Journal: bioRxiv

Article Title: Evolutionarily conserved midbody reorganization precedes ring canal formation during gametogenesis

doi: 10.1101/2022.06.03.494691

Figure Lengend Snippet: (A) Stills from a time-lapse movie of a germarium expressing Pav::GFP and Histone::RFP. In the plane of focus are two actively dividing cells of a four-cell cyst; the planes of division are oriented perpendicular to each other as depicted in the schematic at the lower right of t=0 minutes. Arrowheads indicate the progression of cleavage furrows to ring canals. 1.2% of Pav-labeled structures are midbodies. B) Mouse testis sections were fixed and stained to detect microtubules (MTs, green), MKLP1 (magenta), and DNA (blue). (B-B’) A dividing germ cell at anaphase. (C-C’) MKLP1-labeled midbodies are present at the intercellular bridge connected segregated sister chromatids; 2.4% (n=242) of MKLP1-positive structures are midbodies. (D-D’) Nascent ring canals (arrowheads) are marked by the association of open ring canal with luminal microtubules. (E-F’) Hydra testes were fixed and stained to detect KIF23 (magenta) and DNA (blue). (E-E’) A dividing spermatocyte with enrichment of KIF23 at the intercellular bridge (arrowhead). (F-F’) Ring canals (“RC”) and a midbody (“M”) are present in the field of view; 3.2% (n=572) of KIF23-labeled structures in dividing cells appeared as midbodies

Article Snippet: The following primary antibodies were used: 1:200 rabbit anti-MKLP1 (clone 7C9, Bioss Antibodies, cat. BSM-52401R) and 1:10 mouse anti-alpha Tubulin (clone 4A1, Developmental Studies Hybridoma Bank).

Techniques: Expressing, Labeling, Staining

Journal: Scientific Reports

Article Title: CRISPR-mediated gene targeting of CK1δ/ε leads to enhanced understanding of their role in endocytosis via phosphoregulation of GAPVD1

doi: 10.1038/s41598-020-63669-2

Figure Lengend Snippet: Intracellular localization of endogenous CK1δ-mNG and CK1ε-mNG. ( A–C ) Representative images of fixed HEK293 cells at indicated cell cycle stages producing CK1δ-mNG or CK1ε-mNG stained with ( A ) DAPI and anti-γ-tubulin, ( B ) DAPI and anti-γ-tubulin, or ( C ) DAPI and anti-MKLP1 antibodies. Scale bars, 10 μm. Insets correspond to centrosomes in A and B or the midbody in C. Scale bars, 0.5 μm. ( D ) Representative single z-sections of live-cell images of HEK293 CK1δ-mNG and CK1ε-mNG cells. Yellow arrows indicate examples of vesicle-like structures. Scale bars, 10 μm.

Article Snippet: Cells were incubated with antibodies against γ-tubulin (Sigma-Aldrich, GTU88; 1:500), Flag M2 (Sigma-Aldrich, 1:1500), or MKLP1 (Bethyl laboratory, 1:1000).

Techniques: Staining

Journal: PLoS ONE

Article Title: Gas2l3, a Novel Constriction Site-Associated Protein Whose Regulation Is Mediated by the APC/C Cdh1 Complex

doi: 10.1371/journal.pone.0057532

Figure Lengend Snippet: (A) HeLa cells were transfected with vectors expressing Gas2l3-EGFP, Gas2l3-DM4-EGFP, or EGFP (control). Thirty-two hrs post-transfection, cells were fixed and immunolabeled, as described in . The slides were scanned using a Zeiss AxioImager upright microscope and 100X oil lens for imaging. Transfected (green) cells were counted in order to detect 500 positive daughter cell pairs connected by a midbody (positives). The proportion of attached daughter cells equals 500/ N , where N = the number of transfected cells ( N - 500 = the number of negatives). (B) HeLa cells were transfected with vectors expressing EGFP-tagged Gas2l3 (wt) or Gas2l3-DM4. Thirty-two hours post-transfection, cells were fixed, immunolabeled with anti-MKLP1 (stembody marker) and anti-Tubulin, and stained with DAPI. We used a Zeiss AxioImager Z1 upright microscope and 100X oil lens for imaging.

Article Snippet: The following primary antibodies were used for IF: rabbit polyclonal anti-hGas2l3 (serum, custom-made by Covance), mouse monoclonal and rabbit polyclonal anti-Aurora B (Abcam, ab3609, ab2254), rabbit polyclonal MKLP1 (Gene Tex, GTX30315), mouse monoclonal anti-αTubulin (Abcam, ab7291), mouse monoclonal anti-FLAG® M2 (Sigma-Aldrich, F3165), and mouse monoclonal anti-Myc (DSHB, 9E10).

Techniques: Transfection, Expressing, Immunolabeling, Microscopy, Imaging, Marker, Staining

Journal: STAR protocols

Article Title: A protocol for isolating and imaging large extracellular vesicles or midbody remnants from mammalian cell culture.

doi: 10.1016/j.xpro.2023.102562

Figure Lengend Snippet: Figure 1. Increasing the concentration of PEG6000 distorts the MKLP1-GFP signal and MBR structure (A) Cleared conditioned medium from HeLa-MKLP1-GFP cells was incubated with increasing concentrations of PEG6000 and settled onto coverslips. Increasing PEG concentration resulted in decreased transparency. The scale bar represents 5 mm. (B) Higher PEG6000 concentrations caused an increased, blurred fluorescent background. The coverslips were imaged at 203 magnification on a Revolve microscope. The scale bar represents 100 mm. (C) MBRs isolated from HeLa-MKLP1-GFP cell culture medium by increasing PEG6000 concentrations imaged at 1003 magnification by N-SIM Z projection. The scale bar represents 10 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-MKLP1 Novus Biologicals Cat#NBP2-56923 Mouse anti-CD9 Santa Cruz Biotechnology Cat#SC-13118 Mouse anti-a-tubulin Alexa Fluor 555 Millipore Sigma Cat#05-829X-555 Alexa Fluor 488 goat anti-mouse IgG Jackson ImmunoResearch Cat#115-545-003 Alexa Fluor 647 goat anti-rabbit IgG Jackson ImmunoResearch Cat#111-605-003 Chemicals, peptides, and recombinant proteins DMEM/F12 medium Gibco Cat#11330-057 DMEM Gibco Cat#11965092 Fetal bovine serum (FBS) Fisher Scientific Cat#26140079 Penicillin/Streptomycin Life Technologies Cat#15140-122 Geneticin Gibco Cat#10131-035 Phosphate-buffered saline (PBS) no calcium no magnesium Gibco Cat#14190250 0.25% Trypsin-EDTA (1X) Gibco Cat#25200-056 Poly(ethylene glycol), BioUltra, 6000 (PEG6000) Millipore Sigma Cat#81253 Gold nanoparticles, 30 nm diameter, methyl terminated, PEG 5000 coated Sigma-Aldrich Cat#765732 Poly-L-lysine solution, mol. wt.

Techniques: Concentration Assay, Incubation, Microscopy, Isolation, Cell Culture

Journal: STAR protocols

Article Title: A protocol for isolating and imaging large extracellular vesicles or midbody remnants from mammalian cell culture.

doi: 10.1016/j.xpro.2023.102562

Figure Lengend Snippet: Figure 2. Isolated MBRs are midbodies (MB) formed during the late telophase stage of mitosis (A) Representative images of MBRs isolated from HeLa-CCL-2 cells stained with anti-MKLP1 (magenta) and anti-CD9 (cyan). Images were taken at 603 magnification on a confocal microscope. The scale bar represents 24 mm, and the inset represents an 83 enlargement of the dotted square. The same slide images of isolated MBRs from HeLa-CCL-2 cell culture labeled with anti-MKLP1 (Magenta) and anti-CD9 (Cyan) were taken at 1003 magnification on an N-SIM microscope. The scale bar represents 10 mm, and the inset represents a 43 enlargement of the dotted square. (B) Representative images of mitotic HeLa-CCL-2 cells labeled with anti-MKLP1 (green), anti-ɑ-tubulin 555 (red), and DAPI (gray). The green midbody in the first image attaches two daughter cells in the final stages of mitosis. Green MBR is taken up by a cell in interphase post-mitosis. Images were taken at 1003 magnification on an N-SIM microscope. The scale bar represents 10 mm. (C) Representative images of isolated MBRs from HeLa-CCL-2 cells using transmission electron microscopy confirm the electron-dense property of MBs at 40,0003 magnification. The scale bar represents 500 nm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-MKLP1 Novus Biologicals Cat#NBP2-56923 Mouse anti-CD9 Santa Cruz Biotechnology Cat#SC-13118 Mouse anti-a-tubulin Alexa Fluor 555 Millipore Sigma Cat#05-829X-555 Alexa Fluor 488 goat anti-mouse IgG Jackson ImmunoResearch Cat#115-545-003 Alexa Fluor 647 goat anti-rabbit IgG Jackson ImmunoResearch Cat#111-605-003 Chemicals, peptides, and recombinant proteins DMEM/F12 medium Gibco Cat#11330-057 DMEM Gibco Cat#11965092 Fetal bovine serum (FBS) Fisher Scientific Cat#26140079 Penicillin/Streptomycin Life Technologies Cat#15140-122 Geneticin Gibco Cat#10131-035 Phosphate-buffered saline (PBS) no calcium no magnesium Gibco Cat#14190250 0.25% Trypsin-EDTA (1X) Gibco Cat#25200-056 Poly(ethylene glycol), BioUltra, 6000 (PEG6000) Millipore Sigma Cat#81253 Gold nanoparticles, 30 nm diameter, methyl terminated, PEG 5000 coated Sigma-Aldrich Cat#765732 Poly-L-lysine solution, mol. wt.

Techniques: Isolation, Staining, Microscopy, Cell Culture, Labeling, Transmission Assay, Electron Microscopy

Journal: STAR protocols

Article Title: A protocol for isolating and imaging large extracellular vesicles or midbody remnants from mammalian cell culture.

doi: 10.1016/j.xpro.2023.102562

Figure Lengend Snippet: Figure 3. Comparison of GOLD PEG5000 nanoparticle concentrations in the isolation of midbody remnants (MBRs) (A) HeLa CCL-2 clear conditioned medium was incubated with decreasing concentrations of PEG-coated gold nanoparticles overnight at 4C. MBRs were stained with anti-MKLP1 and imaged at 203 magnification. The scale bar represents 90 mm. (B) HeLa MKLP1-GFP clear conditioned medium was incubated with PEG5000-coated gold nanoparticles at a 1:5000 (v/v) dilution overnight at 4C. MBRs were stained with anti-CD9 and imaged at 203 magnification. The scale bar represents 90 mm. (C) HeLa MKLP1-GFP clear conditioned medium was incubated with PEGylated gold nanoparticles at a 1:5000 dilution (v/v) overnight at 4C. MBRs were stained with anti-CD9 and imaged at 1003 magnification. The scale bar represents 10 mm. Inset shows a 103 zoomed view of the MBR.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-MKLP1 Novus Biologicals Cat#NBP2-56923 Mouse anti-CD9 Santa Cruz Biotechnology Cat#SC-13118 Mouse anti-a-tubulin Alexa Fluor 555 Millipore Sigma Cat#05-829X-555 Alexa Fluor 488 goat anti-mouse IgG Jackson ImmunoResearch Cat#115-545-003 Alexa Fluor 647 goat anti-rabbit IgG Jackson ImmunoResearch Cat#111-605-003 Chemicals, peptides, and recombinant proteins DMEM/F12 medium Gibco Cat#11330-057 DMEM Gibco Cat#11965092 Fetal bovine serum (FBS) Fisher Scientific Cat#26140079 Penicillin/Streptomycin Life Technologies Cat#15140-122 Geneticin Gibco Cat#10131-035 Phosphate-buffered saline (PBS) no calcium no magnesium Gibco Cat#14190250 0.25% Trypsin-EDTA (1X) Gibco Cat#25200-056 Poly(ethylene glycol), BioUltra, 6000 (PEG6000) Millipore Sigma Cat#81253 Gold nanoparticles, 30 nm diameter, methyl terminated, PEG 5000 coated Sigma-Aldrich Cat#765732 Poly-L-lysine solution, mol. wt.

Techniques: Comparison, Isolation, Incubation, Staining

Journal: The Journal of Cell Biology

Article Title: KIF14 and citron kinase act together to promote efficient cytokinesis

doi: 10.1083/jcb.200511061

Figure Lengend Snippet: Citron kinase is required for cytokinesis and for the localization of KIF14 to the midbody. (A) Cell lysates from HeLa cells treated with control or citron kinase (CK) siRNA duplexes for 50 h were Western blotted for citron kinase, KIF14, and α-tubulin as a loading control. (B and C) HeLa cells treated with control or citron kinase siRNA duplexes for 50 h were fixed and stained with antibodies to citron kinase (green) and KIF14 (red). (B) A typical field of KIF14-depleted cells from such an experiment is shown. The number of binucleated cells was counted and is expressed as a percentage in control and citron kinase–depleted cells. (C) Localization of KIF14 in control and citron kinase–depleted cells. DAPI, blue. (D) HeLa cells treated with control, citron kinase, or KIF14 siRNA duplexes for 50 h were fixed and stained with antibodies to MKlp1 (red) and citron kinase or tubulin (green). Arrows on the merged images indicate midbody remnants. Bars, 10 μm.

Article Snippet: Antibodies were obtained as follows: mouse monoclonals to α-tubulin (clone DM1a; Sigma-Aldrich); Aurora B (clone AIM-1; Becton Dickinson); mouse monoclonal to citron kinase (Becton Dickinson); mouse monoclonal to Rho A (clone 55; Becton Dickinson); rabbit polyclonals to MKlp1 SC-867 (0.2 mg/ml; Santa Cruz Biotechnology, Inc.); the MKlp1 motor domain ( ); the KIF4 amino acids 738–1,232; KIF14 (BL358; Bethyl Laboratories Inc.); ECT2 amino acids 1–388; INCENP ( ); astrin amino acids 1,014–1,193; anillin amino acids 417–656; and affinity-purified sheep polyclonal to MKlp2 ( ; ).

Techniques: Control, Western Blot, Staining

Journal: The Journal of Cell Biology

Article Title: KIF14 and citron kinase act together to promote efficient cytokinesis

doi: 10.1083/jcb.200511061

Figure Lengend Snippet: PRC1 forms a central spindle matrix directing kinesin function during cytokinesis. PRC1 is a master regulator of the central spindle interacting with multiple motor proteins that are involved in cytokinesis. Centralspindlin, shown as MKlp1 for simplicity, controls Rho activation and inactivation, whereas KIF14 is needed for citron kinase localization to the central spindle, midbody, and, thus, its site of activity. One function of Rho is to control the activation state of citron kinase. MKlp2 is needed for both the targeting of Plk1 and the chromosomal passenger complex and, thus, for spatial control of both Plk1 and Aurora B. These kinases phosphorylate proteins required for cytokinesis, including components of the centralspindlin complex. The molecular function of KIF4 is less clear, but it too appears to be required for cytokinesis. In the absence of MKlp1 and MKlp2, early events in central spindle and cleavage furrow formation are abnormal. This leads to defects in furrow positioning and contractility. KIF14 and citron kinase are not required for these early events or late events such as midbody formation but are needed for efficient cytokinesis.

Article Snippet: Antibodies were obtained as follows: mouse monoclonals to α-tubulin (clone DM1a; Sigma-Aldrich); Aurora B (clone AIM-1; Becton Dickinson); mouse monoclonal to citron kinase (Becton Dickinson); mouse monoclonal to Rho A (clone 55; Becton Dickinson); rabbit polyclonals to MKlp1 SC-867 (0.2 mg/ml; Santa Cruz Biotechnology, Inc.); the MKlp1 motor domain ( ); the KIF4 amino acids 738–1,232; KIF14 (BL358; Bethyl Laboratories Inc.); ECT2 amino acids 1–388; INCENP ( ); astrin amino acids 1,014–1,193; anillin amino acids 417–656; and affinity-purified sheep polyclonal to MKlp2 ( ; ).

Techniques: Activation Assay, Activity Assay, Control

Journal: Developmental Cell

Article Title: Microtubule-sliding modules based on kinesins EG5 and PRC1-dependent KIF4A drive human spindle elongation

doi: 10.1016/j.devcel.2021.04.005

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used: mouse monoclonal PRC1 (sc-376983, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal EG5 (sc-365681, Santa Cruz Biotechnology), diluted 1:50; mouse monoclonal KIF4A (E-8, sc-365144, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal MKLP-1 (sc-867, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF20A (A300-879A, Bethyl Laboratories, TX, USA), diluted 1:500; rat anti-alpha Tubulin YL1/2 (MA1-80017, Invitrogen, CA, SAD), diluted 1:500; rabbit polyclonal KIF14 (ab3746, Abcam), diluted 1:50; mouse monoclonal KIFC1 (M-63, sc-100947, Santa Cruz Biotechnology), diluted 1:50; rabbit polyclonal KIF18A (A301-080A, Bethyl Laboratories), diluted 1:100 and rabbit polyclonal KIF4A (A301-074A, Bethyl Laboratories), diluted 1:100.

Techniques: Recombinant, Saline, Protease Inhibitor, Transfection, CRISPR, Knock-Out, Control, Plasmid Preparation, Software