Journal: Molecular Systems Biology

Article Title: Programmable CRISPR‐Cas transcriptional activation in bacteria

doi: 10.15252/msb.20199427

Figure Lengend Snippet:

Article Snippet: The GFP‐mScarlet reporter plasmid (pHH39) was constructed by cloning the mScarlet gene from pEB2‐mScarlet‐I (Addgene #104007) under the WJ97 promoter and joined with the weak GFP expression cassette from pWJ89.

Techniques: Recombinant, Sequencing, Reverse Transcription, Software, Mutagenesis, Bacteria, Real-time Polymerase Chain Reaction

Journal: Developmental cell

Article Title: A Combinatorial MAP Code Dictates Polarized Microtubule Transport

doi: 10.1016/j.devcel.2020.01.029

Figure Lengend Snippet: (A) TIRF-M images and kymographs of KIF5B-mScarlet (kinesin-1) at indicated concentrations + 1 mM ATP in the absence and presence of 50 nM sfGFP-MAP7, 50 nM sfGFP-tau, 50 nM sfGFP-MAP2C, 100 nM DCX-sfGFP, 50 nM sfGFP-DCLK1, or 50 nM sfGFP-MAP9. Scale bars: 1 μm (x), 10 sec (y). (B) Quantification of the landing rates of KIF5B-mScarlet + 1 mM ATP in the absence and presence of each MAP or MAP combination (means ± s.d. in motors μm−1min−1nM−1 are: 0.11 ± 0.07 for KIF5B alone (n=134 kymographs from 3 independent trials), 2.72 ± 0.59 for KIF5B + MAP7 (n=83 kymographs from 2 independent trials), 0.03 ± 0.04 for KIF5B + tau (n=100 kymographs from 2 independent trials), 0.01 ± 0.02 for KIF5B + MAP2C (n=93 kymographs from 2 independent trials), 0.02 ± 0.03 for KIF5B + DCX (n=94 kymographs from 2 independent trials), 0.05 ± 0.05 for KIF5B + DCLK1 (n=92 kymographs from 2 independent trials), 0.03 ± 0.05 for KIF5B + MAP9 (n=96 kymographs from 2 independent trials), 0.06 ± 0.07 for KIF5B + MAP7 + DCX (n=114 kymographs from 2 independent trials), 0.07 ± 0.08 for KIF5B + MAP7 + MAP9 (n=70 kymographs from 2 independent trials), and 0.005 ± 0.001 for 10x the concentration of KIF5B (50 pM) + MAP2C (n=20 kymographs from 2 independent trials). All datapoints are plotted with lines indicating means ± s.d. P < 0.0001 (***) calculated by one-way ANOVA with Bonferroni correction. (C) TIRF-M images and kymographs of 5 nM KIF5B-mScarlet + 1 mM ATP in the presence of 50 nM BFP-MAP7 (pink) with 100 nM DCX-sfGFP (green), or 50 nM BFP-MAP7 (pink) with 50 nM sfGFP-MAP9 (green). Scale bars: 1 μm (x), 10 sec (y). (D) Quantification of the percentage of motile KIF5B motors in the absence (70.2 ± 8.6 %; n=169 molecules from 12 kymographs from 3 independent trials) or presence of 50nM MAP7 (76.2 ± 5.5; n=160 molecules from 12 kymographs from 2 independent trials), 50 nM MAP7 with 100 nM DCX (5.1 ± 6.8; n=160 molecules from 8 kymographs from 2 independent trials), or 50 nM MAP7 with 50 nM MAP9 (14.5 ± 16.6; n=111 molecules from 8 kymographs from 2 independent trials). P < 0.0001 (***) and P = 0.0540 (n.s.) calculated by one-way ANOVA with Bonferroni correction.

Article Snippet: METHOD DETAILS Molecular Biology and Biochemistry The cDNAs for protein expression in this study were as follows: human Tau-2N4R (Addgene #16316), human MAP7 (GE Dharmacon MGC Collection # {"type":"entrez-nucleotide","attrs":{"text":"BC025777","term_id":"19343693","term_text":"BC025777"}} BC025777 ), human MAP2 (Transomics # {"type":"entrez-nucleotide","attrs":{"text":"BC172263","term_id":"225000717","term_text":"BC172263"}} BC172263 ), human MAP9 (Transomics # {"type":"entrez-nucleotide","attrs":{"text":"BC146864","term_id":"223461954","term_text":"BC146864"}} BC146864 ), human DCX (Addgene #83928), mouse DCLK1 (Transomics # {"type":"entrez-nucleotide","attrs":{"text":"BC133685","term_id":"126632012","term_text":"BC133685"}} BC133685 ), human KIF5B (aa 1-560; a gift from R. Vale), and human KIF1A (aa 1-393; Addgene # 61665).

Techniques: Concentration Assay

Journal: Developmental cell

Article Title: A Combinatorial MAP Code Dictates Polarized Microtubule Transport

doi: 10.1016/j.devcel.2020.01.029

Figure Lengend Snippet: (A) Sequence alignment comparing loop 12 of kinesin-3 (KIF1A) and kinesin-1 (KIF5B) and the kinesin-1 chimera construct with the K-loop residues inserted into loop 12 of KIF5B for the studies in (B). (B) TIRF-M images and kymographs of 1 nM KIF5B-mScarlet compared with 1 nM KIF5BK (kinesin-1 chimera with the K-loop insertion from kinesin-3) + 1 mM ATP in the absence and presence of 50 nM sfGFP-MAP9 or 50 nM sfGFP-MAP7. Scale bars: 1 μm (x), 5 sec (y). (C) Quantification of the landing rates of KIF5B-mScarlet compared with KIF5BK-mScarlet + 1 mM ATP in the absence and presence of MAP9 or MAP7. KIF5B data are reproduced from Figure 2B. Means ± s.d. in motors μm−1min−1nM−1 are: 5.44 ± 1.39 for KIF5BK alone (n=55 kymographs from 2 independent trials), 6.04 ± 1.39 for KIF5BK + MAP9 (n=56 kymographs from 2 independent trials), and 0.83 ± 0.98 for KIF5BK + MAP7 (n=63 kymographs from 2 independent trials). All datapoints are plotted with lines indicating means ± s.d. P < 0.0001 (***) and P = 0.025 (*) using a student’s t-test. (D) Velocity histograms of KIF5B, KIF5BK, and KIF1A + 1 mM ATP with Gaussian fits. KIF1A data are reproduced from Figure 3B. Mean ± s.d velocities for KIF5B and KIF5BK are 358.2 ± 295.7 nm/sec (n=188 motors from 3 independent trials) and 489.0 ± 164.5 nm/sec (n=241 motors from 2 independent trials), respectively. P < 0.0001 using a student’s t-test for KIF5B vs. KIF5BK. (E) TIRF-M image and kymograph of 1 nM KIF5B-mScarlet (kinesin-1) + 1 mM ATP in the presence of 50 nM sfGFP-MAP7(aa565-610)-MAP9(aa302-647)-chimera. Scale bars: 2 μm (x), 10 sec (y). (F) Quantification of the landing rates of KIF5B-mScarlet + 1 mM ATP in the absence and presence of the MAP7-MAP9-chimera. Means ± s.d. in motors μm−1min−1nM−1 are: 0.11 ± 0.07 for KIF5B alone (reproduced from Figure 1B) and 0.58 ± 0.23 for KIF5B + MAP7-MAP9-chimera (n=22 kymographs from 2 independent trials). (G) Quantification of the percentage of motile KIF5B motors in the absence (70.2 ± 8.6 %; reproduced from Figure 1D) or presence of 50nM MAP7-MAP9-chimera (8.2 ± 4.8; n=1173 molecules from 24 kymographs from 2 independent trials). For (F) and (G), all datapoints are plotted with lines indicating means ± s.d. P < 0.0001 (***) using a student’s t-test.

Article Snippet: METHOD DETAILS Molecular Biology and Biochemistry The cDNAs for protein expression in this study were as follows: human Tau-2N4R (Addgene #16316), human MAP7 (GE Dharmacon MGC Collection # {"type":"entrez-nucleotide","attrs":{"text":"BC025777","term_id":"19343693","term_text":"BC025777"}} BC025777 ), human MAP2 (Transomics # {"type":"entrez-nucleotide","attrs":{"text":"BC172263","term_id":"225000717","term_text":"BC172263"}} BC172263 ), human MAP9 (Transomics # {"type":"entrez-nucleotide","attrs":{"text":"BC146864","term_id":"223461954","term_text":"BC146864"}} BC146864 ), human DCX (Addgene #83928), mouse DCLK1 (Transomics # {"type":"entrez-nucleotide","attrs":{"text":"BC133685","term_id":"126632012","term_text":"BC133685"}} BC133685 ), human KIF5B (aa 1-560; a gift from R. Vale), and human KIF1A (aa 1-393; Addgene # 61665).

Techniques: Sequencing, Construct

Journal: Developmental cell

Article Title: A Combinatorial MAP Code Dictates Polarized Microtubule Transport

doi: 10.1016/j.devcel.2020.01.029

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: METHOD DETAILS Molecular Biology and Biochemistry The cDNAs for protein expression in this study were as follows: human Tau-2N4R (Addgene #16316), human MAP7 (GE Dharmacon MGC Collection # {"type":"entrez-nucleotide","attrs":{"text":"BC025777","term_id":"19343693","term_text":"BC025777"}} BC025777 ), human MAP2 (Transomics # {"type":"entrez-nucleotide","attrs":{"text":"BC172263","term_id":"225000717","term_text":"BC172263"}} BC172263 ), human MAP9 (Transomics # {"type":"entrez-nucleotide","attrs":{"text":"BC146864","term_id":"223461954","term_text":"BC146864"}} BC146864 ), human DCX (Addgene #83928), mouse DCLK1 (Transomics # {"type":"entrez-nucleotide","attrs":{"text":"BC133685","term_id":"126632012","term_text":"BC133685"}} BC133685 ), human KIF5B (aa 1-560; a gift from R. Vale), and human KIF1A (aa 1-393; Addgene # 61665).

Techniques: Plasmid Preparation, Virus, Recombinant, Microscopy, Transfection, Software