Electrical signals in the ER are cell type and stimulus specific with extreme spatial compartmentalization in neurons.

The endoplasmic reticulum (ER) is a tortuous organelle that spans throughout a cell with a continuous membrane containing ion channels, pumps, and transporters. It is unclear if stimuli that gate ER ion channels trigger substantial membrane potential fluctuations and if those fluctuations spread beyond their site of origin. Here, we visualize ER membrane potential dynamics in HEK cells and cultured rat hippocampal neurons by targeting a genetically encoded voltage indicator specifically to the ER membrane. We report the existence of clear cell-type- and stimulus-specific ER membrane potential fluctuations. In neurons, direct stimulation of ER ryanodine receptors generates depolarizations that scale linearly with stimulus strength and reach tens of millivolts. However, ER potentials do not spread beyond the site of receptor activation, exhibiting steep attenuation that is exacerbated by intracellular large conductance K+ channels. Thus, segments of ER can generate large depolarizations that are actively restricted from impacting nearby, contiguous membrane.

All-Optical Electrophysiology in hiPSC-Derived Neurons With Synthetic Voltage Sensors.

Voltage imaging and "all-optical electrophysiology" in human induced pluripotent stem cell (hiPSC)-derived neurons have opened unprecedented opportunities for high-throughput phenotyping of activity in neurons possessing unique genetic backgrounds of individual patients. While prior all-optical electrophysiology studies relied on genetically encoded voltage indicators, here, we demonstrate an alternative protocol using a synthetic voltage sensor and genetically encoded optogenetic actuator that generate robust and reproducible results. We demonstrate the functionality of this method by measuring spontaneous and evoked activity in three independent hiPSC-derived neuronal cell lines with distinct genetic backgrounds.

Foxn4 promotes gene expression required for the formation of multiple motile cilia.

Multiciliated cell (MCC) differentiation involves extensive organelle biogenesis required to extend hundreds of motile cilia. Key transcriptional regulators known to drive the gene expression required for this organelle biogenesis are activated by the related coiled-coil proteins Multicilin and Gemc1. Here we identify foxn4 as a new downstream target of Multicilin required for MCC differentiation in Xenopus skin. When Foxn4 activity is inhibited in Xenopus embryos, MCCs show transient ciliogenesis defects similar to those seen in mutants of Foxj1, a known key regulator of genes required for motile ciliation. RNAseq analysis indicates that Foxn4 co-activates some Foxj1 target genes strongly and many Foxj1 targets weakly. ChIPseq suggests that whereas Foxn4 and Foxj1 frequently bind to different targets at distal enhancers, they largely bind together at MCC gene promoters. Consistent with this co-regulation, cilia extension by MCCs is more severely compromised in foxn4 and foxj1 double mutants than in single mutants. In contrast to Foxj1, Foxn4 is not required to extend a single motile cilium by cells involved in left-right patterning. These results indicate that Foxn4 complements Foxj1 transcriptionally during MCC differentiation, thereby shaping the levels of gene expression required for the timely and complete biogenesis of multiple motile cilia.