Clionamines stimulate autophagy, inhibit Mycobacterium tuberculosis survival in macrophages, and target Pik1.

The pathogen Mycobacterium tuberculosis (Mtb) evades the innate immune system by interfering with autophagy and phagosomal maturation in macrophages, and, as a result, small molecule stimulation of autophagy represents a host-directed therapeutics (HDTs) approach for treatment of tuberculosis (TB). Here we show the marine natural product clionamines activate autophagy and inhibit Mtb survival in macrophages. A yeast chemical-genetics approach identified Pik1 as target protein of the clionamines. Biotinylated clionamine B pulled down Pik1 from yeast cell lysates and a clionamine analog inhibited phosphatidyl 4-phosphate (PI4P) production in yeast Golgi membranes. Chemical-genetic profiles of clionamines and cationic amphiphilic drugs (CADs) are closely related, linking the clionamine mode of action to co-localization with PI4P in a vesicular compartment. Small interfering RNA (siRNA) knockdown of PI4KB, a human homolog of Pik1, inhibited the survival of Mtb in macrophages, identifying PI4KB as an unexploited molecular target for efforts to develop HDT drugs for treatment of TB.

A scalable Drosophila assay for clinical interpretation of human PTEN variants in suppression of PI3K/AKT induced cellular proliferation.

Gene variant discovery is becoming routine, but it remains difficult to usefully interpret the functional consequence or disease relevance of most variants. To fill this interpretation gap, experimental assays of variant function are becoming common place. Yet, it remains challenging to make these assays reproducible, scalable to high numbers of variants, and capable of assessing defined gene-disease mechanism for clinical interpretation aligned to the ClinGen Sequence Variant Interpretation (SVI) Working Group guidelines for 'well-established assays'. Drosophila melanogaster offers great potential as an assay platform, but was untested for high numbers of human variants adherent to these guidelines. Here, we wished to test the utility of Drosophila as a platform for scalable well-established assays. We took a genetic interaction approach to test the function of ~100 human PTEN variants in cancer-relevant suppression of PI3K/AKT signaling in cellular growth and proliferation. We validated the assay using biochemically characterized PTEN mutants as well as 23 total known pathogenic and benign PTEN variants, all of which the assay correctly assigned into predicted functional categories. Additionally, function calls for these variants correlated very well with our recent published data from a human cell line. Finally, using these pathogenic and benign variants to calibrate the assay, we could set readout thresholds for clinical interpretation of the pathogenicity of 70 other PTEN variants. Overall, we demonstrate that Drosophila offers a powerful assay platform for clinical variant interpretation, that can be used in conjunction with other well-established assays, to increase confidence in the accurate assessment of variant function and pathogenicity.

MAPS: machine-assisted phenotype scoring enables rapid functional assessment of genetic variants by high-content microscopy.

BACKGROUND: Genetic testing is widely used in evaluating a patient's predisposition to hereditary diseases. In the case of cancer, when a functionally impactful mutation (i.e. genetic variant) is identified in a disease-relevant gene, the patient is at elevated risk of developing a lesion in their lifetime. Unfortunately, as the rate and coverage of genetic testing has accelerated, our ability to assess the functional status of new variants has fallen behind. Therefore, there is an urgent need for more practical, streamlined and cost-effective methods for classifying variants. RESULTS: To directly address this issue, we designed a new approach that uses alterations in protein subcellular localization as a key indicator of loss of function. Thus, new variants can be rapidly functionalized using high-content microscopy (HCM). To facilitate the analysis of the large amounts of imaging data, we developed a new software toolkit, named MAPS for machine-assisted phenotype scoring, that utilizes deep learning to extract and classify cell-level features. MAPS helps users leverage cloud-based deep learning services that are easy to train and deploy to fit their specific experimental conditions. Model training is code-free and can be done with limited training images. Thus, MAPS allows cell biologists to easily incorporate deep learning into their image analysis pipeline. We demonstrated an effective variant functionalization workflow that integrates HCM and MAPS to assess missense variants of PTEN, a tumor suppressor that is frequently mutated in hereditary and somatic cancers. CONCLUSIONS: This paper presents a new way to rapidly assess variant function using cloud deep learning. Since most tumor suppressors have well-defined subcellular localizations, our approach could be widely applied to functionalize variants of uncertain significance and help improve the utility of genetic testing.

Regulation of the early stages of endoplasmic reticulum inheritance during ER stress.

The endoplasmic reticulum (ER) is one of the largest cytoplasmic organelles in eukaryotic cells and plays a role in many cellular processes, such as the production and quality control of secretory protein, lipid synthesis, and calcium homeostasis. The ER cannot be generated de novo, and thus its proper inheritance during cell division is paramount to the health and survival of the daughter cells. Although previous work has uncovered the cytoskeletal components involved, we still lack a comprehensive understanding of the intricate steps of and the cytoplasmic and membrane-bound components involved in ER inheritance. To directly address these issues, we utilized microfluidics and genetic analyses to show that before nuclear migration, early ER inheritance can be further divided into three distinctive steps. Moreover, we demonstrated that perturbing each of these steps affects the cell's ability to mitigate ER stress. Thus, proper ER inheritance is essential to ensuring a healthy, functional cell.

A Premalignant Cell-Based Model for Functionalization and Classification of PTEN Variants.

As sequencing becomes more economical, we are identifying sequence variations in the population faster than ever. For disease-associated genes, it is imperative that we differentiate a sequence variant as either benign or pathogenic, such that the appropriate therapeutic interventions or surveillance can be implemented. PTEN is a frequently mutated tumor suppressor that has been linked to the PTEN hamartoma tumor syndrome. Although the domain structure of PTEN and the functional impact of a number of its most common tumor-linked mutations have been characterized, there is a lack of information about many recently identified clinical variants. To address this challenge, we developed a cell-based assay that utilizes a premalignant phenotype of normal mammary epithelial cells lacking PTEN. We measured the ability of PTEN variants to rescue the spheroid formation phenotype of PTEN-/- MCF10A cells maintained in suspension. As proof of concept, we functionalized 47 missense variants using this assay, only 19 of which have clear classifications in ClinVar. We utilized a machine learning model trained with annotated genotypic data to classify variants as benign or pathogenic based on our functional scores. Our model predicted with high accuracy that loss of PTEN function was indicative of pathogenicity. We also determined that the pathogenicity of certain variants may have arisen from reduced stability of the protein product. Overall, this assay outperformed computational predictions, was scalable, and had a short run time, serving as an ideal alternative for annotating the clinical significance of cancer-associated PTEN variants. SIGNIFICANCE: Combined three-dimensional tumor spheroid modeling and machine learning classifies PTEN missense variants, over 70% of which are currently listed as variants of uncertain significance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/13/2775/F1.large.jpg.

Sentinel interaction mapping - a generic approach for the functional analysis of human disease gene variants using yeast.

Advances in sequencing technology have led to an explosion in the number of known genetic variants of human genes. A major challenge is to now determine which of these variants contribute to diseases as a result of their effect on gene function. Here, we describe a generic approach using the yeast Saccharomyces cerevisiae to quickly develop gene-specific in vivo assays that can be used to quantify the level of function of a genetic variant. Using synthetic dosage lethality screening, 'sentinel' yeast strains are identified that are sensitive to overexpression of a human disease gene. Variants of the gene can then be functionalized in a high-throughput fashion through simple growth assays using solid or liquid media. Sentinel interaction mapping (SIM) has the potential to create functional assays for the large majority of human disease genes that do not have a yeast orthologue. Using the tumour suppressor gene PTEN as an example, we show that SIM assays can provide a fast and economical means to screen a large number of genetic variants.

Transfer of the Septin Ring to Cytokinetic Remnants in ER Stress Directs Age-Sensitive Cell-Cycle Re-entry.

During cell division, the inheritance of a functional endoplasmic reticulum (ER) is ensured by the endoplasmic reticulum stress surveillance (ERSU) pathway. Activation of ERSU causes the septin ring to mislocalize, which blocks ER inheritance and cytokinesis. Here, we uncover that the septin ring in fact translocates to previously utilized cell division sites called cytokinetic remnants (CRMs). This unconventional translocation requires Nba1, a negative polarity regulator that normally prevents repolarization and re-budding at CRMs. Furthermore, septin ring translocation relies on the recruitment and activation of a key ERSU component Slt2 by Bem1, without activating Cdc42. Failure to transfer all septin subunits to CRMs delays the cell's ability to re-enter the cell cycle when ER homeostasis is restored and hinders cell growth after ER stress recovery. Thus, these deliberate but unprecedented rearrangements of cell polarity factors during ER stress safeguard cell survival and the timely cell-cycle re-entry upon ER stress recovery.

Mechanisms of PTPσ-Mediated Presynaptic Differentiation.

Formation of synapses between neurons depends in part on binding between axonal and dendritic cell surface synaptic organizing proteins, which recruit components of the developing presynaptic and postsynaptic specializations. One of these presynaptic organizing molecules is protein tyrosine phosphatase σ (PTPσ). Although the protein domains involved in adhesion between PTPσ and its postsynaptic binding partners are known, the mechanisms by which it signals into the presynaptic neuron to recruit synaptic vesicles and other necessary components for regulated transmitter release are not well understood. One attractive candidate to mediate this function is liprin-α, a scaffolding protein with well-established roles at the synapse. We systematically mutated residues of the PTPσ intracellular region (ICR) and used the yeast dihydrofolate reductase (DHFR) protein complementation assay to screen for disrupted interactions between these mutant forms of PTPσ and its various binding partners. Using a molecular replacement strategy, we show that disrupting the interaction between PTPσ and liprin-α, but not between PTPσ and itself or another binding partner, caskin, abolishes presynaptic differentiation. Furthermore, phosphatase activity of PTPσ and binding to extracellular heparan sulfate (HS) proteoglycans are dispensable for presynaptic induction. Previous reports have suggested that binding between PTPσ and liprin-α is mediated by the PTPσ membrane-distal phosphatase-like domain. However, we provide evidence here that both of the PTPσ phosphatase-like domains mediate binding to liprin-α and are required for PTPσ-mediated presynaptic differentiation. These findings further our understanding of the mechanistic basis by which PTPσ acts as a presynaptic organizer.

Nursing the Follicles.

In a recent issue of Science, Lei and Spradling (2016) uncover how germ cells differentiate into oocytes in mouse embryos. Mouse germ cells form cysts, in which sister cells nurse the developing oocyte by donating their organelles and cytoplasmic materials.

A conserved endoplasmic reticulum membrane protein complex (EMC) facilitates phospholipid transfer from the ER to mitochondria.

Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER) to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC) has decreased phosphatidylserine (PS) transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER-mitochondria tethering complex called ERMES (the ER-mitochondria encounter structure) are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER-mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM) complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.

Polarization of the endoplasmic reticulum by ER-septin tethering.

Polarization of the plasma membrane (PM) into domains is an important mechanism to compartmentalize cellular activities and to establish cell polarity. Polarization requires formation of diffusion barriers that prevent mixing of proteins between domains. Recent studies have uncovered that the endoplasmic reticulum (ER) of budding yeast and neurons is polarized by diffusion barriers, which in neurons controls glutamate signaling in dendritic spines. The molecular identity of these barriers is currently unknown. Here, we show that a direct interaction between the ER protein Scs2 and the septin Shs1 creates the ER diffusion barrier in yeast. Barrier formation requires Epo1, a novel ER-associated subunit of the polarisome that interacts with Scs2 and Shs1. ER-septin tethering polarizes the ER into separate mother and bud domains, one function of which is to position the spindle in the mother until M phase by confining the spindle capture protein Num1 to the mother ER.

Barriers to uniformity within the endoplasmic reticulum.

Differentiating the endoplasmic reticulum (ER) into different physical domains may help the ER spatially regulate its many functions. For example, ER sheets are highly decorated with ribosomes for protein synthesis, whereas tubules usually correspond to smooth ER. Hence, ER morphology may play direct roles in functional diversification within the ER. The ER also makes direct physical contacts with other organelles, called ER junctions, enabling further functional diversification through input from external sources. In yeast, an ER diffusion barrier has now been discovered at the bud neck that compartmentalizes the ER into bud and mother diffusion domains by restricting the lateral diffusion of ER membrane proteins. Therefore, diffusion barriers also likely contribute to functional diversification within the ER by creating suites of molecular factors within ER diffusion domains.

Plasma membrane--endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis.

Synthesis of phospholipids, sterols and sphingolipids is thought to occur at contact sites between the endoplasmic reticulum (ER) and other organelles because many lipid-synthesizing enzymes are enriched in these contacts. In only a few cases have the enzymes been localized to contacts in vivo and in no instances have the contacts been demonstrated to be required for enzyme function. Here, we show that plasma membrane (PM)--ER contact sites in yeast are required for phosphatidylcholine synthesis and regulate the activity of the phosphatidylethanolamine N-methyltransferase enzyme, Opi3. Opi3 activity requires Osh3, which localizes to PM-ER contacts where it might facilitate in trans catalysis by Opi3. Thus, membrane contact sites provide a structural mechanism to regulate lipid synthesis.

Phosphatidic acid is a pH biosensor that links membrane biogenesis to metabolism.

Recognition of lipids by proteins is important for their targeting and activation in many signaling pathways, but the mechanisms that regulate such interactions are largely unknown. Here, we found that binding of proteins to the ubiquitous signaling lipid phosphatidic acid (PA) depended on intracellular pH and the protonation state of its phosphate headgroup. In yeast, a rapid decrease in intracellular pH in response to glucose starvation regulated binding of PA to a transcription factor, Opi1, that coordinately repressed phospholipid metabolic genes. This enabled coupling of membrane biogenesis to nutrient availability.