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