VoltageFluor (VF) dyes have the potential to optically measure voltage in excitable membranes with the combination of high spatial and temporal resolution essential to better characterize the voltage dynamics of large groups of excitable cells. VF dyes sense voltage with high speed and sensitivity using photoinduced electron transfer (PeT) through a conjugated molecular wire. We show that tuning the driving force for PeT (ΔGPeT + w) through systematic chemical substitution modulates voltage sensitivity, estimate (ΔGPeT + w) values from experimentally measured redox potentials, and validate the voltage sensitivities in patch-clamped HEK cells for 10 new VF dyes. VF2.1(OMe).H, with a 48% ΔF/F per 100 mV, shows approximately 2-fold improvement over previous dyes in HEK cells, dissociated rat cortical neurons, and medicinal leech ganglia. Additionally, VF2.1(OMe).H faithfully reports pharmacological effects and circuit activity in mouse olfactory bulb slices, thus opening a wide range of previously inaccessible applications for voltage sensitive dyes.

Summary Coreceptor CD4 and CD8αβ double negative (DN) TCRαβ+ intraepithelial T cells, although numerous, have been greatly overlooked and their contribution to the immune response is not known. Here we used T cell receptor (TCR) sequencing of single cells combined with retrogenic expression of TCRs, to study the fate and the major histocompatibility complex (MHC) restriction of DN TCRαβ+ intraepithelial T cells. The data show that commitment of thymic precursors to the DN TCRαβ+ lineage is imprinted by their TCR specificity. Moreover, the TCRs they express display a diverse and unusual pattern of MHC restriction that is non-overlapping with that of CD4+ or CD8αβ+ T cells, indicating that they sense antigens that are not recognized by the conventional T cell subsets. The new insights indicate that DN TCRαβ+ T cells form a third lineage of TCRαβ T lymphocytes expressing a variable TCR repertoire, which serve non-redundant immune functions.

Electron microscopy (EM) has long been the main technique to image cell structures with nanometer resolution, but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce “Click-EM,” a labeling technique for correlative light microscopy and EM imaging of non-protein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal “click chemistry” ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of Click-EM in imaging metabolically tagged DNA, RNA, and lipids in cultured cells and neurons, and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes.

Synthesizing, localizing, and stabilizing new protein copies at synapses are crucial factors in maintaining the synaptic changes required for storing long-term memories. PKM recently emerged as a molecule putatively responsible for maintaining encoded memories over time because its presence correlates with late LTP and because its inhibition disrupts LTP in vitro and long-term memory storage in vivo.Here we investigated PKM stability in rat neurons to better understand its role during information encoding and storage. We used TimeSTAMP reporters to track the synthesis and degradation of PKM as well as a related atypical PKC, PKC. These reporters revealed that both PKM and PKC were upregulated after chemical LTP induction; however, these new PKM copies exhibited more rapid turnover than basally produced PKM, particularly in dendritic spines. In contrast to PKM, new PKC copies exhibited elevated stability. Stable information storage over long periods of time is more challenging the shorter the metabolic lifetime of the candidate molecules.

T lymphocytes develop in the thymus into two major lineages characterized by expression of ab and gd TCR. Each lineage can be further subdivided into distinct subsets that differ in TCR specificity, phenotype and function. CD8aa TCRab intraepithelial lymphocytes (T-IEL) located in the epithelium of the small intestine is phenotypically different from conventional T cells. CD8aa T-IEL have been studied for years however the question of their MHC restriction has not been elucidated. Therefore, we decided to clone TCR isolated from naturally arising CD8aa T-IEL and retrovirally express these TCRs in BM chimera. First, we cloned four TCRab that were expressed retrovirally in RagKO BM chimera. Analysis of the chimera showed that all TCR clones gave rise to T cells. The T cell that developed were CD4-CD8b- but CD8a+ and phenotypically identical to CD8aa T-IEL isolated from wild type mice. In addition, they were preferentially found in the gut. In order to define the MHC restriction of these particular TCRs, BM chimeras in various MHC-deficient backgrounds were generated. Subsequent analysis of the chimera for the presence of CD8aaTCRab IEL demonstrated that they are dependent either on Kb or Db MHC I or b2m-dependent MHC I aside from KbDb. In conclusion, our results indicate that CD8aa T-IEL develops from precursors expressing particular TCRs that allow their engagement in this unique T cell lineage. In addition, CD8aa T-IEL can harbor clones selected on different MHC molecules.

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