Bachelor of Science, California State University, Chico (2012)
Doctor of Philosophy, University of California Davis (2018)
Richard Lewis, Postdoctoral Faculty Sponsor
Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in mammalian brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.
View details for DOI 10.7554/eLife.48750
View details for PubMedID 31566565
The voltage-gated K+ channel Kv2.1 serves a major structural role in the soma and proximal dendrites of mammalian brain neurons, tethering the plasma membrane (PM) to endoplasmic reticulum (ER). Although Kv2.1 clustering at neuronal ER-PM junctions (EPJs) is tightly regulated and highly conserved, its function remains unclear. By identifying and evaluating proteins in close spatial proximity to Kv2.1-containing EPJs, we discovered that a significant role of Kv2.1 at EPJs is to promote the clustering and functional coupling of PM L-type Ca2+ channels (LTCCs) to ryanodine receptor (RyR) ER Ca2+ release channels. Kv2.1 clustering also unexpectedly enhanced LTCC opening at polarized membrane potentials. This enabled Kv2.1-LTCC-RyR triads to generate localized Ca2+ release events (i.e., Ca2+ sparks) independently of action potentials. Together, these findings uncover a novel mode of LTCC regulation and establish a unique mechanism whereby Kv2.1-associated EPJs provide a molecular platform for localized somatodendritic Ca2+ signals in mammalian brain neurons.
View details for DOI 10.7554/eLife.49953
View details for PubMedID 31663850
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
The endoplasmic reticulum (ER) and plasma membrane (PM) form junctions crucial to ion and lipid signaling and homeostasis. The Kv2.1 ion channel is localized at ER-PM junctions in brain neurons and is unique among PM proteins in its ability to remodel these specialized membrane contact sites. Here, we show that this function is conserved between Kv2.1 and Kv2.2, which differ in their biophysical properties, modulation, and cellular expression. Kv2.2 ER-PM junctions are present at sites deficient in the actin cytoskeleton, and disruption of the actin cytoskeleton affects their spatial organization. Kv2.2-containing ER-PM junctions overlap with those formed by canonical ER-PM tethers. The ability of Kv2 channels to remodel ER-PM junctions is unchanged by point mutations that eliminate their ion conduction but eliminated by point mutations within the Kv2-specific proximal restriction and clustering (PRC) domain that do not impact their ion channel function. The highly conserved PRC domain is sufficient to transfer the ER-PM junction-remodeling function to another PM protein. Last, brain neurons in Kv2 double-knockout mice have altered ER-PM junctions. Together, these findings demonstrate a conserved in vivo function for Kv2 family members in remodeling neuronal ER-PM junctions that is distinct from their canonical role as ion-conducting channels shaping neuronal excitability.
View details for DOI 10.1091/mbc.E18-05-0337
View details for PubMedID 30091655
View details for PubMedCentralID PMC6233057
Membrane contacts between endoplasmic reticulum (ER) and plasma membrane (PM), or ER-PM junctions, are ubiquitous in eukaryotic cells and are platforms for lipid and calcium signaling and homeostasis. Recent studies have revealed proteins crucial to the formation and function of ER-PM junctions in non-neuronal cells, but little is known of the ER-PM junctions prominent in aspiny regions of mammalian brain neurons. The Kv2.1 voltage-gated potassium channel is abundantly clustered at ER-PM junctions in brain neurons and is the first PM protein that functions to organize ER-PM junctions. However, the molecular mechanism whereby Kv2.1 localizes to and remodels these junctions is unknown. We used affinity immunopurification and mass spectrometry-based proteomics on brain samples from male and female WT and Kv2.1 KO mice and identified the resident ER vesicle-associated membrane protein-associated proteins isoforms A and B (VAPA and VAPB) as prominent Kv2.1-associated proteins. Coexpression with Kv2.1 or its paralog Kv2.2 was sufficient to recruit VAPs to ER-PM junctions. Multiplex immunolabeling revealed colocalization of Kv2.1 and Kv2.2 with endogenous VAPs at ER-PM junctions in brain neurons from male and female mice in situ and in cultured rat hippocampal neurons, and KO of VAPA in mammalian cells reduces Kv2.1 clustering. The association of VAPA with Kv2.1 relies on a "two phenylalanines in an acidic tract" (FFAT) binding domain on VAPA and a noncanonical phosphorylation-dependent FFAT motif comprising the Kv2-specific clustering or PRC motif. These results suggest that Kv2.1 localizes to and organizes neuronal ER-PM junctions through an interaction with VAPs.SIGNIFICANCE STATEMENT Our study identified the endoplasmic reticulum (ER) proteins vesicle-associated membrane protein-associated proteins isoforms A and B (VAPA and VAPB) as proteins copurifying with the plasma membrane (PM) Kv2.1 ion channel. We found that expression of Kv2.1 recruits VAPs to ER-PM junctions, specialized membrane contact sites crucial to distinct aspects of cell function. We found endogenous VAPs at Kv2.1-mediated ER-PM junctions in brain neurons and other mammalian cells and that knocking out VAPA expression disrupts Kv2.1 clustering. We identified domains of VAPs and Kv2.1 necessary and sufficient for their association at ER-PM junctions. Our study suggests that Kv2.1 expression in the PM can affect ER-PM junctions via its phosphorylation-dependent association to ER-localized VAPA and VAPB.
View details for DOI 10.1523/JNEUROSCI.0893-18.2018
View details for PubMedID 30012696
View details for PubMedCentralID PMC6113906
Voltage-gated K+ (Kv) channels play important roles in regulating neuronal excitability. Kv channels comprise four principal ? subunits, and transmembrane and/or cytoplasmic auxiliary subunits that modify diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Kv2.1-containing Kv channels. We show that AMIGO-1 is extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent population of AMIGO-1 outside of that colocalized with these Kv2 ? subunits. AMIGO-1 is coclustered with Kv2 ? subunits at specific plasma membrane (PM) sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This distinct PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 ? subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 ? subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 ? subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is exclusively associated with Kv2 ? subunits, and that Kv2 ? subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering.
View details for DOI 10.3389/fnmol.2018.00001
View details for PubMedID 29403353
View details for PubMedCentralID PMC5780429