STING induces HOIP-mediated synthesis of M1 ubiquitin chains to stimulate NF-κB signaling.

STING activation by cyclic dinucleotides induces IRF3- and NF-κB-mediated gene expression in mammals, as well as lipidation of LC3B at Golgi-related membranes. While mechanisms of the IRF3 response are well understood, the mechanisms of NF-κB activation via STING remain unclear. We report here that STING activation induces linear/M1-linked ubiquitin chain (M1-Ub) formation and recruitment of the LUBAC E3 ligase, HOIP, to LC3B-associated Golgi membranes where ubiquitin is also localized.

STING induces HOIP-mediated synthesis of M1 ubiquitin chains to stimulate NFκB signaling.

STING activation by cyclic dinucleotides in mammals induces IRF3- and NFκB -mediated gene expression, and the lipidation of LC3B at Golgi-related membranes. While mechanisms of the IRF3 response are well understood, the mechanisms of NFκB activation mediated by STING remain unclear. We report that STING activation induces linear/M1-linked ubiquitin chain (M1-Ub) formation and recruitment of the LUBAC E3 ligase, HOIP, to LC3B-associated Golgi membranes where ubiquitin is also localized.

Nix interacts with WIPI2 to induce mitophagy.

Nix is a membrane-anchored outer mitochondrial protein that induces mitophagy. While Nix has an LC3-interacting (LIR) motif that binds to ATG8 proteins, it also contains a minimal essential region (MER) that induces mitophagy through an unknown mechanism. We used chemically induced dimerization (CID) to probe the mechanism of Nix-mediated mitophagy and found that both the LIR and MER are required for robust mitophagy.

TNIP1 inhibits selective autophagy via bipartite interaction with LC3/GABARAP and TAX1BP1.

Mitophagy is a form of selective autophagy that disposes of superfluous and potentially damage-inducing organelles in a tightly controlled manner. While the machinery involved in mitophagy induction is well known, the regulation of the components is less clear. Here, we demonstrate that TNIP1 knockout in HeLa cells accelerates mitophagy rates and that ectopic TNIP1 negatively regulates the rate of mitophagy.

Altered SYNJ2BP-mediated mitochondrial-ER contacts in motor neuron disease.

Synaptojanin 2 binding protein (SYNJ2BP) is an outer mitochondrial membrane protein with a cytosolic PDZ domain that functions as a cellular signaling hub. Few studies have evaluated its role in disease. Here we use induced pluripotent stem cell (iPSC)-derived motor neurons and post-mortem tissue from patients with two hereditary motor neuron diseases, spinal and bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis type 4 (ALS4), and show that SYNJ2BP expression is increased in diseased motor neurons.

Multiomic Analysis Reveals Disruption of Cholesterol Homeostasis by Cannabidiol in Human Cell Lines.

The nonpsychoactive cannabinoid, cannabidiol (CBD), is Food and Dug Administration approved for treatment of two drug-resistant epileptic disorders and is seeing increased use among the general public, yet the mechanisms that underlie its therapeutic effects and side-effect profiles remain unclear. Here, we report a systems-level analysis of CBD action in human cell lines using temporal multiomic profiling. FRET-based biosensor screening revealed that CBD elicits a sharp rise in cytosolic calcium, and activation of AMP-activated protein kinase in human keratinocyte and neuroblastoma cell lines.

Protocol for Analysis and Consolidation of TrackMate Outputs for Measuring Two-Dimensional Cell Motility using Nuclear Tracking.

Collective cellular migration plays a key role in many fundamental biological processes including development, wound healing, and cancer metastasis. To understand the regulation of cell motility, we must be able to measure it easily and consistently under different conditions. Here we describe a method for measuring and quantifying single-cell and bulk motility of HaCaT keratinocytes using a nuclear stain.

Suppression of α-catenin and adherens junctions enhances epithelial cell proliferation and motility via TACE-mediated TGF-α autocrine/paracrine signaling.

Sustained cell migration is essential for wound healing and cancer metastasis. The epidermal growth factor receptor (EGFR) signaling cascade is known to drive cell migration and proliferation. While the signal transduction downstream of EGFR has been extensively investigated, our knowledge of the initiation and maintenance of EGFR signaling during cell migration remains limited.

Linking epigenetic dysregulation, mitochondrial impairment, and metabolic dysfunction in SBMA motor neurons.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disorder caused by a polyglutamine expansion in the androgen receptor (AR). Using gene expression analysis and ChIP sequencing, we mapped transcriptional changes in genetically engineered patient stem cell-derived motor neurons. We found that transcriptional dysregulation in SBMA can occur through AR-mediated histone modification.

Temporal Metabolite, Ion, and Enzyme Activity Profiling Using Fluorescence Microscopy and Genetically Encoded Biosensors.

Living cells employ complex and highly dynamic signaling networks and transcriptional circuits to maintain homeostasis and respond appropriately to constantly changing environments. These networks enable cells to maintain tight control on intracellular concentrations of ions, metabolites, proteins, and other biomolecules and ensure a careful balance between a cell's energetic needs and catabolic processes required for growth. Establishing molecular mechanisms of genetic and pharmacological perturbations remains challenging, due to the interconnected nature of these networks and the extreme sensitivity of cellular systems to their external environment.

Bok regulates mitochondrial fusion and morphology.

Bok (Bcl-2-related ovarian killer) is a member of the Bcl-2 protein family that governs the intrinsic apoptosis pathway, but the cellular role that Bok plays is controversial. Remarkably, endogenous Bok is constitutively bound to inositol 1,4,5-trisphosphate receptors (IPRs) and is stabilized by this interaction. Here we report that despite the strong association with IPRs, deletion of Bok expression by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease)-mediated gene editing does not alter calcium mobilization via IPRs or calcium influx into the mitochondria.

The plant triterpenoid celastrol blocks PINK1-dependent mitophagy by disrupting PINK1's association with the mitochondrial protein TOM20.

A critical function of the PTEN-induced kinase 1 (PINK1)-Parkin pathway is to mediate the clearing of unhealthy or damaged mitochondria via mitophagy. Loss of either PINK1 or Parkin protein expression is associated with Parkinson's disease. Here, using a high-throughput screening approach along with recombinant protein expression and kinase, immunoblotting, and immunofluorescence live-cell imaging assays, we report that celastrol, a pentacyclic triterpenoid isolated from extracts of the medicinal plant , blocks recruitment pof Parkin to mitochondria, preventing mitophagy in response to mitochondrial depolarization induced by carbonyl cyanide -chlorophenylhydrazone or to gamitrinib-induced inhibition of mitochondrial heat shock protein 90 (HSP90).

Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during Selective Autophagy.

Selective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis.

Reciprocal Roles of Tom7 and OMA1 during Mitochondrial Import and Activation of PINK1.

Mutations in PTEN-induced kinase 1 (PINK1) can cause recessive early-onset Parkinson's disease (PD). Import arrest results in PINK1 kinase activation specifically on damaged mitochondria, triggering Parkin-mediated mitophagy. Here, we show that PINK1 import is less dependent on Tim23 than on mitochondrial membrane potential (ΔΨm).

Structural basis of the phosphorylation-independent recognition of cyclin D1 by the SCF ubiquitin ligase.

Ubiquitin-dependent proteolysis of cyclin D1 is associated with normal and tumor cell proliferation and survival. The SCF (Skp1-Cul1-Rbx1-FBXO31) ubiquitin ligase complex mediates genotoxic stress-induced cyclin D1 degradation. Previous studies have suggested that cyclin D1 levels are maintained at steady state by phosphorylation-dependent nuclear export and subsequent proteolysis in the cytoplasm.

Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers.

Histone deacetylase inhibitors (HDACIs) are known to alter gene expression by both up- and down-regulation of protein-coding genes in normal and cancer cells. However, the exact regulatory mechanisms of action remain uncharacterized. Here we investigated genome wide dose-dependent epigenetic and transcriptome changes in response to HDACI largazole in a transformed and a non-transformed cell line.

Sorafenib targets the mitochondrial electron transport chain complexes and ATP synthase to activate the PINK1-Parkin pathway and modulate cellular drug response.

Sorafenib (Nexavar) is a broad-spectrum multikinase inhibitor that proves effective in treating advanced renal-cell carcinoma and liver cancer. Despite its well-characterized mechanism of action on several established cancer-related protein kinases, sorafenib causes variable responses among human tumors, although the cause for this variation is unknown. In an unbiased screening of an oncology drug library, we found that sorafenib activates recruitment of the ubiquitin E3 ligase Parkin to damaged mitochondria.

Long-term live-cell imaging reveals new roles for Salmonella effector proteins SseG and SteA.

Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single-cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time.

Genome-wide analysis of Musashi-2 targets reveals novel functions in governing epithelial cell migration.

The Musashi-2 (Msi2) RNA-binding protein maintains stem cell self-renewal and promotes oncogenesis by enhancing cell proliferation in hematopoietic and gastrointestinal tissues. However, it is unclear how Msi2 recognizes and regulates mRNA targets in vivo and whether Msi2 primarily controls cell growth in all cell types. Here we identified Msi2 targets with HITS-CLIP and revealed that Msi2 primarily recognizes mRNA 3'UTRs at sites enriched in multiple copies of UAG motifs in epithelial progenitor cells.

A biosensor for the activity of the "sheddase" TACE (ADAM17) reveals novel and cell type-specific mechanisms of TACE activation.

Diverse environmental conditions stimulate protein "shedding" from the cell surface through proteolytic cleavage. The protease TACE [tumor necrosis factor-α (TNFα)--converting enzyme, encoded by ADAM17] mediates protein shedding, thereby regulating the maturation and release of various extracellular substrates, such as growth factors and cytokines, that induce diverse cellular responses. We developed a FRET (fluorescence resonance energy transfer)-based biosensor called TSen that quantitatively reports the kinetics of TACE activity in live cells.

PINK1 triggers autocatalytic activation of Parkin to specify cell fate decisions.

Background: The PINK1-Parkin pathway is known to play important roles in regulating mitochondria dynamics, motility, and quality control. Activation of this pathway can be triggered by a variety of cellular stress signals that cause mitochondrial damage. How this pathway senses different levels of mitochondrial damage and mediates cell fate decisions accordingly is incompletely understood.

Perception without self-matching in conditional tag based cooperation.

We consider a model for the evolution of cooperation in a population where individuals may have one of a number of different heritable and distinguishable markers or tags. Individuals interact with each of their neighbors on a square lattice by either cooperating by donating some benefit at a cost to themselves or defecting by doing nothing. The decision to cooperate or defect is contingent on each individual's perception of its interacting partner's tag.