Journal: Nature Communications

Article Title: Repurposing the mitotic machinery to drive cellular elongation and chromatin reorganisation in Plasmodium falciparum gametocytes

doi: 10.1038/s41467-022-32579-4

Figure Lengend Snippet: a Live-cell fluorescence imaging of stage II-V gametocytes transfected with the nuclear localisation signal (NLS)-mCherry reporter. NLS-mCherry (NLS-mCh, red) labels the elongated nucleus, with pointed extensions (yellow arrowheads). Subpellicular (green arrowheads) and nuclear (cyan arrowheads) populations of microtubules are labelled with Tubulin Tracker (TT, cyan). The nucleus contracts to a more spherical morphology and the nuclear and sub-pellicular microtubule populations are disassembled in stage V gametocytes. The chromatin (Hoechst, grayscale) is present as punctate features in the nucleus (white arrowheads). b Live-cell fluorescence imaging of stage II/III gametocytes in the Pf EB1-GFP/NLS-mCherry co-transfectant parasite line. NLS-mCherry (NLS-mCh, red) delineates the nucleus; and Pf EB1-GFP (EB1-GFP, green) is in the same compartment. Chromatin (Hoechst, grayscale) is distributed along the nuclear microtubules. (See Tubulin Tracker (TT, cyan) overlap with Pf EB1-GFP, cyan arrows). Additional images are presented in Supplementary Fig. . c Quantitative analysis of the contour lengths for the Pf EB1-GFP (EB1) and nuclear microtubules (MT) labelled with Tubulin Tracker (mean ± SD is shown; n = 20 cells per stage). Differences in the length of nuclear microtubule features were evaluated by two-way ANOVA Tukey’s test, **** p < 0.0001. The blue dashed line indicates the limit of resolution (250 nm) of the microscope. d Images showing that the nuclear microtubules (as marked by anti-GFP ( Pf EB1-GFP), green) are depolymerised and the subpellicular microtubules (white arrowheads) are modified with polyglutamate (polyE, red) in stage IV gametocytes. Anti-β-tubulin (anti-β-tub, magenta) labels both nuclear and subpellicular microtubules. The chromatin was labelled with DAPI (grayscale). e Quantitative analysis of anti-polyE fluorescence intensity at different stages of development (mean ± SD is shown; n = 15 cells). Differences in polyE fluorescence signal were determined using a paired one-way ANOVA Tukey’s, I vs. II and II vs. IIIa: NS > 0.9999; IIIa vs. IIIb, IIIb vs. IV and IV vs. V: **** p < 0.0001. Differential interference contrast (DIC) images are shown. Scale bars: 2 μm. Additional images are presented in Supplementary Fig. . Source data for Fig. c, e is provided in the Source Data file.

Article Snippet: Parasite chromatin was labelled with DAPI (2 μg/mL in PBS, Thermo Scientific) for 15 minutes at room temperature, followed by washes with PBS buffer.

Techniques: Fluorescence, Imaging, Transfection, Cotransfection, Microscopy, Modification

Journal: PLoS ONE

Article Title: Synthetic modified Fezf2 mRNA (modRNA) with concurrent small molecule SIRT1 inhibition enhances refinement of cortical subcerebral/corticospinal neuron identity from mouse embryonic stem cells

doi: 10.1371/journal.pone.0254113

Figure Lengend Snippet: (A) GFP modRNA expression was dose-dependent in mES-derived pallial-like progenitors at 14 days in vitro (DIV). Native GFP expression was detected in 25–50% of cells after 24hrs; the intensity of expression increased over the range of 0, 2, 4 microgram modRNA transfection. (B) GFP modRNA expression was time-dependent over 48hrs. (C) GFP modRNA was injected into the lateral ventricles (coronal cross-section of mouse embryo forebrain is shown) and directionally electroporated into the dorsal forebrain / pallium (positive paddle above dark blue colored tissue). (D) Following in utero electroporation of GFP modRNA (0.8 microgram / microliter), GFP protein (green) is expressed after eight hours, and is restricted to Ki67-expressing progenitors (red) of the pallium, not in more superficially located post-mitotic neurons, which express MAP2 (blue). (E) Sagittal section of an E17.5 Fezf2 -null mouse following E12.5 in utero electroporation of Fezf2 modRNA (0.8 microgram / microliter) and tdTomato plasmid (1 microgram / microliter) showed targeted tdTomato fluorescence appropriately restricted to the rostral pallium. (F) Higher magnification image showing tdTomato expression in the cortical plate. (G) tdTomato+ axons projected across the cerebral commissures, including anterior commissure (depicted) and corpus callosum in (arrowheads in E). (H) Several tdTomato+ axons projected caudal to the thalamus, indicating partial genetic rescue of E12.5 SCPN, which are not present in Fezf2 -null mice and can only be generated by similar in utero rescue experiments . (I) A small subset of these tdTomato+ axons reached the cerebral peduncles (N = 3), confirming partial rescue of SCPN axonal projections.

Article Snippet: Primary antibodies and dilutions used were: rat antibody to CTIP2 (1:500, Abcam); mouse antibody to SATB2 (1:200, Abcam); rabbit antibody to SIRT1 (1:250, Millipore); rabbit antibody to CTIP1 (1:500, Abcam); rabbit antibody to GFP (1:500, Invitrogen); rabbit antibody to Ki67 (1:500, Abcam); chicken antibody to NESTIN (1:500, Novus Biologicals); mouse antibody to TuJ1 (1:500, Covance); and mouse antibody to MAP2 (1:500, Sigma).

Techniques: Expressing, Derivative Assay, In Vitro, Transfection, Injection, In Utero, Electroporation, Plasmid Preparation, Fluorescence, Generated