EMBO/soda-nel huggingface dataset:
The Spatiotemporal Dynamics and Membranous Features of the Plasmodium Liver Stage Tubovesicular Network (2014)
Figure 1

The multiple features of the Plasmodium berghei liver stage tubovesicular network (LS‐TVN).

A) Live confocal imaging of liver stage P. berghei expressing UIS4-mCherry and cytoplasmic GFP reveals different morphologies of the LS-TVN: elongated membrane clusters (left), vesicles in the host cell cytoplasm (center), and a thin tubule protruding from the PVM (right). Live imaging was performed 20?h after infection of hepatoma cells. Features are marked with white arrowheads. B) The UIS4- and IBIS1-positive tubules rapidly extend from and retract to the PVM. Confocal time-lapse imaging of a UIS4-mCherry-expressing parasite at 22?h (top) and an IBIS1-mCherry-expressing parasite at 26?h (bottom) after infection of hepatoma cells. Tubules are indicated with a yellow arrow, and the time is shown in minutes and seconds. C) Imaging of IBIS1-mCherry-expressing P. berghei reveals similar features of the LS-TVN, i.e. elongated membrane cluster (left), vesicles in the host cell cytoplasm (center), and a membrane tubule (right). Infected cells were fixed 26?h post-infection and imaged by epifluorescence microscopy. Features are indicated with a yellow arrowhead. Scale bars, 10?µm. D) Quantification of the distinct UIS4- and IBIS1-positive membranous structures in fixed infected hepatoma cells at three different time points: 6?h after infection (the period of sporozoite transformation into exo-erythrocytic forms), 24?h after infection (onset of parasite replication and growth), and 48?h after infection (mature liver stages). Values are shown as mean (±?SD) from three separate wells (n?=?100 per well) of one experiment and are representative of two experimental repetitions.
The Spatiotemporal Dynamics and Membranous Features of the Plasmodium Liver Stage Tubovesicular Network (2014)
Figure 2

The morphology of the UIS4-positive LS‐TVN.

Hepatoma cells infected with Plasmodium berghei ANKA parasites were fixed and stained with anti-UIS4 and anti-HSP70 (A and C). A) Some UIS4-positive vesicles are tethered to the PVM as beads-on-a-string. Infected cell fixed 24?h post-infection. B) The UIS4-mCherry features we detect in live imaging of UIS4-mCherry-expressing P. berghei, i.e. elongated membrane clusters (left), vesicles (center) and tubules (right) are also apparent in fixed wildtype parasites stained with anti-UIS4 antibody. Infected cells 24?h post-infection. Scale bars, 10?µm. C) Often the LS-TVN is a composite of vesicles, tubules and membrane clusters. Infected cell 22?h post-infection. D) The LS-TVN detectable in primary hepatocytes infected with UIS4-mCherry-expressing P. berghei also consists of elongated membrane clusters (yellow arrowhead), vesicles (white arrow) and tubules (yellow arrow). Infected cell 18?h post-infection. E) Dynamic tubules (yellow arrow) are detectable with UIS4-mCherry in infected primary hepatocytes 24?h post-infection. Location of parasite is marked with a yellow asterisk. Scale bars, 10?µm.
The Spatiotemporal Dynamics and Membranous Features of the Plasmodium Liver Stage Tubovesicular Network (2014)
Figure 3

Differential labeling of tubules with UIS4 or IBIS1.

Detection of UIS4 and IBIS1 in hepatoma cells infected with IBIS1-3XF transgenic Plasmodium berghei parasites. Cells were fixed 2426?h post-infection and stained with antibodies against UIS4 (green) and the FLAG-tag of IBIS1 (red). A) Confocal microscopy images show a tubule (upper panel) labeled with UIS4 and IBIS1 (white arrowheads), an elongated membrane cluster (middle panel) in which both proteins are present, and vesicles (bottom panel) that are often labeled with both UIS4 and IBIS1 (yellow arrow) and frequently labeled with only one protein (yellow arrowheads). B) Epifluorescence microscopy of IBIS1 and UIS4 reveals that while most tubules are labeled with both proteins as above (white arrowhead), some are labeled exclusively with UIS4 (upper panel) or IBIS1 (bottom panel). Single-labeled tubules were highlighted with white arrows and their frequency in infected cells with tubules is indicated in percent (±?SD), which was calculated from two separate wells 30?h post-infection (n?>?150 infected cells per well).
The Spatiotemporal Dynamics and Membranous Features of the Plasmodium Liver Stage Tubovesicular Network (2014)
Figure 4

The velocities of the UIS4‐ and IBIS1‐positive features of the LS‐TVN.

A–C) Time-lapse imaging of hepatoma cells infected with UIS4-mCherry-expressing Plasmodium berghei was used to determine the velocities of the UIS4-positive features, e.g., a tubule (A), elongated membrane cluster (B) and vesicle (C) were tracked over time. Measured speeds of the dynamic features indicated with white arrows are depicted graphically to the right. Scale bars, 10?µm. D) The velocities of several UIS4- or IBIS1-positive features [tubules (left), elongated membrane clusters (center) and vesicles (right)] were measured over time, and the peak velocity of each was plotted. Plotted data was derived from multiple imaged infected cells: For IBIS1-positive tubules data is derived from three infected cells and for UIS4-positive tubules from 13 infected cells. Elongated membrane cluster speeds were analyzed in at least five infected cells for both UIS4 and IBIS1, and vesicle speeds were analyzed in at least 12 different cells for both protein markers.
The Spatiotemporal Dynamics and Membranous Features of the Plasmodium Liver Stage Tubovesicular Network (2014)
Figure 5

Movement of the LS‐TVN‐associated tubules is dependent on the microtubule network.

A) Plasmodium LS-TVN features persist without an intact host cytoskeleton. Infected cells were treated with nocodazole or cytochalasin D or both prior to fixation. Values are shown as mean (±?SD) from three wells (n?=?100 per well) *p < 0.05 (unpaired two-tailed Student's t-test). B) Cells infected with UIS4-mCherry-expressing parasites were treated with nocodazole for 1.5?h (top) or cytochalasin D for 30 min (bottom) and imaged by confocal microscopy. Imaging occurred 29 and 24 h post-infection, respectively. UIS4-positive tubules, static (top) and dynamic (bottom), are indicated with white arrowheads. Displayed cells are representative of five imaged parasites from at least two different experiments. C) No colocalization between host microtubules and UIS4-positive tubules is detectable. Infected cells were fixed 25 h post-infection and stained with antibodies against UIS4 and Tubulin. Scale bars, 10 µm.
The Spatiotemporal Dynamics and Membranous Features of the Plasmodium Liver Stage Tubovesicular Network (2014)
Figure 6

FRAP analysis reveals that the diffusion of UIS4‐mCherry is restricted in most LS‐TVN extended membrane clusters, but free within the PVM.

Hepatoma cells infected with UIS4-mCherry transgenic parasites were subjected to FRAP analysis. The UIS4-mCherry (red) and cytoplasmic GFP of the parasite (green) are shown before bleaching (pre-bleach) and after bleaching (post-bleach) with a bleach pulse at the location indicated by an arrow. The fluorescence intensity was measured over time within the regions indicated in the images to the right of the graphs. The bleached area was measured along with a disconnected region to control for general bleaching during the course of imaging. A and B) The bleach was applied to elongated membrane clusters of the LS-TVN approximately 27?h post-infection. The UIS4-mCherry signal typically did not recover in the bleached area (A), although it was found to recover in some elongated membrane clusters (B). C) When the bleach was applied to the PVM at 25?h post-infection, the signal recovered rapidly in the bleached region. Upon the application of additional bleach pulses in the same area, the bleached area (region #1 measured graphically) repeatedly recovered, and the remaining PVM-associated UIS4-mCherry signal depleted (region #2) although the LS-TVN-associated signal remained (region #3). When a region of the LS-TVN was subsequently bleached (yellow arrow, region #4), no recovery was observed. The times are shown in minutes and seconds. Scale bars, 10?µm.
The Spatiotemporal Dynamics and Membranous Features of the Plasmodium Liver Stage Tubovesicular Network (2014)
Figure 7

Visualization of UIS4‐positive tubules in infected hepatoma cells by correlative light and electron microscopy (CLEM).

CLEM was performed with hepatoma cells 24?h after infection with UIS4-mCherry transgenic parasites. Dynamic processes of UIS4 were recorded by fluorescence microscopy and fixed for TEM while under observation. A and B) A phase-contrast image containing the infected cell of interest (A) was used for comparison to the EM grid after fixation and preparation of the cells for TEM (B). Green outlining of the cells in the phase-contrast image was used as a tool for comparison and alignment. The insets defined in white are shown in the right panels merged with the fluorescent image of the UIS4-mCherry signal acquired immediately prior to fixation. Scale bars, 10?µm. C) Detection of the UIS4-mCherry signal by widefield fluorescence microscopy in the cell of interest immediately prior to fixation. The location of the parasite is marked with an asterisk, and UIS4-positive features are highlighted with arrowheads. D) Thirty-six TEM sections of this sample were prepared and imaged (Movies S4 and S5). Features corresponding to the UIS4-positive tubule (white arrowhead) were found in sections 1016 and to the UIS4-positive signal adjacent to the PVM (yellow arrowhead) were found in Sections 1629. Sections 11 (top) and 20 (bottom) are displayed to highlight these two features. Scale bars, 1?µm.
The Spatiotemporal Dynamics and Membranous Features of the Plasmodium Liver Stage Tubovesicular Network (2014)
Figure 8

Late endocytic compartments associate with elongated membrane clusters.

A) Hepatoma cells infected with UIS4-mCherry-expressing Plasmodium berghei parasites were fixed 22?h post-infection and stained with antibodies against host cell protein markers of distinct organelles: anti-EEA1 to label early endosomes, anti-CD63 and anti-LAMP1 to label late endosomes and lysosomes, anti-Calnexin to label the endoplasmic reticulum, and anti-Golgin97 to label the Golgi. In all samples, either anti-mCherry or anti-UIS4 was used to amplify the UIS4-mCherry signal. Imaging was carried out by epifluorescence microscopy. In the right panels, fluorescence is merged, and selected regions with areas of interest are shown enlarged. At least 20 infected cells possessing LS-TVN features were visually examined for each condition, and representative images of UIS4-positive elongated membrane clusters (left) and UIS4-positive tubules (right, and indicated with yellow arrows) are displayed. Scale bars, 10?µm. B) Profiling of the red (UIS4) and green (CD63, left or LAMP-1, right) signals along the indicated line within the insets from the images in (A) above.
The Spatiotemporal Dynamics and Membranous Features of the Plasmodium Liver Stage Tubovesicular Network (2014)
Figure 9

LC3 colocalizes with UIS4 on the Plasmodium liver stage PVM and TVN

Hepatoma cells infected with Plasmodium berghei expressing UIS4‐mCherry (top) or IBIS1‐mCherry (bottom) were fixed 22 h or 48 h post‐infection, respectively, and stained with anti‐LC3 to label autophagic membranes. In the right panels, fluorescence is merged, and selected areas of interest are shown enlarged. Scale bars, 10 µm.
Architecture of the active post-translational Sec translocon (2020)
Figure 4

Figure 4. Interaction between Sec62 and Sec63

A. Domain organization of Sec63. The sequence of the acidic patch on the C-terminus - divided into C1 and C2 regions - is shown in the lower panel. The two phosphorylated threonines (T652 and T654) are marked in the C2 region. The J domain of Sec63 is indicated by "J".

B. Pull-down assay of Sec62 domain with different biotin-labeled Sec63 C-terminal peptides. Sec63-C2P denotes di-phosphorylated Sec63-C2.

C. ITC-thermogram obtained from ITC after titration of Sec62 domain with increasing amounts of Sec63-C2P.

D. Difference in HDX between the Sec62 domain in complex with Sec63-C2P and the Sec62 domain alone. The graph depicts the summed means of differences ± 3 SD (n = 3 technical replicates) for each peptide plotted according to the midpoint of each peptide.

E. Crystal structure of Sec62 domain color coded according the difference in HDX as shown in (D). The critical residues constituting the interaction interface between Sec63-C2P and Sec62 are labelled.

F. Pull-down assay of the Sec62 domain and a mutant in the interaction interface (Sec62R51E) with biotin-labeled Sec63-C2P.
Temporal order and precision of complex stress responses in individual bacteria (2019)
Figure 2

Figure 2. Hard limits constrain the timing variability of individual promoters from above and below. A. Single-cell gene expression time traces from the dnaK promoter in response to TET stress, normalized to the median full response. Response times were determined as the time point at which a threshold expression level was reached (see main text and Methods). This threshold was low enough so that most cells exceed it and high enough to avoid false positives due to the low signal-to-noise ratio at time point zero. Brown line: median of all cells. Time traces are from one microcolony. B. Histogram of the response times for the dnaK promoter with mean µt = 1.87 h and standard deviation σt = 0.37 h, and fit of an Erlang distribution with shape parameter n = 37 (black line; see text and Methods). Response times are from two microcolonies. C. Standard deviation σt versus mean response time µt for 23 different promoters (Table EV1) in three antibiotic stress conditions (TMP, TET, and NIT). The standard deviation of the response time σt grows with the mean response time µt and does not fall below a 'precision limit' (dashed line) that increases linearly with a slope ~0.165. The dotted line indicates the upper bound to timing variability where σt = µt, see text. The promoter dnaK under TET has low timing variability, whereas the promoters recA, fpr, osmC, and wrbA under TMP stress have high timing variability. The response time mean and standard deviation are from subsampling of descendants of single cells that were present at the time of stress addition (Methods). Subsampling for each promoter was done from at least 2 microcolonies and the descendants of at least 17 individual cells present at the time of stress addition. D. Schematic of a molecular chain of n events triggered by stress and resulting in measurable fluorescence, according to the statistical kinetics model applied here. The gray arrows indicate possible deviations from a linear chain of events, like feedback, reversibility, or branching, which are also captured in the model. E. Standard deviation σt versus mean response time µt for the LlacO-1 promoter induced with different IPTG concentrations. Note that all cells crossed the defined threshold under the tested IPTG concentrations. The dashed and dotted lines are the same as in C.
Temporal order and precision of complex stress responses in individual bacteria (2019)
Figure 4

Figure 4. Early oxidative stress response under NIT is precisely timed and precedes the SOS response in every single cell. A. Normalized expression from the promoters ybjC and recA in response to NIT stress, measured at the population level using GFP reporters (Zaslaver et al, 2006); 4 μg/mL NIT was added at time zero. Data are taken from (Mitosch et al, 2017). The oxidative stress promoter ybjC clearly precedes the SOS response promoter recA, when measured at the population level (Mitosch et al, 2017). Error bars show standard deviation of three replicate experiments measured on different days. B. Normalized ybjC and recA expression over time in response to the addition of 4 μg/mL NIT at time zero in single cells from one microcolony. Dashed line: threshold used to determine response times (Methods). Lower panels, upper row: Dual-color images of the oxidative stress reporter ybjC (green) and the SOS reporter recA (red) in single cells at 4 different time points after NIT addition in one microcolony. Note that the ybjC signal (green) is stronger at t = 2h, whereas the recA signal (red) is only apparent at t = 4h and 6h. Lower row: Constitutively expressed mCherry used for segmentation. C. Response times for ybjC and recA from individual cells with dual reporters (Figure 3), combined from two microcolonies. All response times for recA are higher than for ybjC and response times for both reporters are strongly correlated (r = 0.74 ± 0.04, p = 3.8 · 10-6). The error of the correlation coefficient is from subsampling of descendants of single cells that were present at the time of NIT addition (Methods).
Temporal order and precision of complex stress responses in individual bacteria (2019)
Figure 5

Figure 5. Early acid stress under TMP is not part of the causal chain of molecular events leading to DNA damage. A. Expression from the gadB and recA promoters, measured at the population level using GFP reporters (Zaslaver et al, 2006) over time; 0.5 μg/mL TMP was added at time zero. The acid stress response (gadB) clearly precedes the SOS response (recA). Error bars show standard deviation of three replicate experiments measured on different days. B. Normalized expression of gadB and recA expression over time in individual cells responding to the addition of 0.5 μg/mL TMP at time zero shown for one microcolony. Dashed line: threshold used to determine response times (Methods). Lower panels, upper row: Dual-color images of gadB (blue) and recA (red) in single cells at 4 different time points. Most cells are predominantly either red or blue, i.e. acid stress and SOS response are typically not both strongly expressed in the same cells. Lower row: Constitutively expressed mCherry used for segmentation. C. Scatter plot of response times for gadB and recA in single cells; there is no clear temporal order and no significant correlation (r = 0.08 ± 0.08, p = 0.68); data is combined from three microcolonies. Error is from subsampling of descendants of single cells present at the time point of TMP addition (Methods). D. Expression of gadB and recA in inosine-supplemented medium, averaged over 13 single cells in a microcolony. Insets: Dual color image of gadB and recA and segmentation image as in B at t = 6h.
Dynamic Assembly of the Exomer Secretory Vesicle Cargo Adaptor Subunits (2015)
Figure 1

Figure 1. Exomer assembles from its subunits on the TGN.

(A) Exomer components localize to different extent to the TGN and to the cytoplasm. Fluorescence images of cells expressing GFP-tagged exomer components and TGN marker Sec7-DsRed. The brightness and contrast were adjusted differently in the GFP and merge images. Bar, 5 µm.

(B) Quantification of the relative TGN to cytoplasm concentration of the GFP-tagged ChAPs in the cell. The mean fluorescence at the TGN and cytoplasm was determined using the Icy imaging software. 20-30 individual cells were analysed. For statistical significance testing the Student’s T test was used.

(C) FCS measures the in vivo mobility of exomer components in the cytoplasm. FCS analysis was performed on the GFP, Chs5-GFP and GFP-tagged ChAPs in the wild-type background (top) and on GFP-tagged ChAPs in the Δchs5 background (bottom). 10-20 FCS measurements were performed for each condition using different cells. The ACF of free GFP is fitted with a one-component anomalous diffusion model. The ACF of Chs5 in WT and ChAPs in WT and Δchs5 background is fitted with a two-component diffusion model. Representative ACF curves (thin full lines) and fits (thick dashed lines) are shown. Estimated diffusion correlation times, diffusion coefficients and fractions are shown in Table 1.

(D) Quantification of the Chs5 independent fraction of ChAPs present in the cytoplasm based on the measurements shown in (C).

(E) Comparison of the residuals of the one-component and two-component fit (arrows).
Dynamic Assembly of the Exomer Secretory Vesicle Cargo Adaptor Subunits (2015)
Figure 2

Figure 2. Exomer components display distinct dynamic behaviour at the TGN compared to classical coat components.

(A) Comparison of the dynamic behavior of the GFP-tagged Chs5 and Bch1 with the GFP-tagged Sec28, Cop1, Clc1 and Apm1. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2.

(B) Binding kinetics of the GFP-tagged Chs5 and ChAPs at the TGN is different. Data processing was carried out as in (A).

(C) Quantification of the relative expression levels of the exomer components in GFP-tagged strains. A representative immunoblot of yeast lysates is shown; Pgk1 serves as loading control. The asterisks depict the analyzed bands. The plot shows average and SD of three independent biological experiments. The relative expression levels are normalized to Chs6 (depicted by the red line).
Dynamic Assembly of the Exomer Secretory Vesicle Cargo Adaptor Subunits (2015)
Figure 3

Figure 3. ChAPs affect differently Chs5 recruitment and stabilization at the TGN.

(A) Chs5 is necessary for recruitment and stable binding of ChAPs at the TGN. Differential centrifugation of cell lysates obtained from Chs6-GFP WT and Δchs5 strain. TCL, total cell lysate; S10, 10,000 g supernatant; P10, 10,000 g pellet; S100, 100,000 g supernatant; P100, 100,000 g pellet. Anp1 serves as the Golgi marker and Pgk1 as the cytoplasm marker. A representative immunoblot of three independent biological experiments is shown.

(B) Diffusional behavior of GFP-tagged ChAPs in the cytoplasm (open triangles, dashed line) and at the TGN (closed circles, full line). The mean of 10-20 FRAP measurements from different cells is shown. Calculated diffusion coefficients are shown in Expanded View Table 1.

(C) Chs5-GFP displays reduced membrane association in the absence of ChAPs. Differential centrifugation of cell lysates obtained from Chs5 GFP WT and ΔΔΔΔ strains. TCL, total cell lysate; S10, 10,000 g supernatant; P10, 10,000 g pellet; S100, 100,000 g supernatant; P100, 100,000 g pellet. Anp1 serves as the Golgi marker and Pgk1 as the cytoplasm marker. A representative immunoblot of three independent biological experiments is shown.

(D) Fluorescence images of cells expressing Chs5-GFP and TGN marker Sec7-DsRed in WT, ChAP and strains. Merged images depict the differences in Chs5-GFP localization to the TGN, cytoplasm and distinct foci (arrows) in these strains. The brightness and contrast were adjusted differently in the merged images. Bar, 5 µm.

(E) Co-localization analysis of Chs5-GFP and TGN marker Sec7-DsRed in WT, ChAP and strains. Mander 1 depicts the relative amount of the GFP signal co-localizing with the overall DsRed signal, and Mander 2 vice versa. Manders coefficients were obtained using the JACoP plug-in for ImageJ after background subtraction (N = 20) and are given in Expanded View Table 2.

(F) Binding kinetics of Chs5-GFP at the TGN in WT, ChAP and strains. The mean of 20-30 FRAP measurements from different cells is shown. Calculated parameters are shown in Table 2. The schematic cartoons depict the predicted majority of the analyzed complexes. The violet ovals represent Chs5 dimer and circles represent ChAPs: green:Bch1, red:Bud7, dark blue:Bch2, light blue:Chs6.