Nicotine dependence is thought to arise in part because nicotine permeates into the endoplasmic reticulum (ER), where it binds to nicotinic receptors (nAChRs) and begins an "inside-out" pathway that leads to up-regulation of nAChRs on the plasma membrane. However, the dynamics of nicotine entry into the ER are unquantified. Here, we develop a family of genetically encoded fluorescent biosensors for nicotine, termed iNicSnFRs. The iNicSnFRs are fusions between two proteins: a circularly permutated GFP and a periplasmic choline-/betaine-binding protein engineered to bind nicotine. The biosensors iNicSnFR3a and iNicSnFR3b respond to nicotine by increasing fluorescence at [nicotine] <1 µM, the concentration in the plasma and cerebrospinal fluid of a smoker. We target iNicSnFR3 biosensors either to the plasma membrane or to the ER and measure nicotine kinetics in HeLa, SH-SY5Y, N2a, and HEK293 cell lines, as well as mouse hippocampal neurons and human stem cell-derived dopaminergic neurons. In all cell types, we find that nicotine equilibrates in the ER within 10 s (possibly within 1 s) of extracellular application and leaves as rapidly after removal from the extracellular solution. The [nicotine] in the ER is within twofold of the extracellular value. We use these data to run combined pharmacokinetic and pharmacodynamic simulations of human smoking. In the ER, the inside-out pathway begins when nicotine becomes a stabilizing pharmacological chaperone for some nAChR subtypes, even at concentrations as low as ∼10 nM. Such concentrations would persist during the 12 h of a typical smoker's day, continually activating the inside-out pathway by >75%. Reducing nicotine intake by 10-fold decreases activation to ∼20%. iNicSnFR3a and iNicSnFR3b also sense the smoking cessation drug varenicline, revealing that varenicline also permeates into the ER within seconds. Our iNicSnFRs enable optical subcellular pharmacokinetics for nicotine and varenicline during an early event in the inside-out pathway.

Nicotine dependence is thought to arise in part because nicotine permeates into the endoplasmic reticulum (ER), where it binds to nicotinic receptors (nAChRs) and begins an "inside-out" pathway that leads to up-regulation of nAChRs on the plasma membrane. However, the dynamics of nicotine entry into the ER are unquantified. Here, we develop a family of genetically encoded fluorescent biosensors for nicotine, termed iNicSnFRs. The iNicSnFRs are fusions between two proteins: a circularly permutated GFP and a periplasmic choline-/betaine-binding protein engineered to bind nicotine. The biosensors iNicSnFR3a and iNicSnFR3b respond to nicotine by increasing fluorescence at [nicotine] <1 µM, the concentration in the plasma and cerebrospinal fluid of a smoker. We target iNicSnFR3 biosensors either to the plasma membrane or to the ER and measure nicotine kinetics in HeLa, SH-SY5Y, N2a, and HEK293 cell lines, as well as mouse hippocampal neurons and human stem cell-derived dopaminergic neurons. In all cell types, we find that nicotine equilibrates in the ER within 10 s (possibly within 1 s) of extracellular application and leaves as rapidly after removal from the extracellular solution. The [nicotine] in the ER is within twofold of the extracellular value. We use these data to run combined pharmacokinetic and pharmacodynamic simulations of human smoking. In the ER, the inside-out pathway begins when nicotine becomes a stabilizing pharmacological chaperone for some nAChR subtypes, even at concentrations as low as ∼10 nM. Such concentrations would persist during the 12 h of a typical smoker's day, continually activating the inside-out pathway by >75%. Reducing nicotine intake by 10-fold decreases activation to ∼20%. iNicSnFR3a and iNicSnFR3b also sense the smoking cessation drug varenicline, revealing that varenicline also permeates into the ER within seconds. Our iNicSnFRs enable optical subcellular pharmacokinetics for nicotine and varenicline during an early event in the inside-out pathway.

The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency.

In conventional biological imaging, diffraction places a limit on the minimal xy distance at which two marked objects can be discerned. Consequently, resolution of target molecules within cells is typically coarser by two orders of magnitude than the molecular scale at which the proteins are spatially distributed. Photoactivated localization microscopy (PALM) optically resolves selected subsets of protect fluorescent probes within cells at mean separations of <25 nanometers. It involves serial photoactivation and subsequent photobleaching of numerous sparse subsets of photoactivated fluorescent protein molecules. Individual molecules are localized at near molecular resolution by determining their centers of fluorescent emission via a statistical fit of their point-spread-function. The position information from all subsets is then assembled into a super-resolution image, in which individual fluorescent molecules are isolated at high molecular densities. In this paper, some of the limitations for PALM imaging under current experimental conditions are discussed.

Recent advances in super-resolution microscopy have pushed the resolution limit of light microscopy closer to that of electron microscopy. However, as they invariably rely on fluorescence, light microscopy techniques only visualize whatever gets labeled. On the other hand, while electron microscopy reveals cellular structures at the highest resolution, it offers no specificity. The information gap between the two imaging modalities can only be bridged by correlative light and electron microscopy (CLEM). Previously we have developed a probe (mEos4) whose fluorescence and photoconversion survive 0.5-1% OsO4 fixation, allowing super-resolution visualization of organelles and fused proteins in the context of resinembedded ultrastructure in both transmission EM (TEM) and scanning EM (SEM) [1,2].

Biotinidase deficiency is the primary enzymatic defect in biotin-responsive, late-onset multiple carboxylase deficiency. Untreated children with profound biotinidase deficiency usually exhibit neurological symptoms including lethargy, hypotonia, seizures, developmental delay, sensorineural hearing loss and optic atrophy; and cutaneous symptoms including skin rash, conjunctivitis and alopecia. Although the clinical features of the disorder markedly improve or are prevented with biotin supplementation, some symptoms, once they occur, such as developmental delay, hearing loss and optic atrophy, are usually irreversible. To prevent development of symptoms, the disorder is screened for in the newborn period in essentially all states and in many countries. In order to better understand many aspects of the pathophysiology of the disorder, we have developed a transgenic biotinidase-deficient mouse. The mouse has a null mutation that results in no detectable serum biotinidase activity or cross-reacting material to antibody prepared against biotinidase. When fed a biotin-deficient diet these mice develop neurological and cutaneous symptoms, carboxylase deficiency, mild hyperammonemia, and exhibit increased urinary excretion of 3-hydroxyisovaleric acid and biotin and biotin metabolites. The clinical features are reversed with biotin supplementation. This biotinidase-deficient animal can be used to study systematically many aspects of the disorder and the role of biotinidase, biotin and biocytin in normal and in enzyme-deficient states.

Abstract ingle molecule localisation microscopy (SMLM), experimentally achieved over a decade ago, has become a routinely used analytical tool across the life sciences. Synergistic advances in probe chemistry, optical physics and data analysis has propelled SMLM into the quantitative realm, enabling unprecedented access to the cellular machinery at the nanoscale. In its early years, SMLM primarily served as a platform for impressive rendered images of sub diffraction scale structures, however more recently a shift towards interrogating SMLM point pattern data in a robust mathematical framework has occurred. A prevalent theme in the SMLM field is the need for quantitative analytical methods, to better understand the underlying processes on which SMLM reports and to extract statistically valid biological insights. Whilst some forms of post processing analytics, for example cluster analysis, have been widely studied, others such as fibre analysis remain in their infancy. Here, we review the current state of the art of cluster analysis and fibre analysis and present new methods for their implementation in both 3D SMLM data sets and multi-colour data.

The scaffolding function of receptor interacting protein kinase 1 (RIPK1) confers intrinsic and extrinsic resistance to immune checkpoint blockades (ICBs) and has emerged as a promising target for improving cancer immunotherapies. To address the challenge posed by a poorly defined binding pocket within the intermediate domain, we harnessed proteolysis targeting chimera (PROTAC) technology to develop a first-in-class RIPK1 degrader, LD4172. LD4172 exhibited potent and selective RIPK1 degradation both in vitro and in vivo. Degradation of RIPK1 by LD4172 triggered immunogenic cell death (ICD) and enriched tumor-infiltrating lymphocytes and substantially sensitized the tumors to anti-PD1 therapy. This work reports the first RIPK1 degrader that serves as a chemical probe for investigating the scaffolding functions of RIPK1 and as a potential therapeutic agent to enhance tumor responses to immune checkpoint blockade therapy.

Protein design is a powerful tool for interrogating the basic requirements for the function of a metal site in a way that allows for the selective incorporation of elements that are important for function. Rubredoxins are small electron transfer proteins with a reduction potential centered near 0 mV (vs normal hydrogen electrode). All previous attempts to design a rubredoxin site have focused on incorporating the canonical CXXC motifs in addition to reproducing the peptide fold or using flexible loop regions to define the morphology of the site. We have produced a rubredoxin site in an utterly different fold, a three-helix bundle. The spectra of this construct mimic the ultraviolet–visible, Mössbauer, electron paramagnetic resonance, and magnetic circular dichroism spectra of native rubredoxin. Furthermore, the measured reduction potential suggests that this rubredoxin analogue could function similarly. Thus, we have shown that an α-helical scaffold sustains a rubredoxin site that can cycle with the desired potential between the Fe(II) and Fe(III) states and reproduces the spectroscopic characteristics of this electron transport protein without requiring the classic rubredoxin protein fold.

Primary aldosteronism (PA) is the most frequent form of secondary hypertension. The identification of germline or somatic mutations in different genes coding for ion channels and defines PA as a channelopathy. These mutations promote activation of calcium signaling, the main trigger for aldosterone biosynthesis.