Doctor of Philosophy, University of California Davis (2018)
Bachelor of Science, University of Oregon (2011)
Neuronal ER-plasma membrane junctions organized by Kv2-VAP pairing recruit Nir proteins and affect phosphoinositide homeostasis.
The Journal of biological chemistry
The association of plasma membrane (PM)-localized voltage-gated potassium (Kv2) channels with endoplasmic reticulum (ER)-localized vesicle-associated membrane protein-associated proteins VAPA and VAPB defines ER-PM junctions in mammalian brain neurons. Here, we used proteomics to identify proteins associated with Kv2/VAP-containing ER-PM junctions. We found that the VAP-interacting membrane-associated phosphatidylinositol (PtdIns) transfer proteins PYK2 N-terminal domain-interacting receptor 2 (Nir2) and Nir3 specifically associate with Kv2.1 complexes. When co-expressed with Kv2.1 and VAPA in HEK293T cells, Nir2 co-localized with cell-surface conducting and nonconducting Kv2.1 isoforms, enhanced by muscarinic-mediated PtdIns(4,5)P2 hydrolysis, leading to dynamic recruitment of Nir2 to Kv2.1 clusters. In cultured rat hippocampal neurons, exogenously expressed Nir2 did not strongly co-localize with Kv2.1, unless exogenous VAPA was also expressed, supporting the notion that VAPA mediates between Kv2.1 and Nir2. Immunolabeling signals of endogenous Kv2.1, Nir2, and VAP puncta were spatially correlated in cultured neurons. Fluorescence-recovery-after-photobleaching experiments revealed that Kv2.1, VAPA, and Nir2 have comparable turnover rates at ER-PM junctions, suggesting that they form complexes at these sites. Exogenous Kv2.1 expression in HEK293T cells resulted in significant differences in the kinetics of PtdIns(4,5)P2 recovery following repetitive muscarinic stimulation, with no apparent impact on resting PtdIns(4,5)P2 or PtdIns4P levels. Finally, the brains of Kv2.1-knockout mice had altered composition of PtdIns lipids, suggesting a crucial role for native Kv2.1-containing ER-PM junctions in regulating PtdIns lipid metabolism in brain neurons. These results suggest that ER-PM junctions formed by Kv2 channel-VAP pairing regulate PtdIns lipid homeostasis via VAP-associated PtdIns transfer proteins.
View details for DOI 10.1074/jbc.RA119.007635
View details for PubMedID 31594866
Linear Integration of ERK Activity Predominates over Persistence Detection in Fra-1 Regulation
2017; 5 (6): 549-+
ERK signaling regulates the expression of target genes, but it is unclear how ERK activity dynamics are interpreted. Here, we investigate this question using simultaneous, live, single-cell imaging of two ERK activity reporters and expression of Fra-1, a target gene controlling epithelial cell identity. We find that Fra-1 is expressed in proportion to the amplitude and duration of ERK activity. In contrast to previous "persistence detector" and "selective filter" models in which Fra-1 expression only occurs when ERK activity persists beyond a threshold duration, our observations demonstrate that the network regulating Fra-1 expression integrates total ERK activity and responds to it linearly. However, exploration of a generalized mathematical model of the Fra-1 coherent feedforward loop demonstrates that it can perform either linear integration or persistence detection, depending on the basal mRNA production rate and protein production delays. Our data indicate that significant basal expression and short delays cause Fra-1 to respond linearly to integrated ERK activity.
View details for DOI 10.1016/j.cels.2017.10.019
View details for Web of Science ID 000418800600007
View details for PubMedID 29199017
View details for PubMedCentralID PMC5746471
Single-Cell Imaging of ERK Signaling Using Fluorescent Biosensors
KINASE SIGNALING NETWORKS
2017; 1636: 35–59
Single-cell analysis of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) provides a means to perform highly detailed kinetic studies, assess heterogeneity between cells, and distinguish the subcellular localization of ERK activity. We describe here the methods needed to perform such measurements in a cell type of the investigator's choosing. We discuss the selection of appropriate reporters and provide detailed methods for stably introducing reporters, collecting live-cell data, and automatically extracting quantitative information from individual cells.
View details for DOI 10.1007/978-1-4939-7154-1_3
View details for Web of Science ID 000428499500004
View details for PubMedID 28730471