Journal: Biochemistry

Article Title: Structural Mass Spectrometry of the α β -Tubulin Dimer Supports a Revised Model of Microtubule Assembly †

doi: 10.1021/bi900200q

Figure Lengend Snippet: Perturbation of Deuterium Labeling for α/ β -Tubulin as a Function of Nucleotide Occupancy at the E-Site for 0 M Urea Treatment

Article Snippet: Perturbation of Deuterium Labeling in the Intradimer Interface Region Comparing GMPCPP Microtubules with GMPCPP Dimer Molecular Dynamics Simulations of Tubulin Dimer The influence of E-site occupancy on subunit orientation and flexibility was tested computationally with molecular dynamics.

Techniques: Labeling, Sequencing

Journal: Biochemistry

Article Title: Structural Mass Spectrometry of the α β -Tubulin Dimer Supports a Revised Model of Microtubule Assembly †

doi: 10.1021/bi900200q

Figure Lengend Snippet: (A) Structural representation of α/β-tubulin (α, pale green; β, cyan) at an oblique angle of the protofilament axis to highlight both the E-site and N-site nucleotides, with increases in deuteration arising from GMPCPP treatment relative to GTP superimposed in red. Nucleotides are represented as green spheres. (B) As in (A), with a face-on view of nucleotide at the E-site. (C) As in (A), with a face-on view of GTP at the N-site.

Article Snippet: Perturbation of Deuterium Labeling in the Intradimer Interface Region Comparing GMPCPP Microtubules with GMPCPP Dimer Molecular Dynamics Simulations of Tubulin Dimer The influence of E-site occupancy on subunit orientation and flexibility was tested computationally with molecular dynamics.

Techniques:

Journal: Biochemistry

Article Title: Structural Mass Spectrometry of the α β -Tubulin Dimer Supports a Revised Model of Microtubule Assembly †

doi: 10.1021/bi900200q

Figure Lengend Snippet: Perturbation of Deuterium Labeling for α/ β -Tubulin as a Function of Nucleotide Occupancy at the E-Site for 0.25 M Urea Treatment

Article Snippet: Perturbation of Deuterium Labeling in the Intradimer Interface Region Comparing GMPCPP Microtubules with GMPCPP Dimer Molecular Dynamics Simulations of Tubulin Dimer The influence of E-site occupancy on subunit orientation and flexibility was tested computationally with molecular dynamics.

Techniques: Labeling, Sequencing

Journal: Biochemistry

Article Title: Structural Mass Spectrometry of the α β -Tubulin Dimer Supports a Revised Model of Microtubule Assembly †

doi: 10.1021/bi900200q

Figure Lengend Snippet: Alternative views of the differential deuteration level data for GDP/GTP at 0.25 M urea, mapped to PDB 1JFF (α-tubulin in pale green, β-tubulin in cyan). (A) Peptides with increased deuteration upon exchange with GDP, and in direct contact with E-site nucleotide, are shown in yellow. All others are shown in red. View is down the z-axis, corresponding to the microtubule protofilament axis viewed from the plus end, where y represents radial direction out from the center of the microtubule. (B) Color scheme as in (A), highlighting the intradimer region.

Article Snippet: Perturbation of Deuterium Labeling in the Intradimer Interface Region Comparing GMPCPP Microtubules with GMPCPP Dimer Molecular Dynamics Simulations of Tubulin Dimer The influence of E-site occupancy on subunit orientation and flexibility was tested computationally with molecular dynamics.

Techniques:

Journal: Biochemistry

Article Title: Structural Mass Spectrometry of the α β -Tubulin Dimer Supports a Revised Model of Microtubule Assembly †

doi: 10.1021/bi900200q

Figure Lengend Snippet: Differential deuteration data for GDP/GTP at 0.25 M urea, mapped to (A) PDB 1JFF and (B) PDB 1SA0. Data associated with the E-site are removed for clarity. GDP-induced increases in deuteration are again shown in red, with α-tubulin in pale green and β-tubulin in cyan. Peptides indicated in A represent the two sequences in contact with both inter and intradimer interface peptides. Colchicine is displayed in B as yellow spheres. (C) Differential SASA data for 1SA0 relative to 1JFF, restricted to the intradimer interface. Red represents >+50% change (colchicine removed for clarity).

Article Snippet: Perturbation of Deuterium Labeling in the Intradimer Interface Region Comparing GMPCPP Microtubules with GMPCPP Dimer Molecular Dynamics Simulations of Tubulin Dimer The influence of E-site occupancy on subunit orientation and flexibility was tested computationally with molecular dynamics.

Techniques:

Journal: Biochemistry

Article Title: Structural Mass Spectrometry of the α β -Tubulin Dimer Supports a Revised Model of Microtubule Assembly †

doi: 10.1021/bi900200q

Figure Lengend Snippet: View of the differential deuteration level data from the E-site for GMPCPP/GTP at 0.25 M urea mapped to PDB 1JFF as in Figure 5. Color scheme follows Figure 5.

Article Snippet: Perturbation of Deuterium Labeling in the Intradimer Interface Region Comparing GMPCPP Microtubules with GMPCPP Dimer Molecular Dynamics Simulations of Tubulin Dimer The influence of E-site occupancy on subunit orientation and flexibility was tested computationally with molecular dynamics.

Techniques:

Journal: Biochemistry

Article Title: Structural Mass Spectrometry of the α β -Tubulin Dimer Supports a Revised Model of Microtubule Assembly †

doi: 10.1021/bi900200q

Figure Lengend Snippet: Perturbation of Deuterium Labeling in the Intradimer Interface Region Comparing GMPCPP Microtubules with GMPCPP Dimer

Article Snippet: Perturbation of Deuterium Labeling in the Intradimer Interface Region Comparing GMPCPP Microtubules with GMPCPP Dimer Molecular Dynamics Simulations of Tubulin Dimer The influence of E-site occupancy on subunit orientation and flexibility was tested computationally with molecular dynamics.

Techniques: Labeling, Sequencing

Journal: Biochemistry

Article Title: Structural Mass Spectrometry of the α β -Tubulin Dimer Supports a Revised Model of Microtubule Assembly †

doi: 10.1021/bi900200q

Figure Lengend Snippet: Proposed model of nucleotide control over αβ-tubulin assembly from an intradimer perspective. The most flexible state of αβ-tubulin (left dimer, symbolized by greater spacing between α- and β-tubulin) is shown in equilibrium with a less flexible state (right dimer, symbolized by reduced spacing between α- and β-tubulin), with the relative population of states dictated at least in part by nucleotide occupancy, as shown. Intradimer flexibility is stabilized by the straight microtubule lattice upon assembly (symbolized by no spacing between α- and β-tubulin). On the basis of the significantly larger change in deuteration upon assembly, we suggest that the energy required for intradimer stabilization of GTP-tubulin (E2) is only slightly less than that required to stabilize GDP-tubulin (E1 + E2).

Article Snippet: Perturbation of Deuterium Labeling in the Intradimer Interface Region Comparing GMPCPP Microtubules with GMPCPP Dimer Molecular Dynamics Simulations of Tubulin Dimer The influence of E-site occupancy on subunit orientation and flexibility was tested computationally with molecular dynamics.

Techniques: Control

Journal: Viruses

Article Title: The Major Capsid Protein, VP1, of the Mouse Polyomavirus Stimulates the Activity of Tubulin Acetyltransferase 1 by Microtubule Stabilization

doi: 10.3390/v12020227

Figure Lengend Snippet: The acetylation of microtubules was elevated during the late phase of infection. ( A – C ) 3T6 cells were infected with MPyV and lysed at the indicated hours post infection (hpi). Lysates were separated by SDS/PAGE, transferred onto membrane, and acetylated tubulin (ac-tub), tubulin (tub), VP1, and GAPDH were stained by specific antibodies. ( D , E ) A graphic illustration of a densitometry analysis of the digital images of Western blots from four independent experiments. Shown is the fold increase relative to mock-infected cells (a representative 24 h time point) ( D ) or infected cells at 8 hpi (representing amount of incoming virus) ( E ). +/− SD. * p < 0.05, ** p < 0.01, determined by the Student’s t test. The changes in total levels of tubulin ( D , red columns) were not significant (ns) according to the analysis using the Student’s t test.

Article Snippet: GMPCPP-stabilized microtubules were grown using a mixture of 1.3 mg/mL tubulin and 2 mM GMPCPP (guanosine-5′-[(α,β)-methyleno]triphosphate, Merck Millipore, Billerica, MA, USA) in a BRB80 buffer (80 mM Pipes, pH = 6.8, 2 mM MgCl 2 , 1 mM EGTA) and incubated for 1 h for the microtubule binding assay or 15 min for the dynamic microtubule assay at 37 °C.

Techniques: Infection, SDS Page, Membrane, Staining, Western Blot, Virus

Journal: Viruses

Article Title: The Major Capsid Protein, VP1, of the Mouse Polyomavirus Stimulates the Activity of Tubulin Acetyltransferase 1 by Microtubule Stabilization

doi: 10.3390/v12020227

Figure Lengend Snippet: VP1 increases the level of microtubules in cells. ( A ) 3T6 cells were infected or ( C ) WOP cells were transfected with the control plasmid (ctrl) or with the plasmid expressing VP1 (VP1). Cells were fractionated into a polymerized (p) and soluble (s) fraction at 40 hpi or 24 hpt. These fractions were applied to SDS-PAGE, immunoblotted, and tubulin was stained by a specific antibody. ( B , D ) A graphic illustration of a densitometry analysis of the digital images of Western blots from three independent experiments. The fold increase in polymerized tubulin relative to the mock-infected/mock-transfected cells is shown. +/− SD. * p < 0.05, ** p < 0.01 determined by Student’s t test.

Article Snippet: GMPCPP-stabilized microtubules were grown using a mixture of 1.3 mg/mL tubulin and 2 mM GMPCPP (guanosine-5′-[(α,β)-methyleno]triphosphate, Merck Millipore, Billerica, MA, USA) in a BRB80 buffer (80 mM Pipes, pH = 6.8, 2 mM MgCl 2 , 1 mM EGTA) and incubated for 1 h for the microtubule binding assay or 15 min for the dynamic microtubule assay at 37 °C.

Techniques: Infection, Transfection, Control, Plasmid Preparation, Expressing, SDS Page, Staining, Western Blot

Journal: Viruses

Article Title: The Major Capsid Protein, VP1, of the Mouse Polyomavirus Stimulates the Activity of Tubulin Acetyltransferase 1 by Microtubule Stabilization

doi: 10.3390/v12020227

Figure Lengend Snippet: VP1 directly binds microtubules and stabilizes them. ( A ) A schematic diagram of a microtubule binding assay with VP1/EGFP-tVP3. ( B ) Time-lapse fluorescence micrographs of rhodamine-labeled microtubules (Rh MT) depolymerizing in the absence or presence of 3.65 µg/mL VP1/EGFP-tVP3. Scale bar, 10 μm. ( C ) Depolymerization rates of taxol-stabilized rhodamine-labeled microtubules in the absence (control) or presence of 3.65 µg/mL VP1/EGFP-tVP3. Control: 2.67 ± 2.07 µm/min ( N = 22). Microtubules + 3.65 µg/mL VP1/EGFP-tVP3: 0.01 ± 0.01 µm/min ( N = 16); values are the mean ± SD. Red lines represent the median values; the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the extreme data points. ( D ) Kymographs showing dynamic microtubules polymerizing with 15 µM free rhodamine-labeled tubulin in the absence or presence of 3.65 or 9.13 µg/mL VP1/EGFP-tVP3. Horizontal scale bar 3 μm; vertical bar 5 min. ( E ) Cumulative polymerization time of both microtubules’ plus and minus tips over 20 min in the absence or presence of 3.65 or 9.13 µg/mL VP1/EGFP-tVP3. Tubulin (15 µM) control: 25.85 ± 5.68 ( N = 9), tubulin (15 µM) + 3.65 µg/mL VP1/EGFP-tVP3: 14.5 ± 8.91 ( N = 6), tubulin (15 µM) + 9.13 µg/mL VP1/EGFP-tVP3: 1.75 ± 2.75 ( N = 6); values are the mean ± SD. Red lines represent median values; the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the extreme data points. ( F ) Coomassie-stained SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of 5 mg/mL unlabeled porcine tubulin, 182.5 μg/mL VP1/EGFP-tVP3, or their mixture, after the VP1-co-sedimentation assay. Supernatant fraction (s) and pellet (p). In ( C ) and ( E ), * p < 0.05, **** p < 0.0001, determined by Student’s t test.

Article Snippet: GMPCPP-stabilized microtubules were grown using a mixture of 1.3 mg/mL tubulin and 2 mM GMPCPP (guanosine-5′-[(α,β)-methyleno]triphosphate, Merck Millipore, Billerica, MA, USA) in a BRB80 buffer (80 mM Pipes, pH = 6.8, 2 mM MgCl 2 , 1 mM EGTA) and incubated for 1 h for the microtubule binding assay or 15 min for the dynamic microtubule assay at 37 °C.

Techniques: Microtubule Binding Assay, Fluorescence, Labeling, Control, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Sedimentation

Journal: Viruses

Article Title: The Major Capsid Protein, VP1, of the Mouse Polyomavirus Stimulates the Activity of Tubulin Acetyltransferase 1 by Microtubule Stabilization

doi: 10.3390/v12020227

Figure Lengend Snippet: VP1 protects microtubules from nocodazole induced depolymerization. WOP cells were transfected with plasmids expressing VP1 and 24 hpt cells were treated with nocodazole (4 µM) for the indicated times. ( A ) After the treatment, soluble tubulin was washed out, the cells were fixed, and polymerized tubulin (tub; red) and VP1 (green) were stained by specific antibodies. Shown are the selected confocal sections. Enlarged details of the cells are presented in insets. Bar: 10 µm. ( B ) After the treatment, cells were fractionated into a soluble tubulin fraction (s) and polymerized tubulin fraction (p). Equal amounts of the fractions were resolved via SDS/PAGE, immunoblotted, and tubulin and VP1 were stained with specific antibodies. ( C ) A graphic illustration of a densitometry analysis of the digital images of Western blots from three independent experiments. The fold change in polymerized tubulin relative to the mock-treated cells ( t = 0 min) is shown. +/− SD; * p ˂ 0.05 determined by Student’s t test.

Article Snippet: GMPCPP-stabilized microtubules were grown using a mixture of 1.3 mg/mL tubulin and 2 mM GMPCPP (guanosine-5′-[(α,β)-methyleno]triphosphate, Merck Millipore, Billerica, MA, USA) in a BRB80 buffer (80 mM Pipes, pH = 6.8, 2 mM MgCl 2 , 1 mM EGTA) and incubated for 1 h for the microtubule binding assay or 15 min for the dynamic microtubule assay at 37 °C.

Techniques: Transfection, Expressing, Staining, SDS Page, Western Blot

Journal: Viruses

Article Title: The Major Capsid Protein, VP1, of the Mouse Polyomavirus Stimulates the Activity of Tubulin Acetyltransferase 1 by Microtubule Stabilization

doi: 10.3390/v12020227

Figure Lengend Snippet: αTAT1 is a part of the VP1–hyperacetylated microtubule complex. ( A , E ) Infected 3T6 cells ( A ) or VP1 expressing cells ( E ) were in situ fractionated; then, an equal amount of washed out material from each fraction was separated via SDS/PAGE and transferred onto the membrane. The presence of VP1 and tubulin (tub) in each fraction was determined by specific antibodies. ( B , F ) A graphic illustration of the densitometry analysis of the digital images of Western blots from two independent experiments. Presented are the fold changes of tubulin in the SDS fraction of the infected ( B ) or VP1-expressing cells ( F ), which were compared with mock-infected or mock-transfected cells. ( C , G ) An equal amount of the SDS fraction from infected (inf) ( C ) or VP1-expressing cells (VP1) ( G ) was resolved on SDS/PAGE, immunoblotted to the membrane and tubulin, and acetylated α-tubulin (ac-tub) was stained by specific antibodies. ( D , H ) A graphic illustration of the densitometry analysis of the digital images of Western blots from two independent experiments. Presented are the fold changes of α-tubulin acetylation ratios in the SDS fraction of the infected ( D ) or VP1-expressing cells ( H ), which were compared with those of the mock-infected or mock-transfected cells. ( I ) Lysates of WOP and WOP-EGFP-αTAT1 were separated by SDS/PAGE, blotted onto the membrane, and EGFP was stained by a specific antibody. ( J ) Infected (inf) or VP1-expessing cells were in situ fractionated, and equal amounts of the pooled fractions and SDS fraction were separated by SDS/PAGE, transferred onto the membrane, and EGFP was stained by a specific antibody; × indicates non-specific antibody staining.

Article Snippet: GMPCPP-stabilized microtubules were grown using a mixture of 1.3 mg/mL tubulin and 2 mM GMPCPP (guanosine-5′-[(α,β)-methyleno]triphosphate, Merck Millipore, Billerica, MA, USA) in a BRB80 buffer (80 mM Pipes, pH = 6.8, 2 mM MgCl 2 , 1 mM EGTA) and incubated for 1 h for the microtubule binding assay or 15 min for the dynamic microtubule assay at 37 °C.

Techniques: Infection, Expressing, In Situ, SDS Page, Membrane, Western Blot, Transfection, Staining

Journal: Viruses

Article Title: The Major Capsid Protein, VP1, of the Mouse Polyomavirus Stimulates the Activity of Tubulin Acetyltransferase 1 by Microtubule Stabilization

doi: 10.3390/v12020227

Figure Lengend Snippet: Acetylated microtubules are dispensable for MPyV infection. ( A ) Lysates of 3T3-wt and αTAT1 KO cells were separated by SDS/PAGE, blotted onto the membrane, and acetylated α-tubulin (ac-tub) and GAPDH were stained by specific antibodies. ( B ) Lysates (40 hpi) of infected 3T3-wt and αTAT1 KO cells were resolved on SDS/PAGE, transferred onto the membrane, and VP1, LT, and GAPDH were stained by specific antibodies. ( C ) A graphic illustration of the densitometry analysis of the digital images of Western blots of three independent experiments. The fold change relative to 3T3-wt cells +/− SD is shown. ( D ) Lysates of 3T6 cells treated for 24 h with 5 µM tubacin were separated by 10% SDS/PAGE, transferred onto a membrane, and acetylated α-tubulin and GAPDH were stained by specific antibodies. ( E , H ) 3T6 ( E ), 3T3-wt, and αTAT1 KO cells ( H ) were infected, and 24 hpi tubacin was added (5 µM). Cells were lysed after 16 h tubacin treatment, lysates were separated by SDS/PAGE and immunoblotted, and VP1, LT, and GAPDH were stained by specific antibodies. ( F , I , J ) Graphic illustration of the densitometry analysis of the digital images of the Western blots of three independent experiments. Shown is the fold change relative to the mock treated 3T6 ( F ), 3T3-wt, ( I ) and αTAT1 KO ( J ) cells +/− SD. ( G , K ) Cells 3T6 ( G ), 3T3-wt, and αTAT1 KO cells ( K ) were infected; then, 24 hpi tubacin was added (5 µM), and, after 24 h of incubation, MPyV virions were isolated. 3T6 cells were infected with equal volumes of the virus isolated from the tubacin treated or mock-treated cells. Cells were fixed at 24 hpi, and the LT antigen was stained by a specific antibody. The values in the graph refer to the fold change in numbers of LT positive cells relative to the mock-treated cells and represent the mean values from three independent experiments +/− SD. ( L ) Lysates of WOP, WOP-EGFP-αTAT1 (αTAT1), and WOP-EGFP expressing cells (EGFP) were separated by SDS/PAGE, blotted onto the membrane, and acetylated α-tubulin, EGFP, and GAPDH were stained by specific antibodies. ( M ) Lysates of infected WOP, WOP-EGFP-αTAT1, and WOP-EGFP expressing cells were performed at 40 hpi and resolved by SDS/PAGE; proteins transferred onto the membrane and VP1 and GAPDH were stained by specific antibodies. ( N ) Graphic illustration of the densitometry analysis of the digital images of Western blots of three independent experiments. Shown is the fold change relative to the WOP cells +/− SD. ( O ) WOP, WOP-EGFP-αTAT1, and WOP-EGFP expressing cells were infected, and 48 hpi MPyV virions were isolated. 3T6 cells were infected with equal volumes of the isolated virus from cells. Cells were fixed at 24 hpi, and the LT antigen was stained by a specific antibody. The values in the graph refer to the fold change in the numbers of LT antigen positive cells relative to WOP cells and represent the mean values of three independent experiments +/− SD × indicate non-specific antibody staining. * p ˂ 0.05, ** p ˂ 0.01, *** p ˂ 0.001 determined by the Student’s t test, ns—not significant.

Article Snippet: GMPCPP-stabilized microtubules were grown using a mixture of 1.3 mg/mL tubulin and 2 mM GMPCPP (guanosine-5′-[(α,β)-methyleno]triphosphate, Merck Millipore, Billerica, MA, USA) in a BRB80 buffer (80 mM Pipes, pH = 6.8, 2 mM MgCl 2 , 1 mM EGTA) and incubated for 1 h for the microtubule binding assay or 15 min for the dynamic microtubule assay at 37 °C.

Techniques: Infection, SDS Page, Membrane, Staining, Western Blot, Incubation, Isolation, Virus, Expressing

Journal: Nature Communications

Article Title: Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

doi: 10.1038/s41467-018-04991-2

Figure Lengend Snippet: Full-length HSET organizes growing MTs into asters. a Schematic of HSET truncations purified in this study. HSET contains two MT-binding domains: an ATP-independent globular tail domain located at the N terminus (amino acid 1–138, brown), and an ATP-dependent conserved kinesin motor domain located at the C terminus (aa 305–673, blue). HSET also contains a coiled-coil stalk domain necessary for dimerization (aa 139–304, black). All constructs contained an N-terminal 6× His tag used for affinity purification. b Aster formation of growing MTs by HSET. 20 µM tubulin (10% Alexa594-labeled, magenta) was mixed in assay buffer with the indicated EGFP-HSET truncation (green) and monitored by time-lapse microscopy at 37 °C. With the exception of EGFP-HSETΔTail (20 nM), all HSET constructs were present at 100 nM. c Bundle formation of nongrowing, GMPCPP-stabilized MTs by HSET. Alexa594-labeled GMPCPP-MTs (10% labeled, 1 µM tubulin in polymer form, magenta) were mixed in assay buffer with the indicated EGFP-HSET truncation (green) and monitored by time-lapse microscopy at 37 °C. HSET concentrations are identical to b . For contrast measurements over time, see Supplementary Figure . For movies, see Supplementary Movies – . For additional EGFP-HSET images on GMPCPP-MTs, see Fig. . Technical replicates of experiments in b , c were repeated n ≥ 3 times for each condition, and representative images are shown. Scale bars, 50 µm

Article Snippet: Alexa594-labeled GMPCPP-MTs (10% labeled, 1 µM tubulin in polymeric form, magenta) were mixed in assay buffer with the indicated motor-QDot complexes (21:7 nM motor :QDots, green) and monitored by time-lapse microscopy at 37 °C.

Techniques: Purification, Binding Assay, Construct, Affinity Purification, Labeling, Time-lapse Microscopy

Journal: Nature Communications

Article Title: Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

doi: 10.1038/s41467-018-04991-2

Figure Lengend Snippet: Soluble (non-MT) tubulin promotes the ability of HSET to drive aster self-organization of GMPCPP-MTs independent of MT polymerization. a EGFP-HSET-driven self-organization of GMPCPP-stabilized MTs with increasing tubulin concentration. Alexa594-labeled GMPCPP-MTs (10% labeled, 1 µM tubulin in polymer form, magenta) were mixed in assay buffer with EGFP-HSET (100 nM, green) and monitored by time-lapse microscopy at 37 °C. Unlabeled tubulin was added to the reaction at the indicated concentration. Technical replicates were repeated N ≥ 3 times for each condition, and representative images are shown. Scale bar, 50 µm. b EGFP-HSET-driven self-organization of GMPCPP-stabilized MTs in the absence of MT polymerization. Experiments were performed identically to a but in the absence of taxol and the presence of saturating colchicine and GDP to prevent polymerization. Technical replicates were repeated N ≥ 2 times for each condition, and representative images are shown. Scale bar, 50 µm

Article Snippet: Alexa594-labeled GMPCPP-MTs (10% labeled, 1 µM tubulin in polymeric form, magenta) were mixed in assay buffer with the indicated motor-QDot complexes (21:7 nM motor :QDots, green) and monitored by time-lapse microscopy at 37 °C.

Techniques: Concentration Assay, Labeling, Time-lapse Microscopy

Journal: Nature Communications

Article Title: Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

doi: 10.1038/s41467-018-04991-2

Figure Lengend Snippet: Soluble (non-MT) tubulin activates processive motility of HSET on single MTs. a Schematic. EGFP-HSET truncations were diluted in P12 buffer and monitored on GMPCPP-stabilized MTs by time-lapse TIRF. b Representative kymographs for time-lapse TIRF images for the indicated constructs at the following concentrations: EGFP-HSET and EGFP-HSETΔMotor, 50 pM. EGFP-HSETΔTail, 250 pM. Distance is on the x -axis (scale bar, 10 µm), and time is on the y -axis (scale bar, 10 s). c Mean-squared displacement (MSD) analysis of particle motion. The reported diffusion constant D is determined from a linear fit over the first 5 s, with the units nm 2 /s: EGFP-HSET: D = 6.3 × 10 4 , n = 206; EGFP-HSETΔMotor: D = 9.4 × 10 4 , n = 197; EGFP-HSETΔTail: D = 0.1 × 10 4 , n = 200. Data are presented as the calculated mean MSD ( y -axis) from two independent experiments over the indicated time intervals ( x -axis) for the indicated n particles ± SEM. d EGFP-HSET in BRB80 + 50 mM KCl was observed in the absence (left) or presence (right) of soluble tubulin and visualized by kymograph ( x -scale bar, distance, 10 µm; y -scale bar, time, 1 min). e Quantification of processive (≥5 s) event frequency as a function of [EGFP-HSET] in the presence (dark green) or absence (light green) of 2 µM tubulin. Data are presented as the number of processive events divided by the total observed MT length multiplied by the observation time for two independent experiments ± SD calculated from N ≥ 10 movies for each condition. Boxes represent first and third quartiles, whiskers represent detection limits, and lines represent median (mean overlaid). f Unlabeled HSET was mixed with 10 nM Cy5-tubulin in BRB80 + 50 mM KCl and observed. Velocities and run lengths of moving Cy5-tubulin particles were determined by kymograph and plotted as histograms. Data are reported as the mean velocity and run length values of n particles from CDF fitting ± the 95% CI from bootstrapping from two independent experiments. g 100 nM Cy5-tubulin (magenta) and 1 nM EGFP-HSET (green) were observed near-simultaneously by high-speed TIRF in BRB80 + 50 mM KCl, and visualized by kymograph ( x -scale bar, distance, 5 µm; y -scale bar, time, 10 s)

Article Snippet: Alexa594-labeled GMPCPP-MTs (10% labeled, 1 µM tubulin in polymeric form, magenta) were mixed in assay buffer with the indicated motor-QDot complexes (21:7 nM motor :QDots, green) and monitored by time-lapse microscopy at 37 °C.

Techniques: Construct, Diffusion-based Assay

Journal: Nature Communications

Article Title: Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

doi: 10.1038/s41467-018-04991-2

Figure Lengend Snippet: Multiple HSET motors conjugated to quantum dots drive self-assembly of GMPCPP-MTs into asters. a EGFP-HSET or EGFP-HSETΔTail was conjugated to streptavidin-QDots via the N-terminal 6× His-tag and a biotin anti-His antibody at a 3:1 ratio and visualized via TIRF. Representative kymographs of EGFP-HSET-QDots (left, 1 nM EGFP-HSET: 0.33 nM QDot) and EGFP-HSETΔTail-QDots (right, 0.5 nM EGFP-HSETΔTail: 0.17 nM QDot) are shown ( x -scale, distance, 5 µm; y -scale, time, 10 s). b – d Velocities ( b ), run lengths ( c ), and end dwell times ( d ) for the indicated constructed conjugated to QDots at a 3:1 ratio (EGFP-HSET, black, EGFP-HSETΔTail, red) were determined by kymograph analysis and plotted as histograms for the population. Data are reported as the mean values (insets) from CDF fitting ± the 95% CI from bootstrapping for the indicated n particles from 2 independent experiments, where N ≥ 4 movies for each condition. Populations for EGFP-HSET-QDots (black, upper) and EGFP-HSETΔTail-QDots (red, lower) are shown. For run length/end dwell times, particles reaching the end of MTs/dissociating immediately (<1 frame) are color-coded on the histograms. e Self-organization of GMPCPP-stabilized MTs by EGFP-HSET-QDots and EGFP-HSETΔTail-QDots. Alexa594-labeled GMPCPP-MTs (10% labeled, 1 µM tubulin in polymeric form, magenta) were mixed in assay buffer with the indicated motor-QDot complexes (21:7 nM motor :QDots, green) and monitored by time-lapse microscopy at 37 °C. The yellow box indicates the field of view depicted in f . Technical replicates were repeated n ≥ 3 times for each condition, and representative images are shown. Scale bar, 50 µm. f Zoomed-in view of the indicated field. The yellow arrow indicates EGFP-HSET-QDots that have accumulated on the minus end of an MT bundle. Time is indicated in min:s. Scale bar, 10 µm

Article Snippet: Alexa594-labeled GMPCPP-MTs (10% labeled, 1 µM tubulin in polymeric form, magenta) were mixed in assay buffer with the indicated motor-QDot complexes (21:7 nM motor :QDots, green) and monitored by time-lapse microscopy at 37 °C.

Techniques: Construct, Labeling, Time-lapse Microscopy

Journal: Current biology : CB

Article Title: Kinesin-3 responds to local microtubule dynamics to target synaptic cargo delivery to the presynapse

doi: 10.1016/j.cub.2018.11.065

Figure Lengend Snippet: A) KIF5C(1-560)-HaloTag motility on dynamic microtubules. Left panel shows full kymograph of dynamic microtubule channel. Insets: upper panel shows early time point when the microtubule is short; Left, overlay of dynamic microtubule (cyan) and GMPCPP-stabilized microtubule seed (magenta) channels, (−) and (+) represent the microtubule minus- and plus-end; Center, KIF5C channel; Right, overlay of a line representing the growing microtubule plus-end on the KIF5C channel. Horizontal scale bars: 10 μm; Vertical scale bars: 30 sec. The plot in the middle represents the location of KIF5C run terminations (blue circles) along a growing dynamic microtubule (dark blue line; microtubule plus-end), respective to the distance to the minus-end of that microtubule. The plot on the right shows the length of KIF5C runs (blue lines) that terminated at the plus-end (dark blue line). The numbers refer to the maximum KIF5C run-length observed in 120 seconds periods over 10 minutes. B) same as (A), but for KIF1A(1-393)-HaloTag. C) Percentage of runs initiating and D) terminating within 2μm of a microtubule plus-end (n=1035-1755 runs from 4-6 microtubules; average±95%CI). E) Circles represent KIF1A (red) and KIF5C (blue) run lengths observed on three representative microtubules and normalized to the maximum length of the microtubule they were observed on. The line traces show the dynamic profile of the representative microtubules along time. See also Figure S5 and S6.

Article Snippet: GMPCPP-microtubule seeds for assays with dynamic microtubules were prepared as above but with 5% biotinylated (Cytoskeleton) and 5% HyLite-647-labeled tubulin (Cytoskeleton).

Techniques:

Journal: Current biology : CB

Article Title: Kinesin-3 responds to local microtubule dynamics to target synaptic cargo delivery to the presynapse

doi: 10.1016/j.cub.2018.11.065

Figure Lengend Snippet: A) Binding and B) quantification of KIF1A, KIF5B, and KIF5C to GMPCPP- and GDP-taxol-stabilized microtubules in the presence of AMP-PNP (n=1004-5855 microtubules per condition; n.s., non-significant, ***p<0.0001; one-way ANOVA with Sidak’s post-hoc test). C) Binding and D) quantification of KIF1A binding to GMPCPP- and GDP-taxol-stabilized microtubules under increasing ionic strength. Circles show mean intensity values; the 95% confidence intervals are too narrow and not visible in the graph. (n=398-2491 microtubules). E) Binding and F) quantification of KIF1A binding to fully tyrosinated and detyrosinated GMPCPP-stabilized microtubules G) under increasing ionic strength. Circles show mean intensity values; the 95% confidence intervals are too narrow and not visible in the graph. (n=2709-6737 microtubules). H) Binding and I) quantification of KIF1A binding to GMPCPP-stabilized microtubules in the presence or absence of EB3 (n=13569-14309 microtubules). J) Representative kymographs and stills depictingKIF1A motors rapidly detaching from a microtubule at the EB1 comet region (asterisks). The stills on the right show one of these events (each time point integrates a 150 msec interval).

Article Snippet: GMPCPP-microtubule seeds for assays with dynamic microtubules were prepared as above but with 5% biotinylated (Cytoskeleton) and 5% HyLite-647-labeled tubulin (Cytoskeleton).

Techniques: Binding Assay

Journal: Current biology : CB

Article Title: Kinesin-3 responds to local microtubule dynamics to target synaptic cargo delivery to the presynapse

doi: 10.1016/j.cub.2018.11.065

Figure Lengend Snippet: A) KIF1A motor domain. The T258M mutation affects a residue located in KIF1A motor domain loop 11. B) Panels and quantification showing KIF1A-WT and -T258M(1-393)-HaloTag binding to GMPCPP- and GDP-taxol-stabilized microtubules. (n=2303-2351 microtubles per group; average±95%CI; **p<0.01,****p<0.0001; Kruskal-Wallis with Dunn’s post-hoc test). C) same as Figure 3A-B and D) same as Figure 3E but for KIF1A-T258M(1-393)-HaloTag, which is represented in orange. E) eCDF showing the location of KIF1A-WT (red) and KIF1A-T258M (orange) run initiation, respective to the plus-end tip. X-intercepts represent the location on the microtubule where run initiations starts to follow a random pattern. (KIF1A-WT, n=1035 runs from 6 microtubules; KIF1A-T258M, n=1628 runs from 5 microtubules). F) Correlation between GDP-taxol/GMPCPP microtubules binding ratio and distance of run initiation to the plus-end tip. (KIF5C: n=1755 runs from 4 microtubules; KIF1A-WT: n=1035 runs from 6 microtubules; KIF1A-A255V: n=702 runs from 4 microtubules; KIF1A-T258M: n=1628 runs from 5 microtubules; KIF1A-R350G: n=596 runs from 4 microtubules; GDP/GMPCPP MT binding ratio – KIF5C-WT: n=5855/4000; KIF1A-WT: n=2491/2303; KIF1A-A255V: n=2130/1046; KIF1A-T258M: n=2351/2351; KIF1A-R350G: n=2106/1162). G) KIF1A-WT rapidly detaches from microtubules once it reaches the microtubule plus-end, whereas KIF1A-T258M frequently lingers at the microtubule plus-end before detaching. See also Figure S5, S6, and Table S1.

Article Snippet: GMPCPP-microtubule seeds for assays with dynamic microtubules were prepared as above but with 5% biotinylated (Cytoskeleton) and 5% HyLite-647-labeled tubulin (Cytoskeleton).

Techniques: Mutagenesis, Binding Assay