EMBO/soda-nel huggingface dataset:
ATRX loss induces telomere dysfunction and necessitates induction of alternative lengthening of telomeres during human cell immortalization (2019)
Figure 5

Figure 5. ATRX deficiency triggers telomeric replication stress and DNA damage response. A, B ATRX depletion induced telomeric replication dysfunction. IMR90 cells at day 25 post infection of lentiviral control or sgA#10 were synchronized by serum starvation and switched to 15% serum-containing medium and assayed by 53BP1/TelG immuno-FISH (A) at indicated time. Arrows denote telomeres that are positive for 53BP1 signals. Scale bar, 15 µm. (B) Percentage of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.

C, D RPA/TRF2 co-immunofluorescence (C) revealed strongly increased telomeric replication stress in U87 cells at day 30 but not day 10 post lentiviral sgA#10 infection (D). Lentiviral control and sgA#10 infected U87 cells at indicated time post-infection were released following thymidine (Thy) synchronization and processed for RPA and TRF2 co-staining. Scale bar, 10 µm. (D) Percentages of cells with > 4 RPA/TRF2 co-localized foci are expressed as means ± s.d., N = 3, unpaired t test.
ATRX loss induces telomere dysfunction and necessitates induction of alternative lengthening of telomeres during human cell immortalization (2019)
Figure 7

Figure 7. ATRX loss dictates telomere maintenance program during immortalization.

A Growth curves show the immortalization processes of lentiviral Rosa control (sgRosa#1 or #2) or sgATRX (sgA#10 or #19) transduced IMR90 cells. TERT transduced IMR90 culture was included as a positive control for immortalization.

B Quantitative RT-PCR of endogenous TERT gene expression revealed that sgATRX but not sgRosa transduced IMR90 cells adopted ALT during immortalization. Data are expressed as means ± s.d., N = 3, unpaired t test.

C Western blots show depletion of ATRX protein expression in the immortalized ALT IMR90 cells.

D C-circle analysis shows increased C-circle formation in the immortalized ALT cells. U2OS was used as a positive control, and reactions lacking DNA or ɸ29 serve as negative controls.

E Telomere restriction fragment (TRF) analysis show enhanced telomeric length heterogeneities in the immortalized ALT cells.

F, G PML/TRF2 co-immunofluorescence (F) shows increased APB formation in the immortalized ALT cells (G). Scale bar, 10 µm. (G) Percentages of APB positive cells are expressed as means ± s.d., N = 3, unpaired t test.

H Predicted model for the ATRX/DAXX loss-induced telomere dysfunction and ALT activation.

EXPANDED VIEW FIGURE LEGENDS
Early endosomes and endosomal coatomer are required for autophagy. (2009)
Figure 1.

siRNA depletion of COPI increases autophagy. (A) siRNA depletion of β′-, β-, and α-COPI (top) and Sec23A and B (bottom) in 293/GFP-LC3 cells for 48 h. Decreased protein levels of the corresponding subunits is shown by immunoblots. (B) Loss of β′-, α-, and β-COP but not subunits of COPII (Sec 23A and B) increase GFP-LC3-II. In A and B, tubulin was the loading control. (C) GFP-LC3–positive vesicles were detectable in 293/GFP-LC3 cells after siRNA depletion of β′-, α-, and β-COP but only basal levels of GFP-LC3–positive vesicles are observed in control siRNA-depleted cells.
Early endosomes and endosomal coatomer are required for autophagy. (2009)
Figure 2.

Time course of COPI depletion shows that Golgi disperses before AVs are formed, but Golgi dispersal does not cause AV formation. (A) COPI subunits were depleted in 293/GFP-LC3 cells and analyzed 24, 36, and 48 h after addition of siRNA. Immunoblotting for β′- and α-COP confirms loss of COP subunits. (B) GFP-LC3-I and -II were monitored by immunoblots in parallel lysates using anti-GFP antibodies at the indicated times. (C) After 24 h siRNA treatment, depletion of β′-COP caused morphological changes and a reduction in perinuclear population of GM130. Box indicates enlarged area. (D) Rab1a/b was depleted using siRNAs for 48 h. Lysates were probed with anti-Rab1 to confirm depletion. (E) β′-COP or Rab1a/b were depleted as in D, and analyzed by indirect immunofluorescence using anti-GM130 and GFP fluorescence. In β′-COP panel, the asterisk indicates cells that did not show an accumulation of GFP-LC3–positive AVs.
Early endosomes and endosomal coatomer are required for autophagy. (2009)
Figure 3.

AV formation after siRNA depletion of COPI requires Atg5 and Atg7. 293/GFP-LC3 cells were incubated with siRNA for β′-, α-COP, or control siRNA alone or combined with siRNA against Atg5 or Atg7 for 48 h, then (A) lysed and analyzed by immunoblot for Atg5, Atg7, GFP-LC3, and tubulin (loading control), or (B) fixed for immunofluorescence analysis using anti-ERGIC-53 antibodies, or GFP fluorescence. In A, the asterisk indicates nonspecific band; in B and C, asterisk indicates cells that did not show a fragmented ERGIC-53.
Early endosomes and endosomal coatomer are required for autophagy. (2009)
Figure 4.

Correlative light-electron microscopy analysis of GFP-LC3 puncta demonstrates they are AVs. (A) Phase, (B) fluorescent, and (C) TEM of the same field of 293/GFP-LC3 cells 48 h after siRNA depletion of α-COP. In B the image was inverted to allow better visualization of the GFP-LC3–positive structures. (D) Higher magnification of cell shown with asterisk in A and C. Top region of cell of interest is enlarged in E to show the two AVs structures indicated by arrows in B. (F and G) Higher magnification of AVs shown by arrows in E.
Early endosomes and endosomal coatomer are required for autophagy. (2009)
Figure 5.

Ub and p62 accumulate in α- and β′-COP–depleted 293/GFP-LC3 cells. (A) 293/GFP-LC3 cells were treated with control or β′-COP siRNA for 48 h, then fixed and labeled with anti-p62 and anti-ubiquitin antibodies for colocalization with GFP-LC3. (B) HEK293 cells treated as in A and labeled with anti-ubiquitin and anti-p62 antibodies. (C) Cryosections from β′-COP–depleted cells were labeled with anti-Ub antibodies followed by 10-nm protein A–gold. AV, autophagosomes.
Early endosomes and endosomal coatomer are required for autophagy. (2009)
Figure 6.

AVs in COPI-depleted cells are not degradative. (A) Long-lived protein degradation was assessed 72 h after transfection with control or β′-COP siRNA in 293/GFP-LC3 cells. Cells were either starved of amino acids for 2 h (St) or incubated in fresh growth medium (Fed) for 2 h. The amount of protein degradation is presented as mean ± SEM. (B) siControl-treated cells and β′- and α-COP were depleted for 48 h, then incubated with Lysotracker red (50 nM) for 30 min before fixation. Merged image shows very little colocalization of GFP-LC3 AV-positive structures. Asterisk shows cell enlarged in inset. (C) After β′- and α-COP siRNA depletion, Magic Red-RR2 (MR) was used to detect active cathepsin B in cells which were fed or starved (St) for 2 h. The intensity of MR labeling was measured in live cells by flow cytometry.
Early endosomes and endosomal coatomer are required for autophagy. (2009)
Figure 7.

GFP-LC3 AVs are LAMP2 positive. (A) 293/GFP-LC3 cells were incubated with control or β′- or α-COP siRNA for 48 h, fixed and labeled with anti-LAMP2 antibodies, followed by Alexa 555 anti–mouse antibodies. After COPI depletion many GFP-LC3–positive AVs are positive for LAMP2. Asterisk indicates cell enlarged in inset. (B) Cryosections of β′-COP-depleted cells labeled with anti-GFP (left) or anti-GFP and anti-LAMP2 (right) followed by protein A–gold as indicated. AV, autophagosomes, M, mitochondria.
Early endosomes and endosomal coatomer are required for autophagy. (2009)
Figure 8.

GFP-LC3–positive AVs colocalize TGN46 and early endocytotic markers after COPI depletion. (A) 293/GFP-LC3 cells were incubated with siRNA for β′-COP or control siRNA (Cont) for 48 h, processed for immunofluorescence, and labeled with antibodies for TGN46. In (B) siRNA-treated cells were fixed and labeled, or starved for 2 h (St), and labeled with antibodies to EEA1. (C) Control-starved HEK293 cells were labeled for endogenous LC3 and EEA1 by double labeling after methanol fixation. (D) After siRNA treatment, rhodamine-labeled dextran was internalized for 20 min, followed by a 40-min chase after which the 293/GFP-LC3 cells were fixed and analyzed by confocal microscopy.
Early endosomes and endosomal coatomer are required for autophagy. (2009)
Figure 9.

Transferrin and EGF trafficking is inhibited by COPI depletion. 293/GFP-LC3 cells were treated with β′-COP siRNA or control siRNA. After 48 h cells were incubated with Alexa 555 transferrin (A) for 2 min, (B) 30 min, or (C) 30 min followed by a 30-min chase, then washed and fixed and analyzed by confocal microscopy. (D and E) 125I-EGF was added to HeLa cells after 48 h of siRNA depletion of β′-COP, or control siRNA for 10 min. The cells were then washed as described in the Materials and methods, and chased for 30, 60, 120, and 180 min (including 10-min incubation). (D) The amount of 125I-EGF internalized was shown as a percentage of the total 125I-EGF taken up in 10 min. (E) The amount of degraded 125I-EGF was determined as a percentage of the total radioactivity present after TCA precipitation over the total 125I-EGF internalized. The experiment was performed three times in duplicate and the data shown is the mean ± SEM. The significance was determined by Student's t test. *, P ≤ 0.05.
Cold-induced calreticulin OsCRT3 conformational changes promote OsCIPK7 binding and temperature sensing in rice (2022)
Figure 2

Figure 2. Subcellular localization of OsCRT3 in N. benthamiana leaves.

A Schematic of the N1-54:mVenus:OsCRT3 construct, with mVenus inserted downstream of the putative transmembrane domain (TMD), between aa 31-51 of OsCRT3. aa: amino acids.

B, C Colocalization of N1-54:mVenus:OsCRT3 with OFP-HDEL. OFP-HDEL is an ER lumen marker harboring a C-terminal HDEL ER-retention sequence. (C) shows a magnification of the box represented in (B). Bar: 20 μm.

D Colocalization analyses along the white arrow in (C).

E, F Colocalization of N1-54:mVenus:OsCRT3 with the ER membrane marker AtCNX1-mCherry. (F) shows a magnification of the box represented in (E). Bar: 20 μm.

G Colocalization analyses along the white arrow in (F).
Cold-induced calreticulin OsCRT3 conformational changes promote OsCIPK7 binding and temperature sensing in rice (2022)
Figure 3

Figure 3. Ca2+ signaling upon cold shock in rice plants.

A Cytosolic free Ca2+ concentrations upon cold treatment of rice callus expressing aequorin in wild type (DJ), oscrt3-1 mutant, and the OsCRT3-complemented transgenic lines (pOsCRT3). The blue background indicates cold treatment of the rice callus.

B Mean maximal Ca2+ influxes in (A). Values are expressed as means ± SD. At least 6 calli were used for each sample. Different letters represent statistically significantly differences among the lines (Fisher's least significant difference test, P < 0.05).

C Cold response of cytosolic free Ca2+ concentration in root cells expressing NES-YC3.6. Superimposition of representative normalized ratio variations for wild-type, oscrt3-1, and the complemented transgenic pOsCRT3 root cells. The rectangle represents the regions of interest (ROIs) for the ratio measurements. The blue background indicates duration of cold treatment. Bar: 50 μm.
Cold-induced calreticulin OsCRT3 conformational changes promote OsCIPK7 binding and temperature sensing in rice (2022)
Figure 4

Figure 4. OsCRT3 interacts with OsCIPK7.

A Diagram of OsCRT3 and OsCIPK7 protein domains.

B Yeast two-hybrid analysis to test the OsCRT3-OsCIPK7 interaction. The interactions between OsCRT3, OsCRT3N, OsCRT3P or OsCRT3C with OsCIPK7, OsCIPK7N, OsCIPK7C, OsCIPK7NAF, or OsCIPK7PPI were verified.

C Pull-down assay of the interaction between OsCRT3 and OsCIPK7.

D Schematic of OsCIPK7-GFP construction.

E Localization of OsCIPK7-GFP in N. benthamiana leaf epidermal cells. OFP-HDEL was used as an ER marker. Bar: 20 μm.

F Magnification of the box represented in (E). Colocalization analyses along the white arrow are shown. Bar: 20 μm.
Cold-induced calreticulin OsCRT3 conformational changes promote OsCIPK7 binding and temperature sensing in rice (2022)
Figure 5

Figure 5. OsCIPK7 interacts with calcineurin B-like proteins OsCBL7/8.

A OsCIPK7 interacts with OsCBLs in a yeast two-hybrid system. Interactions were verified by growing the yeast on selective medium (SD/-Leu-Trp, SD/-Leu-Trp-His-Ade) and conducting β-Galactosidase assays.

B Subcellular localization of OsCBL7 and OsCBL8 in N. benthamiana leaf epidermal cells. Pictures were taken after 2 and 3 days of expression. Bar: 50 μm.

C Bimolecular fluorescence complementation (BiFC) assay confirming the interaction between OsCBL7/8 and OsCIPK7 in N. benthamiana leaf epidermal cells. 19K was used as a negative control. Bar: 50 μm.

D Interactions between OsCIPK7 and OsCBL7 or OsCBL8 were attenuated by OsCRT3 in a yeast three-hybrid assay.

E Constructs and OsCRT3 protein expression level in (D).

F Luciferase Complementation Imaging assay (LCI) to detect the interaction between OsCIPK7-nluc and OsCBL7-cluc or OsCBL8-cluc, with co-expression of N1-54:mVenus:OsCRT3 (mVenus-OsCRT3) or GFP, respectively. Quantification of luciferase activity was measured and shown. Values are mean ± SD. At least 6 leaves were used for each sample. *P < 0.05, Student's t-test. Experiments were repeated at least twice with similar results.

G Immunoblot confirming the expression of OsCIPK7, OsCBL7, OsCBL8, and OsCRT3 in (F) respectively.

H Pull-down assay showing that the interaction between OsCIPK7 and OsCBL7 or OsCBL8 was weakened with increasing amounts of OsCRT3. MBP was used as a negative control. Relative expression analysis was conducted using Image J software.
Cold-induced calreticulin OsCRT3 conformational changes promote OsCIPK7 binding and temperature sensing in rice (2022)
Figure 7

Figure 7. OsCRT3 enhances OsCIPK7 kinase activity.

A OsCIPK7 showed autophosphorylation activity but could not phosphorylate OsCRT3 in vitro. The loaded protein is indicated by red arrows. Autoradiograph of γ-32P-labeled OsCIPK7 indicates autophosphorylation of OsCIPK7.

B OsCIPK7 didn't phosphorylate OsCBL7 or OsCBL8 in vitro. Loaded proteins stained with Coomassie brilliant blue (CBB) are shown in the top panel and indicated by red arrows. Autoradiograph of γ-32P-labeled myelin basic protein and OsCIPK7 are shown in the bottom panel.

C The kinase activity OsCIPK7 was enhanced with increasing concentrations of OsCRT3 protein. SDS-PAGE gel showing CBB-stained OsCRT3, myelin basic protein, and OsCIPK7 (top panel). γ-32P-labeled myelin basic protein and OsCIPK7 substrate phosphorylation (bottom panel). The amount of OsCRT3 added was 0 μg, 0.2 μg, 0.4 μg, 0.6 μg, 0.8 μg in the SDS-PAGE gel lanes, respectively.
Competing interaction partners modulate the activity of Sgs1 helicase during DNA end resection (2019)
Figure 1

Fig 1 - DNA duplex processing by Sgs1

A Sketch of the employed magnetic tweezers assay and DNA processing by Sgs1.

B Observed dsDNA processing patterns of Sgs1, including an overview over consecutive bursts and pauses (upper panel) as well as detailed views into individual bursts containing multiple unwinding events (lower panels). A typical unwinding event of Sgs1 starts with slow, gradual unwinding of the dsDNA followed by DNA rehybridization, that can be almost instant (76% of events, see blue sections in lower right panel) or contain slow rewinding sections (24% of events, see orange sections in lower right panel).

C Histograms of the observed unwinding and rewinding velocities for Sgs1. The mean unwinding rate was 65 ± 2 bp/s (N = 899). The mean rewinding rate was 115 ± 6 bp/s (N = 287). We attribute the large rewinding rate in the absence of RPA to an increased error in inferring the rewinding rate from the short rewinding sections.
Competing interaction partners modulate the activity of Sgs1 helicase during DNA end resection (2019)
Figure 2

Fig 2 - DNA duplex processing by Sgs1 in the presence of RPA

A Sketch of DNA processing by Sgs1 in the presence of RPA.

B Observed dsDNA processing pattern of Sgs1 in the presence of RPA including an overview over successive bursts and pauses (upper panel) and detailed views of single bursts (lower panels). Unwinding events are always followed by slow rewinding events (orange sections).

C Histograms of the observed unwinding and rewinding velocities for Sgs1 in the presence of RPA (brown bars). The mean unwinding rate was 51 ± 3 bp/s (N = 2139). The mean rewinding rate was 66 ± 3 bp/s (N = 2091). White bars show for comparison the distributions of unwinding and rewinding velocities for Sgs1 alone (taken from Fig 1C).

D Force-dependence of the Sgs1 unwinding (black squares) (N = 2139) and rewinding rates (black diamonds) (N = 2091) in the presence of RPA (errors given as standard deviations). Red circles represent the force-dependent DNA opening and closure rates measured for RPA alone (taken from Ref. Kemmerich et al, 2016a. A fit to this data is shown as a solid red line.).
Competing interaction partners modulate the activity of Sgs1 helicase during DNA end resection (2019)
Figure 3

Fig 3 - DNA duplex processing by the combined activity of Sgs1, Dna2 and RPA

A Sketch of DNA processing by the Sgs1-Dna2-RPA complex.

B Typical dsDNA processing event. The dashed red line connects the minima at the end of the rewinding sections (see text for details). The inset shows a DNA unwinding event in the presence of nuclease-dead Dna2 E675A and RPA.

C Histograms of the observed unwinding and rewinding rates (green bars) with mean values of 69 ± 3 bp/s (N = 365) and 74 ± 5 bp/s (N = 282), respectively. White bars show for comparison the unwinding and rewinding velocities of Sgs1 in the presence of RPA (taken from Fig 2C).
Competing interaction partners modulate the activity of Sgs1 helicase during DNA end resection (2019)
Figure 4

Fig 4 - DNA duplex processing by Sgs1 in the presence of Top3-Rmi1

A Sketch of DNA processing by Sgs1 in the presence of Top3-Rmi1.

B dsDNA processing events observed for Sgs1 in the presence of Top3-Rmi1. Typical events consist of periods with slow gradual unwinding typically followed by instant DNA rezipping as seen also for Sgs1 alone.

C Histogram of the unwinding rate of Sgs1 in the presence of Top3-Rmi1 (violet bars) with a mean rate of 86 ± 3 bp/s (N = 1101). For comparison, the distribution of unwinding rates for Sgs1 alone is depicted with white bars (taken from Fig 1C).

D Sketch of DNA processing by Sgs1 in the presence of Top3-Rmi1 and RPA.

E dsDNA processing by Sgs1 in the presence of Top3-Rmi1 and RPA. In contrast to the absence of RPA, DNA closure is seen as a slow rewinding.

F Histograms of unwinding and rewinding rates of Sgs1 in the presence of Top3-Rmi1 and RPA (purple bars). The mean rates for unwinding and rewinding are 66 ± 2 bp/s (N = 502) and 70 ± 4 bp/s (N = 369), respectively. For comparison, the velocity distributions for Sgs1 in the presence of RPA are shown as white bars (taken from Fig 2C).
Binding of IFT22 to the intraflagellar transport complex is essential for flagellum assembly (2019)
Figure 1

Figure 1: IFT22 associates with guanine nucleotides through an unusual G5-dependent mechanism

A. IFT22 nucleotide-binding experiments. Fluorescence measurements using increasing amounts of TbIFT22 and TbIFT22/74342-401/81397-450 core complex incubated with mant-labeled GDP (mant-GDP) or non-hydrolysable GTP / ATP analogs (mant-GMPPNP / mant-AMPPNP). The fluorescence intensity is plotted as a function of protein concentration. Data were fitted to a single-site binding equation for determination of the dissociation constant (Kd). Kd values and standard deviations are calculated from three independent experiments.

B. Structural comparison of GTP-bound HsRab8A (light purple) and TbIFT22 (green) depicted in cartoon representation. Nucleotides are shown as sticks and Mg2+ as balls. Unstructured regions of TbIFT22 are represented with dotted lines. The zoomed-in view shows a superposition of the nucleotide-binding pocket. While classical GTPases form hydrogen bonds between a conserved aspartate of the G4 motif (NKxD) and the guanine base, IFT22 instead utilizes D175 located in the G5 loop.

C. Topology diagrams of a classical Rab GTPase and of IFT22. Positions of the conserved nucleotide-binding G-motifs (G1-G5) as well as switch regions are indicated.

D. Top: Cartoon representation of IFT22 (grey) with positions of two nucleotide-binding mutants highlighted. GTP is shown in stick representation and Mg2+ as a ball. D175 (blue) is the unusual residue binding the guanine base (see also Fig 1B), while S19 (pink) is a conserved residue required for coordination of the Mg2+ cation and is commonly mutated to an asparagine to prevent nucleotide binding. Bottom: Nucleotide-binding experiments of IFT22 nucleotide-binding mutants D175E, D175A and S19N with fluorescently labeled nucleotides. Only the S19N mutation (light pink) abolished IFT22 nucleotide-binding ability completely.

E. Superposition of GTP- (green) and GDP-bound (light green) TbIFT22 structures. Switch regions are marked in yellow and dotted lines indicate disordered loops not modeled in the structures.
Binding of IFT22 to the intraflagellar transport complex is essential for flagellum assembly (2019)
Figure 3

Figure 3: Molecular basis of IFT22 association with IFT74/81 ccVI

A. Cartoon representation of the IFT22-binding site on IFT74/81 ccVI (top left) and surface conservation representation of different orientations of IFT22/74/81 (top right and bottom). IFT74/81 ccVI displays a highly conserved patch at the IFT22-binding interface (black dashed circle). A 180° rotation of IFT22 (bottom right) exhibits a likewise conserved patch at the IFT74/81-binding interface (black dashed circles; position of the IFT74/81 helices is marked with light grey lines). Conserved residues are marked and labeled according to the Tb sequence. Conservation coloring is based on Clustal Omega multiple sequence alignments with H. sapiens, M. musculus, D. rerio, T. brucei, C. reinhardtii and C. elegans sequences (see Appendix Fig S2 and Appendix Fig S3D) and ConSurf conservation grades (Sievers et al, 2011; Landau et al, 2005).

B. Detailed view of the IFT22-IFT74/81 binding site in two perpendicular orientations showing interacting residues in stick representation. Residues provided by IFT22 switch I and II are shown in yellow, whereas IFT22 non-switch region interactions are colored in green. IFT74 is shown in orange and IFT81 in grey. IFT22 residue where mutation abolishes interaction with IFT74/81 are highlighted in red (R43 and A86, see also Fig 4 and Appendix Fig S3E).

C. SDS-PAGE gel of a Ni2+-NTA pulldown using His-tagged TbIFT22 (WT and mutants) and untagged CrIFT25/27/74/81. WT TbIFT22 is able to pull down the Cr tetrameric complex, thus forming a chimeric IFT-B1 pentamer, while both the A86R and S19N mutant fail to bind the complex.