Journal: Scientific Reports

Article Title: The Atlantic Cod MHC I compartment has the properties needed for cross-presentation in the absence of MHC II

doi: 10.1038/s41598-024-76225-z

Figure Lengend Snippet: Atlantic cod MHC I localizes to late endosomes/lysosomes in primary hepatocytes and leukocytes. ( A ) Representative image of an Atlantic cod primary hepatocyte transiently transfected with Atlantic cod MHC I-GFP. Cells were stained using lysotracker red and imaged using an Olympus SpinSR SoRA microscope. Scale bar: 10 μm. Magnification of boxed area is shown as inset. The yellow lines illustrate regions selected for fluorescence intensity profile analysis. ( B ) The graph shows the normalized fluorescence intensity profiles relative to MHC I-GFP and lysotracker represented as mean ± SEM ( n = 90 vesicles from 17 cells from three fish specimens). ( C ) Representative image of Atlantic cod primary hepatocyte transiently co-transfected with MHC I-GFP and mApple-Rab7A. Cells were imaged using an Olympus SpinSR SoRA microscope. Scale bar: 10 μm. Magnification of boxed area is shown as inset. The yellow lines illustrate regions selected for fluorescence intensity profile analysis. ( D ) The graph shows the normalized fluorescence intensity profiles relative to MHC I-GFP and mApple-Rab7A represented as mean ± SEM ( n = 158 vesicles from 24 cells from two fish specimens). ( E ) The graph represents colocalization analysis between Atlantic cod MHC I-GFP and lysotracker or mApple-Rab7A. Colocalization was analysed by calculating Manders’ coefficient in ImageJ. Scatter plot shows the mean ± SEM from n ≥ 18 cells (from at least two fish, colour coded for each experimental repeat). Dots represent individual measurements and are colour coded according to experimental repeat. ( F ) Representative image of an Atlantic cod primary leukocyte transiently transfected with MHC I-GFP and stained using lysotracker red. Cells were imaged using an Olympus SpinSR SoRA microscope. Scale bar: 10 μm. Magnification of boxed area is shown as inset. The yellow lines illustrate regions selected for fluorescence intensity profile analysis. ( G ) The graph shows the normalized fluorescence intensity profiles relative to MHC IGFP and lysotracker represented as mean ( n = 79 vesicles from 8 cells from one fish). ( H ) Representative image of an Atlantic cod primary leukocyte transiently co-transfected with MHC IGFP and mApple-Rab7A. Cells were imaged using an Olympus SpinSR SoRA microscope. Scale bar: 10 μm. Magnification of boxed area is shown as inset. The yellow lines illustrate regions selected for fluorescence intensity profile analysis. The purple asterisk indicates a large Rab7A positive vesicle, with three intralumenal MHC I-positive vesicles. ( I ) The graph shows the normalized fluorescence intensity profiles relative to MHC IGFP and mApple-Rab7A represented as mean ( n = 99 vesicles from 20 cells from one fish). ( J ) Imaris isosurface 3D rendering of the large mApple-Rab7A-positive vesicle containing MHC I-GFP intracellular vesicles from the image in ( H ) (marked with the purple asterisk). The yellow line illustrates the region used to generate the fluorescence intensity plot shown at the right. ( K ) The graph shows the normalized fluorescence intensity profile relative to MHC IGFP and mApple-Rab7A from the vesicle in ( J ).

Article Snippet: Imaris isosurface 3D rendering is shown for the infected cell (right) including magnifications of the insets.

Techniques: Transfection, Staining, Microscopy, Fluorescence

Journal: Scientific Reports

Article Title: The Atlantic Cod MHC I compartment has the properties needed for cross-presentation in the absence of MHC II

doi: 10.1038/s41598-024-76225-z

Figure Lengend Snippet: Francisella noatunensis ssp. noatunensis localizes to and escapes cMIC. ( A ) Representative images of ACL cells transiently co-transfected with MHC I-GFP and BFP-Rab7A before and after infection with Francisella noatunensis ssp . noatunensis ( Fnn ) stable expressing mCherry. Cells were imaged using a Zeiss LSM880 Fast AiryScan microscope. Scale bar: 10 μm. Imaris isosurface 3D rendering is shown for the infected cell (right) including magnifications of the insets. Insect 1 illustrates an intact and non-infected endosome. Insect 2 shows Fnn -containing endosomes that are opened (as visible from the top view), with the internalized bacteria (in blue) protruding from these endosomes (as visible from the side view), thus escaping from the infected endosomes. Scale bar: 10 μm. ( B ) Magnification of the boxed area from ( A ). Scale bar: 1 μm. The graphs show the normalized fluorescence intensity profiles along the two yellow lines (plot 1 and 2) for Fnn -mCherry, MHC I-GFP and BFP-Rab7A. ( C ) The graph represents colocalization analysis between MHC I-GFP and BFP-Rab7A in control or Fnn- mCherry infected ACL cells. Colocalization was analysed by calculating Manders’ coefficient in ImageJ. Scatter plot shows the mean ± SEM from three independent experiments, colour coded for each experimental repeat ( n ≥ 29 cells). Dots represent individual measurements and are colour coded according to experimental repeat.

Article Snippet: Imaris isosurface 3D rendering is shown for the infected cell (right) including magnifications of the insets.

Techniques: Transfection, Infection, Expressing, Microscopy, Bacteria, Fluorescence, Control

Journal: Scientific Reports

Article Title: The Atlantic Cod MHC I compartment has the properties needed for cross-presentation in the absence of MHC II

doi: 10.1038/s41598-024-76225-z

Figure Lengend Snippet: Atlantic cod MHC I co-localize with LRP1 in Atlantic cod hepatocytes and leukocytes. ( A ) Representative image of an Atlantic cod primary hepatocyte transiently transfected with MHC IGFP, fixed, and stained with antibody against LRP1. Cells were imaged using a Zeiss LSM880 Fast AiryScan microscope. Scale bar: 10 μm. Magnification of boxed areas are shown below. The yellow lines in the magnified images illustrate regions selected for fluorescence intensity profile analysis. (B) The graph shows the normalized fluorescence intensity profiles relative to MHC IGFP and LRP1 represented as mean ± SEM ( n = 164 vesicles from 21 cells from three fish specimens). ( C ) Representative image of an Atlantic cod primary leukocyte transiently transfected with MHC IGFP, fixed, and stained with antibody against LRP1. Cells were imaged using a Zeiss LSM880 Fast AiryScan microscope. Scale bar: 5 μm. Magnification of boxed areas are shown below. The yellow lines in the magnified images illustrate regions selected for fluorescence intensity profile analysis. Imaris isosurface 3D rendering of the boxed areas are shown to the right. (D) The graph shows the normalized fluorescence intensity profiles relative to MHC I-GFP and LRP1 represented as mean ( n = 81 vesicles from 11 cells from one fish). ( E ) Representative image of Atlantic cod primary leukocytes transiently transfected with GFP-Rab5, fixed, and stained with antibody against LRP1. Cells were imaged using a Zeiss LSM880 Fast AiryScan microscope. Scale bar: 5 μm. Magnification of the boxed area is shown below. Imaris isosurface 3D rendering of the boxed area is shown to the right.

Article Snippet: Imaris isosurface 3D rendering is shown for the infected cell (right) including magnifications of the insets.

Techniques: Transfection, Staining, Microscopy, Fluorescence

Journal: The EMBO Journal

Article Title: Longitudinal autophagy profiling of the mammalian brain reveals sustained mitophagy throughout healthy aging

doi: 10.1038/s44318-024-00241-y

Figure Lengend Snippet: ( A ) Schematic of a parasagittal brain section highlighting the cerebellum and Purkinje cells analyzed. ( B ) Mitophagy profiling in Purkinje neurons. Confocal photomicrographs from mito -QC brain sections showing representative instances of mitophagy throughout aging in cerebellar Purkinje neurons. Arrowheads indicate events of mitophagy. Scale bar = 20 µm. GCL granule cell layer, PCL Purkinje cell layer, ML molecular layer. ( C ) Macroautophagy profiling in Purkinje neurons. Confocal photomicrographs from auto -QC brain sections showing representative instances of nonselective macroautophagy throughout aging in cerebellar Purkinje neurons. Arrowheads indicate events of autophagy. Scale bar = 20 µm. ( D ) Quantitative analysis of Purkinje cells in mito -QC mice reveals a robust age-dependent increase in the number of acidified mitolysosomes in vivo, in addition to increases in mean mitolysosome size throughout time. One-way ANOVA with Bonferroni post hoc. **** P < 0.0001. n = 47. ( E ) Quantitative analysis of Purkinje cells in auto -QC mice demonstrates the numbers of acidified autolysosomes remain constant throughout aging and do not change in size. One-way ANOVA with Bonferroni post hoc. ns = not significant; P = > 0.9999. n = 27. ( F ) Identification of differentially acidified autolysosomes formed throughout aging within Purkinje cells in vivo. Immunohistochemical analysis of endolysosomal network using LAMP1 antibody identifies distinct lysosomal subpopulations. Shown is a 3D isosurface render generated using IMARIS, alongside confocal photomicrograph insets. Arrowheads indicate LAMP1-mCherry-positive (GFP-negative) colocalization, arrows indicate LAMP1-mCherry-GFP colocalization. Lysosomes are predominantly enriched at the pre-dendritic zones. Scale bar = 5 µm. ( G ) Quantitative analysis reveals an age-dependent increase in the number and mean size of differentially acidified lysosomes (LAMP1-mCherry-GFP-positive structures) over time. Although the overall number of LAMP1-positive endolysosomal structures remains constant throughout mammalian life, this compartment expands in size as a function of age. One-way ANOVA with Bonferroni post hoc. ns = not significant; P > 0.9999, *** P = 0.0002 and **** P = 0.0001. n = 27. ( H ) In vivo transmission electron microscopy (TEM) imaging of young and geriatric mito -QC mice. Scale bar = 1 µm. Quantitation of TEM images reveal an age-dependent increase in lysosomal (L) area, consistent with LAMP1 data in ( G ), with further TEM analysis demonstrating selective alterations in lysosomal area occurs at the somatodendritic zones of Purkinje neurons, while no change occurs at the axonal aspect. Mitochondria (Mit), Lipid droplet (LD). Student’s unpaired two-tailed t test. ** P = 0.0019 and 0.0017; ns = not significant; P = 0.1225. n = 5. Box plots extend from the 25th to the 75th percentiles, with a median line positioned inside the box. Whiskers denote the minimum and maximum values. .

Article Snippet: Shown is a 3D isosurface render generated using IMARIS, alongside confocal photomicrograph insets.

Techniques: In Vivo, Immunohistochemical staining, Generated, Transmission Assay, Electron Microscopy, Imaging, Quantitation Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: Longitudinal autophagy profiling of the mammalian brain reveals sustained mitophagy throughout healthy aging

doi: 10.1038/s44318-024-00241-y

Figure Lengend Snippet: ( A ) Simplified schematic of A9 and A10 DA circuits. Both A9 and A10 neurons reside in the ventral mesencephalon (VM) and have distinct targets within the brain. A9 DA neurons project to the dorsolateral striatum, forming the nigrostriatal pathway that modulates voluntary locomotion, whereas A10 DA neurons project to the ventromedial striatum and forebrain to control reward, motivation, and aversion. ( B , C ) Mitophagy in aging A9 DA neurons. 3D Isosurface renders of young and geriatric mito -QC A9 dopaminergic neurons (scale bar = 5 µm) and representative confocal images of mitophagy events throughout aging, scale bar = 20 µm. Quantitative analysis reveals increased mitophagy levels as a function of age. One-way ANOVA with Bonferroni post hoc. ** P = 0.0014. n = 43. ( D , E ) Macroautophagy in aging A9 DA neurons. Representative maximum projections of autophagy events throughout aging in A9 dopaminergic neurons (scale bar = 20 µm). Quantitative analysis of autophagy in A9 dopaminergic neurons shows no age-dependent alterations in autophagy levels. One-way ANOVA with Bonferroni post hoc. ns = not significant; P > 0.9999. n = 19. ( F , G ) Mitophagy in aging A10 DA neurons. Representative maximum projections of mitophagy events throughout aging in A10 dopaminergic neurons (scale bars = 20 µm). Quantitative analysis of mitophagy in A10 dopaminergic neurons reveals no change between young and geriatric mice. Some fluctuations in mitophagy levels were detected with a modest increase at 15 months. One-way ANOVA with Bonferroni post hoc. ns = not significant; P > 0.9999, * P = 0.024. n = 44. ( H , I ) Macroautophagy in aging A10 DA neurons. Isosurface renders of young and geriatric auto -QC A10 dopaminergic neurons (scale bar = 5 µm). Representative images of autophagy events throughout aging in A10 dopaminergic neurons (scale bar = 20 µm). Quantitative analysis of autophagy in A10 dopaminergic neurons reveals decreased levels of autophagy as a function of age. One-way ANOVA with Bonferroni post hoc. ** P = 0.0076. n = 26. Box plots extend from the 25th to the 75th percentiles, with a median line positioned inside the box. Whiskers denote the minimum and maximum values. .

Article Snippet: Shown is a 3D isosurface render generated using IMARIS, alongside confocal photomicrograph insets.

Techniques: Control