Neutrophil-like cells derived from the HL-60 cell-line as a genetically-tractable model for neutrophil degranulation.

Research on neutrophil biology has been limited by the short life span and limited genetic manipulability of these cells, driving the need for representative and efficient model cell lines. The promyelocytic cell line HL-60 and its subline PLB-985 can be differentiated into neutrophil-like cells (NLCs) and have been used to study neutrophil functions including chemotaxis, phagocytosis, endocytosis, and degranulation. Compared to neutrophils derived from hematopoietic stem cells, NLCs serve as a cost-effective neutrophil model. NLCs derived from both HL-60 and PLB-985 cells have been shown to perform degranulation, an important neutrophil function. However, no study has directly compared the two lines as models for degranulation including their release of different types of mobilizable organelles. Furthermore, Nutridoma, a commercially available supplement, has recently been shown to improve the chemotaxis, phagocytosis, and oxidative burst abilities of NLCs derived from promyelocytic cells, however it is unknown whether this reagent also improves the degranulation ability of NLCs. Here, we show that NLCs derived from both HL-60 and PLB-985 cells are capable of degranulating, with each showing markers for the release of multiple types of secretory organelles, including primary granules. We also show that differentiating HL-60 cells using Nutridoma does not enhance their degranulation activity over NLCs differentiated using Dimethyl Sulfoxide (DMSO) plus Granulocyte-colony stimulating factor (G-CSF). Finally, we show that promyelocytic cells can be genetically engineered and differentiated using these methods, to yield NLCs with a defect in degranulation. Our results indicate that both cell lines serve as effective models for investigating the mechanisms of neutrophil degranulation, which can advance our understanding of the roles of neutrophils in inflammation and immunity.

Phosphate starvation signaling increases mitochondrial membrane potential through respiration-independent mechanisms.

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.

Signaling dynamics distinguish high- and low-priority neutrophil chemoattractant receptors.

Human neutrophils respond to multiple chemoattractants to guide their migration from the vasculature to sites of infection and injury, where they clear pathogens and amplify inflammation. To properly focus their responses during this complex navigation, neutrophils prioritize pathogen- and injury-derived signals over long-range inflammatory signals, such as the leukotriene LTB4, secreted by host cells. Different chemoattractants can also drive qualitatively different modes of migration even though their receptors couple to the same Gαi family of G proteins. Here, we used live-cell imaging to demonstrate that the responses differed in their signaling dynamics. Low-priority chemoattractants caused transient responses, whereas responses to high-priority chemoattractants were sustained. We observed this difference in both primary neutrophils and differentiated HL-60 cells, for downstream signaling mediated by Ca2+, a major regulator of secretion, and Cdc42, a primary regulator of polarity and cell steering. The rapid attenuation of Cdc42 activation in response to LTB4 depended on the phosphorylation sites Thr308 and Ser310 in the carboxyl-terminal tail of its receptor LTB4R in a manner independent of endocytosis. Mutation of these residues to alanine impaired chemoattractant prioritization, although it did not affect chemoattractant-dependent differences in migration persistence. Our results indicate that distinct temporal regulation of shared signaling pathways distinguishes between receptors and contributes to chemoattractant prioritization.

Optogenetic control of receptors reveals distinct roles for actin- and Cdc42-dependent negative signals in chemotactic signal processing.

During chemotaxis, neutrophils use cell surface G Protein Coupled Receptors to detect chemoattractant gradients. The downstream signaling system is wired with multiple feedback loops that amplify weak inputs and promote spatial separation of cell front and rear activities. Positive feedback could promote rapid signal spreading, yet information from the receptors is transmitted with high spatial fidelity, enabling detection of small differences in chemoattractant concentration across the cell. How the signal transduction network achieves signal amplification while preserving spatial information remains unclear. The GTPase Cdc42 is a cell-front polarity coordinator that is predictive of cell turning, suggesting an important role in spatial processing. Here we directly measure information flow from receptors to Cdc42 by pairing zebrafish parapinopsina, an optogenetic G Protein Coupled Receptor with reversible ON/OFF control, with a spectrally compatible red/far red Cdc42 Fluorescence Resonance Energy Transfer biosensor. Using this toolkit, we show that positive and negative signals downstream of G proteins shape a rapid, dose-dependent Cdc42 response. Furthermore, F-actin and Cdc42 itself provide two distinct negative signals that limit the duration and spatial spread of Cdc42 activation, maintaining output signals local to the originating receptors.

A bacteria-derived tail anchor localizes to peroxisomes in yeast and mammalian cells.

Prokaryotes can provide new genetic information to eukaryotes by horizontal gene transfer (HGT), and such transfers are likely to have been particularly consequential in the era of eukaryogenesis. Since eukaryotes are highly compartmentalized, it is worthwhile to consider the mechanisms by which newly transferred proteins might reach diverse organellar destinations. Toward this goal, we have focused our attention upon the behavior of bacteria-derived tail anchors (TAs) expressed in the eukaryote Saccharomyces cerevisiae. In this study, we report that a predicted membrane-associated domain of the Escherichia coli YgiM protein is specifically trafficked to peroxisomes in budding yeast, can be found at a pre-peroxisomal compartment (PPC) upon disruption of peroxisomal biogenesis, and can functionally replace an endogenous, peroxisome-directed TA. Furthermore, the YgiM(TA) can localize to peroxisomes in mammalian cells. Since the YgiM(TA) plays no endogenous role in peroxisomal function or assembly, this domain is likely to serve as an excellent tool allowing further illumination of the mechanisms by which TAs can travel to peroxisomes. Moreover, our findings emphasize the ease with which bacteria-derived sequences might target to organelles in eukaryotic cells following HGT, and we discuss the importance of flexible recognition of organelle targeting information during and after eukaryogenesis.

Evidence for Amino Acid Snorkeling from a High-Resolution, In Vivo Analysis of Fis1 Tail-Anchor Insertion at the Mitochondrial Outer Membrane.

Proteins localized to mitochondria by a carboxyl-terminal tail anchor (TA) play roles in apoptosis, mitochondrial dynamics, and mitochondrial protein import. To reveal characteristics of TAs that may be important for mitochondrial targeting, we focused our attention upon the TA of the Saccharomyces cerevisiae Fis1 protein. Specifically, we generated a library of Fis1p TA variants fused to the Gal4 transcription factor, then, using next-generation sequencing, revealed which Fis1p TA mutations inhibited membrane insertion and allowed Gal4p activity in the nucleus. Prompted by our global analysis, we subsequently analyzed the ability of individual Fis1p TA mutants to localize to mitochondria. Our findings suggest that the membrane-associated domain of the Fis1p TA may be bipartite in nature, and we encountered evidence that the positively charged patch at the carboxyl terminus of Fis1p is required for both membrane insertion and organelle specificity. Furthermore, lengthening or shortening of the Fis1p TA by up to three amino acids did not inhibit mitochondrial targeting, arguing against a model in which TA length directs insertion of TAs to distinct organelles. Most importantly, positively charged residues were more acceptable at several positions within the membrane-associated domain of the Fis1p TA than negatively charged residues. These findings, emerging from the first high-resolution analysis of an organelle targeting sequence by deep mutational scanning, provide strong, in vivo evidence that lysine and arginine can "snorkel," or become stably incorporated within a lipid bilayer by placing terminal charges of their side chains at the membrane interface.

Reduced Glucose Sensation Can Increase the Fitness of Saccharomyces cerevisiae Lacking Mitochondrial DNA.

Damage to the mitochondrial genome (mtDNA) can lead to diseases for which there are no clearly effective treatments. Since mitochondrial function and biogenesis are controlled by the nutrient environment of the cell, it is possible that perturbation of conserved, nutrient-sensing pathways may successfully treat mitochondrial disease. We found that restricting glucose or otherwise reducing the activity of the protein kinase A (PKA) pathway can lead to improved proliferation of Saccharomyces cerevisiae cells lacking mtDNA and that the transcriptional response to mtDNA loss is reduced in cells with diminished PKA activity. We have excluded many pathways and proteins from being individually responsible for the benefits provided to cells lacking mtDNA by PKA inhibition, and we found that robust import of mitochondrial polytopic membrane proteins may be required in order for cells without mtDNA to receive the full benefits of PKA reduction. Finally, we have discovered that the transcription of genes involved in arginine biosynthesis and aromatic amino acid catabolism is altered after mtDNA damage. Our results highlight the potential importance of nutrient detection and availability on the outcome of mitochondrial dysfunction.

Mutation of the mitochondrial large ribosomal RNA can provide pentamidine resistance to Saccharomyces cerevisiae.

Pentamidine is used to treat several trypanosomal diseases, as well as opportunistic infection by pathogenic fungi. However, the relevant targets of this drug are unknown. We isolated dominant mutations providing pentamidine resistance to Saccharomyces cerevisiae, one of which was localized to mitochondrial DNA. Next-generation sequencing revealed alteration of a widely conserved base at the peptidyl transferase center of the mitochondrial 21S ribosomal RNA. Our results provide a potential rationale for the toxicity of this drug to patients, and we discuss whether blockade of mitochondrial translation is the mechanism by which pathogenic fungi or protists are killed by pentamidine.

Activation of the pleiotropic drug resistance pathway can promote mitochondrial DNA retention by fusion-defective mitochondria in Saccharomyces cerevisiae.

Genetic and microscopic approaches using Saccharomyces cerevisiae have identified many proteins that play a role in mitochondrial dynamics, but it is possible that other proteins and pathways that play a role in mitochondrial division and fusion remain to be discovered. Mutants lacking mitochondrial fusion are characterized by rapid loss of mitochondrial DNA. We took advantage of a petite-negative mutant that is unable to survive mitochondrial DNA loss to select for mutations that allow cells with fusion-deficient mitochondria to maintain the mitochondrial genome on fermentable medium. Next-generation sequencing revealed that all identified suppressor mutations not associated with known mitochondrial division components were localized to PDR1 or PDR3, which encode transcription factors promoting drug resistance. Further studies revealed that at least one, if not all, of these suppressor mutations dominantly increases resistance to known substrates of the pleiotropic drug resistance pathway. Interestingly, hyperactivation of this pathway did not significantly affect mitochondrial shape, suggesting that mitochondrial division was not greatly affected. Our results reveal an intriguing genetic connection between pleiotropic drug resistance and mitochondrial dynamics.