Journal: bioRxiv
Article Title: Independent evolution of holocentric centromeres in an early branching apicomplexan parasite
doi: 10.1101/2025.09.30.679541
Figure Lengend Snippet: (A) Diagram of the targeting construct designed to add a 3HA tag and Nluc-P2A-Neo R cassette to the C-terminus of the C. parvum CENH3 ( cgd4_2030 ). (B) HCT-8 cells were infected with CENH3-3HA C. parvum parasites, fixed at 22 hpi, and stained with rat anti-HA and VVL-Biotin, followed by secondary antibodies Alexa Fluor 488 goat anti-rat IgG and Alexa Fluor 647 Streptavidin followed by Hoechst staining. Images were acquired as Z-stacks using LSCM-A and are presented with orthogonal views. Scale bars, 1 μm. (C) Cartoon depiction corresponding to the microscopy image in panel (B). In Cryptosporidium , a late-stage meront contains eight nuclei that have recently completed cytokinesis to form mature merozoites. The CENH3-3HA signal within each nucleus displays a diffuse staining pattern in C. parvum . (D) HCT-8 cells were infected with CENH3-3HA T. gondii parasites, fixed at 24 hours post-infection (hpi), and stained with rat anti-HA and rabbit anti-aldolase (ALD) to visualize the cytosol, followed by secondary antibodies Alexa Fluor 488 goat anti-rat IgG and Alexa Fluor 568 anti-rabbit IgG. Hoechst was used for nuclear staining. Images were acquired as Z-stacks using LSCM-A and are presented with orthogonal views. Scale bars, 1 μm. (E) Cartoon depiction corresponding to the microscopy image in panel (D). In T. gondii tachyzoites, the CENH3-3HA signal within each nucleus displays a discrete staining pattern. (F) HCT-8 cells were infected with excysted C. parvum sporozoites for 2 h, then washed twice to remove extracellular parasites. Cultures were fixed at 30 min increments between 6-9 hpi to identify the first (trophozoites with one nucleus), second (early meronts with 2 nuclei), and third (mid-stage meronts with 4 nuclei) mitotic divisions during the first round of merogony by widefield microscopy. Scale bars, 1 μm. (G) Cultures were infected with CENH3-3HA C. parvum sporozoites for 2 h, washed, and fixed at 6.5 hpi to capture the first round of mitosis. Cultures were stained with rat anti-HA, rabbit anti-centrin-1, and VVL-Biotin, followed by secondary antibodies Alexa Fluor 488 goat anti-rat IgG, Alexa Fluor 568 goat anti-mouse IgG, and Alexa Fluor 647 Streptavidin, and lastly Hoechst. Parasites undergoing mitosis were identified by having two centrin-1 points per nucleus, whereas parasites in interphase had only one centrin-1 point. Images were acquired as Z-stacks using LSCM-A and are presented with orthogonal views. Scale bars, 1 μm.
Article Snippet: 2) The 3HA-pLinker region was amplified from a TK-3HA-CENH3-Nluc-P2A-Neo R -TK plasmid (data not shown in this study), 3) the GBP CDS was amplified from C. parvum genomic DNA, 4) the pLinker-3Ty sequence was amplified from a tagging plasmid previously generated in our lab [ ], and 5) the P2A skip peptide sequence was generated as a gBlock Gene Fragment from Azenta.
Techniques: Construct, Infection, Staining, Microscopy
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![BaDV-2 IGR IRES activity in infectious clone and in cells. ( A ) Bicistronic reporters were transfected into S2 cells followed by mock infection or CrPV infection (MOI = 20). Cells were collected 6 h post transfection. Bicistronic RNAs tested contain the wild-type CrPV, mutant CrPV (CC6214-5 to GG to disrupt PKI base-pairing), wild-type BaDV-2 full-length IGR IRES (WT BaDV-2), BaDV-2 3′ DEL 5, and BaDV “minimal” IRES (double deletion at both 3′ and 5′ end. Luciferase activities were measured and normalized to wild-type CrPV with mock infection. Red and black dots represent data points from three independent experiments. A paired t -test was used to determine the p value and thus the significance levels. “n.s.” denotes the difference is not significant between the control groups and the experimental groups ( p > 0.05). Shown are the averages from at least three independent experiments ± standard deviation. ( B ) Schematic of infectious clone CrPV (CrPV, CrPV_IGR_IRES) and chimeric clones with CrPV IGR IRES replaced with BaDV-2 IGR IRES (BaDV-2 IGR IRES) and with <t>P2A-site</t> added after BaDV-2 IGR IRES (BaDV-2 IGR IRES+ P2A). ( C ) Infectious clone RNAs were incubated in Sf-21 extracts containing [35S]-methionine/cysteine and then monitored by SDS-PAGE analysis. Mock = mock transfection; OS = ORF1Stop; CrPV = pCrPV; BaDV = BaDV-2 IGR IRES; P2A = BaDV-2 IGR IRES + P2A. Shown is a representative gel from at least three independent experiments. ( D ) In vitro transcribed infectious clone RNAs were transfected into S2 cells for 144 h. VP2 expression was detected by immunoblotting. Shown are a representative SDS PAGE gel and the averages from at least three independent experiments ± standard deviation.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5867/pmc11125867/pmc11125867__viruses-16-00695-g006.jpg)
