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.Peer reviewe

Dissecting Endocytic Mechanisms of the Chemoattractant Receptor FPR1 in a Human Neutrophil Model

Neutrophils are key mediators of immune responses and inflammation in the body. Various neutrophil functions including chemotaxis, the rapid, directed migration of neutrophils towards chemical signals, are controlled by the recognition of chemoattractants by a specialized subfamily of G-protein-coupled receptors (GPCRs) including the widely studied formyl-peptide receptor 1 (FPR1) in humans. Receptor internalization prevents excessive activation of downstream ligand-induced signaling pathways, which allows cells to tune their sensitivity and limit inflammatory responses. Moreover, mutants deficient in FPR1-endocytosis migrate longer distances compared to their wild-type counterparts, suggesting that receptor internalization might act as a termination signal for neutrophil chemotaxis. Although precise regulation of chemotactic receptor internalization is critical for proper migration and function of neutrophils, the mechanism of FPR1 internalization and the role of internalization in chemotactic responses remain unclear.Taking genetic and cell biological approaches, we investigated the roles of both classic and unknown endocytosis regulators. Although their roles had been unclear, we found that multiple GPCR kinases (GRKs) and both β-arrestin 1 and 2 are important for FPR1 internalization using a flow-cytometry based endocytosis assay (Chapter 2). Moreover, we performed two parallel genome-wide CRISPR/Cas9 knock-out screens for the regulators of FPR1 surface expression and internalization (Chapter 3). Our results suggested multiple endocytic routes that FPR1 may follow, and this complexity may help explain conflicting data in the field. This screen identified potential new regulators of FPR1 endocytosis, including multiple subunits of endosome-associated protein complexes, small GTPases involved in membrane trafficking, and polarity signaling components. Through our ongoing investigation of some of these hits, we aim to validate and fully understand their involvement in chemoattractant receptor internalization and neutrophil chemotaxis. Better understanding of the roles of these regulators in FPR1 endocytosis and neutrophil function will unravel how the regulation of FPR1 endocytosis contributes to receptor-linked signaling events. Moreover, in Chapter 4, I summarize our findings on how ligand-dependent phosphorylation of another chemoattractant receptor, the LTB4 receptor, regulate its internalization. In conclusion, our efforts to gain insight into the mechanisms and biological consequences of chemoattractant receptor internalization broaden the current knowledge of regulation of these receptors and neutrophil chemotaxis. Accumulating evidence highlights the significance of neutrophil activity in the development of various ailments including inflammatory, autoimmune, and cardiovascular diseases. Gaining mechanistic insight into neutrophil function can help us develop novel strategies to modulate cell state and behavior during migration to control excessive or prolonged inflammation and thus prevent tissue damage

ePAD: An Image Annotation and Analysis Platform for Quantitative Imaging

Medical imaging is critical for assessing the response of patients to new cancer therapies. Quantitative lesion assessment on images is time-consuming, and adopting new promising quantitative imaging biomarkers of response in clinical trials is challenging. The electronic Physician Annotation Device (ePAD) is a freely available web-based zero-footprint software application for viewing, annotation, and quantitative analysis of radiology images designed to meet the challenges of quantitative evaluation of cancer lesions. For imaging researchers, ePAD calculates a variety of quantitative imaging biomarkers that they can analyze and compare in ePAD to identify potential candidates as surrogate endpoints in clinical trials. For clinicians, ePAD provides clinical decision support tools for evaluating cancer response through reports summarizing changes in tumor burden based on different imaging biomarkers. As a workflow management and study oversight tool, ePAD lets clinical trial project administrators create worklists for users and oversee the progress of annotations created by research groups. To support interoperability of image annotations, ePAD writes all image annotations and results of quantitative imaging analyses in standardized file formats, and it supports migration of annotations from various propriety formats. ePAD also provides a plugin architecture supporting MATLAB server-side modules in addition to client-side plugins, permitting the community to extend the ePAD platform in various ways for new cancer use cases. We present an overview of ePAD as a platform for medical image annotation and quantitative analysis. We also discuss use cases and collaborations with different groups in the Quantitative Imaging Network and future directions

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

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

S1 Data -

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.</div

Neutrophil-like cells derived from HL-60s and PLB-985 cells show exocytosis of primary granules.

HL-60 or PLB-985 cells were differentiated into Neutrophil-like cells (NLCs) and tested for exocytosis of primary granules. (A, B) NLCs were stained for differentiation marker CD11b, or an isotype-matched control. CD11b levels are compared to undifferentiated cells as an additional negative control. Data are representative of three experiments. (C, D) NLCs were differentiated and then left unstimulated (unstim), or stimulated with either PMA, or cytochalasin B and fMLP (fMLP). Then, cells were stained with anti-CD63 antibody (C-F) and supernatants were tested for secretion of MPO (G). Panels C and D show representative histograms for one experiment performed in triplicate, and E-G are the average of three independent experiments, each with 3 biological replicates. Error bars show the standard deviation and statistical comparison to unstimulated controls was done using a one-tailed students t-test. There was no statistically significant difference between the stimulated conditions for any panel.</p

Neutrophil-like cells derived from promyelocyte precursors release other mobilizable organelles including secretory vesicles.

HL-60 or PLB-985 cells were differentiated into Neutrophil-like cells (NLCs) with DMSO, plus either G-CSF or Nutridoma and then left unstimulated (unstim) or stimulated with cytochalasin B and fMLP (fMLP). Then, cells were stained with anti-CD107a (A-C) or anti-CD35 (D-F) antibody. Panels A, B, D and E show representative histograms of triplicate samples, and C and F are averages of three independent experiments. Error bars show the standard deviation and statistical comparison between the samples was performed using a three-way ANOVA, after confirming normality and equal variance using the Shapiro-Wilk test and Levene’s test respectively. The stimulated conditions were found to be significant compared to their unstimulated controls for all cell lines and differentiation methods tested, with a p-value of 1 x 10−4 and 1 x 10−6 for Fig 3C and 3F respectively. Two-tailed T-tests between comparisons of interest are also shown.</p

Nutridoma differentiation of promyelocytic cells does not substantially improve degranulation ability.

HL-60 (A-D) or PLB-985 (E-H) cells were differentiated into Neutrophil-like cells (NLCs) with DMSO, in addition to either G-CSF or Nutridoma and then left unstimulated (unstim), or stimulated with cytochalasin B and fMLP (fMLP). Then, cells were stained with anti-CD63 antibody (A-C, E-G) and supernatants were tested for secretion of MPO (D, H). Panels A and E show representative histograms for one experiment performed in triplicate and B-D, F-H are averages of three independent experiments. Error bars show the standard deviation and statistical comparison between treatments was done using a two-tailed student’s t-test.</p

Genetically altered promyelocytic precursors yield Neutrophil-like cells with impaired degranulation functions.

(A) Bar chart shows the average relative RAB27A expression levels calculated using the delta-delta Ct (ΔΔCt) method and normalized to a G6PD control. (B) Measurement of primary granule exocytosis by increased CD63 membrane expression in differentiated PLB-985 cells expressing dCas9-KRAB (control) compared to PLB-985 cells expressing dCas9-KRAB and Rab27a sgRNA. (C) Measurement of degranulation activity through MPO release in differentiated PLB-985 cells expressing dCas9-KRAB (control) compared to PLB-985 cells expressing dCas9-KRAB and Rab27a sgRNA. For each panel, the data are averages of three independent experiments performed in triplicate. Error bars represent the standard deviation and statistical comparison was done using a two-tailed student’s t-test.</p