Expression and Subcellular Localization of Mammalian Formin Fhod3 in the Embryonic and Adult Heart

The formin family proteins play pivotal roles in actin filament assembly via the FH2 domain. The mammalian formin Fhod3 is highly expressed in the heart, and its mRNA in the adult heart contains exons 11, 12, and 25, which are absent from non-muscle Fhod3 isoforms. In cultured neonatal cardiomyocytes, Fhod3 localizes to the middle of the sarcomere and appears to function in its organization, although it is suggested that Fhod3 localizes differently in the adult heart. Here we show, using immunohistochemical analysis with three different antibodies, each recognizing distinct regions of Fhod3, that Fhod3 localizes as two closely spaced bands in middle of the sarcomere in both embryonic and adult hearts. The bands are adjacent to the M-line that crosslinks thick myosin filaments at the center of a sarcomere but distant from the Z-line that forms the boundary of the sarcomere, which localization is the same as that observed in cultured cardiomyocytes. Detailed immunohistochemical and immuno-electron microscopic analyses reveal that Fhod3 localizes not at the pointed ends of thin actin filaments but to a more peripheral zone, where thin filaments overlap with thick myosin filaments. We also demonstrate that the embryonic heart of mice specifically expresses the Fhod3 mRNA isoform harboring the three alternative exons, and that the characteristic localization of Fhod3 in the sarcomere does not require a region encoded by exon 25, in contrast to an essential role of exons 11 and 12. Furthermore, the exon 25-encoded region appears to be dispensable for actin-organizing activities both in vivo and in vitro, albeit it is inserted in the catalytic FH2 domain

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

Effect of the T(D/E)₅XE region of Fhod3 on actin assembly.

<p>(A) Effect of the Fhod3 T(D/E)₅XE region encoded by exon 25 on <i>in vivo</i> actin assembly. HeLa cells were transfected with plasmids encoding Fhod3-ΔN (wt) (amino acids 931–1,586) with or without the T(D/E)₅XE region in the FH2 domain; or plasmids encoding Fhod3-ΔN (I1127A) with or without the T(D/E)₅XE region. Cells were fixed and visualized by GFP-fluorescence (green) or phalloidin staining (red). Scale bar, 10 µm. (B) SDS-PAGE analysis of purified proteins used in an actin polymerization assay. Purified proteins were subjected to 10% SDS-PAGE and stained with <i>Coomassie Brilliant Blue</i>. Positions for marker proteins are indicated in kDa. (C) Effect of the T(D/E)₅XE region of Fhod3 on <i>in vitro</i> actin assembly. G-actin (10% pyrene-labeled) at 2 µM was incubated with 50 nM Fhod3-T(D/E)₅XE(+)-ΔN (wt), Fhod3-T(D/E)₅XE(+)-ΔN (I1127A), Fhod3-T(D/E)₅XE(−)-ΔN (wt), or mDia1-FH1FH2 (amino acids 549–1,175) in the presence of 2 µM profilin I.</p

Localization of Fhod3 in the embryonic heart.

<p>(A) Embryonic mouse cardiomyocytes were subjected to immunofluorescent double staining for endogenous Fhod3 (green) and α-actinin (red). For Fhod3 staining, the anti-Fhod3-(650–802) polyclonal antibodies were used. Bar, 5 µm. (B) Magnified image of single myofibril from immunostained embryonic mouse cardiomyocytes. (C) Sections of mouse embryonic hearts were subjected to immunofluorescent double staining for endogenous Fhod3 (red) and α-actinin (green). For Fhod3 staining, the anti-Fhod3-(C-20) (top panels), the anti-Fhod3-(650–802) (middle panels), and the anti-Fhod3-(873–974) (bottom panels) polyclonal antibodies were used. Bar, 2 µm.</p

Localization of Fhod3 in the adult heart.

<p>(A) Glycerinated fibers from adult mouse hearts were subjected to immunofluorescent double staining for endogenous Fhod3 (red) and α-actinin (green). For Fhod3 staining, the anti-Fhod3-(650–802) polyclonal antibodies were used. Bar, 5 µm. (B) Sections of adult mouse hearts were subjected to immunofluorescent double staining for endogenous Fhod3 (red) and α-actinin (green) followed by phalloidin staining (not shown in merge). For Fhod3 staining, the anti-Fhod3-(650–802), the anti-Fhod3-(873–974), and the anti-Fhod3-(C-20) polyclonal antibodies were used. The anti-Fhod3-(C-20) antibodies used here were pre-adsorbed with an acetone powder of mouse embryonic fibroblast derived from Fhod3 knockout mice. For details, see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034765#s4" target="_blank">Materials and Methods</a>”. Bar, 2 µm. (C) Sections of tissues from a left ventricle of a human heart were subjected to immunofluorescent double staining for endogenous Fhod3 (red) and α-actinin (green). For Fhod3 staining, the anti-Fhod3-(650–802) polyclonal antibodies were used. Bar, 2 µm. (D) Sections of tissues from a left ventricle of two patients (#1 and #2) with idiopathic dilated cardiomyopathy (DCM) and a patients with idiopathic hypertrophic cardiomyopathy (HCM) were subjected to immunofluorescent double staining for endogenous Fhod3 (red) and α-actinin (green). For Fhod3 staining, the anti-Fhod3-(650–802) polyclonal antibodies were used. Bar, 2 µm.</p

Localization of Fhod3 in the heart of Fhod3 transgenic mice.

<p>(A) Sections of adult hearts from transgenic mice that specifically express a high amount of the T(D/E)₅XE-region-deficient Fhod3 protein in the heart were subjected to immunofluorescent double staining for Fhod3 (red) and α-actinin (green), followed by phalloidin staining. For Fhod3 staining, the anti-Fhod3-(C-20) (top panels), the anti-Fhod3-(650–802) (middle panels), or the anti-Fhod3-(873–974) (bottom panels) polyclonal antibodies were used. Bar, 2 µm. (B) Sections of adult hearts from Fhod3 transgenic mice were subjected to immunofluorescent double staining for Fhod3 (red) and myomesin (green). For Fhod3 staining, the anti-Fhod3-(650–802) polyclonal antibodies were used. Bar, 2 µm. (C) Magnified image of single myofibril from immunostained sections of adult hearts of Fhod3 transgenic mice. Sections were stained for α-actinin (green), phalloidin (blue), and Fhod3 or Tmod1 (red). For Fhod3 staining, the anti-Fhod3-(650–802) (top panels), the anti-Fhod3-(873–974) (2nd panels), and the anti-Fhod3-(C-20) (3rd panels) polyclonal antibodies were used. Bar, 2 µm. (D) Fluorescence intensity profiles in a line scan of sarcomeres. Line scan profiles of fluorescence intensities for the anti-α-actinin antibody (green) and the anti-Fhod3-(C-20) or anti-Tmod antibodies (red) are generated from immunofluorescent images shown in <i>C</i>. (E) Distance between the Z-line and Fhod3 and that between the Z-line and Tmod. The distance of the fluorescence peak of Fhod3 or Tmod from that of α-actinin are measured on immunofluorescent images. Box-and-whisker plots indicate 25th percentile (bottom line), median (middle line), 75th percentile (top line), and nearest observations within 1.5 times the interquartile range (whiskers). *, <i>P</i><0.001, Welch's <i>t</i> test. (F) Magnified image of single myofibril from immunostained sections of adult hearts of Fhod3 transgenic mice. Sections were stained for Fhod3 (red), Tmod1 (green), and phalloidin (blue). For Fhod3 staining, the anti-Fhod3-(650–802) polyclonal antibodies were used. (G) Ultrastructural localization of Fhod3. Ultrathin cryosections of adult hearts from Fhod3 transgenic mice were immunolabeled using the anti-Fhod3-(650–802) antibodies, and labeling was detected using gold-conjugated secondary antibodies.</p

Expression of alternatively spliced variants of Fhod3.

<p>The RT-PCR fragments amplified using specific primers flanking exon 25 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034765#pone-0034765-g001" target="_blank">Figure 1B</a>) are subcloned and subjected to sequencing analysis. The number in parenthesis indicates the percentage of each variant in the indicated tissue.</p>*<p>N.D. not determined.</p

Expression of Fhod3 isoforms in the embryonic tissues.

<p>(A) Exon structure of the moue Fhod3 gene. Alternative splicing exons are indicated by gray boxes. (B) Schematic presentation of domain structure of mouse Fhod3. The alternative splicing regions are in black boxes, and primers for RT-PCR analysis are indicated by arrows. (C) Specific primers designed for isoforms derived from the alternative splicing in the C-terminal FH2 domain. The primers ‘S6’ and ‘S7’ are specific for Fhod3 mRNAs with and without exon 25 that encodes the T(D/E)₅XE region (boxed in black), respectively. The primer ‘S8’ is common for both variants. (D) Tissue-specific expression of the “T(D/E)₅XE” exon in mouse embryos. The RT-PCR products using specific primers (shown in B and C) were subjected to agarose-gel electrophoresis. sk. muscle, skeletal muscle. (E) Tissue-specific expression of splicing variants lacking exons 11 and 12. The RT-PCR products (shown in B) were subjected to agarose-gel electrophoresis.</p