We present a protocol to generate highly multiplexed spatial data at cellular and subcellular resolutions using iterative indirect immunofluorescence imaging (4i). We describe streamlined steps for using 4i across fixed cultured cells, formalin-fixed paraffin-embedded (FFPE) tissue sections, and metaphase chromosome spreads. We detail procedures for sample preparation, antibody and DNA staining, immunofluorescence imaging, antibody elution, and image processing. This protocol is adapted for high-throughput analysis of fixed cultured cells and addresses sample-specific challenges such as intrinsic tissue autofluorescence and chromosome fragility. For complete details on the use and execution of this protocol for fixed cultured cells, please refer to Comandante-Lou et al.1.

During aging and in retinal degenerative diseases, vulnerable retinal pigment epithelial (RPE) cells are subject to mitochondrial dysfunction, creating a need for accessibility to tools which can facilitate assessment of the ocular posterior pole bioenergetics. Here, we present a protocol for quantifying mitochondrial respiration in the posterior eye cup (RPE-choroid-sclera) of young and old mice. We describe steps for eye cup dissection, optimization of tissue size, drug concentrations, and cycle conditions using the XF Cell Mito Stress Test.

SUMMARYCilia and the nucleus were two defining features of the last eukaryotic common ancestor. In early eukaryotic evolution, these structures evolved through the diversification of a common membrane-coating ancestor, the protocoatomer. While in cilia, the descendants of this protein complex evolved into parts of the intraflagellar transport complexes and BBSome, the nucleus gained its selectivity by recruiting protocoatomer-like proteins to the nuclear envelope to form the selective nuclear pore complexes. Recent studies show a growing number of proteins shared between the proteomes of the respective organelles, and it is currently unknown how ciliary transport proteins could acquire nuclear functions and vice versa. The nuclear functions of ciliary proteins are still observable today and remain relevant for the understanding of the disease mechanisms behind ciliopathies. In this work, we review the evolutionary history of cilia and nucleus and their respective defining proteins and integrate current knowledge into theories for early eukaryotic evolution. We postulate a scenario where both compartments co-evolved and that fits current models of eukaryotic evolution, explaining how ciliary proteins and nucleoporins acquired their dual functions.

Asymmetric cell divisions (ACDs) generate two daughter cells with identical genetic information but distinct cell fates through epigenetic mechanisms. However, the process of partitioning different epigenetic information into daughter cells remains unclear. Here, we demonstrate that the nucleosome remodeling and deacetylase (NuRD) complex is asymmetrically segregated into the surviving daughter cell rather than the apoptotic one during ACDs in Caenorhabditis elegans. The absence of NuRD triggers apoptosis via the EGL-1-CED-9-CED-4-CED-3 pathway, while an ectopic gain of NuRD enables apoptotic daughter cells to survive. We identify the vacuolar H+-adenosine triphosphatase (V-ATPase) complex as a crucial regulator of NuRD\'s asymmetric segregation. V-ATPase interacts with NuRD and is asymmetrically segregated into the surviving daughter cell. Inhibition of V-ATPase disrupts cytosolic pH asymmetry and NuRD asymmetry. We suggest that asymmetric segregation of V-ATPase may cause distinct acidification levels in the two daughter cells, enabling asymmetric epigenetic inheritance that specifies their respective life-versus-death fates.

Circular RNA (circRNA) has emerged as potential therapeutic targets for cardiovascular diseases. Given the central role of the TGFβ signaling pathway in cardiac remodeling and its potential as a therapeutic target, we hypothesized that a circRNA from this pathway could modulate cardiac remodeling and serve as a heart failure treatment. Therefore, we identified a circRNA, named circSMAD3, that was significantly reduced in murine heart failure models. Functionally, circSMAD3 mitigated cardiomyocyte hypertrophy and inhibited cardiac fibroblast activation in vitro. Mechanistically, circSMAD3 interacts with YBX1, stabilizing it and facilitating its binding to SMAD3 in the nucleus, disrupting the TGFβ/SMAD3 signaling pathway, and ultimately restoring cardiac remodeling. This study highlights circSMAD3 as a promising therapeutic target for heart failure treatment.

Bromodomain protein BRD4 binds to acetylated histones to regulate transcription. BRD4 also drives cancer cell proliferation. However, the role of BRD4 in normal cell growth has remained unclear. Here, we investigated this question by using mouse embryonic fibroblasts with conditional Brd4 knockout (KO). We found that Brd4KO cells grow more slowly than wild type cells; they do not complete replication, fail to achieve mitosis, and exhibit extensive DNA damage throughout all cell cycle stages. BRD4 was required for expression of more than 450 cell cycle genes including genes encoding core histones and centromere/kinetochore proteins that are critical for genome replication and chromosomal segregation. Moreover, we show that many genes controlling R-loop formation and DNA damage response (DDR) require BRD4 for expression. Finally, BRD4 constitutively occupied genes controlling R-loop, DDR and cell cycle progression. In summary, BRD4 epigenetically marks above genes and serves as a master regulator of normal cell growth.

PARP inhibitors (PARPi) are efficacious in BRCA1-null tumors; however, their utility is limited in tumors with functional BRCA1. We hypothesized that pharmacologically reducing BRCA1 protein levels could enhance PARPi effectiveness in BRCA1 wild-type tumors. To identify BRCA1 downregulating agents, we generated reporter cell lines using CRISPR-mediated editing to tag endogenous BRCA1 protein with HiBiT. These reporter lines enable the sensitive measurement of BRCA1 protein levels by luminescence. Validated reporter cells were used in a pilot screen of epigenetic-modifying probes and a larger screen of more than 6,000 compounds. We identified 7 compounds that could downregulate BRCA1-HiBiT expression and synergize with olaparib. Three compounds, N-acetyl-N-acetoxy chlorobenzenesulfonamide (NANAC), A-443654, and CHIR-124, were validated to reduce BRCA1 protein levels and sensitize breast cancer cells to the toxic effects of olaparib. These results suggest that BRCA1-HiBiT reporter cells hold promise in developing agents to improve the clinical utility of PARPi.

Sucrose is the transport form of carbohydrate in plants serving as signal molecule besides nutrition, but the signaling is elusive. Here, neutral invertase 8 (OsNIN8) mutated at G461R into OsNIN8m, which increased its charge and hydrophobicity, decreased hydrolysis of sucrose to 13% and firmer binding to sucrose than the wildtype. This caused downstream metabolites and energy accumulation forming overnutrition. Paradoxically, division of subinitials in longitudinal cell lineages was only about 15 times but more than 100 times in wildtype, resulting in short radicle. Further, mutation of OsNIN8 into deficiency of hydrolysis but maintenance of sucrose binding allowed cell division until ran out of energy showing the association but not hydrolysis gave the signal. Chemically, sucrose binding to OsNIN8 was exothermic but to OsNIN8m was endothermic. Therefore, OsNIN8m lost the signal function owing to change of thermodynamic state. So, OsNIN8 sensed sucrose for cell division besides hydrolyzed sucrose.

Although up to 80% small cell lung cancer (SCLC) patients\' response is good for first-line chemotherapy regimen, most patients develop recurrence of the disease within weeks to months. Here, we report cytostatic effect of leflunomide (Leflu) and teriflunomide (Teri) on SCLC cell proliferation through inhibition of DRP1 phosphorylation at Ser616 and decreased mitochondrial fragmentation. When administered together, Teri and carboplatin (Carbo) act synergistically to significantly inhibit cell proliferation and DRP1 phosphorylation, reduce abundance of intermediates in pyrimidine de novo pathway, and increase apoptosis and DNA damage. Combination of Leflu&Carbo has anti-tumorigenic effect in vivo. Additionally, lurbinectedin (Lur) and Teri potently and synergistically inhibited spheroid growth and depleted uridine and DRP1 phosphorylation in mouse tumors. Our results suggest combinations of Carbo and Lur with Teri or Leflu are efficacious and underscore how the relationship between DRP1/DHODH and mitochondrial plasticity serves as a potential therapeutic target to validate these treatment strategies in SCLC clinical trials.

My path to becoming a scientist has taken many twists and turns. This is perhaps not unusual to hear. Indeed, in discussions with my colleagues it seems that for many of us the path was never a straight one. Certainly, for me there have been moments when my whole world was encompassed by science and at other times, I have felt strongly that my time in science was up. I like to think that as scientists we ask a lot of questions and, for many of us, those questions extend to our very purpose as a scientist. My intention with this article is not to document my career path in detail or to provide very specific advice. Rather, I hope to describe how questions have defined my journey and to inspire others to occasionally pause and ask themselves what a career in science means to them. Today, I am an Assistant Professor at a major Canadian university, and here are the questions I asked along the way.