Localization of the Drosophila Rad9 Protein to the Nuclear Membrane Is Regulated by the C-Terminal Region and Is Affected in the Meiotic Checkpoint

Rad9, Rad1, and Hus1 (9-1-1) are part of the DNA integrity checkpoint control system. It was shown previously that the C-terminal end of the human Rad9 protein, which contains a nuclear localization sequence (NLS) nearby, is critical for the nuclear transport of Rad1 and Hus1. In this study, we show that in Drosophila, Hus1 is found in the cytoplasm, Rad1 is found throughout the entire cell and that Rad9 (DmRad9) is a nuclear protein. More specifically, DmRad9 exists in two alternatively spliced forms, DmRad9A and DmRad9B, where DmRad9B is localized at the cell nucleus, and DmRad9A is found on the nuclear membrane both in Drosophila tissues and also when expressed in mammalian cells. Whereas both alternatively spliced forms of DmRad9 contain a common NLS near the C terminus, the 32 C-terminal residues of DmRad9A, specific to this alternative splice form, are required for targeting the protein to the nuclear membrane. We further show that activation of a meiotic checkpoint by a DNA repair gene defect but not defects in the anchoring of meiotic chromosomes to the oocyte nuclear envelope upon ectopic expression of non-phosphorylatable Barrier to Autointegration Factor (BAF) dramatically affects DmRad9A localization. Thus, by studying the localization pattern of DmRad9, our study reveals that the DmRad9A C-terminal region targets the protein to the nuclear membrane, where it might play a role in response to the activation of the meiotic checkpoint

Localization of the <i>Drosophila</i> Rad9, Hus1 and Rad1 proteins in S₂R+ and follicle cells.

<p>A–G, Confocal images of S₂R+ cells, I–K, Confocal images of follicle cells from egg chambers. (A) S₂R+ cells expressi ng HA-DmHus1 and stained with anti-HA antibodies in red. (B) S₂R+ cells expressing GFP-DmRad1. (C) S₂R+ cells expressing GFP-DmRad9A. (F) S₂R+ cells expressing DmRad9B-GFP. (D) and (G) Staining with anti-lamin antibodies, which mark the nuclear membrane, in red. (E and H) are merged image of (C with differential interference contrast (DIC) image) and (F with a DIC image), respectively. (I) Egg chamber from HA-DmHus1::<i>CY2Gal4</i> transgenic flies. (J) Egg chamber from GFP-DmRad1::<i>CY2Gal4</i> transgenic flies. (K) Egg chamber from FLAG-DmRad9A::<i>CY2Gal4</i> transgenic flies. In both S₂R+ and follicle cells, DmHus1 is found in the cytoplasm, DmRad1 is found throughout the cell and Dm DmRad9A is localized to the nuclear membrane. DmRad9B is localized to the nucleus in S₂R+ cells.</p

Effects of meiotic checkpoint activation on DmRad9A oocyte nuclear membrane localization.

<p>Confocal images of stage 7 egg chambers. (A, E, I and M) are stained for DNA (blue, arrows mark oocyte nucleus DNA, karyosome); arrows mark the oocyte nucleus (karyosome). (B, F, J and N) GFP-DmRad9A (green). (C, G, K and O) are stained with anti-lamin antibodies, which mark the nuclear membrane, in red. (Inset in H, L and P), represents a schematic description of the oocyte nucleus. Red-lamin, green-GFP-DmRad9A and blue-karyosome. (A–H) GFP-DmRad9A:: <i>nosGal 4-VP16</i> egg chamber, E–H are enlargement of the oocyte region from A–D, respectively. (I-L) BAF3A:: GFP-DmRad9A:: <i>nosGal 4-VP16</i> egg chamber. M–P, GFP-DmRad9A:: <i>nosGal 4-VP16; okr^AA</i>/<i>okr^RU</i>.</p

Physical interaction between DmRad9, DmRad1 and DmHus1.

<p>DmRad9 was co-expressed in S2 cells with DmRad1 and DmHus1. A total lysate of S2 cells was extracted and subjected to immunoprecipitation. (A) DmRad1 was immunoprecipitated using anti-GFP antibodies. Anti-HA antibodies were used to detect DmHus1. (B) The same blot as in (A) was probed for FLAG-DmRad9 using anti-FLAG antibodies. (C–F) Confocal images of S₂R+ cells expressing FLAG-DmRad9, GFP-DmRad1 and HA-DmHus1. (G–J) Confocal images of follicle cells from transgenic FLAG-DmRad9::HA-DmHus1::GFP-DmRad1::<i>CY2Gal4</i> flies expressing egg chamber. (C) Staining with anti-FLAG antibodies detecting Flag-DmRad9. (D) Staining with anti-HA antibodies detecting HA-DmHus1. (E) GFP-DmRad1. (F) Merged (C–E). (G) Staining with anti-FLAG antibodies detecting Flag-DmRad9. (H) Staining with anti-HA antibodies detecting HA-DmHus1. (I) GFP-DmRad1. (J) merged G–I. Total protein served as positive control while a sample treated with protein A alone (no beads) served as negative control.</p

Localization of the <i>Drosophila</i> Rad9, Hus1 and Rad1 proteins in mammalian

<p>Human Embryonic Kidney 293 <b>(HEK293).</b> Confocal images of cells expressing (A) GFP-DmHus1, (D) GFP-DmRad1, and (G) GFP-DmRad9A. (B, E and H) Antibody staining of the NUP 414 protein, which recognizes several nucleoporins. (C, F and I) are merged images of (A–B), (D–E), and (G–H), respectively.</p

Identification of the DmRad9A nuclear localization signal.

<p>Confocal images of S₂R+ cells expressing DmRad9A mutated in suspected NLS sequences. (A) DmRad9A mutated at position 287 – 289 (NLS1). (D) DmRad9A mutated Position 300–302 (NLS2). (G) DmRad9A mutated Position 314–316 (NLS3). (B, E and H) stained with anti-lamin antibodies, which mark the nuclear membrane, in red. (C) Merged image of (A) and (B). (F) Merged image of (D) and (E). (I) Merged image of (G) and (H).</p

Effect of interleukin-1 antagonist on growth of children with colchicine resistant or intolerant FMF

Abstract Introduction Familial Mediterranean Fever (FMF) is the most common monogentic autoinflammatory disease. FMF results from mutations in MEFV, which lead to a pro-inflammatory state and increased production of Interleukin 1 beta subunit (IL-1b) by myeloid cells. Despite the overall positive results obtained with anti-IL-1 agents in FMF patients, little is known about the long-term growth impact of these drugs in the pediatric population. Objectives To assess the long-term body weight and height trajectories in children with FMF treated with anti-IL-1 agents. Methods We conducted a retrospective analysis of 646 pediatric FMF patients followed in our center, of whom 22 were treated with either anakinra (36.3%) and/or canakinumab (90.9%). Patients were assessed for demographic, clinical and genetic characteristics and were followed for a mean of 3.05 ± 1.75 years. Data of height and weight percentiles were recorded before and after treatment. Results The most common indication for IL-1 blockers treatment was colchicine resistance (66.6%). Ninety percent of those patients had a moderate or severe disease according to the Pras score and had higher proportion of M694V homozygosity compared with patients who did not require anti IL-1 agents (95.2% vs. 30.5%, p < 0.001). Overall, anakinra and canakinumab resulted in a complete response in 80% of patients and exhibited low rates of adverse effects. We found a significant increase in height and body weight percentiles following treatment (19.6 ± 16% vs. 30.8 ± 23%, p = 0.007, and 29.5 ± 30% vs. 39.1 ± 36%, p = 0.043, respectively). Conclusion Treatment with anti-IL-1 agents in children with FMF is effective and safe and may potentiate long-term growth

ALFY-Controlled DVL3 Autophagy Regulates Wnt Signaling, Determining Human Brain Size.

Primary microcephaly is a congenital neurodevelopmental disorder of reduced head circumference and brain volume, with fewer neurons in the cortex of the developing brain due to premature transition between symmetrical and asymmetrical cellular division of the neuronal stem cell layer during neurogenesis. We now show through linkage analysis and whole exome sequencing, that a dominant mutation in ALFY, encoding an autophagy scaffold protein, causes human primary microcephaly. We demonstrate the dominant effect of the mutation in drosophila: transgenic flies harboring the human mutant allele display small brain volume, recapitulating the disease phenotype. Moreover, eye-specific expression of human mutant ALFY causes rough eye phenotype. In molecular terms, we demonstrate that normally ALFY attenuates the canonical Wnt signaling pathway via autophagy-dependent removal specifically of aggregates of DVL3 and not of Dvl1 or Dvl2. Thus, autophagic attenuation of Wnt signaling through removal of Dvl3 aggregates by ALFY acts in determining human brain size

Case Series of Myocarditis Following mRNA COVID Vaccine Compared to Pediatric Multisystem Inflammatory Syndrome: Multicenter Retrospective Study

Introduction: Since the development of COVID-19 vaccines, more than 4.8 billion people have been immunized worldwide. Soon after vaccinations were initiated, reports on cases of myocarditis following the second vaccine dose emerged. This study aimed to report our experience with adolescent and young adults who developed post-COVID-19 vaccine myocarditis and to compare these patients to a cohort of patients who acquired pediatric inflammatory multisystem syndrome (PIMS/PIMS-TS) post-COVID-19 infection. Methods: We collected reported cases of patients who developed myocarditis following COVID-19 vaccination (Pfizer mRNA BNT162b2) from all pediatric rheumatology centers in Israel and compared them to a cohort of patients with PIMS. Results: Nine patients with post-vaccination myocarditis were identified and compared to 78 patients diagnosed with PIMS. All patients with post-vaccination myocarditis were males who developed symptoms following their second dose of the vaccine. Patients with post-vaccination myocarditis had a shorter duration of stay in the hospital (mean 4.4 &plusmn; 1.9 vs. 8.7 &plusmn; 4.7 days) and less myocardial dysfunction (11.1% vs. 61.5%), and all had excellent outcomes as compared to the chronic changes among 9.2% of the patients with PIMS. Conclusion: The clinical course of vaccine-associated myocarditis appears favorable, with resolution of the symptoms in all the patients in our cohort