pqcxip retroviral vector encoding egfp lamin a c s22a  (Addgene inc)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cytomegalovirus disrupts Lamin A/C to control microtubule-mediated nuclear movement and cell migration

doi: 10.1073/pnas.2507831122

Figure Lengend Snippet: The effects of Lamin A/C mutants and SUN2 expression on actin filaments in HCMV-infected cells. ( A – C ) NHDFs stably expressing eGFP-tagged Lamin A/C variants; WT, nonphosphorylatable alanine mutant (S22A), or head and tail deletion mutant (ΔHC) were infected with TB40/E-UL99-mCherry at MOI 5 for 6 d. Cells were fixed in methanol (which quenches fluorescent proteins) and stained with antibodies against actin (green), gB (red), and GFP (turquoise), while DNA was stained with Hoechst (blue). ( A ) Images are representative of multiple fields of view derived from at least three independent experiments. ( B ) Enlarged examples of merged images from A which illustrate the partial restoration of actin filaments in some infected cells expressing the Lamin A/C S22A mutant; white arrows show examples of clear filaments while red arrows show examples of weak to no filament restoration. ( C ) Quantification of actin caps. n = 150 cells from three independent replicate experiments (wt: 1/50, 2/50, 1/50; S22A: 25/50, 20/50, 22/50, ΔHC: 0/50, 0/50, 1/50). Bars represent mean ± SEM; ns = not significant, **** P < 0.00001, one-way ANOVA test. ( D ) Lamin A/C expression does not restore SUN2 expression in infected cells. Lamin A/C-expressing cells were infected as described in A – C and then fixed and stained with antibodies against SUN2 (green), gB (red), and GFP (turquoise), while DNA was stained with Hoechst (blue). Representative images are shown from three independent replicate experiments. Note that methanol fixation was required for SUN2 imaging and incomplete quenching results in residual detection of concentrated GFP signal with the Lamin A/C-ΔHC variant, but its pattern is distinct and does not interfere with SUN2 detection. ( E and F ) SUN2 expression does not restore actin caps in infected cells. Control NHDFs or NHDFs expressing flag-tagged SUN2 were infected at MOI 5 for 6d then fixed and stained with antibodies against actin (green), gB (red), and Flag (turquoise), while DNA was stained with Hoechst (blue). ( E ) Representative images of uninfected or infected cells. ( F ) Quantification of actin caps in control or SUN2-expressing NHDFs infected with HCMV from three independent replicate experiments (control NHDFs: Mock; 55/35, 50/50, 44/44, or HCMV infected; 0/36, 0/44, 0/40; SUN2-expressing NHDFs: Mock; 47/47, 55/55, 48/48, or HCMV infected; 0/52, 0/47, 0/50). Bars represent mean ± SEM; **** P < 0.0001, two-way ANOVA test.

Article Snippet: Similarly, the pQCXIP retroviral vector encoding eGFP-Lamin A/C S22A was generated by amplifying the S22A variant from the pLPC-S22A-LaminA plasmid (Addgene #69066) using the same primers.

Techniques: Expressing, Infection, Stable Transfection, Mutagenesis, Staining, Derivative Assay, Imaging, Variant Assay, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cytomegalovirus disrupts Lamin A/C to control microtubule-mediated nuclear movement and cell migration

doi: 10.1073/pnas.2507831122

Figure Lengend Snippet: Lamin A/C phosphorylation and inactivation is required for HCMV-induced cell migration. NHDFs stably expressing eGFP control or eGFP-tagged Lamin A/C variants; WT, nonphosphorylatable alanine mutant (S22A), or head and tail deletion mutant (ΔHC) were infected with TB40/E-UL99-mCherry at MOI 2. ( A – C ) Time lapse imaging was performed by acquiring 1 frame per 30 min starting at 120 h.p.i. ( A and B ) Representative stills from Movies S3 and S4 are shown, with the nucleus of infected cells outlined. ( C ) Graph plots the percentage of cells wherein the displacement of the center of the nucleus was above 30 µM. n = 291 cells total from three independent biological replicates, * P < 0.05, ** P < 0.005, *** P < 0.0005, one-way ANOVA test. ( D – F ) Migration of infected cells in trans-well assays. ( D ) Schematic of trans-well assay illustrating cells expressing eGFP-Lamin constructs that accumulate in the nucleus, which is costained with Hoechst after fixation at the end-point of the assay. Cells were infected with HCMV TB40/E UL99-mCherry (MOI = 2) and transferred at 5 d.p.i. to the top of trans-well inserts with 12 µM pores. Migrating cells must pass through these pores to reach the bottom of the inserts. Samples were fixed with 4% PFA at 7 d.p.i., inserts were removed and the top (seeded cells) and bottom (migrated cells) were imaged after staining with Hoechst. ( D ) Representative images of the top and bottom of inserts. An example of a migrated cell at the bottom (white arrow) compared with those that have not migrated from the top is shown in the WT cells. Pores are visible in images of the bottom of wells (orange arrow). In the case of Lamin A/C mutants, out of focus light is visible from cells still at the top of trans-wells (blue arrows). ( E ) The number of infected cells that migrated to the other side of the transwell membrane was counted and WT numbers were set to 100% as the basis for comparisons with effects of Lamin A/C mutants. Total number of migrated cells n = 1,155 for WT, n = 296 for S22A n = 173 for ΔHC from four independent biological replicates, ns = not significant, ** P < 0.005, **** P < 0.00001, one-way ANOVA test.

Article Snippet: Similarly, the pQCXIP retroviral vector encoding eGFP-Lamin A/C S22A was generated by amplifying the S22A variant from the pLPC-S22A-LaminA plasmid (Addgene #69066) using the same primers.

Techniques: Phospho-proteomics, Migration, Stable Transfection, Expressing, Control, Mutagenesis, Infection, Imaging, Construct, Staining, Membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cytomegalovirus disrupts Lamin A/C to control microtubule-mediated nuclear movement and cell migration

doi: 10.1073/pnas.2507831122

Figure Lengend Snippet: Lamin A/C phosphorylation is required for HCMV-induced formation of acetylated microtubule networks. ( A – C ) NHDFs were mock infected or infected with TB40/E-UL99-mCherry at MOI 5 and then treated with DMSO solvent control or 10 µM maribavir (MBV) for 6 d. Samples were fixed and stained with antibodies against acetylated tubulin (green) and gB (red), while DNA was stained with Hoechst (blue). ( A and B ) Representative fields of view derived from at least three independent experiments illustrate the induction of acetylated microtubules during infection along with the organization, emanating from the AC and extending through the cytoplasm, along with the inhibitory effects of MBV treatment. ( C ) Graph plots the percentage of cells with robust microtubule acetylation, bars represent mean ± SEM; n = 198 cells total from three independent experiments, **** P < 0.0001, two-tailed Student’s t test. ( D – F ) NHDFs stably expressing eGFP control or eGFP-tagged Lamin A/C variants; WT, nonphosphorylatable alanine mutant (S22A), or head and tail deletion mutant (ΔHC) were infected with TB40/E-UL99-mCherry at MOI 5 for 6 d. Samples were fixed and stained with antibodies against acetylated tubulin (green), gB (red), and GFP (turquoise), while DNA was stained with Hoechst (blue). ( D and E ) Representative fields of view derived from four independent experiments and associated zoomed Insets in E . illustrate the lack (red arrows) or severe reduction (orange arrows) in the levels of acetylated microtubule networks emanating from the AC into the cytoplasm in infected cells expressing Lamin A/C mutants. Note that methanol fixation was required for microtubule imaging and incomplete quenching results in residual detection concentrated GFP signal with the Lamin A/C-ΔHC variant. ( F ) Graph plots the percentage of cells with robust microtubule acetylation derived from four independent experiments; WT cells; 22/30, 24/30, 21/30, 20/30; S22A cells; 8/30, 6/30, 9/30, 4/30; ΔHC cells; 0/30, 1/30, 2/30, 3/30. Bars represent mean ± SEM; ** P < 0.01, **** P < 0.0001, two-tailed Student’s t test.

Article Snippet: Similarly, the pQCXIP retroviral vector encoding eGFP-Lamin A/C S22A was generated by amplifying the S22A variant from the pLPC-S22A-LaminA plasmid (Addgene #69066) using the same primers.

Techniques: Phospho-proteomics, Infection, Solvent, Control, Staining, Derivative Assay, Two Tailed Test, Stable Transfection, Expressing, Mutagenesis, Imaging, Variant Assay