Calcium Signaling Mechanisms Across Kingdoms.

Calcium (Ca2+) is a unique mineral that serves as both a nutrient and a signal in all eukaryotes. To maintain Ca2+ homeostasis for both nutrition and signaling purposes, the tool kit for Ca2+ transport has expanded across kingdoms of eukaryotes to encode specific Ca2+ signals referred to as Ca2+ signatures. In parallel, a large array of Ca2+-binding proteins has evolved as specific sensors to decode Ca2+ signatures. By comparing these coding and decoding mechanisms in fungi, animals, and plants, both unified and divergent themes have emerged, and the underlying complexity will challenge researchers for years to come. Considering the scale and breadth of the subject, instead of a literature survey, in this review we focus on a conceptual framework that aims to introduce readers to the principles and mechanisms of Ca2+ signaling. We finish with several examples of Ca2+-signaling pathways, including polarized cell growth, immunity and symbiosis, and systemic signaling, to piece together specific coding and decoding mechanisms in plants versus animals.

mTOR Signaling in Growth, Metabolism, and Disease.

The mechanistic target of rapamycin (mTOR) coordinates eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors. Extensive research over the past two decades has established a central role for mTOR in regulating many fundamental cell processes, from protein synthesis to autophagy, and deregulated mTOR signaling is implicated in the progression of cancer and diabetes, as well as the aging process. Here, we review recent advances in our understanding of mTOR function, regulation, and importance in mammalian physiology. We also highlight how the mTOR signaling network contributes to human disease and discuss the current and future prospects for therapeutically targeting mTOR in the clinic.

Eukaryotic cell size regulation and its implications for cellular function and dysfunction.

Depending on cell type, environmental inputs, and disease, the cells in the human body can have widely different sizes. In recent years, it became clear that cell size is a major regulator of cell function. However, we are only beginning to understand how optimization of cell function determines a given cell's optimal size. Here, we review currently known size control strategies of eukaryotic cells, and the intricate link of cell size to intracellular biomolecular scaling, organelle homeostasis and cell cycle progression. We detail the cell size dependent regulation of early development and the impact of cell size on cell differentiation. Given the importance of cell size for normal cellular physiology, cell size control must account for changing environmental conditions. We describe how cells sense environmental stimuli, such as nutrient availability, and accordingly adapt their size by regulating cell growth and cell cycle progression. Moreover, we discuss the correlation of pathological states with misregulation of cell size, and how for a long time, this was considered a downstream consequence of cellular dysfunction. We review newer studies that reveal a reversed causality, with misregulated cell size leading to pathophysiological phenotypes such as senescence and aging. In summary, we highlight important roles of cell size in cellular function and dysfunction, which could have major implications for both diagnostics and treatment in the clinic.

Genome homeostasis defects drive enlarged cells into senescence.

Cellular senescence refers to an irreversible state of cell-cycle arrest and plays important roles in aging and cancer biology. Because senescence is associated with increased cell size, we used reversible cell-cycle arrests combined with growth rate modulation to study how excessive growth affects proliferation. We find that enlarged cells upregulate p21, which limits cell-cycle progression. Cells that re-enter the cell cycle encounter replication stress that is well tolerated in physiologically sized cells but causes severe DNA damage in enlarged cells, ultimately resulting in mitotic failure and permanent cell-cycle withdrawal. We demonstrate that enlarged cells fail to recruit 53BP1 and other non-homologous end joining (NHEJ) machinery to DNA damage sites and fail to robustly initiate DNA damage-dependent p53 signaling, rendering them highly sensitive to genotoxic stress. We propose that an impaired DNA damage response primes enlarged cells for persistent replication-acquired damage, ultimately leading to cell division failure and permanent cell-cycle exit.

Oncogenic signals prime cancer cells for toxic cell overgrowth during a G1 cell cycle arrest.

CDK4/6 inhibitors are remarkable anti-cancer drugs that can arrest tumor cells in G1 and induce their senescence while causing only relatively mild toxicities in healthy tissues. How they achieve this mechanistically is unclear. We show here that tumor cells are specifically vulnerable to CDK4/6 inhibition because during the G1 arrest, oncogenic signals drive toxic cell overgrowth. This overgrowth causes permanent cell cycle withdrawal by either preventing progression from G1 or inducing genotoxic damage during the subsequent S-phase and mitosis. Inhibiting or reverting oncogenic signals that converge onto mTOR can rescue this excessive growth, DNA damage, and cell cycle exit in cancer cells. Conversely, inducing oncogenic signals in non-transformed cells can drive these toxic phenotypes and sensitize the cells to CDK4/6 inhibition. Together, this demonstrates that cell cycle arrest and oncogenic cell growth is a synthetic lethal combination that is exploited by CDK4/6 inhibitors to induce tumor-specific toxicity.

CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence.

Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2- breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients.

mTORC1 stimulates cell growth through SAM synthesis and m6A mRNA-dependent control of protein synthesis.

The mechanistic target of rapamycin complex 1 (mTORC1) regulates metabolism and cell growth in response to nutrient, growth, and oncogenic signals. We found that mTORC1 stimulates the synthesis of the major methyl donor, S-adenosylmethionine (SAM), through the control of methionine adenosyltransferase 2 alpha (MAT2A) expression. The transcription factor c-MYC, downstream of mTORC1, directly binds to intron 1 of MAT2A and promotes its expression. Furthermore, mTORC1 increases the protein abundance of Wilms' tumor 1-associating protein (WTAP), the positive regulatory subunit of the human N6-methyladenosine (m6A) RNA methyltransferase complex. Through the control of MAT2A and WTAP levels, mTORC1 signaling stimulates m6A RNA modification to promote protein synthesis and cell growth. A decline in intracellular SAM levels upon MAT2A inhibition decreases m6A RNA modification, protein synthesis rate, and tumor growth. Thus, mTORC1 adjusts m6A RNA modification through the control of SAM and WTAP levels to prime the translation machinery for anabolic cell growth.

LATS1 controls CTCF chromatin occupancy and hormonal response of 3D-grown breast cancer cells.

The cancer epigenome has been studied in cells cultured in two-dimensional (2D) monolayers, but recent studies highlight the impact of the extracellular matrix and the three-dimensional (3D) environment on multiple cellular functions. Here, we report the physical, biochemical, and genomic differences between T47D breast cancer cells cultured in 2D and as 3D spheroids. Cells within 3D spheroids exhibit a rounder nucleus with less accessible, more compacted chromatin, as well as altered expression of ~2000 genes, the majority of which become repressed. Hi-C analysis reveals that cells in 3D are enriched for regions belonging to the B compartment, have decreased chromatin-bound CTCF and increased fusion of topologically associating domains (TADs). Upregulation of the Hippo pathway in 3D spheroids results in the activation of the LATS1 kinase, which promotes phosphorylation and displacement of CTCF from DNA, thereby likely causing the observed TAD fusions. 3D cells show higher chromatin binding of progesterone receptor (PR), leading to an increase in the number of hormone-regulated genes. This effect is in part mediated by LATS1 activation, which favors cytoplasmic retention of YAP and CTCF removal.

GA dynamics governing nodulation revealed using GIBBERELLIN PERCEPTION SENSOR 2 in Medicago truncatula lateral organs.

During nutrient scarcity, plants can adapt their developmental strategy to maximize their chance of survival. Such plasticity in development is underpinned by hormonal regulation, which mediates the relationship between environmental cues and developmental outputs. In legumes, endosymbiosis with nitrogen fixing bacteria (rhizobia) is a key adaptation for supplying the plant with nitrogen in the form of ammonium. Rhizobia are housed in lateral root-derived organs termed nodules that maintain an environment conducive to Nitrogenase in these bacteria. Several phytohormones are important for regulating the formation of nodules, with both positive and negative roles proposed for gibberellin (GA). In this study, we determine the cellular location and function of bioactive GA during nodule organogenesis using a genetically-encoded second generation GA biosensor, GIBBERELLIN PERCEPTION SENSOR 2 in Medicago truncatula. We find endogenous bioactive GA accumulates locally at the site of nodule primordia, increasing dramatically in the cortical cell layers, persisting through cell divisions and maintaining accumulation in the mature nodule meristem. We show, through mis-expression of GA catabolic enzymes that suppress GA accumulation, that GA acts as a positive regulator of nodule growth and development. Furthermore, increasing or decreasing GA through perturbation of biosynthesis gene expression can increase or decrease the size of nodules, respectively. This is unique from lateral root formation, a developmental program that shares common organogenesis regulators. We link GA to a wider gene regulatory program by showing that nodule-identity genes induce and sustain GA accumulation necessary for proper nodule formation.

How total mRNA influences cell growth.

Experimental observations tracing back to the 1960s imply that ribosome quantities play a prominent role in determining a cell's growth. Nevertheless, in biologically relevant scenarios, growth can also be influenced by the levels of mRNA and RNA polymerase. Here, we construct a quantitative model of biosynthesis providing testable scenarios for these situations. The model explores a theoretically motivated regime where RNA polymerases compete for genes and ribosomes for transcripts and gives general expressions relating growth rate, mRNA concentrations, ribosome, and RNA polymerase levels. On general grounds, the model predicts how the fraction of ribosomes in the proteome depends on total mRNA concentration and inspects an underexplored regime in which the trade-off between transcript levels and ribosome abundances sets the cellular growth rate. In particular, we show that the model predicts and clarifies three important experimental observations, in budding yeast and Escherichia coli bacteria: i) that the growth-rate cost of unneeded protein expression can be affected by mRNA levels, ii) that resource optimization leads to decreasing trends in mRNA levels at slow growth, and iii) that ribosome allocation may increase, stay constant, or decrease, in response to transcription-inhibiting antibiotics. Since the data indicate that a regime of joint limitation may apply in physiological conditions and not only to perturbations, we speculate that this regime is likely self-imposed.

Sfp1 regulates transcriptional networks driving cell growth and division through multiple promoter-binding modes.

The yeast Sfp1 protein regulates both cell division and growth but how it coordinates these processes is poorly understood. We demonstrate that Sfp1 directly controls genes required for ribosome production and many other growth-promoting processes. Remarkably, the complete set of Sfp1 target genes is revealed only by a combination of ChIP (chromatin immunoprecipitation) and ChEC (chromatin endogenous cleavage) methods, which uncover two promoter binding modes, one requiring a cofactor and the other a DNA-recognition motif. Glucose-regulated Sfp1 binding at cell cycle "START" genes suggests that Sfp1 controls cell size by coordinating expression of genes implicated in mass accumulation and cell division.

Regulatory elements coordinating initiation of chromosome replication to the Escherichia coli cell cycle.

Escherichia coli coordinates replication and division cycles by initiating replication at a narrow range of cell sizes. By tracking replisomes in individual cells through thousands of division cycles in wild-type and mutant strains, we were able to compare the relative importance of previously described control systems. We found that accurate triggering of initiation does not require synthesis of new DnaA. The initiation size increased only marginally as DnaA was diluted by growth after dnaA expression had been turned off. This suggests that the conversion of DnaA between its active ATP- and inactive ADP-bound states is more important for initiation size control than the total free concentration of DnaA. In addition, we found that the known ATP/ADP converters DARS and datA compensate for each other, although the removal of them makes the initiation size more sensitive to the concentration of DnaA. Only disruption of the regulatory inactivation of DnaA mechanism had a radical impact on replication initiation. This result was corroborated by the finding that termination of one round of replication correlates with the next initiation at intermediate growth rates, as would be the case if RIDA-mediated conversion from DnaA-ATP to DnaA-ADP abruptly stops at termination and DnaA-ATP starts accumulating.

Increased expression of long non-coding RNA FIRRE promotes hepatocellular carcinoma by HuR-CyclinD1 axis signaling.

There is a critical need to understand the disease processes and identify improved therapeutic strategies for hepatocellular carcinoma (HCC). The long noncoding RNAs (lncRNAs) display diverse effects on biological regulations. The aim of this study was to identify a lncRNA as a potential biomarker of HCC and investigate the mechanisms by which the lncRNA promotes HCC progression using human cell lines and in vivo. Using RNA-Seq analysis, we found that lncRNA FIRRE was significantly upregulated in hepatitis C virus (HCV) associated liver tissue and identified that lncRNA FIRRE is significantly upregulated in HCV-associated HCC compared to adjacent non-tumor liver tissue. Further, we observed that FIRRE is significantly upregulated in HCC specimens with other etiologies, suggesting this lncRNA has the potential to serve as an additional biomarker for HCC. Overexpression of FIRRE in hepatocytes induced cell proliferation, colony formation, and xenograft tumor formation as compared to vector-transfected control cells. Using RNA pull-down proteomics, we identified HuR as an interacting partner of FIRRE. We further showed that the FIRRE-HuR axis regulates cyclin D1 expression. Our mechanistic investigation uncovered that FIRRE is associated with an RNA-binding protein HuR for enhancing hepatocyte growth. Together, these findings provide molecular insights into the role of FIRRE in HCC progression.

ZNT5-6 and ZNT7 play an integral role in protein N-glycosylation by supplying Zn2+ to Golgi α-mannosidase II.

The stepwise addition of monosaccharides to N-glycans attached to client proteins to generate a repertoire of mature proteins involves a concerted action of many glycosidases and glycosyltransferases. Here, we report that Golgi α-mannosidase II (GMII), a pivotal enzyme catalyzing the first step in the conversion of hybrid- to complex-type N-glycans, is activated by Zn2+ supplied by the early secretory compartment-resident ZNT5-ZNT6 heterodimers (ZNT5-6) and ZNT7 homodimers (ZNT7). Loss of ZNT5-6 and ZNT7 function results in marked accumulation of hybrid-type and complex/hybrid glycans with concomitant reduction of complex- and high-mannose-type glycans. In cells lacking the ZNT5-6 and ZNT7 functions, the GMII activity is substantially decreased. In contrast, the activity of its homolog, lysosomal mannosidase (LAMAN), is not decreased. Moreover, we show that the growth of pancreatic cancer MIA PaCa-2 cells lacking ZNT5-6 and ZNT7 is significantly decreased in a nude mouse xenograft model. Our results indicate the integral roles of ZNT5-6 and ZNT7 in N-glycosylation and highlight their potential as novel target proteins for cancer therapy.

Knockout of endoplasmic reticulum-localized molecular chaperone HSP90.7 impairs seedling development and cellular auxin homeostasis in Arabidopsis.

The Arabidopsis endoplasmic reticulum-localized heat shock protein HSP90.7 modulates tissue differentiation and stress responses; however, complete knockout lines have not been previously reported. In this study, we identified and analyzed a mutant allele, hsp90.7-1, which was unable to accumulate the HSP90.7 full-length protein and showed seedling lethality. Microscopic analyses revealed its essential role in male and female fertility, trichomes and root hair development, proper chloroplast function, and apical meristem maintenance and differentiation. Comparative transcriptome and proteome analyses also revealed the role of the protein in a multitude of cellular processes. Particularly, the auxin-responsive pathway was specifically downregulated in the hsp90.7-1 mutant seedlings. We measured a much-reduced auxin content in both root and shoot tissues. Through comprehensive histological and molecular analyses, we confirmed PIN1 and PIN5 accumulations were dependent on the HSP90 function, and the TAA-YUCCA primary auxin biosynthesis pathway was also downregulated in the mutant seedlings. This study therefore not only fulfilled a gap in understanding the essential role of HSP90 paralogs in eukaryotes but also provided a mechanistic insight on the ER-localized chaperone in regulating plant growth and development via modulating cellular auxin homeostasis.

The binding affinity-dependent inhibition of cell growth and viability by DNA sulfur-binding domains.

Increasing evidence suggests that DNA phosphorothioate (PT) modification serves several purposes in the bacterial host, and some restriction enzymes specifically target PT-DNA. PT-dependent restriction enzymes (PDREs) bind PT-DNA through their DNA sulfur binding domain (SBD) with dissociation constants (KD) of 5 nM~1 µM. Here, we report that SprMcrA, a PDRE, failed to dissociate from PT-DNA after cleavage due to high binding affinity, resulting in low DNA cleavage efficiency. Expression of SBDs in Escherichia coli cells with PT modification induced a drastic loss of cell viability at 25°C when both DNA strands of a PT site were bound, with one SBD on each DNA strand. However, at this temperature, SBD binding to only one PT DNA strand elicited a severe growth lag rather than lethality. This cell growth inhibition phenotype was alleviated by raising the growth temperature. An in vitro assay mimicking DNA replication and RNA transcription demonstrated that the bound SBD hindered the synthesis of new DNA and RNA when using PT-DNA as the template. Our findings suggest that DNA modification-targeting proteins might regulate cellular processes involved in DNA metabolism in addition to being components of restriction-modification systems and epigenetic readers.

Coordinated activities of Myosin Vb isoforms and mTOR signaling regulate epithelial cell morphology during development.

The maintenance of epithelial architecture necessitates tight regulation of cell size and shape. However, mechanisms underlying epithelial cell size regulation remain poorly understood. We show that the interaction of Myosin Vb with Rab11 prevents the accumulation of apically derived endosomes to maintain cell-size, whereas that with Rab10 regulates vesicular transport from the trans-Golgi. These interactions are required for the fine-tuning of the epithelial cell morphology during zebrafish development. Furthermore, the compensatory cell growth upon cell-proliferation inhibition involves a preferential expansion of the apical domain, leading to flatter epithelial cells, an efficient strategy to cover the surface with fewer cells. This apical domain growth requires post-trans-Golgi transport mediated by the Rab10-interacting Myosin Vb isoform, downstream of the mTOR-Fatty Acid Synthase (FASN) axis. Changes in trans-Golgi morphology indicate that the Golgi synchronizes mTOR-FASN-regulated biosynthetic input and Myosin Vb-Rab10 dependent output. Our study unravels the mechanism of polarized growth in epithelial cells and delineates functions of Myosin Vb isoforms in cell size regulation during development.

UBA5 inhibition restricts lung adenocarcinoma via blocking macrophage M2 polarization and cisplatin resistance.

UBA5, a ubiquitin-like activated enzyme involved in ufmylation and sumoylation, presents a viable target for pancreatic and breast cancer treatments, yet its role in lung adenocarcinoma (LUAD) remains underexplored. This study reveals UBA5's tumor-promoting effect in LUAD, as evidenced by its upregulation in patients and positive correlation with TNM stages. Elevated UBA5 levels predict poor outcomes for these patients. Pharmacological inhibition of UBA5 using DKM 2-93 significantly curtails the growth of A549, H1299, and cisplatin-resistant A549 (A549/DDP) LUAD cells in vitro. Additionally, UBA5 knockdown via shRNA lentivirus suppresses tumor growth both in vitro and in vivo. High UBA5 expression adversely alters the tumor immune microenvironment, affecting immunostimulators, MHC molecules, chemokines, receptors, and immune cell infiltration. Notably, UBA5 expression correlates positively with M2 macrophage infiltration, the predominant immune cells in LUAD. Co-culture experiments further demonstrate that UBA5 knockdown directly inhibits M2 macrophage polarization and lactate production in LUAD. Moreover, in vivo studies show reduced M2 macrophage infiltration following UBA5 knockdown. UBA5 expression is also associated with increased tumor heterogeneity, including tumor mutational burden, microsatellite instability, neoantigen presence, and homologous recombination deficiency. Experiments indicate that UBA5 overexpression promotes cisplatin resistance in vitro, whereas UBA5 inhibition enhances cisplatin sensitivity in both in vitro and in vivo settings. Overall, these findings suggest that targeting UBA5 inhibits LUAD by impeding cancer cell proliferation, M2 macrophage polarization, and cisplatin resistance.

PALB2-mutated human mammary cells display a broad spectrum of morphological and functional abnormalities induced by increased TGFß signaling.

Heterozygous mutations in any of three major genes, BRCA1, BRCA2 and PALB2, are associated with high-risk hereditary breast cancer susceptibility frequently seen as familial disease clustering. PALB2 is a key interaction partner and regulator of several vital cellular activities of BRCA1 and BRCA2, and is thus required for DNA damage repair and alleviation of replicative and oxidative stress. Little is however known about how PALB2-deficiency affects cell function beyond that, especially in the three-dimensional setting, and also about its role during early steps of malignancy development. To answer these questions, we have generated biologically relevant MCF10A mammary epithelial cell lines with mutations that are comparable to certain clinically important PALB2 defects. We show in a non-cancerous background how both mono- and biallelically PALB2-mutated cells exhibit gross spontaneous DNA damage and mitotic aberrations. Furthermore, PALB2-deficiency disturbs three-dimensional spheroid morphology, increases the migrational capacity and invasiveness of the cells, and broadly alters their transcriptome profiles. TGFß signaling and KRT14 expression are enhanced in PALB2-mutated cells and their inhibition and knock down, respectively, lead to partial restoration of cell functions. KRT14-positive cells are also more abundant with DNA damage than KRT14-negative cells. The obtained results indicate comprehensive cellular changes upon PALB2 mutations, even in the presence of half dosage of wild type PALB2 and demonstrate how PALB2 mutations may predispose their carriers to malignancy.

The mTORC2 signaling network: targets and cross-talks.

The mechanistic target of rapamycin, mTOR, controls cell metabolism in response to growth signals and stress stimuli. The cellular functions of mTOR are mediated by two distinct protein complexes, mTOR complex 1 (mTORC1) and mTORC2. Rapamycin and its analogs are currently used in the clinic to treat a variety of diseases and have been instrumental in delineating the functions of its direct target, mTORC1. Despite the lack of a specific mTORC2 inhibitor, genetic studies that disrupt mTORC2 expression unravel the functions of this more elusive mTOR complex. Like mTORC1 which responds to growth signals, mTORC2 is also activated by anabolic signals but is additionally triggered by stress. mTORC2 mediates signals from growth factor receptors and G-protein coupled receptors. How stress conditions such as nutrient limitation modulate mTORC2 activation to allow metabolic reprogramming and ensure cell survival remains poorly understood. A variety of downstream effectors of mTORC2 have been identified but the most well-characterized mTORC2 substrates include Akt, PKC, and SGK, which are members of the AGC protein kinase family. Here, we review how mTORC2 is regulated by cellular stimuli including how compartmentalization and modulation of complex components affect mTORC2 signaling. We elaborate on how phosphorylation of its substrates, particularly the AGC kinases, mediates its diverse functions in growth, proliferation, survival, and differentiation. We discuss other signaling and metabolic components that cross-talk with mTORC2 and the cellular output of these signals. Lastly, we consider how to more effectively target the mTORC2 pathway to treat diseases that have deregulated mTOR signaling.