Journal: eLife

Article Title: A first order phase transition mechanism underlies protein aggregation in mammalian cells

doi: 10.7554/eLife.39695

Figure Lengend Snippet: Plotted is the energy of cluster formation ( - L o g ( P ( n ) as described in versus n for clusters in normal growth conditions for (i) MCF10A cells with clusters marked with Synphilin (circles), (ii) Neuro2A cells with clusters marked by alpha Synuclein (stars). For the range of values < n ~ 1000000 where the Neuro2A data set begins to exhibit a high noise level due to smaller size of data set, the two distributions are indistinguishable. Log refers to the natural log (base ‘e’). Untreated cell data is from the normalized distribution of 10,000 clusters from 10 cells, Neuro2A data from 3000 clusters from 5 cells.

Article Snippet: For MCF10A –alpha Synuclein cell line – alpha Synuclein-Dendra2 plasmid, was transiently transfected using extremegene9 and selected using Kanamycin resistance cassette in MCF10A (ATCC, USA) cells.

Techniques:

Journal: eLife

Article Title: A first order phase transition mechanism underlies protein aggregation in mammalian cells

doi: 10.7554/eLife.39695

Figure Lengend Snippet:

Article Snippet: For MCF10A –alpha Synuclein cell line – alpha Synuclein-Dendra2 plasmid, was transiently transfected using extremegene9 and selected using Kanamycin resistance cassette in MCF10A (ATCC, USA) cells.

Techniques: Transfection, Construct, Generated, Plasmid Preparation, Sequencing, Software

Journal: The Biochemical journal

Article Title: N-terminal acetylation and methylation differentially affect the function of MYL9

doi: 10.1042/BCJ20180638

Figure Lengend Snippet: (A) WT MYL9 (SSK) has been found in Nα-methylated and Nα-acetylated forms. The S3P (SPK) MYL9 mutant is Nα-methylated, and the K4Q (SSQ) MYL9 mutant is Nα-acetylated. In vivo, the initiator methionine of WT MYL9 is cleaved to reveal the N-terminal sequence shown. (B–G) in vitro methyltransferase and acetyltransferase assays were performed to determine the KM of NRMT1 or NAA10 (the catalytic subunit of NatA) when using peptides corresponding to the 14 N-terminal amino acids (after Met cleavage) of WT and mutant MYL9 as substrates. (B) The KM of NRMT1 with WT (SSK) MYL9 was determined to be 28.8 μM. (C) NAA10 with WT (SSK) MYL9 had a KM of 0.7 μM. (D) SPK MYL9 was confirmed to be a preferred substrate of NRMT1 with a KM of 0.7 μM. (E) NAA10 showed no activity with SPK MYL9 up to 40 μM. (F) SSQ MYL9 Figure 1. N-terminal mutants of MYL9 select for Nα-methylation or Nα-acetylation. Part 2 of 2 was not a substrate of NRMT1, showing no activity up to 160 μM. (G) The KM of NAA10 with SSQ MYL9 was 5.0 μM. n = 3 for all experiments. All error bars represent standard deviation.

Article Snippet: To generate the C-terminally tagged MYL9-Dendra2 fusion protein, the MYL9 protein ORF (GE Dharmacon) was amplified and subcloned into the pDendra2-N vector (Clontech, Mountain View, CA) using XhoI and HindIII restriction sites.

Techniques: Methylation, Mutagenesis, In Vivo, Sequencing, In Vitro, Activity Assay, Standard Deviation

Journal: The Biochemical journal

Article Title: N-terminal acetylation and methylation differentially affect the function of MYL9

doi: 10.1042/BCJ20180638

Figure Lengend Snippet: (A,B) HCT116 cells were transduced with WT or mutant MYL9-FLAG and transwell migration assays were performed. (A) Western Blotting confirms that MYL9 variants were expressed at equal levels. β-actin was used as a loading control. (B) Only cells expressing the nonmethylatable SSQ MYL9 mutant showed significantly greater migration than nontransduced control cells (P = 0.01), indicating enhanced cytoskeletal activity (Control — 1.00, WT — 1.27 ± 0.10, SPK — 1.52 ± 0.25, SSQ — 1.81 ± 0.26). (C) Representative blot showing increased pS19 of the nonmethylatable SSQ MYL9 mutant after calcimycin treatment. FLAG blot used to determine total MYL9 protein levels. (D) SSQ MYL9 had a significantly greater ratio of pS19 to total protein than SPK MYL9 (P < 0.05; WT — 0.30 ± 0.06, SPK — 0.21 ± 0.12, SSQ — 0.58 ± 0.13). (E) Representative blot showing increased pS19 of WT MYL9 in cells that lack Nα-methylation (Me3). (F) WT MYL9 in NRMT1 KO cells had a significantly greater ratio of pS19 to total protein than WT MYL9 in control cells (P < 0.005; Control — 0.47 ± 0.01, NRMT1 KO — 0.81 ± 0.08). One-way ANOVA with Tukey’s multiple comparisons test was used for analysis of results in B and D. Results in F were analyzed by a Student’s two-tailed t-test. n = 3 for all experiments. All error bars represent standard deviation.

Article Snippet: To generate the C-terminally tagged MYL9-Dendra2 fusion protein, the MYL9 protein ORF (GE Dharmacon) was amplified and subcloned into the pDendra2-N vector (Clontech, Mountain View, CA) using XhoI and HindIII restriction sites.

Techniques: Transduction, Mutagenesis, Migration, Western Blot, Expressing, Activity Assay, Methylation, Two Tailed Test, Standard Deviation

Journal: The Biochemical journal

Article Title: N-terminal acetylation and methylation differentially affect the function of MYL9

doi: 10.1042/BCJ20180638

Figure Lengend Snippet: (A) Fluorescence images of cells expressing Dendra2 alone. (B) The sum fluorescence intensity of Dendra2 only expressing cells is plotted over 48 h. Individual cells show decreased RFP intensity as cell division occurs, but the sum intensity over the field of view remains constant. (C) Fluorescent images of cells expressing WT or mutant MYL9-Dendra2 out to 24 h. (D,E) Fluorescent decay for WT, SPK, and SSQ MYL9 was plotted and fit to a model of one-phase exponential decay. SPK and SSQ MYL9 were each fit to an individual model of decay and a model of decay shared with WT MYL9. Individual and shared fit models were then evaluated by an extra sum-of-squares F test. For both SPK and SSQ MYL9 it was determined that a shared model of decay with WT MYL9 was as effective as an individual model, indicating no effect on stability. The half-life of MYL9 was determined to be 16.4 h. Scale bars are 100 μM. n = 3–4 for all experiments. All error bars represent standard deviation.

Article Snippet: To generate the C-terminally tagged MYL9-Dendra2 fusion protein, the MYL9 protein ORF (GE Dharmacon) was amplified and subcloned into the pDendra2-N vector (Clontech, Mountain View, CA) using XhoI and HindIII restriction sites.

Techniques: Fluorescence, Expressing, Mutagenesis, Standard Deviation

Journal: The Biochemical journal

Article Title: N-terminal acetylation and methylation differentially affect the function of MYL9

doi: 10.1042/BCJ20180638

Figure Lengend Snippet: (A) NIH 3T3 cells expressing the Nα-methylation deficient SSQ mutant of MYL9 showed considerable cell spreading, with lamellipodia and filopodia (black arrows) readily observable. Scale bar is 100 μM. (B) Cells expressing SSQ MYL9 covered significantly greater area than all other cell lines at 45 (P < 0.01) and 60 (P < 0.001) minutes as determined by two-way random measures ANOVA with Tukey’s multiple comparisons test (all measurements μm2; 45 min: Control — 96.7 ± 6.1, WT MYL9 — 96.4 ± 14.6, SPK MYL9 — 98.4 ± 22.1, SSQ MYL9 — 154.1 ± 25.2; 60 min: Control — 115.1 ± 12.6, WT MYL9 — 113.6 ± 18.7, SPK MYL9 — 113.0 ± 28.9, SSQ MYL9 — 188.3 ± 30.0) (n = 3). (C) Equal expression of MYL9 variants was confirmed through Western Blotting. FLAG was used as a measure of MYL9-FLAG expression. GAPDH was used as a loading control. All error bars represent standard deviation.

Article Snippet: To generate the C-terminally tagged MYL9-Dendra2 fusion protein, the MYL9 protein ORF (GE Dharmacon) was amplified and subcloned into the pDendra2-N vector (Clontech, Mountain View, CA) using XhoI and HindIII restriction sites.

Techniques: Expressing, Methylation, Mutagenesis, Western Blot, Standard Deviation

Journal: The Biochemical journal

Article Title: N-terminal acetylation and methylation differentially affect the function of MYL9

doi: 10.1042/BCJ20180638

Figure Lengend Snippet: (A) Cells expressing the Nα-methylation enriched SPK mutant of MYL9 show significantly greater transcription of ICAM1 than all other TNFα stimulated cell lines. Results are shown as fold-change compared with untreated, nontransduced cells, and one-way ANOVA with Tukey’s multiple comparisons test was performed for statistical analysis (P < 0.001; Control — 9.03 ± 1.50, WT — 6.35 ± 1.36, SPK — 23.71 ± 3.21, SSQ — 4.18 ± 1.23). (B) The induction of ICAM1 transcription by SPK MYL9 was significantly decreased in methylation-deficient NRMT1 KO cells. Results are shown as fold-change compared with treated control cells, and a Student’s two-tailed t-test was performed for statistical analysis (P < 0.005; Control — 1.00, NRMT1 KO — 0.46 ± 0.11). (C) All MYL9-Dendra2 protein variants showed equal increases in nuclear localization after TNFα treatment (P < 0.05). Control Dendra2 protein showed no change in localization upon treatment. Two-way ANOVA with Bonferroni’s multiple comparisons test was used for analysis of results (Dendra2-only no treatment (No Tx) — 0.59 ± 0.01, TNFα — 0.58 ± 0.01; WT No Tx — 0.41 ± 0.005, TNFα — 0.50 ± 0.04; SPK No Tx — 0.42 ± 0.03, TNFα — 0.50 ± 0.01; SSQ No Tx — 0.41 ± 0.005, TNFα — 0.48 ± 0.005). Scale bars are 100 μM. (D) ChIP experiments showing the Nα-methylation enriched SPK MYL9-FLAG mutant was the only protein to result in detectable enrichment of the ICAM1 promoter sequence over input. The PLK promoter served as an off-target control sequence. n = 3 for all experiments. All error bars represent standard deviation.

Article Snippet: To generate the C-terminally tagged MYL9-Dendra2 fusion protein, the MYL9 protein ORF (GE Dharmacon) was amplified and subcloned into the pDendra2-N vector (Clontech, Mountain View, CA) using XhoI and HindIII restriction sites.

Techniques: Expressing, Methylation, Mutagenesis, Two Tailed Test, Sequencing, Standard Deviation

Journal: The Biochemical journal

Article Title: N-terminal acetylation and methylation differentially affect the function of MYL9

doi: 10.1042/BCJ20180638

Figure Lengend Snippet: Nα-PTMs of MYL9 were not specifically bound by any of the tested (A) methyl or (B) acetyl reader domains. Nonspecific binding was observed for all forms of MYL9 peptide to the Tudor domain from the LBR protein, which is consistently recognized nonspecifically. (C) The SILAC LC–MS/MS screen identified few proteins whose interactions increased with Nα-modified MYL9. However, both Nα-mono and trimethylation led to widespread blocking of interactions. (D) Only six proteins showed up-regulated interaction with Nα-modified MYL9. (E) Many of the hundreds of proteins that showed down-regulated interaction with Nα-methylated MYL9 are associated with cytoskeletal regulation. (F) Western blots of pulldowns did not confirm the increased interactions with PSMD7 and HEXIM2, but did confirm the decrease in Cofilin-1 interaction with methylated MYL9.

Article Snippet: To generate the C-terminally tagged MYL9-Dendra2 fusion protein, the MYL9 protein ORF (GE Dharmacon) was amplified and subcloned into the pDendra2-N vector (Clontech, Mountain View, CA) using XhoI and HindIII restriction sites.

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Modification, Blocking Assay, Methylation, Western Blot

Journal:

Article Title: Investigating Intracellular Dynamics of FtsZ Cytoskeleton with Photoactivation Single-Molecule Tracking

doi: 10.1529/biophysj.108.128751

Figure Lengend Snippet: Principle of the photoactivation single-molecule tracking method. (A) The microscope setup. The photoactivation laser excites the sample under TIR condition. The fluorescence excitation laser illuminates the whole depth of the sample. (B) A segment of the fluorescence trajectory taken from E. coli cells expressing FtsZ-Dendra2 chimeric protein. The unactivated molecules are illustrated in gray, the activated in red, and the photobleached in black. The first image in the time-series is the differential interference contrast (DIC) image. The rest are fluorescence images shown every 400 ms. The scale bar represents 1 μm.

Article Snippet: The ftsZ gene coding sequence is directly amplified from E. coli chromosome DNA and dendra2 DNA is purchased from Axxora (San Diego, CA).

Techniques: Microscopy, Fluorescence, Expressing

Journal:

Article Title: Investigating Intracellular Dynamics of FtsZ Cytoskeleton with Photoactivation Single-Molecule Tracking

doi: 10.1529/biophysj.108.128751

Figure Lengend Snippet: Localization of FtsZ-Dendra2 proteins in E. coli cells. (A) Green fluorescence from the unactivated proteins in E. coli cells. Each cell is highlighted by the rectangular boxes. The scale bar represents 2 μm. (B) A schematic representation of the FtsZ localization pattern observed in literature using fluorescence microscopy. The central dark ring represents the Z-ring. The helix represents the FtsZ molecules outside the Z-ring. (C) Chimeric construct of ftsZ-dendra2 expression. SD, Shine-Dalgarno sequence.

Article Snippet: The ftsZ gene coding sequence is directly amplified from E. coli chromosome DNA and dendra2 DNA is purchased from Axxora (San Diego, CA).

Techniques: Fluorescence, Microscopy, Construct, Expressing, Sequencing

Journal:

Article Title: Investigating Intracellular Dynamics of FtsZ Cytoskeleton with Photoactivation Single-Molecule Tracking

doi: 10.1529/biophysj.108.128751

Figure Lengend Snippet: The distribution of single FtsZ-Dendra2 molecules' stepping sizes at 200-ms time-interval. The results showed two distinct populations. The solid curves are least-square fitting of the distribution to the function form of The first term represented the stationary population of the molecules. The second represented the mobile population. The two terms of the equation are plotted separately for clarity.

Article Snippet: The ftsZ gene coding sequence is directly amplified from E. coli chromosome DNA and dendra2 DNA is purchased from Axxora (San Diego, CA).

Techniques:

Journal:

Article Title: Investigating Intracellular Dynamics of FtsZ Cytoskeleton with Photoactivation Single-Molecule Tracking

doi: 10.1529/biophysj.108.128751

Figure Lengend Snippet: Distribution of the single molecule photobleaching times. Histogram of FtsZ-Dendra2 photobleaching time is measured from >3000 molecules under excitation density of 450 W/cm2. The straight line represented the single exponential fit of the histogram.

Article Snippet: The ftsZ gene coding sequence is directly amplified from E. coli chromosome DNA and dendra2 DNA is purchased from Axxora (San Diego, CA).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Global proteomics of Ubqln 2-based murine models of ALS

doi: 10.1074/jbc.RA120.015960

Figure Lengend Snippet: Validation and proteasome dependence of top hits in HEK 293 cells . A , western blot demonstrating similar changes in TRIM32, PEG10, and RTL8 protein levels in HEK 293 cells lacking UBQLN1, 2, and 4 (labeled TKO). B , WT or triple knockout (TKO) HEK 293 cells were treated with bortezomib plus epoxomicin (50 nM each) or chloroquine (50 μM), for 0, 3, 6, or 12 h to inhibit proteasomal or lysosomal degradation, respectively. Inhibition of degradation was confirmed with either poly-ubiquitin (Ub) accumulation and free ubiquitin (Ub) depletion or LC3-I/II conversion, respectively. Western blots were probed for top UBQLN2 hits. C–E , WT or TKO HEK 293 cells were transfected with constructs bearing C-terminal fusions of ( C ) PEG10-RF1/2, ( D ) RTL8C, and ( E ) TRIM32 with Dendra2 fluorophore with an IRES-CFP transfection control. After 48 h, cells were exposed to 60 s of intense blue light to induce photoconversion of Dendra2 and samples were taken at the indicated timepoints for quantitation of the RFP/CFP ratio via flow cytometry. Lines of best fit to biphasic decay were plotted in Prism for each time course. n = 4 independent experiments for each construct. RTL8 overexp., overexposure of RTL8 blot.

Article Snippet: All three genes were then cloned into LifeAct-Dendra2 from Addgene (plasmid ID: 54694) replacing LifeAct as an N-terminal fusion with Gibson cloning (SGI).

Techniques: Western Blot, Labeling, Triple Knockout, Inhibition, Transfection, Construct, Quantitation Assay, Flow Cytometry

Journal: Experimental & Molecular Medicine

Article Title: Leukocyte invasion of the brain after peripheral trauma in zebrafish ( Danio rerio )

doi: 10.1038/s12276-022-00801-4

Figure Lengend Snippet: a Experimental setup. dpf, day post-fertilization. b–d Light-sheet imaging showed a significantly increased number of coro1a + leukocytes in the zebrafish whole brain/midbrain and forebrain with peripheral trauma (PT) at 1 day post-trauma (dpt) but not at 2 dpt compared with the control (CTRL). Independent t test, **, p < 0.01. Scale bar, 40 µm (whole brain), 20 µm (forebrain). f forebrain. m, midbrain. h, hindbrain. Asterisk, autofluorescence of pigment. e–f Phospho-histone H3 (PH3) staining indicated no difference in the number of cells in mitosis in the brain between CTRL and PT. Independent t test. Scale bar, 20 µm. g , h qPCR but not light-sheet imaging showed significantly increased expression or the number of mpx1 + neutrophils in the head at 1 dpt. Independent t test, **, p < 0.01. Scale bar, 40 µm. Asterisk, autofluorescence of pigment. i–j Light-sheet imaging and qPCR showed significantly increased expression or the number of mepg1 + macrophages in the head at 1 dpt. Independent t test, **, p < 0.01. Scale bar, 40 µm. k Flow cytometry detected an increased number of mepg1 + macrophages in the head after peripheral trauma. ( l ) No difference in rag1 + lymphocytes (red arrow) was found in whole-mount in situ hybridization (WISH) between CTRL and PT. Scale bar, 40 µm. m qPCR showed significantly increased expression of lcp1 + leukocytes in the head after peripheral trauma. Independent t test, **, p < 0.01. n Experimental setup for dendra2 photoconversion and peripheral leukocyte tracking. o–p A significantly elevated number of photoconverted magenta+ leukocytes (white arrow) in the brains of zebrafish after peripheral trauma. Independent t test, **, p < 0.01. Scale bar, 40 µm.

Article Snippet: The Tg(mpeg1:dendra2) zebrafish line was generated by using the Tol2-mpeg1-dendra2 plasmid (Addgene, 51462) with in vitro transcribed Tol2 transposase mRNA (Addgene, 31831).

Techniques: Imaging, Control, Staining, Expressing, Flow Cytometry, In Situ Hybridization

Journal: Experimental & Molecular Medicine

Article Title: Leukocyte invasion of the brain after peripheral trauma in zebrafish ( Danio rerio )

doi: 10.1038/s12276-022-00801-4

Figure Lengend Snippet: a Experimental setup. hpt, hours post-trauma. b , c The expression of il1b + and il6 + cells (red arrow) in zebrafish larvae was measured by WISH at 3 hpt. Scale bar, 200 µm. d qPCR showed that the mRNA levels of various cytokines or chemokines changed in the traumatic site/tail and the brain/head compared to CTRL. Independent t test, *, p < 0.05, **, p < 0.01. e WISH showed increased il1b + cells (red arrow) in the brain/head, body/trunk, and traumatic site/tail of zebrafish larvae at 24 hpt. Scale bar, 100 µm. h, head. b, body. t, tail. f Design of the transcription activator-like effector nucleases (TALEN) targeting zebrafish il1b . g , h il1b mutation ( il1b Mut ) was confirmed by the restriction fragment length polymorphism (RFLP) assay and Sanger sequencing (−7 bp). ( i ) qPCR showed that the mRNA levels of various cytokines were decreased in il1b Mut zebrafish brains compared to CTRL brains. Independent t test, **, p < 0.01. j–l il1b Mut rescued the increased mpeg1 + macrophages or coro1a + leukocytes in the zebrafish brain at 24 hpt. Two-way ANOVA with Tukey’s HSD post hoc test, **, p < 0.01 compared to CTRL. Scale bar, 40 µm.

Article Snippet: The Tg(mpeg1:dendra2) zebrafish line was generated by using the Tol2-mpeg1-dendra2 plasmid (Addgene, 51462) with in vitro transcribed Tol2 transposase mRNA (Addgene, 31831).

Techniques: Expressing, TALENs, Mutagenesis, RFLP Assay, Sequencing

Journal: Experimental & Molecular Medicine

Article Title: Leukocyte invasion of the brain after peripheral trauma in zebrafish ( Danio rerio )

doi: 10.1038/s12276-022-00801-4

Figure Lengend Snippet: a Experimental setup. b, d Light-sheet imaging showed an increased number and percentage of the colocalization of coro1a + leukocytes and AO + apoptotic cells in the zebrafish brain at 4 dpt. Independent t test, **, p < 0.01 compared with CTRL. c, e Light-sh e et imaging showed an increased number of AO + apoptotic cells in the zebrafish brain at 1 dpt but not at 2 dpt. Independent t test, **, p < 0.01 compared to CTRL. f Light-sheet imaging showed that mpeg1 + macrophages returned to the CHT at 2 dpt. Two-way ANOVA with Tukey’s HSD post hoc test, **, p < 0.01 compared to CTRL at 1 dpt, #, p < 0.05 compared to CTRL at 2 dpt. Scale bar, 5 µm. g Schematic showing that apoptosis in the brain is associated with leukocyte distribution. h–k il1b mutation rescued the increased number of AO + or TUNEL + apoptotic cells in the zebrafish brain at 1 dpi. Two-way ANOVA with Tukey’s HSD post hoc test, **, p < 0.01 compared to CTRL. Scale bar, 40 µm.

Article Snippet: The Tg(mpeg1:dendra2) zebrafish line was generated by using the Tol2-mpeg1-dendra2 plasmid (Addgene, 51462) with in vitro transcribed Tol2 transposase mRNA (Addgene, 31831).

Techniques: Imaging, Mutagenesis, TUNEL Assay

Journal: Experimental & Molecular Medicine

Article Title: Leukocyte invasion of the brain after peripheral trauma in zebrafish ( Danio rerio )

doi: 10.1038/s12276-022-00801-4

Figure Lengend Snippet: a , b Time-lapse light-sheet imaging showed the phagocytosis of AO high and AO low cells (white arrow or arrowhead) by coro1a + leukocytes in the zebrafish brain at 1 dpt. c , d Neutral red staining showed microglial lysosome impairment in zebrafish brains with or without il1b mutation at 1 dpt. Two-way ANOVA with Tukey’s HSD post hoc test, **, p < 0.01 compared to CTRL. Scale bar, 40 µm. e , f LysoTracker staining showed mpeg1 + microglial lysosome impairment in the zebrafish brain at 1 dpt or under il1b mutation. Two-way ANOVA with Tukey’s HSD post hoc test, **, p < 0.01 compared to CTRL. Scale bar, 20 µm.

Article Snippet: The Tg(mpeg1:dendra2) zebrafish line was generated by using the Tol2-mpeg1-dendra2 plasmid (Addgene, 51462) with in vitro transcribed Tol2 transposase mRNA (Addgene, 31831).

Techniques: Imaging, Staining, Mutagenesis