ensconsin map7  (Proteintech)

Journal: bioRxiv

Article Title: Two-step interphase microtubule disassembly aids spindle morphogenesis

doi: 10.1101/126342

Figure Lengend Snippet: A ) Representative time-lapse confocal images (x-y maximum projection) of HeLa cells during mitotic entry stably expressing H2B-mRFP and mEGFP-α-tubulin, and transiently overexpressing Rap1* treated with control siRNA (upper panel) or Cep192 siRNA (lower panel). Inserts show regions zoomed in inverted grayscale. B ) Changes in centrosomal and non-centrosomal microtubule levels relative to NEP for control siRNA (upper panel, 5 cells) and for Cep192 siRNA (lower panel, 5 cells) 2 independent experiments. Measurements were made as in , except mean of α-tubulin-GFP signal around a centrosome was calculated semi-automatically in H2B-mRFP mEGFP-α-tubulin HeLa stable cell line transiently overexpressing Rap1* (see Materials and Methods for details). Graphs show means and SD. C ) Changes in non-centrosomal microtubule levels relative to NEP. Median of α-tubulin-GFP signal measured as described in for control siRNA (blue, 5 cells) and Cep192 siRNA cells (brown, 5 cells), 2 independent experiments as in 2 B. D ) Schematic representation of Ensconsin structure. Coiled Coil domains are shown in light green. E ) Representative confocal images (x-y maximum projection) of fixed HeLa cells stained to show that Ensconsin is removed from microtubules in prophase. Ensconsin in red, α-Tubulin in green and DAPI in blue (11 prophase cells, >30 interphase cells, 2 independent experiments). F ) Representative confocal images of fixed HeLa cells overexpressing Rap1* stained to show that Ensconsin re-localizes at the microtubules upon Cdk1 inhibition with R0-3306. Ensconsin in red, α-Tubulin in green and DAPI in blue (9 DMSO and 10 R0-3306 cells, 2 independent experiments). G ) Representative confocal images of fixed HeLa cells overexpressing Rap1* stained to show that overexpressed Wt-EMTB-mCherry as well as a non-phosphorylatable mutant (A-EMTB-mCherry) remain bound to microtubules in interphase, whereas the phospho-mimetic (E-EMTB-mCherry) remains largely cytoplasmic. α-Tubulin in green, mCherry in red. Inserts show regions zoomed in inverted grayscale (9 cells Wt-EMTB-mCherry, 14 cells A-EMTB-mCherry, 10 cells E-EMTB-mCherry, 1 experiment). H ) Representative time-lapse confocal images (x-y maximum projection) of flat (Rap1*) HeLa cells stably expressing GFP-α-tubulin and wild type Ensconsin microtubule binding domanin (Wt-EMTB-mCherry, upper panel) or its non-phosphorylatable mutant (A-EMTB-mCherry, lower panel) to show that the non-phosphorylatable mutant stays associated with microtubules during prophase, leading to delay in microtubule disassembly at mitotic entry. Inserts show regions zoomed in inverted grayscale. White arrows indicate to microtubule clumps formed due to a failure to remove Ensconsin from microtubules before NEP. Black arrows indicate to interphase microtubules just before or after NEP (18 cells Wt-EMTB-mcherry, 15 cells A-EMTB-mcherry, 5 independentexperiments). In overlay images, signal intensities were adjusted to remove cytoplasmic background signal. Scale bars represent 10μm.

Article Snippet: Primary antibodies were used at the following dilutions: tubulin 1:1000 (DM1A, Sigma-Aldrich), Ensconsin (MAP7, Proteintech, 13446-1-AP) 1:100–200, mCherry 1:500 (Abcam).

Techniques: Stable Transfection, Expressing, Control, Staining, Inhibition, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Two-step interphase microtubule disassembly aids spindle morphogenesis

doi: 10.1101/126342

Figure Lengend Snippet: A ) Changes in non-centrosomal microtubule levels relative to NEP. Variance of α-tubulin-GFP signal measured as described in for control siRNA (blue, 5 cells) and Cep192 siRNA cells (brown, 5 cells) 2 experiments as in . B ) Western blot showing Ensconsin knockdown induced using three different siRNAs targeting Ensconsin. C ) Representative confocal images (x-y maximum projection) of fixed HeLa cells treated with control siRNA and three siRNAs targeting Ensconsin. Inserts show regions zoomed in overlays (>10 cells per condition, 1 experiment). D ) Representative confocal images (x-y maximum projection) of fixed MCF10A cells stained to show that Ensconsin is removed from microtubules in prophase compared to interphase. Ensconsin in red, aTubulin in green and DAPI in blue. Inserts show regions zoomed in overlays, in which intensities were adjusted to remove cytoplasmic background signal (6 prophase cells, >20 interphase cells,1 experiment). E ) Representative confocal images of fixed HeLa cells overexpressing Rap1* in prophase stained to show that the microtubule binding domain of Ensconsin (Wt-EMTB-mCherry) as well as a corresponding phospho-mimetic mutant (E-EMTB-mCherry) are largely cytoplasmic in prophase, whereas the non-phosphorylatable form (A-EMTB-mCherry) localizes to the microtubules. aTubulin in green, Wt-EMTB-mCherry, A-EMTB-mCherry, E-EMTB-mCherry in red. Inserts are zoomed, shown in inverted greyscale or in overlays, where signal intensities were adjusted to remove cytoplasmic background signal (3 cells per condition, 1 experiment). Scale bars represent 10μm.

Article Snippet: Primary antibodies were used at the following dilutions: tubulin 1:1000 (DM1A, Sigma-Aldrich), Ensconsin (MAP7, Proteintech, 13446-1-AP) 1:100–200, mCherry 1:500 (Abcam).

Techniques: Control, Western Blot, Knockdown, Staining, Binding Assay, Mutagenesis