisphosphate receptor or IP3R1), a resident SER protein. The focus of this study was to identify the receptor on the surface of the SER for myosin Va. A yeast two-hybrid screen and immunoprecipitation analyses identified two potential receptor components that interact with both myosin Va and IP3R1: phospholipase C beta 4 (PLCβ4) and receptor-for-activated-kinase 1 (RACK1). Initial work has shown that both PLCβ4 and RACK1 are enriched in dendritic spines, and live cell imaging has shown that RACK1, like myosin Va, localizes at the tip of SER tubules that are moving into spines. Furthermore, the dendritic spines of PNs from IP3R1-null mice are devoid of SER, arguing that IP3R1 is required for the myosin Va-dependent translocation of SER into spines. Finally, the enrichment of PLCβ4 and RACK1 in dendritic spines is disrupted in both myosin Va knockdown cells and IP3R1-null cells. Together, these data suggest that myosin Va may be recruited to the SER via the IP3 receptor, with PLCβ4 and/or RACK1 providing a bridge linking the myosin to the receptor. Myosin Va, a processive class V motor, is involved in the transport of diverse cargos, including melanosomes, endoplasmic reticulum (ER), and mRNAs. The creation of tools to identify proteins that interact with myosin Va should increase our understanding of the cellular processes supported by this myosin. Towards that end, we report here the generation of a tandem affinity purification (TAP) tag knockin mouse at the MYO5A locus. A recombineering-based approach was used to insert via homologous recombination a TAP-tag composed of the IgG binding domain of Protein A, a TEV cleavage site, and the FLAG epitope tag into MYO5A locus immediately after the initiation codon. Mice homozygous for the knockin allele, which express the TAP-tagged version of myosin Va (TAP-MyoVa) exclusively and under the control of the endogenous MYO5A promoter, exhibit normal coat color and no evidence of ataxia, arguing that TAP-MyoVa functions normally. Consistently, the dendritic spines of Purkinje neurons isolated from this mouse are fully loaded with ER, in contrast to the spines of Purkinje neurons from dilute (myosin Va null) mice, which are devoid of ER. Similarly, melanosomes are distributed normally in melanocytes from the TAP-tagged myosin Va mouse, in contrast to melanocytes from dilute mice, where the organelles are concentrated in the cell center. Moreover, introduction of a CMV promoter-driven TAP-Tag myosin Va construct into dilute melanocytes rescues melanosome distribution. Given this clear evidence that TAP-MyoVa is fully functional, we purified TAP-MyoVa and associated proteins directly from juvenile mouse cerebella excised from TAP-tagged mice and subjected the samples to mass spectroscopic analyses. Importantly, elutes contained several known myosin Va binding partners (list), further verifying that TAP-MyoVa is fully functional. Moreover, we found numerous novel interacting proteins. The mouse model created here should facilitate the identification of novel myosin Va binding partners, which in turn should advance our understanding of the roles played by this myosin in vivo.
| Wagner, Wolfgang; Brenowitz, Stephan D; Hammer 3rd, John A (2011) Myosin-Va transports the endoplasmic reticulum into the dendritic spines of Purkinje neurons. Nat Cell Biol 13:40-8 |
| Wagner, Wolfgang; McCroskery, Seumas; Hammer 3rd, John A (2011) An efficient method for the long-term and specific expression of exogenous cDNAs in cultured Purkinje neurons. J Neurosci Methods 200:95-105 |
