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Dynein ATPase

Dynein ATPase is a complex molecular motor protein that plays a crucial role in cellular transport and motility.
It utilizes the energy of ATP hydrolysis to power the movement of organelles, chromosomes, and other cellular components along microtubules.
Dynein ATPase is essential for a wide range of biological processes, including ciliary and flagellar beating, mitotic spindle formation, and neuronal transport.
Understanding the structure, function, and regulation of Dynein ATPase is a key area of research in cell biology and biophysics, with implications for human health and disease.
PubCompre.ai's AI-driven platform can help optimize your Dynein ATPase research protocols, enhance reproducibility, and streamline your workflow to improve scientific outcomes.

Most cited protocols related to «Dynein ATPase»

Cloning and Expression of Dynein Complex

Cited 24 times

The following genes were codon optimised for expression in Sf9 cells and synthesised commercially (Epoch Life Science): DHC (DYNC1H1, accession number NM_001376.4), DIC (DYNC1I2, IC2C, AF134477), DLIC (DYNC1LI2, LIC2, NM_006141.2), Tctex (DYNLT1, Tctex1, NM_006519.2), LC8 (DYNLL1, LC8-1, NM_003746.2) and Robl (DYNLRB1, Robl1, NM_014183.3). The DYNC1H1 gene was fused to a His-ZZ-LTLT tag (Reck-Peterson et al, 2006 (link)) and inserted into pACEBac1 (Vijayachandran et al, 2013 (link)). Ligation-independent infusion (Clontech) cloning was used to seamlessly insert a SNAPf tag (New England Biolabs) to generate pDyn1. Genes for IC2C, LIC2, Tctex1, LC8 and Robl1 were assembled into pIDC (Vijayachandran et al, 2013 (link)), with each expression cassette separated by 30 bp linkers consisting of random sequence and a unique restriction site, to generate pDyn2. pDyn1 and pDyn2 were fused using an in vitro Cre reaction (New England Biolabs) to form pDyn3. The presence of all six dynein genes was verified by PCR.
The mouse Bicd2 (NM_029791.4) gene was codon optimised for Sf9 expression and synthesised commercially (Epoch Life Science). Sequence coding for the N-terminal 400 amino acids of BICD2 was amplified by PCR and cloned into pOmniBac (Vijayachandran et al, 2013 (link)) (modified to fuse a cassette encoding a His-ZZ-LTLT-GFP tag to the 5′ end of the inserted gene) by infusion cloning.
For cloning purposes, we used Phusion polymerase (New England Biolabs) in the supplied high-fidelity buffer. To verify the presence of genes in plasmids or bacmids, we used Quickload Taq 2× master mix (New England Biolabs). Both were used according to the manufacturer's guidelines in a Verity 96-well thermal cycler (Applied Biosystems).

Schlager M.A., Hoang H.T., Urnavicius L., Bullock S.L, & Carter A.P. (2014). In vitro reconstitution of a highly processive recombinant human dynein complex. The EMBO Journal, 33(17), 1855-1868.

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Publication 2014

Amino Acid Sequence Buffers Codon Dynein ATPase EPOCH protocol Genes Genes, vif Ligation Mice, Laboratory Plasmids Sf9 Cells

Purification of Dynein Complexes

Cited 19 times

For purification of dynein complexes, a frozen pellet of 250-ml insect cell culture was thawed on ice and resuspended in lysis buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 1 mM DTT, 0.1 mM ATP, 10% (v/v) glycerol, 2 mM PMSF) supplemented with protease inhibitors (Complete-EDTA Free, Roche Applied Science) to a final volume of 25 ml. Cells were lysed in a 40-ml dounce-type tissue grinder (Wheaton) using 20–30 strokes. The lysate was cleared by centrifugation (504,000 g, 45 min, 4°C; Type 70 Ti Rotor, Beckman Coulter) and added to 3–5 ml pre-washed IgG Sepharose 6 FastFlow beads (GE Healthcare) in a 2.5 × 10 cm Econo-Column (Bio-Rad) and incubated on a roller for 2–6 h. After incubation, the dynein complexes bound to IgG Sepharose beads were washed with 50 ml lysis buffer and 50 ml TEV buffer (50 mM Tris–HCl pH 7.4, 148 mM KAc, 2 mM MgAc, 1 mM EGTA, 10% (v/v) glycerol, 0.1 mM ATP, 1 mM DTT). To fluorescently label the SNAPf tag, dynein coated beads were incubated with either SNAP-Cell TMR-Star or SNAP-Surface Alexa Fluor 647 substrate (New England Biolabs) as described below (see also Supplementary Fig S5). Subsequently, the beads were resuspended in TEV buffer (final volume 5–15 ml) with 50–100 μl TEV protease (4 mg/ml) and incubated at 4°C on a roller overnight. After TEV cleavage, the beads were removed and the protein of interest concentrated in a 100 K molecular weight cut-off concentrator (Amicon Ultracel, Merck-Millipore) to 1–5 mg/ml. TEV protease was removed by size-exclusion chromatography using a TSKgel G4000SW_XL column with a TSKgel SW_XL guard column (TOSOH Bioscience) equilibrated in GF150 buffer (25 mM HEPES pH 7.4, 150 mM KCl, 1 mM MgCl₂, 5 mM DTT, 0.1 mM ATP) or a Superose 6 PC 3.2/30 equilibrated in GF50 buffer (25 mM HEPES pH 7.4, 50 mM KCl, 1 mM MgCl₂, 5 mM DTT, 0.1 mM ATP) using an Ettan LC system (GE Healthcare). Peak fractions were collected, pooled and concentrated to 0.5–10 mg/ml using Amicon concentrators as described above. All purification steps were performed at 4°C. The purification of native pig brain dynein, dynactin and recombinant BICD2N is described in the Supplementary Information.
SDS–PAGE was performed using Novex 4–12% Bis–Tris precast gels using either MOPS or MES buffer (Life Technologies). Gels were stained with either the Coomassie-based reagent Instant Blue (Expedeon) or SYPRO Ruby (Life Technologies) and imaged using a Gel Doc XR+ system with Image Lab 4.0 software (Bio-Rad). Protein concentrations were measured using Quick Start Bradford dye (Bio-Rad) and an Ultrospec 2100 Pro spectrophotometer (Amersham). The proteins were flash frozen in liquid nitrogen and stored at −80°C. Dynein was frozen in the presence of approximately 10% (v/v) glycerol.

Schlager M.A., Hoang H.T., Urnavicius L., Bullock S.L, & Carter A.P. (2014). In vitro reconstitution of a highly processive recombinant human dynein complex. The EMBO Journal, 33(17), 1855-1868.

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Publication 2014

Alexa Fluor 647 Bistris Brain Buffers Cell Culture Techniques Cells Centrifugation Cerebrovascular Accident Coomassie blue Cytokinesis Dynactin Subunit 1 Dynein ATPase Edetic Acid Egtazic Acid Freezing Glycerin HEPES Histocompatibility Testing immunoglobulin G-sepharose Insecta Magnesium Chloride Molecular Sieve Chromatography morpholinopropane sulfonic acid Nitrogen Protease Inhibitors Proteins SDS-PAGE Sodium Chloride Sypro Ruby TEV protease Tromethamine

Structural Analysis of Dynein Motor Domains

Cited 15 times

Dynein motor domain constructs were purified as described above and diluted into EM assay buffer (50 mM Tris HCl pH 8, 150 mM KOAc, 2 mM MgOAc, 1 mM EGTA and 0.1 mM DTT) to a final concentration of 30 nM. Negative staining EM was carried out either in the presence of 3 mM Mg.ADP (+ADP) or 3 mM Mg.ATP and 3 mM Na₃VO₄ (+ADP.Vi) on plasma-cleaned carbon film on 400-square-mesh copper grids (Electron Microscopy Sciences). The samples were stained with 2% (w/v) uranyl acetate. Electron micrographs (Extended Data Fig. 8a) were recorded on a Gatan Orius SC200B CCD fitted to a FEI Tecnai G2 Spirit transmission electron microscope operating at 120 kV. Data were collected at ~1 μm underfocus, with a pixel size of 3.29 Å and an estimated dose of 20 electrons/Å² during 1s exposures. Automated particle picking was done in EMAN2.10a^{40 (link)} using the Swarm boxing tool. Subsequent particle analysis was performed using RELION^{41 (link)}. Autopicked particles were subjected to 2D classification to identify incorrectly picked particles which were manually checked and removed from the dataset. The remaining particles were classified into 10 classes which was sufficient to represent all observed views. Each class was then subclassified into 50 subclasses. Noisy subclasses were discarded and those remaining contained sufficient signal to noise to clearly identify the stalk and linker-GFP (Extended Data Fig. 8b). The ImageJ azimuthal average plugin (http://rsb.info.nih.gov/ij/plugins/azimuthal-average.html) was used to integrate the intensity values surrounding the outside of the motor domain with 1° bin width. This generated a plot with two peaks corresponding to the intensity for the stalk and GFP. Fitting using the sum of two Gaussian functions (Igor Pro 6.3, http://www.wavemetrics.com/products/products.html) was used to measure the angle between them. All experiments were done in triplicate. The angle distribution, using a 10° bin width, was visualised by either histogram or rose plot. The number of particles used were: WT+ADP: 3151, 9914, 7534; WT+ADP.Vi: 8710, 5793, 5284; ΔAAA2L H2 + PS-I 8664, 3408, 9220; ΔAAA4L PS-I 9835, 10799, 9955; R1413A + E2028A: 7994, 2445, 11581; ΔAAA3L H2-S3: 6535, 12185, 6399.

Schmidt H., Zalyte R., Urnavicius L, & Carter A.P. (2014). Structure of human cytoplasmic dynein-2 primed for its powerstroke. Nature, 518(7539), 435-438.

Publication 2014

Biological Assay Buffers Carbon Copper Dynein ATPase Egtazic Acid Electron Microscopy Electrons Plasma Stalking Transmission Electron Microscopy Tromethamine uranyl acetate

Genetic Manipulation of Ustilago maydis

Cited 12 times

The U. maydis strains AB33nRFP, AB33paGRab5a, AB33GT_Peb1R, AB33G₃Dyn2, and AB33G₃Dyn2_ChRab5a were described previously (Schuchardt et al., 2005 (link); Lenz et al., 2006 (link); Schuster et al., 2011a (link), 2011b (link)). The orientation of MT was investigated in strain AB33EB1Y and in strain AB5Dyn2^ts_ Peb1Y, which was generated by homologous integration of plasmid pPeb1Y_N (Lenz et al., 2006 (link)) into the strains AB33 and AB5Dyn2^ts.
To visualize the MT minus ends, a 1013–base pair fragment near the 3′ end of the grc1 gene (γ-tubulin ring complex 1; RefSeq accession number: XP_757621.1), followed by egfp and the nos terminator, the hygromycin resistance cassette, and 1061 base pairs of the downstream sequence were cloned into a cloning vector resulting in plasmid pGrc1G. The plasmid pGrc1G was digested with BsrGI and two additional copies of gfp were introduced as BsrGI fragments, resulting in the plasmid pGrc1-3G. The plasmid pGrc1-3G was digested with DraI and integrated homologously into the grc1 locus of strain AB33, resulting in AB33Grc1-3G. The plasmid potefGFPTub1 (Steinberg et al., 2001 (link)) was digested with NcoI and NdeI to remove the gfp gene and replace it with mCherry gene, resulting in plasmid po_mChTub1. The plasmid popGRab5a (Schuster et al., 2011b) was digested with BamHI and BsrGI to remove the pagfp gene and replace it with pa_mCherry gene, resulting in the plasmid popa_mChRab5a.The plasmids po_mChTub1 and popa_mChRab5a were digested with SspI and integrated at the succinate dehydrogenase locus of strain AB33Grc1-3G, resulting in AB33Grc1-3G__mChTub1 and AB33Grc1-3G_pa_mChRab5a, respectively.
To analyze MT bundles, plasmid potefGFPTub1 (Steinberg et al., 2001 (link)) was ectopically introduced into strain AB33, resulting in AB33GT. The strain AB33GT_Peb1R was obtained by homologous integration of plasmid pPeb1R_N (Lenz et al., 2006 (link)) into AB33GT. The strain AB5Dyn2^ts_GT was generated by ectopic integration of plasmid potefGFPTub1 in the strain AB5Dyn2^ts. For colocalization studies of dynein and MT, plasmid potefGFPTub1 was ectopically integrated into strain AB33G₃Dyn2, resulting in AB33G₃Dyn2_GT. For strain AB33_ΔKin3_r Dyn1_GRab5a, the phleomycin resistance cassette of plasmid pcrgDyn1 (Lenz et al., 2006 (link)) was replaced by the hygromycin resistance cassette, resulting in plasmid pcrgDyn1-H. This plasmid was transformed in strain AB33_ΔKin3_GRab5a. potagRRab5a was generated by replacing the paGFP in plasmid popaGRab5a (Schuster et al., 2011b (link)) with TagRFP (Evrogen, Moscow, Russia). The resulting plasmid potagRRab5a was linearized with AgeI for homologous integration at the succinate dehydrogenase locus of strain AB33ΔKin3_Kin3^tsG, resulting in AB33ΔKin3_ Kin3^tsG_tagRRab5a.
To visualize Kin3-GFP, a 1036–base pair fragment near the 3′ end of the gene, followed by egfp and the nos terminator, the hygromycin resistance cassette, and 1032 base pairs of the downstream sequence were cloned into a cloning vector, resulting in plasmid pKin3G_H. The plasmid was integrated into the native kin3 locus, resulting in strain AB33Kin3G. All strains and plasmids used in this study are summarized in Table 1.

Schuster M., Kilaru S., Fink G., Collemare J., Roger Y, & Steinberg G. (2011). Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array. Molecular Biology of the Cell, 22(19), 3645-3657.

Publication 2011

Cloning Vectors Dynamin I Dynein ATPase Genes Genes, vif hygromycin A Phleomycins Plasmids SDHD protein, human Strains Tubulin

Visualizing Kinesin-Dynein Interactions

Cited 10 times

Flow chambers were assembled using cover glasses (Corning no. 1½) and Taxol-stabilized microtubules containing 10% Alexa488- and biotin-tubulin were immobilized on the glass surface with Biotin-BSA and streptavidin as described (Derr et al., 2012 (link); Huang et al., 2012 (link)). Atto647-labeled Kip2 (0.01–0.05 nM) was added with Bik1 (100 nM) and Bim1 (5 nM) as indicated, in BRB80 supplemented with 1 mM Mg-ATP, 1 mM DTT, 20 μM taxol, 2.5 mg/ml casein, and an oxygen scavenging system. Motility assays were imaged as described (Derr et al., 2012 (link); Huang et al., 2012 (link)). Motor velocities and run lengths were calculated from kymographs. For landing rate quantification, Kip2 was analyzed at 0.01 and 0.02 nM in the presence or absence of Bik1 (100 nM) and Bim1 (5 nM). Landing events were determined from kymographs and the rate was calculated as the number of events per micron of microtubule per nM Kip2 per minute.
For visualizing the colocalization of weak dynein with Kip2, assay chambers were prepared as for dynamic microtubule assays except taxol-stabilized microtubules were attached to the biotin-PEG surface instead of GMP-CPP stabilized seeds, and all buffers contained 20 μM taxol. The final reaction contained the weak dynein construct (2 nM), Lis1 (2 nM), Bik1 (2 nM) and Kip2-Atto647 (0.2 nM).

Roberts A.J., Goodman B.S, & Reck-Peterson S.L. (2014). Reconstitution of dynein transport to the microtubule plus end by kinesin. eLife, 3, e02641.

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Publication 2014

Biological Assay Biotin Buffers Caseins Cell Motility Assays Classical Lissencephaly Debility Dynein ATPase Eyeglasses Kymography Microtubules Oxygen Plant Embryos Streptavidin Taxol Tubulin

Most recents protocols related to «Dynein ATPase»

Cryo-EM Sub-tomogram Particle Analysis

After the metadata preparation, the sub-tomogram particles were made in RELION4 (Zivanov et al, 2022 ) with the binning factor of six. Local 3D refinement with a mask covering one 96-nm MTD was performed. After refinement, the features pf 24-nm repeat of outer dynein arm was observed (Fig EV6D). A smaller mask only covering the inner dynein arm and radial spoke was created for the focused classification without alignment (Fig EV6E). After classification, equivalent 96-nm MTD repeat classes were separated (Fig EV6E). One of the classes was selected for the future process. New sub-tomogram particles with binning factor three and corresponding 10.2 Å of pixel size were generated. Another local refinement was performed with a 96-nm MTD mask. Resolution was estimated using the Fourier shell correlation (FSC) at a cut-off of 0.143 in RELION4 (Zivanov et al., 2022 ). Details of acquisition parameters and particle numbers are summarized in Table EV3.

Hwang J.Y., Chai P., Nawaz S., Choi J., Lopez-Giraldez F., Hussain S., Bilguvar K., Mane S., Lifton R.P., Ahmad W., Zhang K, & Chung J.J. (2023). LRRC23 truncation impairs radial spoke 3 head assembly and sperm motility underlying male infertility. bioRxiv.

Publication Preprint 2023

Dynein ATPase Tomography

Quantitative Gene Expression Analysis via qPCR

Gene expression was evaluated via quantitative real-time polymerase chain reaction (qPCR) according to standard protocols previously reported by our group (Pacheco et al., 2014 (link); Morais et al., 2016 (link)). Briefly, after cell treatment, the total RNA was extracted using the RNeasy kit (Qiagen, Netherlands) followed by DNase treatment, Then, cDNA strands were constructed using a SuperScript^® III First-Strand Synthesis kit (InvitrogenTM, Life Technologies Inc., United States), according to manufacturer’s instructions. Finally, the samples were applied in a SYBR^® green (Applied Biosystems, United States)-based reaction with specific and validated primers to evaluate mRNA levels of genes coding for dynein chains. RNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and quantified using the Livak 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)). The experiments were conducted by Step One Plus^® PCR real-time system (Applied Biosystems, United States).

Morais K.L., Ciccone L., Stura E., Alvarez-Flores M.P., Mourier G., Driessche M.V., Sciani J.M., Iqbal A., Kalil S.P., Pereira G.J., Marques-Porto R., Cunegundes P., Juliano L., Servent D, & Chudzinski-Tavassi A.M. (2023). Structural and functional properties of the Kunitz-type and C-terminal domains of Amblyomin-X supporting its antitumor activity. Frontiers in Molecular Biosciences, 10, 1072751.

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Publication 2023

Anabolism Cells Deoxyribonuclease I DNA, Complementary Dynein ATPase Gene Expression Genes Glyceraldehyde-3-Phosphate Dehydrogenases Oligonucleotide Primers One-Step dentin bonding system Real-Time Polymerase Chain Reaction RNA, Messenger SYBR Green I

Comprehensive Cellular Signaling Pathway Analysis

Cortisol, corticosterone, BSA, DAPI, α-tubulin antibody (T6074), melatonin (M5250), PD98059 (027K2176), 5-azacytidine (5-aza, A2385) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP, C2759) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Lamin A/C (sc-376248), β-actin (sc-47778), MT2 (sc-28453), dynein (sc-13524), NUP62 (sc-48373), importin β (sc-137016), BNIP3 (sc-56167), c-MYC (sc-40), IgG mouse (sc-2025), p-ERK (sc-7383), ERK (sc-94), JNK (sc-7345), p-JNK (sc-6254), p-p38 (sc-166182), and PINK1 (sc-33796) antibodies were acquired from Santa Cruz (Paso Robles, CA, USA). MT1 (NBP1-71113), FKBP4 (NB110-96874), FKBP5 (NBP1-84676), LC3 (NB100-2220) and NIX (NBP1-88558) were purchased from Novus Biologicals (Littleton, CO, USA). TOMM20 (ab56783), p-GR (ab55189), DNMT1 (ab19905), DNMT3a (ab23565), and SP1 (ab227383) antibodies were obtained from Abcam (Cambridge, MA, USA). Cleaved caspase 3 (9661 S), DNMT3b (67259 S), GR (12041 S), 5-methylcytosine (5-mc, 28692 S), p38 (9212 S), IgG rabbit (2729 S), and p-c-MYC ser62 (13748 S) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). HSP90 antibody (PA3-013) was obtained from Thermo Fisher (Rockford, IL, USA).

Kim M.J., Choi G.E., Chae C.W., Lim J.R., Jung Y.H., Yoon J.H., Park J.Y, & Han H.J. (2023). Melatonin-mediated FKBP4 downregulation protects against stress-induced neuronal mitochondria dysfunctions by blocking nuclear translocation of GR. Cell Death & Disease, 14(2), 146.

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Publication 2023

5-Methylcytosine Actins alpha-Tubulin Antibodies Azacitidine Biological Factors Carbonyl Cyanide m-Chlorophenyl Hydrazone Caspase 3 Corticosterone DAPI DNMT1 protein, human DNMT3B protein, human Dynein ATPase FKBP5 protein, human HSP90 Heat-Shock Proteins Hydrocortisone Immunoglobulins Importins LMNA protein, human Melatonin Mus Novus Oncogenes, myc PD 98059 Rabbits tacrolimus binding protein 4 TOMM20 protein, human

Multimodal Imaging of Mitotic Chromosome Dynamics

Fig. S1 shows results related to Fig. 1 (description of PP4 mutants). Fig. S2 shows results related to Fig. 2 (nuclear levels of kinetochore components and kinetochore dynein localization) and Fig. 3. (quantification of sister centromere resolution and mitotic chromosome morphology). Fig. S3 shows results related to Fig. 3 (cohesin levels on mitotic chromosomes) and Fig. 4 (validation of RNAi penetrance for molecular replacement and further characterization of hcp-4 mutants). Table S1 lists C. elegans genotypes. Table S2 lists sequences targeted by CRISPR/Cas9. Table S3 list oligonucleotides for dsRNA production. Video 1 shows embryos co-expressing GFP::TBG-1 and GFP::HIS-58 related to Fig. 1 B. Video 2 shows embryos co-expressing GFP::TBB-2 and mCherry::HIS-11 related to Fig. 1 E. Video 3 shows embryos co-expressing GFP::TBG-1 and GFP::HIS-58 related to Fig. 2 A. Video 4 shows embryos expressing GFP::HCP-4 related to Fig. 2 E. Video 5 shows embryos expressing HIM-10::GFP related to Fig. 2 G. Video 6 shows an embryo co-expressing SMK-1::GFP and mCherry::HIS-58 related to Fig. 2 I. Video 7 shows embryos co-expressing GFP::HCP-3 and mCherry::HIS-58 related to Fig. 3 A. Video 8 shows embryos co-expressing SMK-1::GFP or SMK-1::GFP(Y179A) together with mCherry::HIS-58 related to Fig. 4 F. Video 9 shows embryos co-expressing TBG-1::GFP and GFP::HIS-11 related to Fig. 5 A. Video 10 shows embryos co-expressing TBG-1::GFP and GFP::HIS-11 related to Fig. 5 E.

Rocha H., Simões P.A., Budrewicz J., Lara-Gonzalez P., Carvalho A.X., Dumont J., Desai A, & Gassmann R. (2023). Nuclear-enriched protein phosphatase 4 ensures outer kinetochore assembly prior to nuclear dissolution. The Journal of Cell Biology, 222(3), e202208154.

Publication 2023

Caenorhabditis elegans Centromere Chromosomes Clustered Regularly Interspaced Short Palindromic Repeats cohesins Dynein ATPase Embryo Genotype Kinetochores Oligonucleotides RNA, Double-Stranded RNA Interference

Quantifying Ndel1 Binding Affinities

The binding affinity of Ndel1 constructs for dynein and Lis1 was determined by coupling Ndel1 to 25 μL of Magne HaloTag Beads (Promega) in 2 mL Protein Lo Bind Tubes (Eppendorf) using the following protocol. Beads were washed twice with 1 mL of GF150 without ATP supplemented with 10% glycerol and 0.1% NP40. Ndel1 was diluted in this buffer to 0, 30, 60, 90, 120, 300 and 600 nM. 25 μL of diluted Ndel1 was added to the beads and gently shaken for one hour. 20 μL of supernatant were then analyzed via SDS-PAGE to confirm complete depletion of Ndel1. The Ndel1-conjugated beads were washed once with 1 mL GF150 with 10% glycerol and 0.1% NP40 and once with 1mL of binding buffer (30 mM HEPES [pH 7.4], 2 mM magnesium acetate, 1 mM EGTA, 10% glycerol, 1 mM DTT, 1 mg/mL casein, 0.1% NP40, 1mM ADP) supplemented with 36 mM KCl (or 150mM KCl for high salt Lis1 curves). 5 nM dynein or Lis1 was diluted in binding buffer, which resulted in binding buffer with 36 mM KCl (or 150mM KCl for high salt Lis1 curves). 25 μL of the dynein or Lis1 mixture was added to the beads pre-bound with Ndel1 and gently agitated for 45 minutes. After incubation 20 μL of the supernatant was removed, and 6.67 μL of NuPAGE^® LDS Sample Buffer (4X) and 1.33 μL of Beta-mercaptoethanol was added to each. The samples were boiled for 5 minutes before running on a 4-12% NuPAGE Bis-Tris gel. Depletion was determined using densitometry in ImageJ. Binding curves were fit in Prism9 (Graphpad) with a nonlinear regression for one site binding with Bmax set to 1.
Experiments measuring binding between dynein, Lis1 and Ndel1 were performed with the same method and buffers as above. For the experiment with Lis1 on the beads 50nM of Halo-Lis1 was conjugated to the beads and incubated with 5nM dynein with and without 100nM of WT and E48A Ndel1. For the experiment with Ndel1 on the beads 30nM of Halo-Ndel1 was conjugated to beads and incubated with 5nM dynein in the presence and absence of 30nM Lis1.
For experiments investigating the competition between Ndel1 and dynactin/activating adaptor the methods and buffers were the same as above. 90nM Ndel1 was conjugated to 30μL of NEBExpress^® Ni-NTA Magnetic Beads (NEB) and incubated with 5nM dynein. The appropriate samples were further supplemented with 10nM dynactin and 150nM of wither BicD2 or NINL.

Garrott S.R., Gillies J.P., Siva A., Little S.R., Jbeily R.E, & DeSantis M.E. (2023). Ndel1 modulates dynein activation in two distinct ways. bioRxiv.

Publication Preprint 2023

2-Mercaptoethanol Binding Sites Bistris Buffers Caseins Classical Lissencephaly Densitometry Dynactin Subunit 1 Dynein ATPase Egtazic Acid Glycerin HaloTag HEPES magnesium acetate MM 36 Promega Proteins SDS-PAGE Sodium Chloride

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More about "Dynein ATPase"

Dynein ATPase, also known as cytoplasmic dynein or just dynein, is a crucial molecular motor protein that plays a vital role in various cellular processes.
This complex enzyme utilizes the energy released from ATP hydrolysis to power the movement of organelles, chromosomes, and other cellular components along microtubules.
Dynein ATPase is essential for a wide range of biological functions, including ciliary and flagellar beating, mitotic spindle formation, and neuronal transport.
Understanding the structure, function, and regulation of this motor protein is a key area of research in cell biology and biophysics, with implications for human health and disease.
Researchers often use tools like the EnzChek Phosphate Assay Kit to measure dynein ATPase activity, while compounds such as Nocodazole can be employed to disrupt microtubule dynamics and study the effects on dynein-mediated transport.
The 74.1 mouse monoclonal IC antibody-conjugated agarose is a common tool for immunoprecipitating and purifying dynein complexes, which can then be analyzed using techniques like Alexa Fluor 488 fluorescence labeling.
Transfection reagents like Lipofectamine 2000 are often used to manipulate the expression of dynein subunits and associated proteins, while IgG Sepharose beads can be utilized for pull-down experiments to identify dynein-interacting partners.
The small molecule Blebbistatin is known to inhibit myosin II activity, which can be used to investigate the interplay between dynein and other motor proteins.
Cutting-edge imaging techniques, such as those enabled by the Ti-E microscope, allow researchers to visualize and study the dynamic behavior of dynein in living cells.
Additionally, the Ultrafree-MC VV filter can be employed to remove unwanted components from dynein-containing samples, facilitating further analysis and purification.
By leveraging these tools and techniques, scientists can gain deeper insights into the complex mechanisms underlying dynein ATPase function, ultimately contributing to our understanding of essential cellular processes and their implications for human health.