Correlative Light and Electron Microscopy for the Investigation of Muscle Disease

<div>In order to investigate the biology of nemaline myopathy, a muscle disease characterised by the formation of nemaline (rod-like) aggregates, we created a zebrafish model.  To generate the model we expressed a disease causing form of ACTA1 (ACTA1D286G) tagged with enhanced green fluorescent protein within the muscle. This allowed the visualisation of disease onset and progression in the living animal. This led to the discovery of how and where the characteristic aggregates form. In order to confirm that the fluorescent aggregates observed in the fish corresponded to those observed in patients using electron microscopy we carried out correlative light and electron microscopy on the zebrafish disease model. This allowed visualisation of both the fluorescent protein and the electron dense aggregates. The raw data from these experiments are provided.</div

Additional file 1: of Testing of therapies in a novel nebulin nemaline myopathy model demonstrate a lack of efficacy

Supplementary data for Testing of therapies in a novel nebulin nemaline myopathy model demonstrates and lack of efficacy. Figure S1: Characterisation of Tg(neb-/-; Lifeact-eGFP) fish. Figure S2-S4: Toxicity analyses for treatment of wildtype zebrafish with taurine, L-carnitine and creatine. Figure S5-S6: Quantification of distance travelled and average speed at 6 dpf. Figure S7: Quantification of the phenotypic severity at 6 dpf. Figure S8: Characterisation of facial muscles at 6 dpf. (PDF 7847 kb

Isolation of epithelial cell subsets in the human lung.

(A) EpCAM-positive, lineage- (CD45, CD31, CD140b, and CD235a) negative cells from proximal and distal lung samples are subdivided by their expression of CD166, CD49f, and T1α to collect CD49fhiT1α+CD166mid (P5), CD49fmidT1α-CD166hi (P6), and CD49fmidT1α-CD166mid (P10) from proximal samples and P6 and P10 from distal samples. Representative image from a 63-y-old male exsmoker. n = 121 patients; 21–85 y old; male and female; never-, ex-, and current smokers. (B) Quantitative PCR (qPCR) analyses of lung lineage markers in sorted lung cells for n = 3 patients (a 65-y-old male exsmoker, a 47-y-old female exsmoker, and a 72-y-old male current smoker). Student’s t test. (C) Intracellular staining for differentiated lung epithelial cell markers analysed by fluorescence-activated cell sorting (FACS). n = 3 patients (a 47-y-old female, smoking status unknown; a 72-y-old male, smoking status unknown; and a 69-y-old male exsmoker). Student’s t test. (D) Pie charts showing relative cellular composition of large airway (proximal) and small airway (distal) luminal cell populations isolated from human lungs, as determined by intracellular FACS staining. n = 3 patients (a 47-y-old female, smoking status unknown; a 72-y-old male, smoking status unknown; and a 69-y-old male exsmoker). (E) Representative electron micrographs of proximal P5, proximal P10, and distal P10 from a 67-y-old male exsmoker. Black arrows indicate keratin filaments, and black arrowheads indicate mitochondria. White arrowheads indicate lamellar bodies, and white arrows indicate microvilli. Inset: high magnification of a lamellar body. Scale bar = 1 μm. n = 3 patients (a 67-y-old male exsmoker; a 61-y-old male never smoker; and a 75-y-old female, smoking status unknown). The underlying data for panels B, C, and D can be found in the S1 Data file.</p

Lung BSCs have a rapid yet error-prone response to DNA damage through nonhomologous end joining (NHEJ).

This allows the cells to continue to survive and proliferate after DNA injury and may eventually result in the accumulation of genetic lesions that lead to SqCC formation. In contrast, AT2 progenitor cells have an insufficient response to DNA damage, resulting in cell death.</p

Human and mouse BSCs express markers of nonhomologous end joining.

<p>(A) Immunohistochemistry for RAD51, an early marker of homologous recombination, on WT mouse trachea and lung 1 h post γ-irradiation (6 Gy). The insert is a positive control, a mammary tumour from a MMTV-cre;Brca1^fl/flp53^+/- mouse. Black arrows indicate RAD51-positive nuclei. Representative images from <i>n</i> = 3 mice at each time point. Scale bar = 100 μm. (B) Expression of key genes in the NHEJ repair pathway in human BSCs and AT2 cells. <i>n</i> = 3 patients (a 64-y-old male exsmoker, an 83-y-old male exsmoker, and a 53-y-old male current smoker). RPKM, reads per kilobase per million mapped reads. Paired <i>t</i> test. (C) Immunofluorescence staining of phospho-DNA-PKcs and T1α in human airways and alveoli of three patients. Patient 1, a 56-y-old male smoker; patient 2, a 69-y-old female exsmoker; patient 3, a 70-y-old male smoker. Inset, isotype control. Scale bar = 20 μm. (D) Immunofluorescence staining of phospho-DNA-PKcs and T1α in trachea and lung of WT mice following IR (6 Gy). Representative images of one of <i>n</i> = 3 mice at each time point. Inset, isotype control. Scale bar = 20 μm. The underlying data for panel B can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000731#pbio.2000731.s009" target="_blank">S1 Data</a> file.</p

BSCs use error-prone nonhomologous end joining to repair DNA double-strand breaks.

<p>(A) Immunofluorescence staining of γH2AX and T1α in the lungs and tracheas of WT and SCID^<i>prkdc</i> mice that are nonirradiated or 1, 4 or 8 h post irradiation (6 Gy). Representative images of <i>n</i> = 3 mice at each time point. Arrows indicate γH2AX⁺ T1α⁺ BSCs. Scale bar = 20 μm. (B) Representative FACS plots showing the expression of γH2AX in EpCAM⁺ lung epithelial cells and T1α⁺ tracheal BSCs in WT and SCID^<i>prkdc</i> mice 0, 4, and 7 h following IR (6 Gy). The timing corresponds to the number of hours between time of irradiation and generation of single-cell suspension for FACS analysis. (C) Percentage of γH2AX-positive cells in WT and SCID^<i>prkdc</i> mice in EpCAM⁺ lung epithelial cells and T1α⁺ tracheal BSCs 0, 4, and 7 h following irradiation. <i>n</i> = 6 animals per group. Student’s <i>t</i> test. The timing corresponds to the number of hours between time of irradiation and generation of single-cell suspension for FACS analysis. (D) Immunofluorescence staining of cleaved caspase 3 (CC3), T1α, and 4′,6-diamidino-2-phenylindole (DAPI) in WT and SCID^<i>prkdc</i> tracheas that are nonirradiated or 4, 24, or 96 h post irradiation (6 Gy). Representative images of <i>n</i> = 3 mice at each time point. Arrows indicate CC3⁺ T1α⁺ BSCs. Scale bar = 20 μm. (E) FACS detection of cells in subG1 in tracheal BSCs (T1α⁺) cells isolated from WT or SCID^<i>prkdc</i> mice 24 h post irradiation (6 Gy). <i>n</i> = 7 mice for WT mice and <i>n</i> = 12 for SCID^<i>prkdc</i> mice. Student’s <i>t</i> test. The underlying data for panels C and E can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000731#pbio.2000731.s009" target="_blank">S1 Data</a> file.</p

image_1_Helicobacter pylori Outer Membrane Vesicle Size Determines Their Mechanisms of Host Cell Entry and Protein Content.PDF

Gram-negative pathogens ubiquitously shed outer membrane vesicles (OMVs) that play a central role in initiating and regulating pathogenesis in the host. Due to their highly inflammatory nature, OMVs are extensively being examined for their role in mediating disease in addition to their applications in innovative vaccines. A key mechanism whereby OMVs mediate inflammation and disease progression is dependent on their ability to enter host cells. Currently, the role of OMV size on determining their mechanism of cellular entry and their protein composition remains unknown. In this study, we examined the mechanisms whereby OMV size regulates their mode of entry into epithelial cells, in addition to their protein cargo and composition. We identified that a heterogeneous sized population of Helicobacter pylori OMVs entered epithelial cells via macropinocytosis, clathrin, and caveolin-dependent endocytosis. However, smaller OMVs ranging from 20 to 100 nm in size preferentially entered host cells via caveolin-mediated endocytosis. Whereas larger OMVs ranging between 90 and 450 nm in size entered host epithelial cells via macropinocytosis and endocytosis. Most importantly, we identified the previously unknown contribution that OMV size has on determining their protein content, as fewer and less diverse bacterial proteins were contained within small OMVs compared to larger OMVs. Collectively, these findings identify the importance of OMV size in determining the mechanisms of OMV entry into host cells, in addition to regulating their protein cargo, composition, and subsequent immunogenicity. These findings have significant implications in broadening our understanding of the bacterial regulation of virulence determinants and immunogenic proteins associated with OMVs, their role in mediating pathogenesis and in refining the design and development of OMV-based vaccines.</p

table_1_Helicobacter pylori Outer Membrane Vesicle Size Determines Their Mechanisms of Host Cell Entry and Protein Content.PDF

Gram-negative pathogens ubiquitously shed outer membrane vesicles (OMVs) that play a central role in initiating and regulating pathogenesis in the host. Due to their highly inflammatory nature, OMVs are extensively being examined for their role in mediating disease in addition to their applications in innovative vaccines. A key mechanism whereby OMVs mediate inflammation and disease progression is dependent on their ability to enter host cells. Currently, the role of OMV size on determining their mechanism of cellular entry and their protein composition remains unknown. In this study, we examined the mechanisms whereby OMV size regulates their mode of entry into epithelial cells, in addition to their protein cargo and composition. We identified that a heterogeneous sized population of Helicobacter pylori OMVs entered epithelial cells via macropinocytosis, clathrin, and caveolin-dependent endocytosis. However, smaller OMVs ranging from 20 to 100 nm in size preferentially entered host cells via caveolin-mediated endocytosis. Whereas larger OMVs ranging between 90 and 450 nm in size entered host epithelial cells via macropinocytosis and endocytosis. Most importantly, we identified the previously unknown contribution that OMV size has on determining their protein content, as fewer and less diverse bacterial proteins were contained within small OMVs compared to larger OMVs. Collectively, these findings identify the importance of OMV size in determining the mechanisms of OMV entry into host cells, in addition to regulating their protein cargo, composition, and subsequent immunogenicity. These findings have significant implications in broadening our understanding of the bacterial regulation of virulence determinants and immunogenic proteins associated with OMVs, their role in mediating pathogenesis and in refining the design and development of OMV-based vaccines.</p

Human lung BSCs sustain less DNA damage than alveolar progenitor cells.

<p>(A) Multidimensional scaling plot of expression profiles of human lung epithelial subsets from three patients (a 64-y-old male exsmoker, an 83-y-old male exsmoker, and a 53-y-old male current smoker). Distances represent the leading log2-fold change. (B) Gene ontology (GO) terms associated with DNA repair or the cell cycle are significantly up-regulated in BSCs compared to AT2 cells by rotation gene set tests (ROAST) (<i>p</i> < 0.02). Each pair of bars corresponds to a relevant (GO) term. Bars show the proportion of genes associated with the GO term that are more highly expressed in BSCs (orange) or in AT2 cells (blue), as determined by limma’s roast function. (C) Expression of key genes in the DNA repair pathway in BSCs relative to AT2 cells. RPKM, reads per kilobase per million mapped reads. <i>n</i> = 3 patients; a 64-y-old male exsmoker, an 83-y-old male exsmoker, and a 53-y-old male current smoker. Paired <i>t</i> test. (D) Immunofluorescence staining of γH2AX (green) and T1α (purple) in whole human lung fragments that are nonirradiated (control) or 1 or 24 h post irradiation (6 Gy). <i>n</i> = 2 patients (a 60-y-old female never smoker and a 78-y-old female never smoker). Scale bar = 20 μm. The underlying data for panels A, B, and C can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000731#pbio.2000731.s009" target="_blank">S1 Data</a> file.</p

Human lung BSC and AT2 populations have progenitor activity.

<p>(A) Representative images of colonies grown from lung epithelial subsets in a 3-D in vitro assay; a 64-y-old male current smoker. Black scale bar = 500 μm; white scale bar = 100 μm. (B) Representative immunofluorescence staining of keratin 5 (KRT5) (a 57-y-old male exsmoker) and pro-surfactant protein C (proSFTPC) (a 54-y-old female exsmoker) in BSC and AT2 colonies. <i>n</i> = 13 patients; 39–78 y old; current, ex-, and never smokers. Scale bar = 100 μm. (C) Colony-forming capacity of sorted lung epithelial cells; <i>n</i> = 24 patients for proximal lung samples and <i>n</i> = 27 patients for distal lung samples; 21–85 y old; male and female; never, ex-, and current smokers. Student’s <i>t</i> test. (D) Representative images of human BSC and AT2 cell colonies from an exsmoker patient (a 57-y-old male) compared to a never-smoker patient (a 71-y-old female). Scale bar = 500 μm. (E) Linear regression analysis of the number of human BSCs (r² = 0.2) or AT2 colonies (r² = 0.3) versus number of years of patient tobacco smoking. <i>n</i> = 21 patients for basal colonies and <i>n</i> = 23 patients for AT2 colonies, 21–83 y old, male and female. The underlying data for panels C and E can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000731#pbio.2000731.s009" target="_blank">S1 Data</a> file.</p