Ultrastructural relationship of the phagophore with surrounding organelles

<div><p>Phagophore nucleates from a subdomain of the endoplasmic reticulum (ER) termed the omegasome and also makes contact with other organelles such as mitochondria, Golgi complex, plasma membrane and recycling endosomes during its formation. We have used serial block face scanning electron microscopy (SB-EM) and electron tomography (ET) to image phagophore biogenesis in 3 dimensions and to determine the relationship between the phagophore and surrounding organelles at high resolution. ET was performed to confirm whether membrane contact sites (MCSs) are evident between the phagophore and those surrounding organelles. In addition to the known contacts with the ER, we identified MCSs between the phagophore and membranes from putative ER exit sites, late endosomes or lysosomes, the Golgi complex and mitochondria. We also show that one phagophore can have simultaneous MCSs with more than one organelle. Future membrane flux experiments are needed to determine whether membrane contacts also signify lipid translocation.</p></div

Quantitative Proteomics of Extracellular Vesicles Released from Human Monocyte-Derived Macrophages upon β‑Glucan Stimulation

Fungal infections (mycoses) are common diseases of varying severity that cause problems, especially to immunologically compromised people. Fungi express a variety of pathogen-associated molecular patterns on their surface including β-glucans, which are important immunostimulatory components of fungal cell walls. During stimulatory conditions of infection and colonization, besides intensive intracellular response, human cells actively communicate on the intercellular level by secreting proteins and other biomolecules with several mechanisms. Vesicular secretion remains one of the most important paths for the proteins to exit the cell. Here, we have used high-throughput quantitative proteomics combined with bioinformatics to characterize and quantify vesicle-mediated protein release from β-glucan-stimulated human macrophages differentiated in vitro from primary blood monocytes. We show that β-glucan stimulation induces vesicle-mediated protein secretion. Proteomic study identified 540 distinct proteins from the vesicles, and the identified proteins show a proteomic signature characteristic for their cellular origin. Importantly, we identified several receptors, including cation-dependent mannose-6-phosphate receptor, macrophage scavenger receptor, and P2X7 receptor, that have not been identified from vesicles before. Proteomic data together with detailed pathway and network analysis showed that integrins and their cytoplasmic cargo proteins are highly abundant in extracellular vesicles released upon β-glucan stimulation. In conclusion, the present data provides a solid basis for further studies on the functional role of vesicular protein secretion upon fungal infection

Effect on the expression of selected proteins of Gimap6 ablation in vitro from GIMAP6^fl/flERT2Cre CD4⁺ T cells.

<p>Panel A) Enriched CD4⁺ cell fractions were prepared from spleens of ERT2Cre or GIMAP6^fl/flERT2Cre mice. The cells were maintained in vitro for five days in the presence or absence of 200 nM 4HT. Cell lysates were prepared and analysed by SDS polyacrylamide gel electrophoresis with the indicated antibodies. Panel B) Enriched CD4⁺ cell fractions were prepared from spleens of GIMAP6^fl/flERT2Cre mice. The cells were maintained in vitro in the presence or absence of 4HT for the indicated times before preparation of cell lysates and western analysis with the indicated antibodies. The electrophoretic mobilities of marker proteins are indicated. Panel C) Enriched CD4⁺ cell fractions were prepared and incubated with or without 200 nM 4HT for 4 days. TBK1 inhibitor BX795 (1 μM) was then added to the cells as indicated and incubation continued for 6h. Cell lysates were prepared and proteins analysed by western blotting. In all panels, the electrophoretic mobility of marker proteins is indicated. All results were confirmed in at least two experiments.</p

GIMAP6 relocalises to autophagosomes on cell starvation.

<p>HEK293 cells stably expressing an N-terminally myc-tagged variant of mouse GIMAP6 were either left untreated or were treated with starvation buffer for 90 minutes. They were then fixed and stained with either (A) a mixture of rat monoclonal antibodies to mouse GIMAP6 and a rabbit polyclonal antibody to MAP1LC3B, or (B) the same mixture of rat monoclonal antibodies to GIMAP6 and a rabbit polyclonal antibody to SQSTM1, each followed by fluorochrome-conjugated secondary antibodies. GIMAP6 is shown false-coloured green and MAP1LC3B or SQSTM1, red. Scale bar represents 10 μm.</p

Activation of CD4⁺ splenocytes from GIMAP6^fl/flCD2Cre mice is comparable with those from GIMAP6^fl/fl mice.

<p>A) CD4⁺ enriched naïve splenocytes from GIMAP6^fl/fl and GIMAP6^fl/flCD2Cre mice were activated by maintenance on anti-CD3 antibody-coated plates in medium containing anti-CD28 and IL2 or were maintained in medium without activation. After 72h, cells were stained to measure expression of CD25, CD62L and CD44 to assess the degree of activation. On the histograms dotted lines indicate naïve cells and solid lines, activated cells. B) The median fluorescence intensities of the individual stains from three mice in each group is presented graphically. Unpaired 2-tailed Student’s t-test P values ns p>0.05 ***p<0.001; ****p<0.0001. The experiment was performed twice.</p

The reduced number of peripheral CD4⁺ T cells in GIMAP6^fl/flCD2Cre mice reflects an increase in apoptosis.

<p>A) Splenocytes isolated from GIMAP6^fl/fl and GIMAP6^fl/flCD2Cre animals were stained with fluorescent antibodies to CD4 and CD24. The left-hand panel shows the distribution of CD24 staining on CD4⁺ cells from single mice of the two genotypes (GIMAP6^fl/fl–dotted line; GIMAP6^fl/flCD2Cre–solid line). The right-hand panel shows the median fluorescence intensity of CD24 staining of CD4⁺ cells. n = 4; **P<0.01. B) Freshly isolated splenocytes were stained with an APC-conjugated antibody to CD4 and PE- conjugated annexin V. The cells were then washed and stained with DAPI before flow analysis. The two dot-plots show typical staining patterns for a control (C) and knockout (KO) animal. The quadrants are as follows: Q1 –annexin V^- DAPI⁺; Q2 –annexin V⁺ DAPI⁺; Q3 –annexin V⁺ DAPI^-; Q4 –annexin V^- DAPI^-. The right-hand panel summarises the percentage of annexin V⁺ cells as a percentage of total DAPI^- CD4⁺ T cells for five animals in each group. ns P>0.05; ** P<0.01. C) CD4⁺ enriched naive splenocytes from GIMAP6^fl/fl and GIMAP6^fl/flCD2Cre mice were activated by maintenance on anti-CD3 antibody-coated plates in medium containing anti-CD28 and IL2 or were maintained in medium without activation as described in the Materials and Methods section. After 24 h cells were harvested and stained as in (B). The percentage of CD4⁺ T cells in each quadrant is indicated. n = 3 for each group; ns P>0.05; ** P<0.01.</p

CD4⁺ cells from GIMAP6^fl/flCD2Cre mice show an increased size and a small increase in memory-like characteristics compared with GIMAP6 ^fl/fl mice.

<p>Panel A—The diameters of CD4⁺, CD8⁺ and B cell populations isolated from spleens of GIMAP6^fl/fl and GIMAP6^fl/flCD2Cre animals were measured using an electronic counter (CASY® Cell Counter + Analyser System—Schärfe System GMBH). Panel B–total splenic or lymph node-derived cells were stained for CD4 and then with antibodies to CD62L and CD44. Naïve cells were identified as CD62L⁺CD44^-and memory cells as CD62L^-CD44⁺. Graphs show each population expressed as a percentage of CD4⁺ cells. n = 6. P values as follows: **P<0.001 **** P<0.0001; n = 6 for each group (unpaired 2-tailed Student’s t test).</p

CD8⁺ cells from GIMAP6 ^fl/fl CD2Cre mice show a decrease in memory-like characteristics compared with GIMAP6 ^fl/fl mice.

<p>Total splenic or lymph node-derived cells were stained for CD8 and then with an antibody to CD44. The extent of staining of the CD8⁺ cells by CD44 for each group of cells is represented by histograms. For graphical analysis, CD44⁺ cells were gated as indicated from the histograms and plotted as a percentage of total CD8⁺ cells. n = 6. P values *p<0.05, **p<0.01.</p

CD4 and CD8 cell analysis in GIMAP6^fl/flCD2Cre mice.

<p>Total cell suspensions were prepared from thymi, spleens and lymph nodes, were stained for CD4 and CD8 and analysed by flow cytometry. Nucleated cells were gated on the basis of FSC versus SSC, live versus dead and then for CD4 and CD8. Left-hand panels for each tissue show the gated cells. Nomenclature on the plots is: CD4 SP–single-positive CD4 cells; CD8 –single-positive CD8 cells; DP–cells positive for CD4 and CD8; DN–cells negative for both CD4 and CD8; CD4—CD4 cells; CD8 –CD8 cells. In each case, the left-most plot is from GIMAP6^fl/fl mice and the right hand plot from GIMAP6^fl/flCD2Cre mice. The graphs to the right show the calculated percentage of each cell population. GIMAP6^fl/fl mice are indicated by (C) and GIMAP6^fl/flCD2Cre by (KO). P values as follows: ns P>0.05; **** P<0.0001. n = 5–7 (unpaired 2-tailed Student’s t test).</p

CD4⁺ T cells from GIMAP6^fl/flCD2Cre mice have increased cytoplasmic area, mitochondrial volume fraction and number of autophagic vacuoles.

<p>Electron micrographic images derived from CD4⁺ T cells were prepared and analysed as described in the Materials and Methods section. Panel A—Electron micrographs showing examples of CD4 cells purified from GIMAP6^fl/fl mice (micrographs a and b) and GIMAP6^fl/flCD2Cre mice (micrographs c and d). The arrows on micrographs c and d indicate autophagosomes. Micrograph e shows a higher power of a cell from a GIMAP6^fl/flCD2Cre animal to illustrate mitochondria (m) and a mitochondrial spheroid (*). Panel B—Calculated mitochondrial/cytoplasmic volume ratios (n = 160 cell profiles for cells from GIMAP6^fl/fl mice (C) and n = 158 cell profiles for GIMAP6^fl/flCD2Cre mice (KO) Panel C–Measured cytoplasmic area per nucleated cell profile (n = 78 nucleated cell profiles from GIMAP6^fl/fl mice (C) and n = 73 nucleated cell profiles for GIMAP6^fl/flCD2Cre mice (KO). For Panels B and C ****p<0.0001 (Mann Whitney U test). Panel D–Tabulation of the total analysed cytoplasmic area and the number of autophagic vacuoles observed from CD4⁺ T cells from GIMAP6^fl/fl mice (160 cell profiles) and GIMAP6^fl/flCD2Cre mice (158 cell profiles).</p