Ackerman Institute for the Family

Smads and their transcription factor partners mediate the transcriptional responses of target cells to secreted ligands of the transforming growth factor-β (TGF-β) family, including those of the conserved bone morphogenetic protein (BMP) family, yet only a small number of direct target genes have been well characterized. In C. elegans, the BMP2/4 ortholog DBL-1 regulates multiple biological functions, including body size, via a canonical receptor-Smad signaling cascade. Here, we identify functional binding sites for SMA-3/Smad and its transcriptional partner SMA-9/Schnurri based on ChIP-seq peaks (identified by modEncode) and expression differences of nearby genes identified from RNA-seq analysis of corresponding mutants. We found that SMA-3 and SMA-9 have both overlapping and unique target genes. At a genome-wide scale, SMA-3/Smad acts as a transcriptional activator, whereas SMA-9/Schnurri direct targets include both activated and repressed genes. Mutations in sma-9 partially suppress the small body size phenotype of sma-3, suggesting some level of antagonism between these factors and challenging the prevailing model for Schnurri function. Functional analysis of target genes revealed a novel role in body size for genes involved in one-carbon metabolism and in the endoplasmic reticulum (ER) secretory pathway, including the disulfide reductase dpy-11. Our findings indicate that Smads and SMA-9/Schnurri have previously unappreciated complex genetic and genomic regulatory interactions that in turn regulate the secretion of extracellular components like collagen into the cuticle to mediate body size regulation.

Smads and their transcription factor partners mediate the transcriptional responses of target cells to secreted ligands of the Transforming Growth Factor-β (TGF-β) family, including those of the conserved bone morphogenetic protein (BMP) family, yet only a small number of direct target genes have been well characterized. In C. elegans, the BMP2/4 ortholog DBL-1 regulates multiple biological functions, including body size, via a canonical receptor-Smad signaling cascade. Here, we identify functional binding sites for SMA-3/Smad and its transcriptional partner SMA-9/Schnurri based on ChIP-seq peaks (identified by modEncode) and expression differences of nearby genes identified from RNA-seq analysis of corresponding mutants. We found that SMA-3 and SMA-9 have both overlapping and unique target genes. At a genome-wide scale, SMA-3/Smad acts as a transcriptional activator, whereas SMA-9/Schnurri direct targets include both activated and repressed genes. Mutations in sma-9 partially suppress the small body size phenotype of sma-3, suggesting some level of antagonism between these factors and challenging the prevailing model for Schnurri function. Functional analysis of target genes revealed a novel role in body size for genes involved in one-carbon metabolism and in the endoplasmic reticulum (ER) secretory pathway, including the disulfide reductase dpy-11. Our findings indicate that Smads and SMA-9/Schnurri have previously unappreciated complex genetic and genomic regulatory interactions that in turn regulate the secretion of extracellular components like collagen into the cuticle to mediate body size regulation.

Spectrins are membrane cytoskeletal proteins generally thought to function as heterotetramers comprising two α-spectrins and two β-spectrins. They influence cell shape and Hippo signaling, but the mechanism by which they influence Hippo signaling has remained unclear. We have investigated the role and regulation of the Drosophila β-heavy spectrin (βH-spectrin, encoded by the karst gene) in wing imaginal discs. Our results establish that βH-spectrin regulates Hippo signaling through the Jub biomechanical pathway due to its influence on cytoskeletal tension. While we find that α-spectrin also regulates Hippo signaling through Jub, unexpectedly, we find that βH-spectrin localizes and functions independently of α-spectrin. Instead, βH-spectrin co-localizes with and reciprocally regulates and is regulated by myosin. In vivo and in vitro experiments support a model in which βH-spectrin and myosin directly compete for binding to apical F-actin. This competition can explain the influence of βH-spectrin on cytoskeletal tension and myosin accumulation. It also provides new insight into how βH-spectrin participates in ratcheting mechanisms associated with cell shape change.

The Ajuba LIM protein Jub mediates regulation of Hippo signaling by cytoskeletal tension through interaction with the kinase Warts and participates in feedback regulation of junctional tension through regulation of the cytohesin Steppke. To investigate how Jub interacts with and regulates its distinct partners, we investigated the ability of Jub proteins missing different combinations of its three LIM domains to rescue jub phenotypes and to interact with α-catenin, Warts and Steppke. Multiple regions of Jub contribute to its ability to bind α-catenin and to localize to adherens junctions in Drosophila wing imaginal discs. Co-immunoprecipitation experiments in cultured cells identified a specific requirement for LIM2 for binding to Warts. However, in vivo, both LIM1 and LIM2, but not LIM3, were required for regulation of wing growth, Yorkie activity, and Warts localization. Conversely, LIM2 and LIM3, but not LIM1, were required for regulation of cell shape and Steppke localization in vivo, and for maximal Steppke binding in co-immunoprecipitation experiments. These observations identify distinct functions for the different LIM domains of Jub.

The aim of this report is to present the root cause analysis on failed patient‐specific quality assurance (QA) measurements of pencil beam scanning (PBS) protons; referred to as PBS‐QA measurement. A criterion to fail a PBS‐QA measurement is having a <95% passing rate in a 3.0%‐3.0 mm gamma index analysis. Clinically, we use a two‐dimensional (2D) gamma index analysis to obtain the passing rate. The IBA MatriXX PT 2D detection array with finite size ionization chamber was utilized. A total of 2488 measurements performed in our PBS beamline were cataloged. The percentage of measurements for the sites of head/neck, breast, prostate, and other are 53.3%, 22.7%, 10.5%, and 13.5%, respectively. The measurements with a passing rate of 100 to >94%, 94 to >88%, and <88% were 93.6%, 5.6%, and 0.8%, respectively. The percentage of failed measurements with a <95% passing rate was 10.9%. After removed the user errors of either re‐measurement or re‐analysis, 8.1% became acceptable. We observed a feature of >3% per mm dose gradient with respect to depth on the failed measurements. We utilized a 2D/three‐dimensional (3D) gamma index analysis toolkit to investigate the effect of depth dose gradient. By utilizing this 3D toolkit, 43.1% of the failed measurements were improved. A feature among measurements that remained sub‐optimal after re‐analysis was a sharp >3% per mm lateral dose gradient that may not be well handled using the detector size of 5.0 mm in‐diameter. An analysis of the sampling of finite size detectors using one‐dimensional (1D) error function showed a large dose deviation at locations of low‐dose areas between two high‐dose plateaus. User error, large depth dose gradient, and the effect of detector size are identified as root causes. With the mitigation of the root causes, the goals of patient‐specific QA, specifically detecting actual deviation of beam delivery or identifying limitations of the dose calculation algorithm of the treatment planning system, can be directly related to failure of the PBS‐QA measurements.

Meiosis in female oocytes lacks centrosomes, the microtubule-organizing centers. In Drosophila oocytes, meiotic spindle assembly depends on the chromosomal passenger complex (CPC). To investigate the mechanisms that regulate Aurora B activity, we examined the role of protein phosphatase 2A (PP2A) in Drosophila oocyte meiosis. We found that both forms of PP2A, B55 and B56, antagonize the Aurora B spindle assembly function, suggesting that a balance between Aurora B and PP2A activity maintains the oocyte spindle during meiosis I. PP2A-B56, which has a B subunit encoded by two partially redundant paralogs, wdb and wrd, is also required for maintenance of sister chromatid cohesion, establishment of end-on microtubule attachments, and metaphase I arrest in oocytes. WDB recruitment to the centromeres depends on BUBR1, MEI-S332 and kinetochore protein SPC105R. Although BUBR1 stabilizes microtubule attachments in Drosophila oocytes, it is not required for cohesion maintenance during meiosis I. We propose at least three populations of PP2A-B56 regulate meiosis, two of which depend on SPC105R and a third that is associated with the spindle.

Abstract Despite the importance of fertilization, the molecular basis of sperm-egg interaction is not well understood. In a forward genetics screen for fertility mutants inCaenorhabditis eleganswe identifiedspe-51. Mutant worms make sperm that are unable to fertilize the oocyte but otherwise normal by all available measurements. Thespe-51gene encodes a secreted protein that includes an immunoglobulin (Ig)-like domain and a hydrophobic sequence of amino acids. The SPE-51 protein acts cell-autonomously and localizes to the surface of the spermatozoa. This is the first example of a secreted protein required for the interactions between the sperm and egg with genetic validation for a specific function in fertilization. Our analyses of these genes begin to build a paradigm for sperm-secreted or reproductive tract-secreted proteins that coat the sperm surface and influence their survival, motility, and/or the ability to fertilize the egg.

Abstract Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPO). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, “bind-unwind-load-and-lock,” model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.Significance statementTranscription initiation, the first step and most important step in gene expression for all organisms, involves unwinding of promoter DNA by RNA polymerase (RNAP) to form an open complex (RPo); this step also underpins transcriptional regulation and serves as an antibiotic target. Despite decades of research, the mechanism of promoter DNA unwinding has remained unresolved. Here, we solve this puzzle by using single-molecule fluorescence to directly monitor conformational changes in the promoter DNA and RNAP in real time during RPo formation. We show that RPo forms via a “bind-unwind-load-and-lock” mechanism, where the promoter unwinds outside the RNAP cleft, the unwound template DNA loads into the cleft, and RNAP “locks” the template DNA in place by closing the RNAP clamp module.

The COVID‐19 pandemic has convulsed human communities across the globe like no previous event in history. Family therapists, paradoxically, given the core of their work is with systems, are also experiencing upheaval in professional and personal lives, trying to work amidst a society in chaos. This paper offers a collection of reflections by systemic and family therapists from diverse cultures and contexts penned in the midst of the pandemic. The main intention in distilling these narratives is to preserve the ‘cultural diversity’ and ‘ecological position’ of the contributors, guided by phenomenology, cultural ecology, and systemic worldviews of ‘experiencing.’ The second intention is to ‘unite’ promoting solidarity in this isolating situation by bringing each story together, creating its own metaphor of a family: united, connected, stronger. As a cross‐cultural family practitioner, with a strong mission for collaboration, the lead author acknowledges the importance of Context – the nation and location of the experience; Culture – the manner in which culture impacts on experience; Collaboration – enhancing partnership, enriching knowledge, and mapping the journey’s direction; and Connectedness – combating isolation while enhancing unity. Since the key transmission of culture is through language, raw reflections were sought initially in the practitioners’ own language, which were translated for an English‐speaking readership. These narratives are honest and rich descriptions of the authors’ lived experiences, diverse and distinctive. The contributors trust colleagues will find these reflections helpful, validating and acknowledging the challenges of this unique period in history.