Transient proliferation by reversible YAP and mitogen-control of the cyclin D1/p27 ratio.

Hippo-YAP signaling orchestrates epithelial tissue repair and is therefore an attractive target in regenerative medicine. Yet it is unresolved how YAP integrates with mitogen signaling and contact inhibition to control the underlying transient proliferative response. Here we show that reduced contact inhibition, increased mitogen signaling, and YAP-TEAD activation converge on increasing the nuclear cyclin D1/p27 protein ratio during G1 phase, towards a threshold ratio that dictates whether individual cells enter or exit the cell cycle.

Context-dependent regulation of lipid accumulation in adipocytes by a HIF1α-PPARγ feedback network.

Hypoxia-induced upregulation of HIF1α triggers adipose tissue dysfunction and insulin resistance in obese patients. HIF1α closely interacts with PPARγ, the master regulator of adipocyte differentiation and lipid accumulation, but there are conflicting results regarding how this interaction controls the excessive lipid accumulation that drives adipocyte dysfunction. To directly address these conflicts, we established a differentiation system that recapitulated prior seemingly opposing observations made across different experimental settings.

Early enforcement of cell identity by a functional component of the terminally differentiated state.

How progenitor cells can attain a distinct differentiated cell identity is a challenging problem given the fluctuating signaling environment in which cells exist and that critical transcription factors are often not unique to a differentiation process. Here, we test the hypothesis that a unique differentiated cell identity can result from a core component of the differentiated state doubling up as a signaling protein that also drives differentiation. Using live single-cell imaging in the adipocyte differentiation system, we show that progenitor fat cells (preadipocytes) can only commit to terminally differentiate after up-regulating FABP4, a lipid buffer that is highly enriched in mature adipocytes.

Cortisol and cardiometabolic disease: a target for advancing health equity.

Stress, in both intrinsic psychosocial and extrinsic physical environmental forms, can impact the development of, and outcomes in, cardiovascular disease (CVD) through allostatic load. Cortisol is a core hormonal mediator of allostatic load produced in response to various stresses. Alterations in morning serum cortisol and daily diurnal cortisol have been associated with adiposity, dyslipidemia, incident diabetes, and CVDs such as hypertension.

The circadian clock mediates daily bursts of cell differentiation by periodically restricting cell-differentiation commitment.

Most mammalian cells have an intrinsic circadian clock that coordinates metabolic activity with the daily rest and wake cycle. The circadian clock is known to regulate cell differentiation, but how continuous daily oscillations of the internal clock can control a much longer, multiday differentiation process is not known. Here, we simultaneously monitor circadian clock and adipocyte-differentiation progression live in single cells.

Flattening of circadian glucocorticoid oscillations drives acute hyperinsulinemia and adipocyte hypertrophy.

Disruption of circadian glucocorticoid oscillations in Cushing's disease and chronic stress results in obesity and adipocyte hypertrophy, which is believed to be a main source of the harmful effects of obesity. Here, we recapitulate stress due to jet lag or work-life imbalances by flattening glucocorticoid oscillations in mice. Within 3 days, mice achieve a metabolic state with persistently high insulin, but surprisingly low glucose and fatty acids in the bloodstream, that precedes a more than 2-fold increase in brown and white adipose tissue mass within 3 weeks.

Molecular Competition in G1 Controls When Cells Simultaneously Commit to Terminally Differentiate and Exit the Cell Cycle.

Terminal differentiation is essential for the development and maintenance of tissues in all multi-cellular organisms and is associated with permanent exit from the cell cycle. Failure to permanently exit the cell cycle can result in cancer and disease. However, the molecular mechanisms and timing that coordinate differentiation commitment and cell cycle exit are not yet understood.

Matrix stiffness induces a tumorigenic phenotype in mammary epithelium through changes in chromatin accessibility.

In breast cancer, the increased stiffness of the extracellular matrix is a key driver of malignancy. Yet little is known about the epigenomic changes that underlie the tumorigenic impact of extracellular matrix mechanics. Here, we show in a three-dimensional culture model of breast cancer that stiff extracellular matrix induces a tumorigenic phenotype through changes in chromatin state.

Expression variation and covariation impair analog and enable binary signaling control.

Due to noise in the synthesis and degradation of proteins, the concentrations of individual vertebrate signaling proteins were estimated to vary with a coefficient of variation (CV) of approximately 25% between cells. Such high variation is beneficial for population-level regulation of cell functions but abolishes accurate single-cell signal transmission. Here, we measure cell-to-cell variability of relative protein abundance using quantitative proteomics of individual eggs and cultured human cells and show that variation is typically much lower, in the range of 5-15%, compatible with accurate single-cell transmission.

A Transcriptional Circuit Filters Oscillating Circadian Hormonal Inputs to Regulate Fat Cell Differentiation.

Glucocorticoid and other adipogenic hormones are secreted in mammals in circadian oscillations. Loss of this circadian oscillation pattern correlates with obesity in humans, raising the intriguing question of how hormone secretion dynamics affect adipocyte differentiation. Using live, single-cell imaging of the key adipogenic transcription factors CEBPB and PPARG, endogenously tagged with fluorescent proteins, we show that pulsatile circadian hormone stimuli are rejected by the adipocyte differentiation control system.

Heterogeneous Ribosomes Preferentially Translate Distinct Subpools of mRNAs Genome-wide.

Emerging studies have linked the ribosome to more selective control of gene regulation. However, an outstanding question is whether ribosome heterogeneity at the level of core ribosomal proteins (RPs) exists and enables ribosomes to preferentially translate specific mRNAs genome-wide. Here, we measured the absolute abundance of RPs in translating ribosomes and profiled transcripts that are enriched or depleted from select subsets of ribosomes within embryonic stem cells.

Measuring Gli2 Phosphorylation by Selected Reaction Monitoring Mass Spectrometry.

Phosphorylation is an important mechanism by which Gli proteins are regulated. When the Hedgehog (Hh) pathway is activated, multiple serine and threonine residues of Gli2 are dephosphorylated, while at least one residue undergoes phosphorylation. These changes in phosphorylation have functional relevance for the transcriptional activity of Gli proteins, as shown by in vitro and in vivo assays on Gli mutants lacking the phosphorylated residues.

A dynamic picture of protein behavior in cells.

Protein dynamics in livings cells can now be studied by fully automated, high-throughput fluorescence correlation spectroscopy.

Using SRM-MS to quantify nuclear protein abundance differences between adipose tissue depots of insulin-resistant mice.

Insulin resistance (IR) underlies metabolic disease. Visceral, but not subcutaneous, white adipose tissue (WAT) has been linked to the development of IR, potentially due to differences in regulatory protein abundance. Here we investigate how protein levels are changed in IR in different WAT depots by developing a targeted proteomics approach to quantitatively compare the abundance of 42 nuclear proteins in subcutaneous and visceral WAT from a commonly used insulin-resistant mouse model, Lepr(db/db), and from C57BL/6J control mice.

The proteome of cholesteryl-ester-enriched versus triacylglycerol-enriched lipid droplets.

Within cells, lipids are stored in the form of lipid droplets (LDs), consisting of a neutral lipid core, surrounded by a phospholipid monolayer and an outer layer of protein. LDs typically accumulate either triacylglycerol (TAG) and diacylglycerol or cholesteryl ester (CE), depending on the type of tissue. Recently, there has been an increased interest in the proteins that surround LDs.

Controlling low rates of cell differentiation through noise and ultrahigh feedback.

Mammalian tissue size is maintained by slow replacement of de-differentiating and dying cells. For adipocytes, key regulators of glucose and lipid metabolism, the renewal rate is only 10% per year. We used computational modeling, quantitative mass spectrometry, and single-cell microscopy to show that cell-to-cell variability, or noise, in protein abundance acts within a network of more than six positive feedbacks to permit pre-adipocytes to differentiate at very low rates.

Gli protein activity is controlled by multisite phosphorylation in vertebrate Hedgehog signaling.

Gli proteins are transcriptional effectors of the Hedgehog (Hh) pathway in both normal development and cancer. We describe a program of multisite phosphorylation that regulates the conversion of Gli proteins into transcriptional activators. In the absence of Hh ligands, Gli activity is restrained by the direct phosphorylation of six conserved serine residues by protein kinase A (PKA), a master negative regulator of the Hh pathway.

The E3 ubiquitin ligase UBE3C enhances proteasome processivity by ubiquitinating partially proteolyzed substrates.

To maintain protein homeostasis, cells must balance protein synthesis with protein degradation. Accumulation of misfolded or partially degraded proteins can lead to the formation of pathological protein aggregates. Here we report the use of destabilizing domains, proteins whose folding state can be reversibly tuned using a high affinity ligand, as model substrates to interrogate cellular protein quality control mechanisms in mammalian cells using a forward genetic screen.

Consecutive positive feedback loops create a bistable switch that controls preadipocyte-to-adipocyte conversion.

Adipogenesis, or the conversion of proliferating preadipocytes into nondividing adipocytes, is an important part of the vertebrate weight-maintenance program. It is not yet understood how and when an irreversible transition occurs into a distinct state capable of accumulating lipid. Here, we use single-cell fluorescence imaging to show that an all-or-none switch is induced before lipid accumulation occurs.

Neuropilins are positive regulators of Hedgehog signal transduction.

The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. Here we show that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development.

Parallel adaptive feedback enhances reliability of the Ca2+ signaling system.

Despite large cell-to-cell variations in the concentrations of individual signaling proteins, cells transmit signals correctly. This phenomenon raises the question of what signaling systems do to prevent a predicted high failure rate. Here we combine quantitative modeling, RNA interference, and targeted selective reaction monitoring (SRM) mass spectrometry, and we show for the ubiquitous and fundamental calcium signaling system that cells monitor cytosolic and endoplasmic reticulum (ER) Ca(2+) levels and adjust in parallel the concentrations of the store-operated Ca(2+) influx mediator stromal interaction molecule (STIM), the plasma membrane Ca(2+) pump plasma membrane Ca-ATPase (PMCA), and the ER Ca(2+) pump sarco/ER Ca(2+)-ATPase (SERCA).

Comprehensive identification of PIP3-regulated PH domains from C. elegans to H. sapiens by model prediction and live imaging.

Phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol(3,4,5)-trisphosphate (PIP3) control cell growth, migration, and other processes by recruiting proteins with pleckstrin homology (PH) domains and possibly other domains to the plasma membrane (PM). However, previous experimental and structural work with PH domains left conflicting evidence about which ones are PIP3 regulated. Here we used live-cell confocal imaging of 130 YFP-conjugated mouse PH domains and found that 20% translocated to the PM in response to receptor-generated PIP3 production.

siRNA screen of the human signaling proteome identifies the PtdIns(3,4,5)P3-mTOR signaling pathway as a primary regulator of transferrin uptake.

Background: Iron uptake via endocytosis of iron-transferrin-transferrin receptor complexes is a rate-limiting step for cell growth, viability and proliferation in tumor cells as well as non-transformed cells such as activated lymphocytes. Signaling pathways that regulate transferrin uptake have not yet been identified.

Results: We surveyed the human signaling proteome for regulators that increase or decrease transferrin uptake by screening 1,804 dicer-generated signaling small interfering RNAs using automated quantitative imaging.

Rab10, a target of the AS160 Rab GAP, is required for insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane.

GLUT4 trafficking to the plasma membrane of muscle and fat cells is regulated by insulin. An important component of insulin-regulated GLUT4 distribution is the Akt substrate AS160 rab GTPase-activating protein. Here we show that Rab10 functions as a downstream target of AS160 in the insulin-signaling pathway that regulates GLUT4 translocation in adipocytes.

Single cell imaging of PI3K activity and glucose transporter insertion into the plasma membrane by dual color evanescent wave microscopy.

Many signaling events involve the translocation of signaling molecules to or from the plasma membrane; however, suitable techniques to quantify the temporal relationships between such signaling events are lacking. Here, we describe an evanescent wave microscopy technique that allows parallel measurement of the recruitment and dissociation of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) labeled proteins to and from the plasma membrane in individual living cells. The selective excitation of fluorescence in a zone less than 100 nm above a cover glass enables selective imaging within the plasma membrane of adherent cells, with markedly improved resolution, lower background, and minimal phototoxicity compared to confocal microscopy and other microscopy-based assays.