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Stimulator of IFN genes type I (STING-Type I) IFN signaling in myeloid cells plays a critical role in effective antitumor immune responses, but STING agonists as monotherapy have shown limited efficacy in clinical trials. The mechanisms that downregulate STING signaling are not fully understood. Here, we report that protein phosphatase 2A (PP2A), with its specific B regulatory subunit Striatin 4 (STRN4), negatively regulated STING-Type I IFN in macrophages. Mice with macrophage PP2A deficiency exhibited reduced tumor progression. The tumor microenvironment showed decreased immunosuppressive and increased IFN-activated macrophages and CD8+ T cells. Mechanistically, we demonstrated that Hippo kinase MST1/2 was required for STING activation. STING agonists induced dissociation of PP2A from MST1/2 in normal macrophages, but not in tumor conditioned macrophages. Furthermore, our data showed that STRN4 mediated PP2A binding to and dephosphorylation of Hippo kinase MST1/2, resulting in stabilization of YAP/TAZ to antagonize STING activation. In human patients with glioblastoma (GBM), YAP/TAZ was highly expressed in tumor-associated macrophages but not in nontumor macrophages. We also demonstrated that PP2A/STRN4 deficiency in macrophages reduced YAP/TAZ expression and sensitized tumor-conditioned macrophages to STING stimulation. In summary, we demonstrated that PP2A/STRN4-YAP/TAZ has, in our opinion, been an unappreciated mechanism that mediates immunosuppression in tumor-associated macrophages, and targeting the PP2A/STRN4-YAP/TAZ axis can sensitize tumors to immunotherapy.

Calcium dyshomeostasis is an early critical event in neurodegeneration as exemplified by Alzheimer's (AD), Huntington's (HD) and Parkinson's (PD) diseases. Neuronal calcium homeostasis is maintained by a diversity of ion channels, buffers, calcium-binding protein effectors, and intracellular storage in the endoplasmic reticulum, mitochondria, and lysosomes. The function of these components and compartments is impacted by the toxic hallmark proteins of AD (amyloid beta and Tau), HD (huntingtin) and PD (alpha-synuclein) as well as by interactions with downstream calcium-binding proteins, especially calmodulin. Each of the toxic hallmark proteins (amyloid beta, Tau, huntingtin, and alpha-synuclein) binds to calmodulin. Multiple channels and receptors involved in calcium homeostasis and dysregulation also bind to and are regulated by calmodulin. The primary goal of this review is to show the complexity of these interactions and how they can impact research and the search for therapies. A secondary goal is to suggest that therapeutic targets downstream from calcium dyshomeostasis may offer greater opportunities for success. [ABSTRACT FROM AUTHOR]

Precise signalling between pollen tubes and synergid cells in the ovule initiates fertilization in flowering plants1. Contact of the pollen tube with the ovule triggers calcium spiking in the synergids2,3 that induces pollen tube rupture and sperm release. This process, termed pollen tube reception, entails the action of three synergid-expressed proteins in Arabidopsis: FERONIA (FER), a receptor-like kinase; LORELEI (LRE), a glycosylphosphatidylinositol-anchored protein; and NORTIA (NTA), a transmembrane protein of unknown function4-6. Genetic analyses have placed these three proteins in the same pathway; however, it remains unknown how they work together to enable synergid-pollen tube communication. Here we identify two pollen-tube-derived small peptides7 that belong to the rapid alkalinization factor (RALF) family8 as ligands for the FER-LRE co-receptor, which in turn recruits NTA to the plasma membrane. NTA functions as a calmodulin-gated calcium channel required for calcium spiking in the synergid. We also reconstitute the biochemical pathway in which FER-LRE perceives pollen-tube-derived peptides to activate the NTA calcium channel and initiate calcium spiking, a second messenger for pollen tube reception. The FER-LRE-NTA trio therefore forms a previously unanticipated receptor-channel complex in the female cell to recognize male signals and trigger the fertilization process.

Seven major neurodegenerative diseases and their variants share many overlapping biomarkers that are calmodulin-binding proteins: Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), frontotemporal lobar dementia (FTD), Huntington's disease (HD), Lewy body disease (LBD), multiple sclerosis (MS), and Parkinson's disease (PD). Calcium dysregulation is an early and persistent event in each of these diseases, with calmodulin serving as an initial and primary target of increased cytosolic calcium. Considering the central role of calcium dysregulation and its downstream impact on calcium signaling, calmodulin has gained interest as a major regulator of neurodegenerative events. Here, we show that calmodulin serves a critical role in neurodegenerative diseases via binding to and regulating an abundance of biomarkers, many of which are involved in multiple neurodegenerative diseases. Of special interest are the shared functions of calmodulin in the generation of protein biomarker aggregates in AD, HD, LBD, and PD, where calmodulin not only binds to amyloid beta, pTau, alpha-synuclein, and mutant huntingtin but also, via its regulation of transglutaminase 2, converts them into toxic protein aggregates. It is suggested that several calmodulin binding proteins could immediately serve as primary drug targets, while combinations of calmodulin binding proteins could provide simultaneous insight into the onset and progression of multiple neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Contractile actomyosin bundles are key force-producing and mechanosensing elements in muscle and non-muscle tissues. Whereas the organization of muscle myofibrils and mechanism regulating their contractility are relatively well-established, the principles by which myosin-II activity and force-balance are regulated in non-muscle cells have remained elusive. We show that Caldesmon, an important component of smooth muscle and non-muscle cell actomyosin bundles, is an elongated protein that functions as a dynamic cross-linker between myosin-II and tropomyosin-actin filaments. Depletion of Caldesmon results in aberrant lateral movement of myosin-II filaments along actin bundles, leading to irregular myosin distribution within stress fibers. This manifests as defects in stress fiber network organization and contractility, and accompanied problems in cell morphogenesis, migration, invasion, and mechanosensing. These results identify Caldesmon as critical factor that ensures regular myosin-II spacing within non-muscle cell actomyosin bundles, and reveal how stress fiber networks are controlled through dynamic cross-linking of tropomyosin-actin and myosin filaments.

A multifactorial syndrome, Alzheimer’s disease is the main cause of dementia, but there is no existing therapy to prevent it or stop its progression. One of the earliest events of Alzheimer’s disease is the disruption of calcium homeostasis but that is just a prelude to the disease’s devastating impact. Calcium does not work alone but must interact with downstream cellular components of which the small regulatory protein calmodulin is central, if not primary. This review supports the idea that, due to calcium dyshomeostasis, calmodulin is a dominant regulatory protein that functions in all stages of Alzheimer’s disease, and these regulatory events are impacted by amyloid beta. Amyloid beta not only binds to and regulates calmodulin but also multiple calmodulin-binding proteins involved in Alzheimer’s. Together, they act on the regulation of calcium dyshomeostasis, neuroinflammation, amyloidogenesis, memory formation, neuronal plasticity and more. The complex interactions between calmodulin, its binding proteins and amyloid beta may explain why many therapies have failed or are doomed to failure unless they are considered.

The Saccharomyces cerevisiae spindle pole body (SPB) serves as the sole microtubule-organizing center of the cell, nucleating both cytoplasmic and nuclear microtubules. Yeast pericentrin, Spc110, binds to and activates the γ-tubulin complex via its N terminus, allowing nuclear microtubule polymerization to occur. The Spc110 C terminus links the γ-tubulin complex to the central plaque of the SPB by binding to Spc42, Spc29, and calmodulin (Cmd1). Here, we show that overexpression of the C terminus of Spc110 is toxic to cells and correlates with its localization to the SPB. Spc110 domains that are required for SPB localization and toxicity include its Spc42-, Spc29-, and Cmd1-binding sites. Overexpression of the Spc110 C terminus induces SPB defects and disrupts microtubule organization in both cycling and G2/M arrested cells. Notably, the two mitotic SPBs are affected in an asymmetric manner such that one SPB appears to be pulled away from the nucleus toward the cortex but remains attached via a thread of nuclear envelope. This SPB also contains relatively fewer microtubules and less endogenous Spc110. Our data suggest that overexpression of the Spc110 C terminus acts as a dominant-negative mutant that titrates endogenous Spc110 from the SPB causing spindle defects.

Mitogen-activated protein kinase kinase kinase kinases (MAP4Ks) constitute a mammalian STE20-like serine/threonine kinase subfamily. Recent studies provide substantial evidence for MAP4K family kinases in the Hippo pathway regulation, suggesting a broad role of MAP4Ks in human physiology and diseases. However, a comprehensive analysis of the regulators and effectors for this key kinase family has not been fully achieved. Using a proteomic approach, we define the protein-protein interaction network for human MAP4K family kinases and reveal diverse cellular signaling events involving this important kinase family. Through it, we identify a STRIPAK complex component, STRN4, as a generic binding partner for MAP4Ks and a key regulator of the Hippo pathway in endometrial cancer development. Taken together, the results of our study not only generate a rich resource for further characterizing human MAP4K family kinases in numerous biological processes but also dissect the STRIPAK-mediated regulation of MAP4Ks in the Hippo pathway.

Thrombin, a procoagulant protease, cleaves and activates protease-activated receptor-1 (PAR1) to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC), an anticoagulant protease, activates PAR1 through a distinct cleavage site and promotes anti-inflammatory responses, prosurvival, and endothelial barrier stabilization. The distinct tethered ligands formed through cleavage of PAR1 by thrombin versus APC result in unique active receptor conformations that bias PAR1 signaling. Despite progress in understanding PAR1 biased signaling, the proteins and pathways utilized by thrombin versus APC signaling to induce opposing cellular functions are largely unknown. Here, we report the global phosphoproteome induced by thrombin and APC signaling in endothelial cells with the quantification of 11,266 unique phosphopeptides using multiplexed quantitative mass spectrometry. Our results reveal unique dynamic phosphoproteome profiles of thrombin and APC signaling, an enrichment of associated biological functions, including key modulators of endothelial barrier function, regulators of gene transcription, and specific kinases predicted to mediate PAR1 biased signaling. Using small interfering RNA to deplete a subset of phosphorylated proteins not previously linked to thrombin or APC signaling, a function for afadin and adducin-1 actin binding proteins in thrombin-induced endothelial barrier disruption is unveiled. Afadin depletion resulted in enhanced thrombin-promoted barrier permeability, whereas adducin-1 depletion completely ablated thrombin-induced barrier disruption without compromising p38 signaling. However, loss of adducin-1 blocked APC-induced Akt signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 biased signaling in endothelial cells.

Calcium dyshomeostasis is an early critical event in neurodegeneration as exemplified by Alzheimer’s (AD), Huntington’s (HD) and Parkinson’s (PD) diseases. Neuronal calcium homeostasis is maintained by a diversity of ion channels, buffers, calcium-binding protein effectors, and intracellular storage in the endoplasmic reticulum, mitochondria, and lysosomes. The function of these components and compartments is impacted by the toxic hallmark proteins of AD (amyloid beta and Tau), HD (huntingtin) and PD (alpha-synuclein) as well as by interactions with downstream calcium-binding proteins, especially calmodulin. Each of the toxic hallmark proteins (amyloid beta, Tau, huntingtin, and alpha-synuclein) binds to calmodulin. Multiple channels and receptors involved in calcium homeostasis and dysregulation also bind to and are regulated by calmodulin. The primary goal of this review is to show the complexity of these interactions and how they can impact research and the search for therapies. A secondary goal is to suggest that therapeutic targets downstream from calcium dyshomeostasis may offer greater opportunities for success.

Alpha-adducin (Add1) is a critical component of the actin-spectrin network in erythrocytes, acting to cap the fast-growing, barbed ends of actin filaments, and recruiting spectrin to these junctions. Add1 is highly expressed in T cells, but its role in T-cell activation has not been examined. Using a conditional knockout model, we show that Add1 is necessary for complete activation of CD4+ T cells in response to low levels of antigen but is dispensable for CD8+ T cell activation and response to infection. Surprisingly, costimulatory signals through CD28 were completely abrogated in the absence of Add1. This study is the first to examine the role of actin-capping in T cells, and it reveals a previously unappreciated role for the actin cytoskeleton in regulating costimulation.

Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to signaling, and uncovers a ciliary protein that underlies development and human disease.

Ciliated muconodular papillary tumor (CMPT) is a rare pulmonary tumor, typically occurring in middle-aged and elderly individuals. The molecular mutation spectrum of CMPT remains insufficiently explored. Commonly known driver gene alterations include KRAS , BRAF , EGFR , and ALK rearrangement. This report details the clinicopathological features of 2 patients presenting with CMPT as pulmonary nodules during clinical examinations. Microscopic analysis revealed tumors with glandular or papillary structures, consisting of mucinous cells, ciliated columnar cells, and basal cells. Notably, both patients exhibited STRN::ALK fusion, a finding not previously associated with CMPT. STRN::ALK fusion serves as a target for therapy in various tumors, including non-small cell lung cancer, thyroid cancer, and colon cancer. Consequently, we conducted a review of relevant literature, summarizing the clinicopathological and molecular characteristics of CMPT to facilitate further research. Our insights enhance the understanding of this uncommon tumor and contribute to the expansion of its molecular alteration spectrum., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Salivary gland carcinomas are a heterogeneous group of rare tumors. There is no established standard of care therapy for metastatic disease. We describe the case of a patient with metastatic salivary gland adenocarcinoma harboring STRN-ALK translocation, with tumor response and clinical benefit from anaplastic lymphoma kinase ( ALK ) inhibition. Our patient experienced clinical benefit from first and second generation ALK inhibition in a chemotherapy refractory tumor. Tumor mutation profiling can identify mutations that may render tumors sensitive to targeted therapy with tyrosine kinase inhibitors., (Copyright© Board of Regents of the University of Wisconsin System and The Medical College of Wisconsin, Inc.)

Purpose: To describe the clinical and genetic features, and explore the natural history of retinopathy associated with IQCB1 variants in children and adults with retinopathy., Design: Retrospective cohort study at a single tertiary care referral center., Methods: The study recruited 19 patients with retinopathy, harboring likely disease-causing variants in IQCB1. Demographic data and clinical presentation, best corrected visual acuity (BCVA), fundus appearance, optical coherence tomography (OCT) and autofluorescence features, electroretinography (ERG) and molecular genetics are reported., Results: Ten patients had best corrected visual acuity better than 1.0 LogMAR, and BCVA remained stable till the last review. Seven patients had a vision of hand movements or worse in at least one eye at presentation. There was no correlation found between age of onset and severity of vision loss. Nine patients (47.4%) had a diagnosis of end-stage renal failure at presentation. The other 10 patients (52.6%) had a diagnosis of non-syndromic IQCB1-retinopathy and maintained normal renal function until the last follow-up. The mean age at diagnosis of renal failure was 26.3 ±19.8 years. OCT showed ellipsoid zone (EZ) disruption with foveal sparing in 8/13 patients. All patients had stable OCT findings. Full-field ERGs in four adults revealed a severe cone-rod dystrophy and three children had extinguished ERGs. We identified 17 IQCB1 variants, all predicted to cause loss of function., Conclusion: IQCB1-retinopathy is a severe early-onset cone-rod dystrophy. The dissociation between severely decreased retinal function and relative preservation of retinal structure over a wide age window makes the disease a candidate for gene therapy., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Parasitic nematodes infect over 1 billion people worldwide and cause some of the most common neglected tropical diseases. Despite their prevalence, our understanding of the biology of parasitic nematodes has been limited by the lack of tools for genetic intervention. In particular, it has not yet been possible to generate targeted gene disruptions and mutant phenotypes in any parasitic nematode. Here, we report the development of a method for introducing CRISPR-Cas9-mediated gene disruptions in the human-parasitic threadworm Strongyloides stercoralis. We disrupted the S. stercoralis twitchin gene unc-22, resulting in nematodes with severe motility defects. Ss-unc-22 mutations were resolved by homology-directed repair when a repair template was provided. Omission of a repair template resulted in deletions at the target locus. Ss-unc-22 mutations were heritable; we passed Ss-unc-22 mutants through a host and successfully recovered mutant progeny. Using a similar approach, we also disrupted the unc-22 gene of the rat-parasitic nematode Strongyloides ratti. Our results demonstrate the applicability of CRISPR-Cas9 to parasitic nematodes, and thereby enable future studies of gene function in these medically relevant but previously genetically intractable parasites.

Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.

African black-footed cats (Felis nigripes) are endangered wild felids. One male and full-sibling female African black-footed cat developed vision deficits and mydriasis as early as 3 months of age. The diagnosis of early-onset progressive retinal atrophy (PRA) was supported by reduced direct and consensual pupillary light reflexes, phenotypic presence of retinal degeneration, and a non-recordable electroretinogram with negligible amplitudes in both eyes. Whole genome sequencing, conducted on two unaffected parents and one affected offspring was compared to a variant database from 51 domestic cats and a Pallas cat, revealed 50 candidate variants that segregated concordantly with the PRA phenotype. Testing in additional affected cats confirmed that cats homozygous for a 2 base pair (bp) deletion within IQ calmodulin-binding motif-containing protein-1 (IQCB1), the gene that encodes for nephrocystin-5 (NPHP5), had vision loss. The variant segregated concordantly in other related individuals within the pedigree supporting the identification of a recessively inherited early-onset feline PRA. Analysis of the black-footed cat studbook suggests additional captive cats are at risk. Genetic testing for IQCB1 and avoidance of matings between carriers should be added to the species survival plan for captive management.

We performed a genome-wide association study of 6447 bipolar disorder (BD) cases and 12 639 controls from the International Cohort Collection for Bipolar Disorder (ICCBD). Meta-analysis was performed with prior results from the Psychiatric Genomics Consortium Bipolar Disorder Working Group for a combined sample of 13 902 cases and 19 279 controls. We identified eight genome-wide significant, associated regions, including a novel associated region on chromosome 10 (rs10884920; P=3.28 × 10-8) that includes the brain-enriched cytoskeleton protein adducin 3 (ADD3), a non-coding RNA, and a neuropeptide-specific aminopeptidase P (XPNPEP1). Our large sample size allowed us to test the heritability and genetic correlation of BD subtypes and investigate their genetic overlap with schizophrenia and major depressive disorder. We found a significant difference in heritability of the two most common forms of BD (BD I SNP-h2=0.35; BD II SNP-h2=0.25; P=0.02). The genetic correlation between BD I and BD II was 0.78, whereas the genetic correlation was 0.97 when BD cohorts containing both types were compared. In addition, we demonstrated a significantly greater load of polygenic risk alleles for schizophrenia and BD in patients with BD I compared with patients with BD II, and a greater load of schizophrenia risk alleles in patients with the bipolar type of schizoaffective disorder compared with patients with either BD I or BD II. These results point to a partial difference in the genetic architecture of BD subtypes as currently defined.

In nature, plants are frequently exposed to drought and bacterial pathogens simultaneously. However, information on how the drought and defence pathways interact and orchestrate global transcriptional regulation is limited. Here, we show that moderate drought stress enhances the susceptibility of Arabidopsis thaliana to Pseudomonas syringae pv. tomato DC3000. Using transcriptome meta‐analysis, we found that drought and bacterial stress antagonistically modulate a large set of genes predominantly involved in salicylic acid (SA) and abscisic acid (ABA) signalling networks. We identified that the levels of SA and ABA are dynamically regulated during the course of stress. Importantly, under combined stress, drought through the ABA pathway downregulates the induction of Calmodulin‐binding Protein 60 g (CBP60g) and Systemic Acquired Resistance Deficient 1 (SARD1), two transcription factors crucial for SA production upon bacterial infection. We also identified an important role of NPR1‐LIKE PROTEIN 3 and 4 (NPR3/4) transcriptional repressors in the drought‐mediated negative regulation of CBP60g/SARD1 expression. Using a genetic approach, we show that CBP60g/SARD1 expression is the key determinant of plant defence against bacterial pathogens under combined stress. Thus, these transcription factors act as critical nodes for the crosstalk between drought and bacterial stress signalling under combined stress in plants. Summary statement: Moderate drought attenuates plant defence responses against bacterial pathogens by suppressing the expression of CBP60g and SARD1, which encode two crucial transcription factors involved in activating salicylic acid machinery. [ABSTRACT FROM AUTHOR]

Key Message: BIG regulates ground tissue formative divisions by bridging the auxin gradient with SHR abundance in Arabidopsis roots. The formative divisions of cortex/endodermis initials (CEIs) and CEI daughter cells (CEIDs) in Arabidopsis roots are coordinately controlled by the longitudinal auxin gradient and the radial SHORT ROOT (SHR) abundance. However, the mechanism underlying this coordination remains poorly understood. In this study, we demonstrate that BIG regulates ground tissue formative divisions by bridging the auxin gradient with SHR abundance. Mutations in BIG gene repressed cell cycle progression, delaying the formative divisions within the ground tissues and impairing the establishment of endodermal and cortical identities. In addition, we uncovered auxin's suppressive effect on BIG expression, triggering CYCLIND6;1 (CYCD6;1) activation in an SHR-dependent fashion. Moreover, the degradation of RETINOBLASTOMA-RELATED (RBR) is jointly regulated by BIG and CYCD6;1. The loss of BIG function led to RBR protein accumulation, detrimentally impacting the SHR/SCARECROW (SCR) protein complex and the CEI/CEID formative divisions. Collectively, these findings shed light on a fundamental mechanism wherein BIG intricately coordinates the interplay between SHR/SCR and auxin, steering ground tissue patterning within Arabidopsis root tissue., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Anaplastic thyroid cancer (ATC) is one of the deadliest human cancers and represents <2% of thyroid carcinomas. A therapeutic target for ATC is represented by anaplastic lymphoma kinase (ALK) rearrangements, involved in tumor growth. Crizotinib is an oral small-molecule tyrosine kinase inhibitor of the ALK, MET, and ROS1 kinases, approved in ALK-positive non-small cell lung cancer. Until now, the effect of crizotinib in "primary human ATC cells" (pATCs) with transforming striatin (STRN)-ALK fusion has not been reported in the literature. In this study, we aimed to obtain pATCs with STRN-ALK in vitro and evaluate the in vitro antineoplastic action of crizotinib. Thyroid surgical samples were obtained from 12 ATC patients and 6 controls (who had undergone parathyroidectomy). A total of 10/12 pATC cultures were obtained, 2 of which with transforming STRN-ALK fusion (17%). Crizotinib inhibited proliferation, migration, and invasion and increased apoptosis in 3/10 pATC cultures (2 of which with/1 without STRN-ALK), particularly in those with STRN-ALK. Moreover, crizotinib significantly inhibited the proliferation of AF cells (a continuous cell line obtained from primary ATC cells). In conclusion, the antineoplastic activity of crizotinib has been shown in human pATCs (with STRN-ALK) in preclinical studies in vitro, opening the way to future clinical evaluation in these patients.

The three striatins (STRN, STRN3, STRN4) form the core of STRiatin-Interacting Phosphatase and Kinase (STRIPAK) complexes. These place protein phosphatase 2A (PP2A) in proximity to protein kinases thereby restraining kinase activity and regulating key cellular processes. Our aim was to establish if striatins play a significant role in cardiac remodelling associated with cardiac hypertrophy and heart failure. All striatins were expressed in control human hearts, with up-regulation of STRN and STRN3 in failing hearts. We used mice with global heterozygote gene deletion to assess the roles of STRN and STRN3 in cardiac remodelling induced by angiotensin II (AngII; 7 days). Using echocardiography, we detected no differences in baseline cardiac function or dimensions in STRN+/- or STRN3+/- male mice (8 weeks) compared with wild-type littermates. Heterozygous gene deletion did not affect cardiac function in mice treated with AngII, but the increase in left ventricle mass induced by AngII was inhibited in STRN+/- (but not STRN3+/-) mice. Histological staining indicated that cardiomyocyte hypertrophy was inhibited. To assess the role of STRN in cardiomyocytes, we converted the STRN knockout line for inducible cardiomyocyte-specific gene deletion. There was no effect of cardiomyocyte STRN knockout on cardiac function or dimensions, but the increase in left ventricle mass induced by AngII was inhibited. This resulted from inhibition of cardiomyocyte hypertrophy and cardiac fibrosis. The data indicate that cardiomyocyte striatin is required for early remodelling of the heart by AngII and identify the striatin-based STRIPAK system as a signalling paradigm in the development of pathological cardiac hypertrophy., (© 2024 The Author(s).)

There is strong evidence that changes in the actin/spectrin-based cortical cytoskeleton of outer hair cells (OHCs) regulate their motile responses as well as cochlear amplification, the process that optimizes the sensitivity and frequency selectivity of the mammalian inner ear. Since a RhoA/protein kinase C (PKC)-mediated pathway is known to inhibit the actin-spectrin interaction in other cell models, we decided to investigate whether this signaling cascade could also participate in the regulation of OHC motility. We used high-speed video microscopy and confocal microscopy to explore the effects of pharmacological activation of PKCα, PKCβI, PKCβII, PKCδ, PKCε, and PKCζ with lysophosphatidic acid (LPA) and their inhibition with bisindolylmaleimide I, as well as inhibition of RhoA and Rho-associated protein kinase (ROCK) with C3 and Y-27632, respectively. Motile responses were induced in isolated guinea pig OHCs by stimulation with an 8 V/cm external alternating electrical field as 50 Hz bursts of square wave pulses (100 ms on/off). We found that LPA increased expression of PKCα and PKCζ only, with PKCα, but not PKCζ, phosphorylating the cytoskeletal protein adducin of both Ser-726 and Thr-445. Interestingly, however, inhibition of PKCα reduced adducin phosphorylation only at Ser-726. We also determined that LPA activation of a PKCα-mediated signaling pathway simultaneously enhanced OHC electromotile amplitude and cell shortening, and facilitated RhoA/ROCK/LIMK1-mediated cofilin phosphorylation. Altogether, our results suggest that PKCα-mediated signals, probably via adducin-mediated inhibition of actin-spectrin binding and cofilin-mediated depolymerization of actin filaments, play an essential role in the homeostatic regulation of OHC motility and cochlear amplification.

The mir-34/449 family consists of six homologous miRNAs at three genomic loci. Redundancy of miR-34/449 miRNAs and their dominant expression in multiciliated epithelia suggest a functional significance in ciliogenesis. Here we report that mice deficient for all miR-34/449 miRNAs exhibited postnatal mortality, infertility and strong respiratory dysfunction caused by defective mucociliary clearance. In both mouse and Xenopus, miR-34/449-deficient multiciliated cells (MCCs) exhibited a significant decrease in cilia length and number, due to defective basal body maturation and apical docking. The effect of miR-34/449 on ciliogenesis was mediated, at least in part, by post-transcriptional repression of Cp110, a centriolar protein suppressing cilia assembly. Consistent with this, cp110 knockdown in miR-34/449-deficient MCCs restored ciliogenesis by rescuing basal body maturation and docking. Altogether, our findings elucidate conserved cellular and molecular mechanisms through which miR-34/449 regulate motile ciliogenesis.

Increased fatty acid synthesis is required to meet the demand for membrane expansion of rapidly growing cells. ATP-citrate lyase (ACLY) is upregulated or activated in several types of cancer, and inhibition of ACLY arrests proliferation of cancer cells. Here we show that ACLY is acetylated at lysine residues 540, 546, and 554 (3K). Acetylation at these three lysine residues is stimulated by P300/calcium-binding protein (CBP)-associated factor (PCAF) acetyltransferase under high glucose and increases ACLY stability by blocking its ubiquitylation and degradation. Conversely, the protein deacetylase sirtuin 2 (SIRT2) deacetylates and destabilizes ACLY. Substitution of 3K abolishes ACLY ubiquitylation and promotes de novo lipid synthesis, cell proliferation, and tumor growth. Importantly, 3K acetylation of ACLY is increased in human lung cancers. Our study reveals a crosstalk between acetylation and ubiquitylation by competing for the same lysine residues in the regulation of fatty acid synthesis and cell growth in response to glucose.

An estimated 50 million dengue virus (DENV) infections occur annually and more than forty percent of the human population is currently at risk of developing dengue fever (DF) or dengue hemorrhagic fever (DHF). Despite the prevalence and potential severity of DF and DHF, there are no approved vaccines or antiviral therapeutics available. An improved understanding of DENV immune evasion is pivotal for the rational development of anti-DENV therapeutics. Antagonism of type I interferon (IFN-I) signaling is a crucial mechanism of DENV immune evasion. DENV NS5 protein inhibits IFN-I signaling by mediating proteasome-dependent STAT2 degradation. Only proteolytically-processed NS5 can efficiently mediate STAT2 degradation, though both unprocessed and processed NS5 bind STAT2. Here we identify UBR4, a 600-kDa member of the N-recognin family, as an interacting partner of DENV NS5 that preferentially binds to processed NS5. Our results also demonstrate that DENV NS5 bridges STAT2 and UBR4. Furthermore, we show that UBR4 promotes DENV-mediated STAT2 degradation, and most importantly, that UBR4 is necessary for efficient viral replication in IFN-I competent cells. Our data underscore the importance of NS5-mediated STAT2 degradation in DENV replication and identify UBR4 as a host protein that is specifically exploited by DENV to inhibit IFN-I signaling via STAT2 degradation.

Kinesin-like calmodulin binding protein (KCBP), a Kinesin-14 family motor protein, is involved in the structural organization of microtubules during mitosis and trichome morphogenesis in plants. The molecular mechanism of microtubule bundling by KCBP remains unknown. KCBP binding to microtubules is regulated by Ca(2+)-binding proteins that recognize its C-terminal regulatory domain. In this work, we have discovered a new function of the regulatory domain. We present a crystal structure of an Arabidopsis KCBP fragment showing that the C-terminal regulatory domain forms a dimerization interface for KCBP. This dimerization site is distinct from the dimerization interface within the N-terminal domain. Side chains of hydrophobic residues of the calmodulin binding helix of the regulatory domain form the C-terminal dimerization interface. Biochemical experiments show that another segment of the regulatory domain located beyond the dimerization interface, its negatively charged coil, is unexpectedly and absolutely required to stabilize the dimers. The strong microtubule bundling properties of KCBP are unaffected by deletion of the C-terminal regulatory domain. The slow minus-end directed motility of KCBP is also unchanged in vitro. Although the C-terminal domain is not essential for microtubule bundling, we suggest that KCBP may use its two independent dimerization interfaces to support different types of bundled microtubule structures in cells. Two distinct dimerization sites may provide a mechanism for microtubule rearrangement in response to Ca(2+) signaling since Ca(2+)- binding proteins can disengage KCBP dimers dependent on its C-terminal dimerization interface.

Objective: Our goal was to find metabolism-related lncRNAs that were associated with osteoporosis (OP) and construct a model for predicting OP progression using these lncRNAs., Methods: The GEO database was employed to obtain gene expression profiles. The WGCNA technique and differential expression analysis were used to identify hypoxia-related lncRNAs. A Lasso regression model was applied to select 25 hypoxia-related genes, from which a classification model was created. Its robust classification performance was confirmed with an area under the ROC curve close to 1, as verified on the validation set. Concurrently, we constructed a ceRNA network based on these genes to unveil potential regulatory processes. Biologically active compounds of STZYD were identified using the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP) database. BATMAN was used to identify its targets, and we obtained OP-related genes from Malacards and DisGeNET, followed by identifying intersection genes with metabolism-related genes. A pharmacological network was then constructed based on the intersecting genes. The pharmacological network was further integrated with the ceRNA network, resulting in the creation of a comprehensive network that encompasses herb-active components, pathways, lncRNAs, miRNAs, and targets. Expression levels of hypoxia-related lncRNAs in mononuclear cells isolated from peripheral blood of OP and normal patients were subsequently validated using quantitative real-time PCR (qRT-PCR). Protein levels of RUNX2 were determined through a western blot assay., Results: CBFB, GLO1, NFKB2 and PIK3CA were identified as central therapeutic targets, and ADD3-AS1, DTX2P1-UPK3BP1-PMS2P11, TTTY1B, ZNNT1 and LINC00623 were identified as core lncRNAs., Conclusions: Our work uncovers a possible therapeutic mechanism for STZYD, providing a potential therapeutic target for OP. In addition, a prediction model of metabolism-related lncRNAs of OP progression was constructed to provide a reference for the diagnosis of OP patients., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Lung squamous cell carcinoma (LUSC) is one of the subtypes of lung cancer (LC) that contributes to approximately 25%-30% of its prevalence. Cancer-associated fibroblasts (CAFs) are key cellular components of the TME, and the large number of CAFs in tumour tissues creates a favourable environment for tumour development. However, the function of CAFs in the LUSC is complex and uncertain. First, we processed the scRNA-seq data and classified distinct types of CAFs. We also identified prognostic CAFRGs using univariate Cox analysis and conducted survival analysis. Additionally, we assessed immune cell infiltration in CAF clusters using ssGSEA. We developed a model with a significant prognostic correlation and verified the prognostic model. Furthermore, we explored the immune landscape of LUSC and further investigated the correlation between malignant features and LUSC. We identified CAFs and classified them into three categories: iCAFs, mCAFs and apCAFs. The survival analysis showed a significant correlation between apCAFs and iCAFs and LUSC patient prognosis. Kaplan-Meier analysis showed that patients in CAF cluster C showed a better survival probability compared to clusters A and B. In addition, we identified nine significant prognostic CAFRGs (CLDN1, TMX4, ALPL, PTX3, BHLHE40, TNFRSF12A, VKORC1, CST3 and ADD3) and subsequently employed multivariate Cox analysis to develop a signature and validate the model. Lastly, the correlation between CAFRG and malignant features indicates the potential role of CAFRG in promoting tumour angiogenesis, EMT and cell cycle alterations. We constructed a CAF prognostic signature for identifying potential prognostic CAFRGs and predicting the prognosis and immunotherapeutic response for LUSC. Our study may provide a more accurate prognostic assessment and immunotherapy targeting strategies for LUSC., (© 2024 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Aim: To place the consequences of calcineurin inhibition in a cardiovascular context., Methods: Literature review coupled with personal encounters., Results: Calcineurin is a calcium-binding and calmodulin-binding protein that is conserved across evolution from yeast to mammals. The enzyme functions as a calcium-dependent, calmodulin-stimulated protein phosphatase. Its role in regulating physiology has largely been elucidated by observing calcineurin inhibition. Calcineurin inhibition transformed organ transplantation from an experiment into a therapy and made much of general immunotherapy possible. The function of this phosphatase and how its inhibition leads to toxicity concern us to this date. Initial research from patients and animal models implicated a panoply of factors contributing to hypertension and vasculopathy. Subsequently, the role of calcineurin in regulating the effective fluid volume, sodium reabsorption, and potassium and hydrogen ion excretion was elucidated by investigating calcineurin inhibition. Understanding the regulatory effects of calcineurin on endothelial and vascular smooth muscle cell function has also made substantial progress. However, precisely how the increase in systemic vascular resistance arises requires further mechanistic research., Conclusion: Calcineurin inhibition continues to save lives; however, options to counteract the negative effects of calcineurin inhibition should be vigorously pursued., (© 2024 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

As a key regulator of development, organ size, tissue homeostasis and cancer, the Hippo pathway is tightly regulated by various growth-related signaling events. Among them, energy stress activates the Hippo pathway to inhibit its downstream effector YAP1. Our recent work reported a YAP1-independent role of the Hippo pathway in promoting macroautophagy/autophagy and cell survival in response to energy stress, a process mediated by Hippo kinase MAP4K2. MAP4K2 interacts with and phosphorylates MAP1LC3A/LC3A at S87, which in turn drives autophagosome-lysosome fusion via the RAB3GAP-RAB18 axis. Energy stress activates MAP4K2 by reducing its association with the Hippo phosphatase complex STRIPAK component STRN4. Moreover, MAP4K2 is highly expressed in head and neck cancer, while MAP4K2 and its mediated autophagy are required for head and neck cancer development. Taken together, our study not only reveals a noncanonical role of the Hippo pathway in energy stress response, but also suggests Hippo kinase MAP4K2 as a potential therapeutic target for head and neck cancer treatment. Abbreviation: AMPK: 5'-AMP-activated protein kinase; Atg8: autophagy related 8; LATS1: large tumor suppressor 1; LIR: microtubule-associated protein 1 light chain 3-interacting region; MAP1LC3A/LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP4K2: mitogen-activated protein kinase kinase kinase kinase 2; PPP2/PP2A: protein phosphatase 2; RAB3GAP: RAB3 GTPase activating protein; RAB18: RAB18, member RAS oncogene family; SLMAP: sarcolemma associated protein; STK3/MST2: serine/threonine kinase 3; STK4/MST1: serine/threonine kinase 4; STRIPAK: striatin-interacting phosphatase and kinase; STRN4: striatin, calmodulin binding protein 4; SQSTM1/p62: sequestosome 1; TEAD: TEA domain family member; ULK1: unc-51 like kinase 1; WWTR1/TAZ: WW domain containing transcription regulator 1; YAP1: yes-associated protein 1.

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies and frequent cause of cancer-related death worldwide. Long non-coding RNAs (lncRNAs) play regulatory roles and serve as biomarkers of multiple cancers, including ESCC. Our previous studies have confirmed that lncRNA Kinectin 1 antisense RNA 1 (KTN1-AS1) is highly expressed in ESCC and exerts oncogene function through RBBP4/HDAC1 complex., Objective: Our present study focused on exploring a novel molecular mechanism of KTN1-AS1 in ESCC., Methods: In this study, qRT-PCR assay, Western blot assay, Luciferase reporter assay, and RNA immunoprecipitation assay were conducted., Results: We found that KTN1-AS1 could bind to miR-885-5p in ESCC cells, and miR-885-5p was low expressed in ESCC. Overexpression of miR-885-5p inhibited esophageal cancer cells proliferation and invasion in vitro. Mechanistic analysis demonstrated that miR-885-5p specifically targeted striatin 3 (STRN3), and KTN1-AS1/miR-885-5p promoted the EMT process by Hippo pathway in STRN3/YAP1 dependent manner., Conclusion: To sum up, KTN1-AS1 facilitates ESCC progression by acting as a ceRNA for miR-885-5p to regulate STRN3 expression and the Hippo pathway, and KTN1-AS1 maybe used as a promising therapeutic target for ESCC., (© 2023. The Author(s) under exclusive licence to The Genetics Society of Korea.)

J proteins are a diverse family of co-chaperones that cooperate with heat shock protein 70 (Hsp70) to coordinate protein quality control, especially in response to cellular stress. Current models suggest that individual J proteins might play roles in recruiting Hsp70s to specific functions, such as maintaining cell wall integrity or promoting ribosome biogenesis. However, relatively few stresses have been used to test this model and, as a result, only a few specific activities have been identified. To expand our understanding of the J protein network, we used a synthetic lethal approach in which 11 Saccharomyces cerevisiae deletion strains were treated with 12 well-characterized chemical inhibitors. The results defined new roles for specific J proteins in major signaling pathways. For example, an important role for Swa2 in cell wall integrity was identified and activities of the under-explored Jjj1, Apj1, Jjj3 and Caj1 proteins were suggested. More generally, these findings support a model in which some J proteins, such as Ydj1 and Zuo1, play "generalist" roles, while others, such as Apj1 and Jjj2, are "specialists", having roles in relatively few pathways. Together, these results provide new insight into the network of J proteins.

17β-Estradiol (E2) is the main estrogenic hormone in the body and exerts many cardiovascular protective effects. Via three receptors known to date, including estrogen receptors α (ERα) and β (ERβ) and the G protein-coupled estrogen receptor 1 (GPER, aka GPR30), E2 regulates numerous calcium-dependent activities in cardiovascular tissues. Nevertheless, effects of E2 and its receptors on components of the calcium signaling machinery (CSM), the underlying mechanisms, and the linked functional impact are only beginning to be elucidated. A picture is emerging of the reciprocality between estrogen biology and Ca2+ signaling. Therein, E2 and GPER, via both E2-dependent and E2-independent actions, moderate Ca2+-dependent activities; in turn, ERα and GPER are regulated by Ca2+ at the receptor level and downstream signaling via a feedforward loop. This article reviews current understanding of the effects of E2 and its receptors on the cardiovascular CSM and vice versa with a focus on mechanisms and combined functional impact. An overview of the main CSM components in cardiovascular tissues will be first provided, followed by a brief review of estrogen receptors and their Ca2+-dependent regulation. The effects of estrogenic agonists to stimulate acute Ca2+ signals will then be reviewed. Subsequently, E2-dependent and E2-independent effects of GPER on components of the Ca2+ signals triggered by other stimuli will be discussed. Finally, a case study will illustrate how the many mechanisms are coordinated to moderate Ca2+-dependent activities in the cardiovascular system.

Microtubules are nucleated in vivo by gamma-tubulin complexes. The 300-kDa gamma-tubulin small complex (gamma-TuSC), consisting of two molecules of gamma-tubulin and one copy each of the accessory proteins Spc97 and Spc98, is the conserved, essential core of the microtubule nucleating machinery. In metazoa multiple gamma-TuSCs assemble with other proteins into gamma-tubulin ring complexes (gamma-TuRCs). The structure of gamma-TuRC indicated that it functions as a microtubule template. Because each gamma-TuSC contains two molecules of gamma-tubulin, it was assumed that the gamma-TuRC-specific proteins are required to organize gamma-TuSCs to match 13-fold microtubule symmetry. Here we show that Saccharomyces cerevisiae gamma-TuSC forms rings even in the absence of other gamma-TuRC components. The yeast adaptor protein Spc110 stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins. The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

Microtubules are nucleated in vivo by gamma-tubulin complexes. The 300-kDa gamma-tubulin small complex (gamma-TuSC), consisting of two molecules of gamma-tubulin and one copy each of the accessory proteins Spc97 and Spc98, is the conserved, essential core of the microtubule nucleating machinery. In metazoa multiple gamma-TuSCs assemble with other proteins into gamma-tubulin ring complexes (gamma-TuRCs). The structure of gamma-TuRC indicated that it functions as a microtubule template. Because each gamma-TuSC contains two molecules of gamma-tubulin, it was assumed that the gamma-TuRC-specific proteins are required to organize gamma-TuSCs to match 13-fold microtubule symmetry. Here we show that Saccharomyces cerevisiae gamma-TuSC forms rings even in the absence of other gamma-TuRC components. The yeast adaptor protein Spc110 stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins. The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

Adducins are cytoskeletal actin-binding proteins (alpha, beta, gamma) that function as heterodimers and heterotetramers and are encoded by distinct genes. Experimental and clinical evidence implicates alpha- and beta-adducin variants in hypertension and renal dysfunction. Here, we have addressed the role of alpha- and beta-adducin on glomerular function and disease using beta-adducin null mice, congenic substrains for alpha- and beta-adducin from the Milan hypertensive (MHS) and Milan normotensive (MNS) rats and patients with IgA nephropathy. Targeted deletion of beta-adducin in mice reduced urinary protein excretion, preceded by an increase of podocyte protein expression (phospho-nephrin, synaptopodin, alpha-actinin, ZO-1, Fyn). The introgression of polymorphic MHS beta-adducin locus into MNS (Add2, 529R) rats was associated with an early reduction of podocyte protein expression (nephrin, synaptopodin, alpha-actinin, ZO-1, podocin, Fyn), followed by severe glomerular and interstitial lesions and increased urinary protein excretion. These alterations were markedly attenuated when the polymorphic MHS alpha-adducin locus was also present (Add1, 316Y). In patients with IgA nephropathy, the rate of decline of renal function over time was associated to polymorphic beta-adducin (ADD2, 1797T, rs4984) with a significant interaction with alpha-adducin (ADD1, 460W, rs4961). These findings suggest that adducin genetic variants participate in the development of glomerular lesions by modulating the expression of specific podocyte proteins.

Adducins are cytoskeletal actin-binding proteins (alpha, beta, gamma) that function as heterodimers and heterotetramers and are encoded by distinct genes. Experimental and clinical evidence implicates alpha- and beta-adducin variants in hypertension and renal dysfunction. Here, we have addressed the role of alpha- and beta-adducin on glomerular function and disease using beta-adducin null mice, congenic substrains for alpha- and beta-adducin from the Milan hypertensive (MHS) and Milan normotensive (MNS) rats and patients with IgA nephropathy. Targeted deletion of beta-adducin in mice reduced urinary protein excretion, preceded by an increase of podocyte protein expression (phospho-nephrin, synaptopodin, alpha-actinin, ZO-1, Fyn). The introgression of polymorphic MHS beta-adducin locus into MNS (Add2, 529R) rats was associated with an early reduction of podocyte protein expression (nephrin, synaptopodin, alpha-actinin, ZO-1, podocin, Fyn), followed by severe glomerular and interstitial lesions and increased urinary protein excretion. These alterations were markedly attenuated when the polymorphic MHS alpha-adducin locus was also present (Add1, 316Y). In patients with IgA nephropathy, the rate of decline of renal function over time was associated to polymorphic beta-adducin (ADD2, 1797T, rs4984) with a significant interaction with alpha-adducin (ADD1, 460W, rs4961). These findings suggest that adducin genetic variants participate in the development of glomerular lesions by modulating the expression of specific podocyte proteins.

The Saccharomyces cerevisiae centrosome or spindle pole body (SPB) is a dynamic structure that is remodeled in a cell cycle dependent manner. The SPB increases in size late in the cell cycle and during most cell cycle arrests and exchanges components during G1/S. We identified proteins involved in the remodeling process using a strain in which SPB remodeling is conditionally induced. This strain was engineered to express a modified SPB component, Spc110, which can be cleaved upon the induction of a protease. Using a synthetic genetic array analysis, we screened for genes required only when Spc110 cleavage is induced. Candidate SPB remodeling factors fell into several functional categories: mitotic regulators, microtubule motors, protein modification enzymes, and nuclear pore proteins. The involvement of candidate genes in SPB assembly was assessed in three ways: by identifying the presence of a synthetic growth defect when combined with an Spc110 assembly defective mutant, quantifying growth of SPBs during metaphase arrest, and comparing distribution of SPB size during asynchronous growth. These secondary screens identified four genes required for SPB remodeling: NUP60, POM152, and NCS2 are required for SPB growth during a mitotic cell cycle arrest, and UBC4 is required to maintain SPB size during the cell cycle. These findings implicate the nuclear pore, urmylation, and ubiquitination in SPB remodeling and represent novel functions for these genes.

17β-Estradiol (E2) is the main estrogenic hormone in the body and exerts many cardiovascular protective effects. Via three receptors known to date, including estrogen receptors α (ERα) and β (ERβ) and the G protein-coupled estrogen receptor 1 (GPER, aka GPR30), E2 regulates numerous calcium-dependent activities in cardiovascular tissues. Nevertheless, effects of E2 and its receptors on components of the calcium signaling machinery (CSM), the underlying mechanisms, and the linked functional impact are only beginning to be elucidated. A picture is emerging of the reciprocality between estrogen biology and Ca2+ signaling. Therein, E2 and GPER, via both E2-dependent and E2-independent actions, moderate Ca2+-dependent activities; in turn, ERα and GPER are regulated by Ca2+ at the receptor level and downstream signaling via a feedforward loop. This article reviews current understanding of the effects of E2 and its receptors on the cardiovascular CSM and vice versa with a focus on mechanisms and combined functional impact. An overview of the main CSM components in cardiovascular tissues will be first provided, followed by a brief review of estrogen receptors and their Ca2+-dependent regulation. The effects of estrogenic agonists to stimulate acute Ca2+ signals will then be reviewed. Subsequently, E2-dependent and E2-independent effects of GPER on components of the Ca2+ signals triggered by other stimuli will be discussed. Finally, a case study will illustrate how the many mechanisms are coordinated to moderate Ca2+-dependent activities in the cardiovascular system. [ABSTRACT FROM AUTHOR]