Journal: Nature Communications

Article Title: CENP-E activation by Aurora A and B controls kinetochore fibrous corona disassembly

doi: 10.1038/s41467-023-41091-2

Figure Lengend Snippet: a High temporal resolution spinning-disk confocal time-series of GFP-CENP-E T422A at KTs in U2OS cells. Time: min:sec. Scale bar: 10 μm. Red and white arrowheads indicate a CENP-E T422A comet. b Quantification of GFP-CENP-E T422A stripping velocity represented with mean ± SD. A total of 11 comets in 9 cells from 3 independent experiments were analyzed. c Representative time-series of U2OS GFP-CENP-E WT/T422A cells entering mitosis in the absence of MTs. Time: hour:min. Scale bar: 10 μm. d Time-lapse images of GFP-CENP-E T422A reloading at KTs after MT depolymerization. Time: hour:min Scale bars: 10 μm. e Quantification of GFP-CENP-E T422A at the KTs over time after the addition of nocodazole. Average KT intensities in cells are represented with mean ± SD. N (number of KTs, number of cells, number of independent experiments): t = 0 min (520, 26, 3), t = 10 min (525, 226, 3). Statistical significance was determined by the Mann–Whitney U -test (unpaired, two-tailed; no normal distribution). p values are indicated. f Representative maximum intensity-projected confocal images of expanded fibrous coronas in HeLa cells transiently transfected with GFP-CENP-E WT/T422A. Insets display single KT pairs with expanded coronas. Scale bar: 5 μm; scale bar in insets: 1 μm. g Quantification of corona volume and GFP intensity normalized to CENP-C intensity at KTs. Average KT intensities in cells are represented with mean ± SD. N (number of coronas, number of cells, number of independent experiments) - Corona volume: WT (1038, 29, 3), T422A (1063, 30, 3); KT intensity: WT (899, 40, 3), T422A (781, 38, 3). Statistical significance was determined by the Mann–Whitney U -test (unpaired, two-tailed; no normal distribution). h Structured illumination microscopy (SIM) images of phosphorylated CENP-E at expanded coronas of RPE cells, detected using the anti-phospho-T422 antibody. Scale bar: 5 μm; scale bar in insets: 1 μm. i 3D surface rendering model of the fibrous corona represented in h . j Intensity profile plot of 40 coronas from 10 different cells from 2 independent experiments as represented in h . Thick and dashed lines represent averages and SD, respectively.

Article Snippet: For the generation of stable U2OS GFP-CENP-E/CENP-A-mCherry and HeLa DHC-GFP/CENP-A-mCherry cell lines, we performed lentiviral infection of the U2OS GFP-CENP-E (this study) and HeLa DHC-GFP (gift from Iain Cheeseman, Whitehead Institute for Biomedical Research, Cambridge, USA) cell lines using pLenti CENP-A-mCherry vector (Addgene #89767), followed by fluorescence activated cell-sorting (FACS) of mCherry-positive cells.

Techniques: Stripping Membranes, MANN-WHITNEY, Two Tailed Test, Transfection, Microscopy

Journal: Nature Communications

Article Title: CENP-E activation by Aurora A and B controls kinetochore fibrous corona disassembly

doi: 10.1038/s41467-023-41091-2

Figure Lengend Snippet: a Spinning-disk confocal time-series of HeLa cells stably expressing DHC-GFP/CENP-A-mCherry and labeling of DNA with SiR-DNA undergoing the indicated treatments. The red arrow shows the presence of dynein in one KT in early metaphase. Relative time from NEB is represented. Time scale: hour:min. Scale bar: 10 μm. b DHC-GFP dynamics at KTs after NEB. Dots and dashed lines represent mean values and SD, respectively. The solid thick line represents the fitted curve. N (number of cells, number of independent experiments): siCTRL (13, 2), siCENP-E (14, 2). c Representative maximum intensity-projected point-scanning confocal images of U2OS WT/T422A monopoles immunostained against the indicated proteins. Scale bar: 10 μm. d Fluorescence intensity profile plots of the indicated proteins (representing the region depicted by a white dashed line in c ). e Quantification of spindle pole accumulation of indicated proteins in U2OS GFP-CENP-E WT and T422A cells represented in c . Values are plotted with mean ± SD. N (number of cells, number of independent experiments): GFP-CENP-E in WT (51, 3), GFP-CENP-E in T422A (55, 3), SPDLY in WT (54, 3), SPDLY in T422A (52, 3), ZW10 in WT (33, 3), ZW10 in T422A (34, 3), MAD1 in WT (43, 3), MAD1 in T422A (39, 3), BUBR1 in WT (50, 3), BUBR1 in T422A (49, 3). Statistical significance was determined by the Mann–Whitney U -test (unpaired, two-tailed; no normal distribution). p values are indicated.

Article Snippet: For the generation of stable U2OS GFP-CENP-E/CENP-A-mCherry and HeLa DHC-GFP/CENP-A-mCherry cell lines, we performed lentiviral infection of the U2OS GFP-CENP-E (this study) and HeLa DHC-GFP (gift from Iain Cheeseman, Whitehead Institute for Biomedical Research, Cambridge, USA) cell lines using pLenti CENP-A-mCherry vector (Addgene #89767), followed by fluorescence activated cell-sorting (FACS) of mCherry-positive cells.

Techniques: Stable Transfection, Expressing, Labeling, Fluorescence, MANN-WHITNEY, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Parkinson’s disease-linked D620N VPS35 knockin mice manifest tau neuropathology and dopaminergic neurodegeneration

doi: 10.1073/pnas.1814909116

Figure Lengend Snippet: Axonal damage in D620N VPS35 KI mice. Representative photomicrographs of striatum and substantia nigra from 13-mo-old VPS35 D620N/WT , VPS35 D620N/D620N , and control VPS35 WT/WT mice indicating ( A ) APP immunohistochemistry revealing APP-positive spheroids ( black arrowheads ), a marker of axonal damage. (Scale bars: 100 μm.) Insets show high-power images of the boxed regions. (Scale bars: 50 μm.) ( B ) Gallyas silver staining revealing degenerating neuritic processes ( red arrows ). (Scale bar: 50 μm.) ( C ) Bar graph indicating quantitation of silver-positive black neurites in the striatum. Data are expressed as the percent of total pixels per image (mean ± SEM, n = 5 or 6 animals per genotype). *** P < 0.001 or **** P < 0.0001 by one-way ANOVA with Bonferroni’s post hoc test compared with WT/WT mice.

Article Snippet: Human V5-tagged VPS35 plasmids were obtained from Addgene (no. 21691; ref. ) and previously described ( ).

Techniques: Immunohistochemistry, Marker, Silver Staining, Quantitation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Parkinson’s disease-linked D620N VPS35 knockin mice manifest tau neuropathology and dopaminergic neurodegeneration

doi: 10.1073/pnas.1814909116

Figure Lengend Snippet: Hippocampal tau abnormalities in D620N VPS35 KI mice. Representative photomicrographs of hippocampus from 13-mo-old VPS35 D620N/WT , VPS35 D620N/D620N , and control VPS35 WT/WT mice indicating immunohistochemical staining with antibodies to phosphorylation-specific tau (AT8) or abnormal conformation-specific tau (MC1). AT8- and MC1-positive immunoreactivity, including abnormal somatodendritic labeling, is markedly increased in the hippocampus of D620N/WT and D620N/D620N mice compared with WT mice. Insets represent high-power images from the boxed regions in the hippocampal CA1 and CA3 subregions. (Scale bars: 100 μm.)

Article Snippet: Human V5-tagged VPS35 plasmids were obtained from Addgene (no. 21691; ref. ) and previously described ( ).

Techniques: Immunohistochemical staining, Staining, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Parkinson’s disease-linked D620N VPS35 knockin mice manifest tau neuropathology and dopaminergic neurodegeneration

doi: 10.1073/pnas.1814909116

Figure Lengend Snippet: Tau abnormalities in nigral dopaminergic neurons of D620N VPS35 KI mice. Immunofluorescent confocal colocalization analysis of the SNpc from 13-mo-old VPS35 D620N/WT , VPS35 D620N/D620N , and control VPS35 WT/WT mice colabeled with ( A ) Tau5 (total tau), ( C ) AT8, or ( E ) MC1 antibodies (green) and the dopaminergic neuronal marker TH (red). Merged images indicate the accumulation of Tau5-, AT8-, and MC1-positive immunofluorescent signals within the somatodendritic compartment of nigral dopaminergic neurons of D620N/WT and D620N/D620N mice. (Scale bars: 15 μm.) Bar graphs indicating CTCF values (mean ± SEM, n = 4 animals per group) of ( B ) Tau5, ( D ) AT8, or ( F ) MC1 fluorescence intensity in TH-positive dopaminergic neurons. *** P < 0.001 by one-way ANOVA with Bonferroni’s post hoc test compared with WT/WT mice.

Article Snippet: Human V5-tagged VPS35 plasmids were obtained from Addgene (no. 21691; ref. ) and previously described ( ).

Techniques: Marker, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Parkinson’s disease-linked D620N VPS35 knockin mice manifest tau neuropathology and dopaminergic neurodegeneration

doi: 10.1073/pnas.1814909116

Figure Lengend Snippet: Lethal neurodegenerative phenotype of human A53T-α-Syn transgenic mice is independent of D620N VPS35. Kaplan–Meier survival curves of A53T-α-Syn and A53T-α-Syn/VPS35 KI mice were generated by monitoring cohorts of all genotypes ( VPS35 WT/WT /αSyn A53T , n = 21; VPS35 D620N/WT /αSyn A53T , n = 48; VPS35 D620N/D620N /αSyn A53T , n = 23) until mice had to be euthanized due to the onset of terminal disease. There is no significant difference between these three genotypes by log-rank (Mantel–Cox) test ( P = 0.4601), as indicated. VPS35 WT/WT ( n = 32), VPS35 D620N/WT ( n = 41), and VPS35 D620N/D620N ( n = 12) mice exhibit normal survival up to 24 mo of age.

Article Snippet: Human V5-tagged VPS35 plasmids were obtained from Addgene (no. 21691; ref. ) and previously described ( ).

Techniques: Transgenic Assay, Generated

Journal: Journal of neurochemistry

Article Title: The tumor suppressor p53 guides GluA1 homeostasis through Nedd4-2 during chronic elevation of neuronal activity.

doi: 10.1111/jnc.13271

Figure Lengend Snippet: Fig. 2 Mdm2-p53 signaling mediates chronically elevated neuronal activity- induced glutamate receptor subunit 1 (GluA1) ubiquitination and down-regulation. (a) western blots of GluA1 and glutamate receptor subunit 2 (GluA2) from vehicle- or PTX-treated WT cortical neurons (n = 4). (b) western blots of GluA1 and p53 from WT cortical neurons lentivirally transfected with V5-p53 (over-expression) or p53 shRNA (n = 3). (c) western blots of Ubiquitin (Ub) or GluA1 after GluA1 immunoprecipitation from PTX- and/or Nutlin-3-treated WT cortical neurons (n = 4). (d) western blots of GluA1 and GluA2 from PTX- and/or Nutlin-3-treated WT cortical neurons (n = 4). Plotted is the average of experimental groups after normalizing to Gapdh and to vehicle-treated groups, except for (c) which is normalized to immunoprecipitated GluA1 and then to vehicle-treated groups. (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: The pCI-HANedd4-1, pCI-HA-Nedd4-2, pCI-SEP-GluA1, pcDNA-PSD-95, pLenti6/V5-p53_wt p53, and pLKO-p53-shRNA-427 plasmids were from Addgene, Cambridge, MA, USA.

Techniques: Activity Assay, Ubiquitin Proteomics, Western Blot, Transfection, Over Expression, shRNA, Immunoprecipitation

Journal: bioRxiv

Article Title: Targeting mitophagy in SRSF2 mutant hematologic malignancies

doi: 10.1101/2023.06.25.546449

Figure Lengend Snippet: (A) Venn diagram of genes differentially expressed (blue) or differentially spliced (green) in CHIR-treated WT , SRSF2 P95H/+ and SF3B1 K700E/+ K562 cells relative to DMSO-treated controls. (B) Sashimi plots of PINK1 in WT , SRSF2 P95H/+ and SF3B1 K700E/+ cells treated with DMSO or CHIR (left). Bar graph shows intron 6 retention as a percentage of total transcripts (right). (C) PINK1 mRNA levels in WT , SRSF2 P95H/+ and SF3B1 K700E/+ cells treated with DMSO or 3uM CHIR for 24 h detected by RT-qPCR using primers that span exons 1 and 2 (mean ± SD, n = 3). **: p <0.001. (D) PINK1 nascent transcript detection by RT-qPCR using primers for intron 1 (left) and intron 5 (right) in WT , SRSF2 P95H/+ and SF3B1 K700E/+ cells treated with DMSO or 3uM CHIR for 24 h. (E-F) Representive RT-PCR with primers spanning exon 6 (E6), intron 6 (I6), and exon 7 (E7) of PINK1 in WT , SRSF2 P95H/+ , SF3B1 K700E/+ K562 cells (E) and primary AML cells, CD34 + cells from healthy donors, and CD34 + cells from CMML patients (F) treated with DMSO or 3uM CHIR for 24h. M: DNA marker. PCR product with retained intron 6 is 568bp and product for spliced exon6-7 (without retained intron) is 206bp. (G) RT-PCR analysis of PINK1 splicing in WT , GSK3A/B DKO, and GSK3A/B DKO cells overexpressing GSK-3β (HEK293T).

Article Snippet: PINK1 (pLenti6-DEST PINK1-V5 WT, 13320), WT (pRRL_SRSF2_WT_mCherry, 84020), and P95H SRSF2 (pRRL_SRSF2_P95H_mCherry, 84023) lentiviral overexpression constructs were purchased from Addgene.

Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Marker

Journal: bioRxiv

Article Title: Targeting mitophagy in SRSF2 mutant hematologic malignancies

doi: 10.1101/2023.06.25.546449

Figure Lengend Snippet: (A) Representive RT-PCR results of PINK1 splicing in TF-1 cells expressing either WT or P95H mutant SRSF2 treated with DMSO or 1µM CHIR for 24h. (B) Violin plot of OPTN , ULK1 and TOMM7 normalized expression in AML patients in the TCGA dataset (n = 405) with or without mutations in SF3B1 . Statistical analysis was performed using two-tailed Mann-Whitney test.

Article Snippet: PINK1 (pLenti6-DEST PINK1-V5 WT, 13320), WT (pRRL_SRSF2_WT_mCherry, 84020), and P95H SRSF2 (pRRL_SRSF2_P95H_mCherry, 84023) lentiviral overexpression constructs were purchased from Addgene.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Mutagenesis, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Targeting mitophagy in SRSF2 mutant hematologic malignancies

doi: 10.1101/2023.06.25.546449

Figure Lengend Snippet: (A) Heat map shows z-scored expression of mitophagy related genes in WT and SRSF2 P95H/+ K562 cells. (B) Representative confocal images of mitochondria (green, TOMM20 + ) and lysosomes (red, LAMP1 + ) in K562 WT , or SRSF2 P95H/+ cells treated with DMSO or 3uM CHIR for 2d (left). Bar plot shows quantification of the co-localization of mitochondria with lysosomes (n=5 fields for each group. N=3). (C) Representative TEM images of WT and SRSF2 P95H/+ K562 cells treated with DMSO or 3µM CHIR for 2d (left). Bar plot (right) shows quantification of the number of autophagic vacuoles per cell. Each dot represents one cell. (D) Immunoblotting of PINK1 and β-catenin in WT and SRSF2 P95H/+ cells treated with DMSO or 3 uM CHIR for indicated times. (E) Violin plot of OPTN , ULK1 and TOMM7 normalized expression in AML patients in the TCGA dataset (n = 581) with or without mutations in SRSF2 . Statistical analysis was performed using two-tailed Mann-Whitney test. (F) Overall survival (OS) of AML patients (n = 106) separated into two subgroups based on high and low expression of OPTN in publicly available TCGA datasets. Group cutoff for high and low expression was set to median (50%). Statistical analysis was performed using Mantel-Cox test.

Article Snippet: PINK1 (pLenti6-DEST PINK1-V5 WT, 13320), WT (pRRL_SRSF2_WT_mCherry, 84020), and P95H SRSF2 (pRRL_SRSF2_P95H_mCherry, 84023) lentiviral overexpression constructs were purchased from Addgene.

Techniques: Expressing, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Targeting mitophagy in SRSF2 mutant hematologic malignancies

doi: 10.1101/2023.06.25.546449

Figure Lengend Snippet: (A-B) Gene ontology enrichment analysis of differentially spliced genes in SRSF2 mutant cells versus WT K562 cells (A) and primary CMML and AML patient samples (B). Bar graphs show significantly dysregulated pathways that are associated with mitochondrial functions, with Fisher’s exact test -log[q-value] on X-axis. (C) Venn diagram showing overlap of proteins differentially expressed in SRSF2 P95H/+ versus WT K562 cells and MitoCarta 3.0 database. (D) Bar graph shows Log2 ratio of selected mitochondrial protein in SRSF2 P95H/+ vs. SRSF2 +/+ K562 cells. (E-G) Quantification of mitochondrial parameters in WT and SRSF2 P95H/+ cells (mean ± SD, n = 3). E, mitotracker green (MTG). F, Mitochondrial DNA copy number. Data are shown as ratio of mitochondrial DNA (mt-ND1) to nuclear DNA (B2M) in wt and SRSF2 P95H/+ cells. G, MMP per mitochondrion. (H) Basal, maximal, and complex I-linked mitochondrial respiratory capacity was measured in WT and SRSF2 P95H/+ cells. Maximal respiratory capacity was measured after FCCP injection. Complex I-linked respiration was measured by sequential addition of the complex I-linked substrates pyruvate, malate, and glutamate (P/M/G) and ADP. Oxygen flux expressed as respiration per million cells [pmol/(s·10 6 cells], mean ± SD of N = 3 independent cultures. Each sample was measured in duplicate. (I) Analysis of mitochondrial depolarization in WT and SRSF2 P95H/+ cells treated with DMSO or 2µM CCCP for 24h using JC-1 staining, in which a high Red + :Green + ratio indicates high MMP (n=3). (J) Representive RT-PCR results of PINK1 splicing in WT and SRSF2 P95H/+ cells treated with DMSO, 3µM CHIR or indicated concentrations of CCCP for 24h. (K) RT-qPCR analysis of PINK1 mRNA levels in WT and SRSF2 P95H/+ cells treated with DMSO, 3µM CHIR, or 2µM CCCP for 24 h.

Article Snippet: PINK1 (pLenti6-DEST PINK1-V5 WT, 13320), WT (pRRL_SRSF2_WT_mCherry, 84020), and P95H SRSF2 (pRRL_SRSF2_P95H_mCherry, 84023) lentiviral overexpression constructs were purchased from Addgene.

Techniques: Mutagenesis, Injection, Staining, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Targeting mitophagy in SRSF2 mutant hematologic malignancies

doi: 10.1101/2023.06.25.546449

Figure Lengend Snippet: (A-B) Quantification of mitochondrial parameters in WT and SRSF2 P95H/+ cells treated with DMSO or 3µM CHIR (mean ± SD, n = 3). A, MMP per mitochondrion. B, MTG. (C) GSEA showing mitochondrial related enrichment plots for CHIR versus DMSO treated WT and SRSF2 P95H/+ cells. (D) Schematic of generating WT and SRSF2 P95H/+ stable lines overexpressing PINK1 for apoptosis assay with or without CHIR. (E) Percentages of viable, early-, and late-apoptotic cells in PINK1 overexpressing WT and SRSF2 P95H/+ cells treated with DMSO or 3µM CHIR in vitro for 8d (mean ± SD, n=3). (F) Proposed model for dependency of SRSF2 mutant leukemias on PINK1-mediated mitophagy and preferential cytotoxicity of GSK-3i and mitophagy inhibitor Chloroquine. (G) Quantification of mitochondrial mass by MTG staining in WT and SRSF2 P95H/+ cells treated with vehicle or indicated concentrations of Chloroquine for 6d (mean ± SD, n=3). (H) Percentage of viable cells based on 7-AAD and Annexin V flow cytometric analysis of WT and SRSF2 P95H/+ cells treated with vehicle or 20µM Chloroquine in vitro for 8d (mean ± SD, n=3). (I) Schematic representation of lentiviral delivery of SRSF2 wt or SRSF2 P95H to primary AML cells for Chloroquine treatment followed by apoptosis assay. (J) Representive flow cytometric analysis (left) and quantification (right) of apoptosis in primary cells from patient with AML overexpressing either WT or SRSF2 P95H , as measured by Annexin V and 7-AAD staining in absence or presence of 15µM Chloroquine for 4d.

Article Snippet: PINK1 (pLenti6-DEST PINK1-V5 WT, 13320), WT (pRRL_SRSF2_WT_mCherry, 84020), and P95H SRSF2 (pRRL_SRSF2_P95H_mCherry, 84023) lentiviral overexpression constructs were purchased from Addgene.

Techniques: Apoptosis Assay, In Vitro, Mutagenesis, Staining