Junctions between cortical endoplasmic reticulum (cER) and the plasma membrane are a subtle but ubiquitous feature in mammalian cells; however, very little is known about the functions and molecular interactions that are associated with neuronal ER-plasma-membrane junctions. Here, we report that Kv2

To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding the linear HA epitope with high affinity and specificity in vivo. The resulting probe, called the HA frankenbody, can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA-tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful tool for imaging protein dynamics in vivo.

Kv2.1 surface clusters in transfected HEK cells and hippocampal neurons are shown to be trafficking platforms involved in potassium channel movement to and from the cell surface. This work is the first to define stable cell surface sites for ion channel delivery and retrieval at the cell surface.

The Kv2.1 voltage-gated K+ channel is found both freely diffusing over the plasma membrane and concentrated in micron-sized clusters localized to the soma, proximal dendrites and axon initial segment of hippocampal neurons. In transfected HEK cells, Kv2.1 channels within cluster microdomains are non-conducting. Using TIRF microscopy the number of GFP-tagged Kv2.1 channels on the HEK cell surface was compared to K+ channel conductance measured by whole-cell voltage-clamp of the same cell. This approach indicated that as channel density increases non-clustered channels cease conducting. At the highest density observed, only 4% of all channels were conducting. Mutant Kv2.1 channels that fail to cluster also possessed the non-conducting state with 17% conducting K+ at higher surface densities. The non-conducting state was specific to Kv2.1 as Kv1.4 was always conducting regardless of the cell-surface expression level. Anti-Kv2.1 immuno-fluorescence intensity, standardized to Kv2.1 surface density in transfected HEK cells, was used to determine the expression levels of endogenous Kv2.1 in cultured rat hippocampal neurons. Endogenous Kv2.1 levels were compared to the number of conducting channels determined by whole-cell voltage clamp. Only 13 and 27% of the endogenous Kv2.1 was conducting in neurons cultured for 14 and 20 days, respectively. Together these data indicate that the non-conducting state depends primarily on surface density as opposed to cluster location and that this non-conducting state also exists for native Kv2.1 found in cultured hippocampal neurons. This excess of Kv2.1 protein relative to K+ conductance further supports a non-conducting role for Kv2.1 in excitable tissues.

Cereal Chem. 83(3):255-258A negative relationship between dough strength and dough extensibility would pose a problem for breeding hard wheats, as both dough strength and dough extensibility are desirable. We derived 77 recombinant inbred lines (RIL) from a cross between hard red spring wheat cultivars McNeal and Thatcher. McNeal produces flour with stronger dough and lower extensibility than does Thatcher. RIL were evaluated for strength-related properties using mixograph analysis and extensibility parameters using the Kieffer attachment to the TA.XT2 texture analyzer. Additionally, the RIL were test baked. Measurements using the mixograph and the Kieffer attachment were highly heritable. Maximum dough extensibility (Ext max ) was negatively correlated with resistance to extension (R max ) (r = -0.74) and with mixograph tolerance (r = -0.45). Loaf volume was correlated with both R max (r = 0.42) and area under the extensigraph curve (r = 0.44) based on partial correlation analysis adjusted for protein differences. Ext max was negatively correlated with loaf volume (r = -0.26). The McNeal allele for polymorphism at the Gli1-B1 locus on chromosome 1BS caused high dough-mixing tolerance and low dough extensibility. Our results suggest that traditional selection criteria in hard red spring wheat, including tolerance to dough mixing and high loaf volume, may result in reduced dough extensibility.