Journal: bioRxiv

Article Title: Microglial 25-hydroxycholesterol mediates neuroinflammation and neurodegeneration in a tauopathy mouse model

doi: 10.1101/2023.09.08.556884

Figure Lengend Snippet: A) Representative images from a double stain of the DAM markers ApoE (green) and Clec7a (red) in 9.5-month old wild type (WT; n=5), Ch25h KO (CKO; n=5), PS19 (T; n=20) and PS19/Ch25h KO (TCKO; n=20) mouse brain sections. Percentage of area covered by ApoE immunoreactivity (B) and ApoE immunoreactivity in Clec7a positive cells (C) was quantified in the hippocampus. D) Representative images from a double stain of Trem2 (red) and Clec7a (green). Percentage of area covered by Trem2 immunoreactivity (E) and ApoE immunoreactivity in Clec7a positive cells (F) was quantified in the hippocampus. G) Representative images of homeostatic microglia immunostained with P2ry12 in the hippocampus (Scale bar 50 µm). Total P2ry12 immunoreactivity area was analyzed using Imaris (H). Scale bar 30 µm. Data expressed as mean ± SD. One-way ANOVA with Tukey’s post hoc test (two-sided) was used for all statistical analysis *p<0.05, **p<0.01, p<0.001, ****p<0.0001.

Article Snippet: Tissue was then incubated overnight at 4°C with primary antibodies CD68 (rat monoclonal, 1:400; Biorad, MCA1957), Iba1 (rabbit polyclonal, 1:1000; FUJIFILM Wako’s, 019-19741), GFAP (chicken polyclonal, 1:1000; Abcam, ab4674), Clec7a or Dectin-1 (rat monoclonal, 1:100; Invivogen, mabg-mdect), Trem2 (sheep polyclonal, 1:400; R&D systems, AF1729), ApoE (mouse monoclonal HJ6.3, 1:1000; from D. Holtzman); Tmem119 (rabbit polyclonal, 1:250; Cell Signaling, 90840S), P2ry12 (rat monoclonal, 1;100; Biolegend, 848002), Cd11b (rat monoclonal, 1:400; BioLegend, 101202), p-STAT3 (rabbit polyclonal, 1:500; Cell Signaling, 9145S), p-p65 NF-kB (rabbit polyclonal, 1:3000; Cell Signaling, 3033S), MC1 antibody (mouse monoclonal, 1:500: gift from Peter Davis), CD3 (rat monoclonal, 1:500; Invitrogen, 14-0032-82).

Techniques: Staining

Journal: Nature Communications

Article Title: Astroglial exosome HepaCAM signaling and ApoE antagonization coordinates early postnatal cortical pyramidal neuronal axon growth and dendritic spine formation

doi: 10.1038/s41467-023-40926-2

Figure Lengend Snippet: Representative images ( a ) and quantification ( b ) of βIII-tubulin + neuronal axon (white arrows) length following treatment of cortical neurons with A-Exo. or A-Exo. mixed with flowthrough (FT) from the SEC column; 0.2x and 0.5x FT each is concentrated from 2- or 5-ml exosome-free ACM, respectively. Number of neurons quantified in each group shown in the graph (7–9 neurons/replicate, 3 biological replicates)/group; Scale bar: 100 μm; c Representative (from >5 replicates) immunoblot of different apolipoproteins in all eluted fractions (500 μl/fraction, pooled as indicated) of ACM (100 ml) from SEC with optimal exposure. Unconcentrated elution (15 μl/sample) was run on immunoblot; Representative images ( d ) and quantification ( e ) of βIII-tubulin + neuronal axon (white arrows) length following co-treatment of cortical neurons with A-Exo. and different dose of hAPOE3. Number of neurons quantified in each group shown in the graph (4–5 neurons/replicate, 3 biological replicates)/group; Scale bar: 100 μm; f Quantification of βIII-tubulin + neuronal axon length following co-treatment of A-Exo. with common hAPOE isoforms. Number of neurons quantified in each group shown in the graph (10–12 neurons/replicate, 2 biological replicates)/group; Representative images ( g ) and quantification ( h ) of βIII-tubulin + neuronal axon (white arrows) length in control cortical neurons (i) or neurons treated with A-Exo. (ii) and A-Exo. mixed with WT (iii) or ApoE KO (iv) FT, respectively. Scale bar: 100 μm; Number of neurons quantified in each group shown in the graph (7–9 neurons/replicate, 3 biological replicates)/group; Representative images ( i ) and quantification ( j ) of βIII-tubulin + neuronal axon (white arrows) length in control (i) cortical neurons or neurons treated with WT (ii) or ApoE KO (iii) A-Exo. Number of neurons quantified in each group shown in the graph (4–8 neurons/replicate, 3 biological replicates)/group; Scale bar: 100 μm; 1 μg A-Exo. was used in each treatment. p values in ( b , e , f , h , j ) were calculated using one-way ANOVA followed by a Tukey post hoc test; n.s. not significant. Data are presented as mean values ± SEM.

Article Snippet: We only observed modestly reduced CST axon growth (average ~600 μm shorter, Fig. ) but not statistically significant ( p = 0.36, Fig. ) in ApoE KO pups compared to WT pups.

Techniques: Western Blot

Journal: Nature Communications

Article Title: Astroglial exosome HepaCAM signaling and ApoE antagonization coordinates early postnatal cortical pyramidal neuronal axon growth and dendritic spine formation

doi: 10.1038/s41467-023-40926-2

Figure Lengend Snippet: Representative image of VGluT1 and PSD95 staining in cortical neuronal cultures ( a ) and quantification of VGluT1 ( b ) and PSD95 density ( c ) on neurites following ACM treatment. Control cortical neurons (i) and neurite (iv), cortical neurons (ii) and dendrite (v) treated with WT ACM, and cortical neurons (iii) and dendrite (vi) treated with ApoE KO ACM; Scale bar: 20 μm (i–iii) and 10 μm (iv–vi); n = 16 neurons (8 neurons/replicate, 2 biological replicates)/group; Representative confocal and Imaris images of apical ( d ) and basal ( e ) dendrites and spines of layer V pyramidal neurons from motor cortex of Thy1-YFP + and Thy1-YFP + ApoE −/− mice (P30). Dendrites and spines were traced and quantified in Imaris. Scale bars: 10 μm; Quantification of apical ( f ) and basal ( g ) dendrites of layer V pyramidal neurons from motor cortex of Thy1-YFP + and Thy1-YFP + ApoE −/− mice (P30). n = 5 mice/group; Representative neuron image ( h ) and 3D Sholl analysis ( i ) of layer V pyramidal neurons from motor cortex of Thy1-YFP + and Thy1-YFP + ApoE −/− mice. Scale bar: 20 μm; n = 5 mice/group; Representative images ( j ) and quantification ( k ) of CM-DiI-labeled CST axons in the spinal cord of WT (i) and ApoE KO (ii) mice. Orange arrows indicate the pyramidal decussation; yellow lines indicate the beginning and ending points for the CST axon length measurement; Scale bar: 1 mm; n = 8 mice for WT and 9 mice for ApoE KO; p value in ( f , g , k ) determined by two-tailed t- test; p values in ( b , c ) determined using the one-way ANOVA followed by a Tukey post hoc test; p values in ( i ) determined using the multiple t -test. Data are presented as mean values ± SEM.

Article Snippet: We only observed modestly reduced CST axon growth (average ~600 μm shorter, Fig. ) but not statistically significant ( p = 0.36, Fig. ) in ApoE KO pups compared to WT pups.

Techniques: Staining, Labeling, Two Tailed Test

Journal: Molecular Cell

Article Title: How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication

doi: 10.1016/j.molcel.2023.06.035

Figure Lengend Snippet: Structure of Pol α-primase in the budding yeast replisome (A) Domain architecture of yeast Pol α-primase. exo, exonuclease domain; cat, catalytic domain; CIP, Ctf4-interacting peptide; NTD, N-terminal domain; CTD, C-terminal domain; PDE, phosphodiesterase domain; OB, oligonucleotide/oligosaccharide-binding domain. (B) Composite cryo-EM map of the budding yeast Pol α-primase associated replisome bound to replication fork DNA containing a 60 nucleotide 5′ flap. Density for Ctf4 is not observed in this map. The map was derived from combining individual focused refinements and is colored according to chain occupancy. (C and D) Atomic model of the budding yeast Pol α-primase associated replisome lacking Ctf4 derived from cryo-EM data displayed in (B). Regions of CMG that physically interact with Pol α-primase are colored. (E) Focused view of the Pri1 catalytic subunit of primase, showing how it is positioned above the exit channel for lagging-strand template ssDNA. (F) Cryo-EM reconstruction displaying continuous density for lagging-strand template ssDNA extending from the point of dsDNA strand separation toward the active site region of Pri1. Map colored by chain occupancy with the density assigned to the lagging-strand template post-strand separation colored manually. (G) Schematic illustrating the organization of Pol α-primase in the budding yeast replisome. The path of lagging-strand template ssDNA visualized in the structure immediately following strand separation is illustrated (solid pink line). The putative path of the lagging-strand template between the Pri1 and Pol1 active sites is also illustrated (dashed pink line).

Article Snippet: This configuration more closely resembles the architecture of human Pol α-primase bound to CST (CTC1-STN1-TEN1) and telomeric ssDNA ( I), and a very recent structure of a human Pol α-primase elongation complex, than the human Pol α-primase apo structure.

Techniques: Binding Assay, Cryo-EM Sample Prep, Derivative Assay

Journal: Molecular Cell

Article Title: How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication

doi: 10.1016/j.molcel.2023.06.035

Figure Lengend Snippet: Cryo-EM statistics

Article Snippet: This configuration more closely resembles the architecture of human Pol α-primase bound to CST (CTC1-STN1-TEN1) and telomeric ssDNA ( I), and a very recent structure of a human Pol α-primase elongation complex, than the human Pol α-primase apo structure.

Techniques:

Journal: Molecular Cell

Article Title: How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication

doi: 10.1016/j.molcel.2023.06.035

Figure Lengend Snippet: The structural basis for Pol α-primase recruitment to the budding yeast replisome (A) Schematic of the budding yeast replisome highlighting Pol α-primase-binding sites (red circles labeled a–e). (B) Atomic model highlighting the interfaces between Pri2 NTD (green) and the Mcm5 (blue) zinc finger (site a) and Mcm3 (cyan) N-terminal helical domain (site b). Residues colored yellow with side chains displayed represent those targeted for mutational analysis. (C) Multiple sequence alignment indicating the conservation of Mcm3 residues contacting Pri2 NTD (site b), colored according to conservation. Stars correspond to the Mcm3 residues colored yellow in (B) that were mutated. (D) Atomic model highlighting the interface between the Pol12 NTD (green) and the Mcm3 (cyan) AAA+ domain in the MCM C-tier (site c). (E) Atomic model highlighting the interface between the Pri2 Nterm (green) and the Psf2 subunit of GINS (brown) (site d). (F) Atomic model showing how Pri2-F2 projects into a hydrophobic pocket on Psf2, colored as in (E). (G) Multiple sequence alignment of Pri2 Nterm residues contacting Psf2. The alignment is grouped into fungal and metazoan sequences and colored according to conservation. Stars indicate residues mutated to alanine in the Pri2-AAA mutant.

Article Snippet: This configuration more closely resembles the architecture of human Pol α-primase bound to CST (CTC1-STN1-TEN1) and telomeric ssDNA ( I), and a very recent structure of a human Pol α-primase elongation complex, than the human Pol α-primase apo structure.

Techniques: Binding Assay, Labeling, Sequencing, Mutagenesis

Journal: Molecular Cell

Article Title: How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication

doi: 10.1016/j.molcel.2023.06.035

Figure Lengend Snippet: Pol α-primase CMG binding sites are critical for DNA replication (A) Summary of Pol α-primase and Cdt1-Mcm2-7 mutants and the interaction sites that are targeted. CR, charge reversal. (B) Schematic of the DNA template and anticipated products for origin-dependent budding yeast in vitro DNA replication reactions. (C and D) Denaturing agarose gel analysis of origin-dependent DNA replication reactions performed as illustrated in (B) for 20 min. (E–G) Diploid budding yeast cells of the indicated genotype were sporulated and the resulting tetrads were dissected and grown on YPD medium for 3 days at 25°C. Dissections that displayed abnormal segregation patterns were cropped from plate images.

Article Snippet: This configuration more closely resembles the architecture of human Pol α-primase bound to CST (CTC1-STN1-TEN1) and telomeric ssDNA ( I), and a very recent structure of a human Pol α-primase elongation complex, than the human Pol α-primase apo structure.

Techniques: Binding Assay, In Vitro, Agarose Gel Electrophoresis

Journal: Molecular Cell

Article Title: How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication

doi: 10.1016/j.molcel.2023.06.035

Figure Lengend Snippet: Structure of Pol α-primase in a human replisome assembled on fork DNA with a 60-nt 5′ flap (A) Composite cryo-EM map of the human replisome containing Pol α-primase, assembled on forked DNA containing a 60 nucleotide 5′ flap ( Figure S1 A). The map was derived from combining individual focused refinements and is colored according to chain occupancy. (B) Atomic model for the human Pol α-primase associated replisome, derived from cryo-EM data displayed in (A). Regions of CMG that interact directly with Pol α-primase are colored. (C) Focused view of PRIM1 showing its position at the mouth of the exit channel for lagging-strand ssDNA.

Article Snippet: This configuration more closely resembles the architecture of human Pol α-primase bound to CST (CTC1-STN1-TEN1) and telomeric ssDNA ( I), and a very recent structure of a human Pol α-primase elongation complex, than the human Pol α-primase apo structure.

Techniques: Cryo-EM Sample Prep, Derivative Assay

Journal: Molecular Cell

Article Title: How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication

doi: 10.1016/j.molcel.2023.06.035

Figure Lengend Snippet: Structure of Pol α-primase in a human replisome assembled on fork DNA with a 15-nt 5′ flap (A) Cryo-EM reconstruction of the Pol α-primase associated human replisome engaged on a DNA fork containing a 15 nucleotide 5′ flap. Map colored according to subunit occupancy. (B) Atomic model for the human Pol α-primase associated replisome, derived from cryo-EM data displayed in (A). Regions of CMG that interact directly with Pol α-primase are colored. (C and D) Comparison of Pol α-primase from human replisomes bound to forked DNA with a 15 nt 5′ flap (C) and 60 nt 5′ flap (D) as illustrated.

Article Snippet: This configuration more closely resembles the architecture of human Pol α-primase bound to CST (CTC1-STN1-TEN1) and telomeric ssDNA ( I), and a very recent structure of a human Pol α-primase elongation complex, than the human Pol α-primase apo structure.

Techniques: Cryo-EM Sample Prep, Derivative Assay, Comparison

Journal: Molecular Cell

Article Title: How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication

doi: 10.1016/j.molcel.2023.06.035

Figure Lengend Snippet: Structural basis for Pol α-primase recruitment to the human replisome for priming (A) Schematic of the human replisome engaged by Pol α-primase. Red circled labels indicate protein-protein interaction sites between Pol α-primase and the replisome. (B) Atomic model highlighting the interface between PRIM2 NTD (green) and the MCM3 (cyan) N-terminal helical domain (site b). Residues colored yellow with side chains displayed are those targeted for mutational analysis. (C) Atomic model highlighting the interfaces between PRIM2 Nterm and the PSF2 subunit of GINS (site d) and the POLA2 N-terminal helix (residues 96–114) and both PSF1 and SLD5 (site g). (D) Zoomed in view of the PRIM2 Nterm :PSF2 interface (site d). (E) Table summarizing the protein-protein interfaces between Pol α-primase and the replisome in both budding yeast and human. Each discrete site is assigned a letter identifier corresponding to the labeling in (A) and Figure 2 A. (F) Schematic of the forked DNA template and anticipated products of in vitro DNA replication with purified human proteins. (G) Denaturing agarose gel analysis of an in vitro DNA replication reaction performed as in (A) with the indicated proteins for 20 min.

Article Snippet: This configuration more closely resembles the architecture of human Pol α-primase bound to CST (CTC1-STN1-TEN1) and telomeric ssDNA ( I), and a very recent structure of a human Pol α-primase elongation complex, than the human Pol α-primase apo structure.

Techniques: Labeling, In Vitro, Purification, Agarose Gel Electrophoresis

Journal: Molecular Cell

Article Title: How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication

doi: 10.1016/j.molcel.2023.06.035

Figure Lengend Snippet:

Article Snippet: This configuration more closely resembles the architecture of human Pol α-primase bound to CST (CTC1-STN1-TEN1) and telomeric ssDNA ( I), and a very recent structure of a human Pol α-primase elongation complex, than the human Pol α-primase apo structure.

Techniques: Virus, Recombinant, Protease Inhibitor, Staining, Derivative Assay, Purification, Software, Electron Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Targeting apolipoprotein E and N-terminal amyloid β-protein precursor interaction improves cognition and reduces amyloid pathology in Alzheimer’s mice

doi: 10.1016/j.jbc.2023.104846

Figure Lengend Snippet: 6KApoEp treatment dampens amyloidogenic APP processing without altering BACE1 expression . A , Western blot is shown using anti-N-terminal amyloid-β 1–17 ( Aβ ) monoclonal antibody ( mAb 82E1 ), which detects amyloidogenic APP cleavage fragments, including phospho-C99 ( pC99 ) and nonphospho-C99 ( C99 ) as well as Aβ monomer and oligomers. Western blot is also shown using anti-C-terminal BACE1 (β-secretase) polyclonal antibody ( pAb BACE1 ). Actin is included as a loading control, and densitometry values are indicated below each lane. Equal amounts of total protein were loaded per lane. B and C , densitometry data are shown for ratios of pC99, C99, or Aβ to actin. D , abundance of Aβ oligomers in the detergent-soluble brain homogenate fraction (measured by sandwich ELISA) is shown. E , densitometry data are shown for ratios of BACE1 to actin. Data were obtained from APP/PS1/E2 mice that received vehicle ( APP/PS1/E2-V ) or 6KApoEp ( APP/PS1/E2-6K A poEp ), APP/PS1/E3 mice that received vehicle ( APP/PS1/E3-V ) or 6KApoEp ( APP/PS1/E3-6K A poEp ), and APP/PS1/E4 mice that received vehicle ( APP/PS1/E4-V ) or 6KApoEp ( APP/PS1/E4-6K A poEp ) for 3 months starting at 12 months of age. Western blotting data for ( B , C , and E ) included each mouse ( n = 4 per group with two of each sex), and quantitative data were averaged. Sandwich ELISA data for ( D ) included each mouse ( n = 8 per group with four of each sex), and measured data were averaged. Statistical comparisons for ( B ) are between groups for each protein. Statistical comparisons for ( C – E ) are between groups. ∗∗∗ p ≤ 0.001 for APP/PS1/E2-V or APP/PS1/E3-V versus APP/PS1/E4-V mice; †† p < 0.01; ††† p ≤ 0.001 for each 6KApoEp- versus each vehicle-treated APP/PS1/E2/E3/E4 mice ( Tables S16–S19 ). V , vehicle.

Article Snippet: Electrophoresed proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) that were blocked at ambient temperature for 1 h. Membranes were then hybridized at ambient temperature for 1 h with primary antibodies as follows: anti-N-terminal APP pAb (1:2000 dilution, IBL); anti-sodium-potassium ATPase mAb (1:20,000 dilution, EP1845Y; abcam), anti-heat-shock protein 90 mAb (1:1000 dilution, C45G5; Cell Signaling Technology), anti-N-terminal Aβ 1–16 mAb (1:500 dilution, 82E1; IBL); anti-C-terminal-BACE1 pAb (1:400 dilution; IBL); anti-phospho-p44/42 MAPK mAb (1:2000 dilution, D13.14.4E; Cell Signaling Technology); anti-p44/42 MAPK mAb (1:3000 dilution, 137F5; Cell Signaling Technology); anti-phospho-p38 MAPK (Thr180/Tyr182) mAb (1:1000 dilution, D3F9; Cell Signaling Technology); anti-p38 MAPK pAb (1:1500 dilution, Cell Signaling Technology); anti-C-terminal apoE pAb, (1:1000 dilution, A299; IBL); anti-human apoE LDLR binding domain pAb (1:1000 dilution; IBL), anti-DLK/MAP3K12 pAb (1:1500 dilution, GeneTex, Inc), or anti-β-actin mAb (1:5000 dilution, 13E5; Cell Signaling Technology; as a loading control).

Techniques: Expressing, Western Blot, Sandwich ELISA, Mouse Assay

Journal: The Journal of Biological Chemistry

Article Title: Targeting apolipoprotein E and N-terminal amyloid β-protein precursor interaction improves cognition and reduces amyloid pathology in Alzheimer’s mice

doi: 10.1016/j.jbc.2023.104846

Figure Lengend Snippet: 6KApoEp inhibits apoE-N-terminal APP interaction . A and B , brain homogenates from the vehicle-treated and 6KApoEp-treated APP/PS1/E2/E3/E4 mouse groups were immunoprecipitated with anti-N-terminal APP polyclonal antibody ( pAb , A ) or anti-C-terminal apoE pAb ( B ), and apoE ( A ) and APP ( holo , B ) were determined by Western blotting with anti-C-terminal apoE pAb or anti-N-terminal APP pAb. The left six lanes denote precipitates in each blot. The right six lanes denote inputs. IgG H , immunoglobulin heavy chain; IgG L , immunoglobulin light chain.

Article Snippet: Electrophoresed proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) that were blocked at ambient temperature for 1 h. Membranes were then hybridized at ambient temperature for 1 h with primary antibodies as follows: anti-N-terminal APP pAb (1:2000 dilution, IBL); anti-sodium-potassium ATPase mAb (1:20,000 dilution, EP1845Y; abcam), anti-heat-shock protein 90 mAb (1:1000 dilution, C45G5; Cell Signaling Technology), anti-N-terminal Aβ 1–16 mAb (1:500 dilution, 82E1; IBL); anti-C-terminal-BACE1 pAb (1:400 dilution; IBL); anti-phospho-p44/42 MAPK mAb (1:2000 dilution, D13.14.4E; Cell Signaling Technology); anti-p44/42 MAPK mAb (1:3000 dilution, 137F5; Cell Signaling Technology); anti-phospho-p38 MAPK (Thr180/Tyr182) mAb (1:1000 dilution, D3F9; Cell Signaling Technology); anti-p38 MAPK pAb (1:1500 dilution, Cell Signaling Technology); anti-C-terminal apoE pAb, (1:1000 dilution, A299; IBL); anti-human apoE LDLR binding domain pAb (1:1000 dilution; IBL), anti-DLK/MAP3K12 pAb (1:1500 dilution, GeneTex, Inc), or anti-β-actin mAb (1:5000 dilution, 13E5; Cell Signaling Technology; as a loading control).

Techniques: Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Targeting apolipoprotein E and N-terminal amyloid β-protein precursor interaction improves cognition and reduces amyloid pathology in Alzheimer’s mice

doi: 10.1016/j.jbc.2023.104846

Figure Lengend Snippet: 6KApoEp is detected in brains from 6KApoEp-treated APP/PS1/E2/E3/E4 mice, and is stable in mouse plasma for 24 h at 37 °C. A , Western blots are shown using anti-low-density lipoprotein receptor ( LDLR ) binding domain (residues 133–152 of human apoE) polyclonal antibody ( pAb ) that is the matching epitope as 6KApoEp except for 6K. The right five lanes denote a series of 6KApoEp calibration peptides ( i.e. , 20, 40, 80, 160, and 320 ng). Brain homogenates were obtained from APP/PS1/E2 mice that received vehicle ( APP/PS1/E2-V ) or 6KApoEp ( APP/PS1/E2-6KApoEp ), APP/PS1/E3 mice that received vehicle ( APP/PS1/E3-V ) or 6KApoEp ( APP/PS1/E3-6KApoEp ), and APP/PS1/E4 mice that received vehicle ( APP/PS1/E4-V ) or 6KApoEp ( APP/PS1/E4-6KApoEp ) for 3 months starting at 12 months of age. B , Western blotting data for the calibration graph were obtained from a series of 6KApoEp calibration peptides ( i.e. , 20, 40, 80, 160, and 320 ng). Western blotting data for 3 bars were obtained from APP/PS1/E2 mice that received 6KApoEp ( APP/PS1/E2-6KApoEp ), APP/PS1/E3 mice that received 6KApoEp ( APP/PS1/E3-6KApoEp ), and APP/PS1/E4 mice that received 6KApoEp ( APP/PS1/E4-6KApoEp ) for 3 months starting at 12 months of age. Western blotting data for ( B ) included each mouse ( n = 4 per group with two of each sex), and quantitative data were averaged. C , the stability of 6KApoEp (40 ng) in mouse plasma was examined with a time of incubation ( i.e. , 0, 3, 6, 12, and 24 h) at 37 °C. D , the experiment was performed four times, and quantitative data were averaged ( Table S23 ).

Article Snippet: Electrophoresed proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) that were blocked at ambient temperature for 1 h. Membranes were then hybridized at ambient temperature for 1 h with primary antibodies as follows: anti-N-terminal APP pAb (1:2000 dilution, IBL); anti-sodium-potassium ATPase mAb (1:20,000 dilution, EP1845Y; abcam), anti-heat-shock protein 90 mAb (1:1000 dilution, C45G5; Cell Signaling Technology), anti-N-terminal Aβ 1–16 mAb (1:500 dilution, 82E1; IBL); anti-C-terminal-BACE1 pAb (1:400 dilution; IBL); anti-phospho-p44/42 MAPK mAb (1:2000 dilution, D13.14.4E; Cell Signaling Technology); anti-p44/42 MAPK mAb (1:3000 dilution, 137F5; Cell Signaling Technology); anti-phospho-p38 MAPK (Thr180/Tyr182) mAb (1:1000 dilution, D3F9; Cell Signaling Technology); anti-p38 MAPK pAb (1:1500 dilution, Cell Signaling Technology); anti-C-terminal apoE pAb, (1:1000 dilution, A299; IBL); anti-human apoE LDLR binding domain pAb (1:1000 dilution; IBL), anti-DLK/MAP3K12 pAb (1:1500 dilution, GeneTex, Inc), or anti-β-actin mAb (1:5000 dilution, 13E5; Cell Signaling Technology; as a loading control).

Techniques: Western Blot, Binding Assay, Incubation

Journal: bioRxiv

Article Title: Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

doi: 10.1101/2023.05.08.539880

Figure Lengend Snippet: ( A ) Anti-Myc co-IPs of Myc-tagged POT1 proteins and FLAG-tagged CST from co-transfected 293T cells showing that mPOT1b interacts with human CST better than human POT1. Immunoblots were probed with anti-Myc and anti-FLAG antibodies. ( B ) Design of constructs encoding chimeric swaps between mPOT1b (red) and human POT1 (blue). ( C ) CST binding by chimeric human-mouse POT1 proteins. Anti-Myc co-IPs of Myc-tagged POT1 constructs, and FLAG-tagged CST from co-transfected 293T cells showing that aa 323-326 of mPOT1b are important for the CST interaction (comparison of HMb4 and HMb5). Immunoblots were probed with anti-Myc and anti-FLAG antibodies. ( D ) Sequence alignment of a region in the POT1 hinge (aa 309-330) in which the insertion of ESDL enhances the CST interaction. ( E ) Purified POT1/TPP1 and CST proteins and cartoon schematics used for fluorescent size-exclusion chromatography (FSEC) analysis. (F-G) FSEC analysis of the interaction between CST and POT1(WT)/GFP-TPP1 (blue) or POT1(ESDL)/GFP-TPP1 (red) in the absence ( F ) or presence ( G ) of telomeric ssDNA. Traces without CST are shown as dashed lines and color key is the same for both panels. RFU: relative fluorescence units.

Article Snippet: Cryo-EM map and model of the apo CST–POT1(ESDL)/TPP1 complex.

Techniques: Transfection, Western Blot, Construct, Binding Assay, Sequencing, Purification, Size-exclusion Chromatography, Fluorescence

Journal: bioRxiv

Article Title: Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

doi: 10.1101/2023.05.08.539880

Figure Lengend Snippet: Residue numbering labels correspond to the wild-type human POT1 sequence. Alpha helices are indicated as thick dashed lines and beta strands are indicated with arrows. The large red arrow points to the ESDL insertion, which is boxed in red. The alignment was calculated using MUSCLE with default settings and colored using the Clustal X color scheme.

Article Snippet: Cryo-EM map and model of the apo CST–POT1(ESDL)/TPP1 complex.

Techniques: Sequencing

Journal: bioRxiv

Article Title: Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

doi: 10.1101/2023.05.08.539880

Figure Lengend Snippet: ( A ) FSEC analysis of the binding between increasing concentrations of CST and POT1(ESDL)/TPP1 in the absence of telomeric ssDNA showing the concentration dependence of the interaction. RFU: relative fluorescence units. ( B ) Native glycerol gradient analysis of ssDNA-bound CST–POT1(ESDL)/TPP1. Pooled fractions are indicated with an asterisk. (Top) Coomassie-stained SDS-PAGE gel (4-12% Bis-Tris gel run in MOPS-SDS buffer, Invitrogen) of glycerol gradient fractions. (Middle) SYBR Gold-stained native PAGE (4-20% TBE gel run in 0.5x TB buffer, Invitrogen). (Bottom) Pooled fractions. ( C ) Domain architecture of components used to reconstitute an apo CST–POT1(ESDL)/TPP1 complex. TPP1 is fused to the N-terminus of Stn1 and retains part of the TPP1 serine-rich linker. ( D ) Coomassie-stained SDS-PAGE gel (4-12% Bis-Tris gel run in MOPS-SDS buffer, Invitrogen) showing the purified CST– POT1(ESDL)/TPP1 fusion complex used for structural analysis.

Article Snippet: Cryo-EM map and model of the apo CST–POT1(ESDL)/TPP1 complex.

Techniques: Binding Assay, Concentration Assay, Fluorescence, Staining, SDS Page, Clear Native PAGE, Purification

Journal: bioRxiv

Article Title: Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

doi: 10.1101/2023.05.08.539880

Figure Lengend Snippet: ( A ) Domain organization of CST and POT1(ESDL)/TPP1 subunits. Regions not observed in the cryo-EM maps are shown at 50% opacity. Dashed lines indicate regions not included in the expression construct. *The TPP1 serine-rich linker was included in the apo complex but not the ssDNA-bound complex. OB: oligosaccharide/oligonucleotide-binding domain; ARODL: Acidic Rpa1 OB-Binding Domain-like 3-helix bundle; wH: winged helix-turn-helix domain; HJRL: Holliday junction resolvase-like domain; RD: POT1 recruitment domain; TID: TIN2-interacting domain. ( B ) Cryo-EM reconstruction of apo CST–POT1(ESDL)/TPP1 at 3.9-Å resolution (see also ). ( C ) Cryo-EM reconstruction of ssDNA-bound CST–POT1(ESDL)/TPP1 at 4.3-Å resolution (see also ). ( D - E ) Atomic models for the apo and ssDNA-bound CST– POT1(ESDL)/TPP1 complexes, respectively . See also Movies S1 and S2.

Article Snippet: Cryo-EM map and model of the apo CST–POT1(ESDL)/TPP1 complex.

Techniques: Cryo-EM Sample Prep, Expressing, Construct, Binding Assay

Journal: bioRxiv

Article Title: Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

doi: 10.1101/2023.05.08.539880

Figure Lengend Snippet: (Left) Coomassie-stained SDS-PAGE gels (4-12% Bis-Tris gel run in MOPS-SDS buffer, Invitrogen) of proteins used in negative-stain EM analysis. (Middle) Representative negative-stain EM micrographs and top 25 reference-free 2D-class averages (sorted by number of particles per class from most populated class at top left to least populated class at bottom right) of each complex. (Right) Selected 2D averages zoomed to show CST features. Additional density attributable to the addition of POT1(ESDL)/TPP1 is indicated with red arrowheads.

Article Snippet: Cryo-EM map and model of the apo CST–POT1(ESDL)/TPP1 complex.

Techniques: Staining, SDS Page

Journal: bioRxiv

Article Title: Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

doi: 10.1101/2023.05.08.539880

Figure Lengend Snippet: ( A ) Representative motion-corrected micrograph and ( B ) enlarged view of the area marked in (A) with selected particles circled. ( C ) Representative 2D-class averages show high-resolution features and different orientations. ( D ) Cryo-EM image-processing pipeline used for the apo CST–POT1(ESDL)/TPP1 complex, including supervised 3D classification with noise decoy classes. Several rounds of heterogeneous refinement were used to select for particles with well-resolved Stn1 C and POT1 OB1/2 . ( E ) Final map of the apo CST–POT1(ESDL)/TPP1 complex with POT1(ESDL)/TPP1 colored as in for reference. ( F ) Directional FSC plots and sphericity values of the reconstruction calculated using the 3D-FSC server ( https://3dfsc.salk.edu/ ). ( G ) Plot of the angular distribution of particles in the final reconstruction. ( H ) Local resolution estimates of the apo CST–POT1(ESDL)/TPP1 map.

Article Snippet: Cryo-EM map and model of the apo CST–POT1(ESDL)/TPP1 complex.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

doi: 10.1101/2023.05.08.539880

Figure Lengend Snippet: ( A - B ) Gold-standard (blue), model-vs-map (red) FSC curves for apo ( A ) and ssDNA-bound ( B ) CST–POT1(ESDL)/TPP1 reconstructions. The map resolution was estimated using the gold-standard FSC and a cut-off criterion of 0.143. The similarity between the resolution estimate of the gold-standard FSC (0.143 cut-off) and resolution estimate of the model vs map FSC (0.5 cut-off) suggests no substantial over-fitting. ( C - D ) Cryo-EM map densities for each subunit indicating quality of fit for the apo ( C ) and ssDNA-bound ( D ) CST–POT1(ESDL)/TPP1 models.

Article Snippet: Cryo-EM map and model of the apo CST–POT1(ESDL)/TPP1 complex.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

doi: 10.1101/2023.05.08.539880

Figure Lengend Snippet: ( A ) Alternative cryo-EM image-processing pipeline used for the ssDNA-bound CST– POT1(ESDL)/TPP1 complex. This pipeline was used to select for classes with CST bound to ssDNA that also contained POT1 OB-C . Briefly, classes matching the supervised reference of this conformation were pooled, but further 3D classification or heterogenous refinement could not isolate a high-resolution group of particles solely in the desired conformation. ( B ) When processing the particles without clear density for POT1 OB-1/2 and Stn1 C , the TPP1 OB-fold could be visualized at low contouring thresholds. Coloring the map of an intermediate step shows an unaccounted-for cylindrical density reminiscent of an OB fold. ( C ) Deletion of the TPP1 OB-fold does not affect the CST–POT1(ESDL)/TPP1 interaction. Protein used and FSEC analysis of CST–POT1(ESDL)/TPP1(ΔOB) interaction in the absence (top) and presence (bottom) of telomeric ssDNA. Traces without CST are shown as dashed lines. RFU: relative fluorescence units.

Article Snippet: Cryo-EM map and model of the apo CST–POT1(ESDL)/TPP1 complex.

Techniques: Cryo-EM Sample Prep, Fluorescence

Journal: bioRxiv

Article Title: Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

doi: 10.1101/2023.05.08.539880

Figure Lengend Snippet: ( A ) Representative motion-corrected micrograph and ( B ) enlarged view of the area marked in (A) with selected particles circled. ( C ) Representative 2D-class averages show high-resolution features and different orientations. ( D ) Cryo-EM image-processing pipeline used for the CST– POT1(ESDL)/TPP1–ssDNA complex, including supervised 3D classification with noise decoy classes. Focused 3D classification with a mask was used to select for particles with well-resolved Stn1 C and POT1 OB1/2 . ( E ) Final map of the CST–POT1(ESDL)/TPP1–ssDNA complex with ssDNA–POT1(ESDL)/TPP1 colored as in for reference. ( F ) Directional FSC plots and sphericity values of the reconstruction calculated using the 3D-FSC server ( https://3dfsc.salk.edu/ ). ( G ) Plot of the angular distribution of particles in the final reconstruction. ( H ) Local resolution estimates of the CST–POT1(ESDL)/TPP1–ssDNA map.

Article Snippet: Cryo-EM map and model of the apo CST–POT1(ESDL)/TPP1 complex.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

doi: 10.1101/2023.05.08.539880

Figure Lengend Snippet: ( A ) Left panels: Structure of apo CST–POT1(ESDL)/TPP1 (left) and close-up view of the interaction of the POT1 hinge with Ctc1 OB-D (right). Right panels: Surface electrostatic potential analysis of the POT1(ESDL) hinge interacting with Ctc1 OB-D . The POT1(ESDL) hinge is shown in cartoon (left) and surface (right) representation with electrostatic potential colored from red (−10 k B T/e) to white (0 k B T/e) to blue (+10 k B T/e) (see also ). ( B ) Increasing negative charge of amino acids in the POT1 hinge enhances the interaction with CST. Anti-Myc co-IPs of Myc-tagged POT1 constructs and FLAG-tagged CST from co-transfected 293T cells. Immunoblots were probed with anti-Myc and anti-FLAG antibodies. ( C ) FSEC analysis of the interaction between CST and POT1(WT)/GFP-TPP1 (top, blue), POT1(SED)/GFP-TPP1 (middle, purple), and POT1(ESDL)/GFP-TPP1 (bottom, red) in the presence (right) or absence (left) of telomeric ssDNA . Traces without CST are shown as dashed lines, and traces containing POT1/TPP1 that have been treated with λ protein phosphatase (λPP) are shown in orange. RFU: relative fluorescence units.

Article Snippet: Cryo-EM map and model of the apo CST–POT1(ESDL)/TPP1 complex.

Techniques: Construct, Transfection, Western Blot, Fluorescence

Journal: bioRxiv

Article Title: Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

doi: 10.1101/2023.05.08.539880

Figure Lengend Snippet: ( A ) Electrostatic surface from with map density of hinge shown as mesh. (Right) 180° rotation view of POT1(ESDL) hinge showing negative charge on Ctc1-facing side and map density. ( B ) Coomassie blue-stained SDS-PAGE gel (4-12% Bis-Tris gel run in MOPS-SDS buffer, Invitrogen) showing proteins used in the FSEC analysis in . ( C ) Kinase assay with HeLa nuclear extract on a peptide-scanning array containing peptides corresponding to POT1, mPOT1b, and mPOT1a hinge regions.

Article Snippet: Cryo-EM map and model of the apo CST–POT1(ESDL)/TPP1 complex.

Techniques: Staining, SDS Page, Kinase Assay

Journal: bioRxiv

Article Title: Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

doi: 10.1101/2023.05.08.539880

Figure Lengend Snippet: ( A ) Comparison of CST-bound POT1 OB-3 /TPP1 (this study, colored) to unbound POT1 OB-3 /TPP1 (PDB 5H65 /5UN7 , grayscale). ( B ) Close-up views of sites of interest at or near the interface between POT1 and Ctc1 (see also ). Residues colored in red are mutated in CP. (i) Self-interaction between POT1 Ser322 and Arg367. Salt bridges are shown as yellow dashed lines. CP mutation S322L is predicted to disrupt this salt bridge and affect phosphorylation of the hinge. (ii) Self-interaction between POT1 hinge and C-terminus. (iii) Localization of Gly503 in the hydrophobic core of Ctc1 ARODL predicts a destabilizing effect of the G503R CP mutation. (iv) Primary hydrophobic interface between POT1 hinge, POT1 OB-3 , and Ctc1 ARODL . CP mutation H484P is predicted to disrupt the hydrophobic stacking interactions with POT1 Pro603 and Ctc1 His488 and Pro483. (v) Salt bridge between POT1 hinge residue Glu325 and Ctc1 OB-D residue Arg624. ( C ) Interaction between POT1 OB-2 and Ctc1 at the CST ssDNA-binding interface. Comparison of CST–POT1(ESDL)/TPP1 structure (this study, colored) to ssDNA-bound CST structure (PDB 6W6W , gray with DNA colored). Ctc1 aa 909-927 are modeled as poly-alanine stubs. ( D ) Negative-stain EM 2D averages (left) of ssDNA-bound CST–POT1(ESDL)/TPP1 showing three major conformations depicted by the cartoon schematics (right). 2D averages are flipped or rotated by 90° increments from into similar orientations for ease of comparison and are sorted by number of particles per class from most populated class at top left to least populated class at bottom right. The 2D averages that correspond to each cartoon state are indicated with black, gray, or white circles. The scale bar represents 333 Å (See also ).

Article Snippet: Cryo-EM map and model of the apo CST–POT1(ESDL)/TPP1 complex.

Techniques: Mutagenesis, Binding Assay, Staining

Journal: bioRxiv

Article Title: Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

doi: 10.1101/2023.05.08.539880

Figure Lengend Snippet: ( A ) Same as in but individual panels include the cryo-EM map density for the apo CST– POT1(ESDL)/TPP1 complex shown as white mesh. ( B ) Anti-Strep co-IPs of Strep-tagged POT1 constructs and FLAG-tagged CST from co-transfected 293T cells showing that the corresponding S328L CP mutation in mPOT1b disrupts the CST interaction. Immunoblots were probed with anti-Strep and anti-FLAG antibodies. ( C ) Anti-Myc co-IPs of Myc-tagged mPOT1b and FLAG-tagged CST (wild-type or bearing the G503R mutation) from co-transfected 293T cells showing that the CST G503R CP mutation disrupts the mPOT1b interaction. Immunoblots were probed with anti-Myc and anti-FLAG antibodies.

Article Snippet: Cryo-EM map and model of the apo CST–POT1(ESDL)/TPP1 complex.

Techniques: Cryo-EM Sample Prep, Construct, Transfection, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

doi: 10.1101/2023.05.08.539880

Figure Lengend Snippet: ( A ) Superposition of CST–POT1(ESDL)/TPP1 with the CST–Polα/Primase recruitment complex (RC, left) and pre-initiation complex (PIC, right). The CST–POT1(ESDL)/TPP1–ssDNA complex is shown as an opaque surface, and CST–Polα/Primase complexes are shown in cartoon representation with transparent surfaces. Orthogonal views show that POT1(ESDL)/TPP1 binding does not obstruct the major interface of the RC, but POT1 OB-1/2 and Stn1 C would interfere with binding of the POLA1 catalytic core to the CST ssDNA-binding site in the PIC (see also ). ( B ) Model for the telomeric recruitment and regulation of CST–Polα/Primase by shelterin. Phosphorylated POT1 recruits CST–Polα/Primase in an auto-inhibited, RC-like state. CST–Polα/Primase is then held in the auto-inhibited state during the steps of 5’-end resection and telomerase-mediated G-strand extension. Dephosphorylation of POT1 releases CST–Polα/Primase into the PIC, allowing fill-in to begin.

Article Snippet: Cryo-EM map and model of the apo CST–POT1(ESDL)/TPP1 complex.

Techniques: Binding Assay, De-Phosphorylation Assay

Journal: bioRxiv

Article Title: Structural basis of CST-Polα/Primase recruitment and regulation by POT1 at telomeres

doi: 10.1101/2023.05.08.539880

Figure Lengend Snippet: ( A ) Multiple views of CST–Polα/Primase in RC (left) and PIC (right) conformations. CST– Polα/Primase structures are shown in cartoon representation with a transparent surface. ( B ) Superposition of the CST–POT1(ESDL)/TPP1–ssDNA structure with the structures of the RC (left, see also Movie S4) and PIC (right, see also Movie S3) complexes showing additional views compared to . The clash between the POT1 HJRL and PRIM2 is indicated. ( C ) Multi-body analysis of CST–Polα/Primase in the RC conformation. Polα/Primase was designated body 1 and CST was designated body 2 with the corresponding masks shown. Histograms of the projections of the relative orientations onto the corresponding components show a unimodal distribution, consistent with continuous flexibility rather than discrete states. The first three principal components accounted for 61% of the variance in the data. Reconstructed maps from the extreme ends are shown in red and blue for each of the first three principal components with arrows indicating the direction of motion (see also Movies S5-7). ( D ) PAE plots of the top two (of 5) ranked AlphaFold-Multimer models for Ctc1–POLA2. Green arrowheads indicate high confidence in the position prediction of POLA2 relative to Ctc1. All 5 top-ranked models predicted the interaction with high confidence. ( E ) AlphaFold-Multimer model of Ctc1 bound to POLA2 NTD .

Article Snippet: Cryo-EM map and model of the apo CST–POT1(ESDL)/TPP1 complex.

Techniques: