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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. YAP increases this ratio indirectly, in concert with mitogen signaling, by increasing EGFR and other receptors that signal primarily through ERK. After a delay, contact inhibition suppresses YAP activity which gradually downregulates mitogen signaling and the cyclin D1/p27 ratio. Increasing YAP activity by ablating the suppressor Merlin/NF2 reveals a balancing mechanism in which YAP suppression and contact inhibition of proliferation can be recovered but only at higher local cell density. Thus, critical for tissue repair, robust proliferation responses result from the YAP-induced and receptor-mediated prolonged increase in the cyclin D1/p27 ratio, which is only reversed by delayed suppression of receptor signaling after contact inhibition of YAP., Competing Interests: Declaration of interests The authors declare no competing interests.

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. Using single-cell imaging and coarse-grained mathematical modeling, we show how HIF1α can both promote and repress lipid accumulation during adipogenesis. Our model predicted and our experiments confirmed that the opposing roles of HIF1α are isolated from each other by the positive-feedback-mediated upregulation of PPARγ that drives adipocyte differentiation. Finally, we identify three factors: strength of the differentiation cue, timing of hypoxic perturbation, and strength of HIF1α expression changes that, when considered together, provide an explanation for many of the previous conflicting reports., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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. Upon induction of adipogenesis in mouse preadipocyte cells, we show that after a long delay, cells first abruptly start to engage a positive feedback between CEBPA and PPARG before then engaging, after a second delay, a positive feedback between FABP4 and PPARG. These sequential positive feedbacks both need to engage in order to drive PPARG levels past the threshold for irreversible differentiation. In the last step before commitment, PPARG transcriptionally increases FABP4 expression while fatty acid-loaded FABP4 increases PPARG activity. Together, our study suggests a control principle for robust cell identity whereby a core component of the differentiated state also promotes differentiation from its own progenitor state., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Bahrami-Nejad et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 review examines the role of cortisol as a key mechanistic link between stress physiology and cardiometabolic disease. Importantly, we discuss the role of targeting cortisol through pharmacological, behavioral, and environmental interventions to advance health equity in cardiometabolic disease., Competing Interests: Declaration of interests The authors have no conflicts of interest to declare., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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. Strikingly, we find a bursting behavior in the cell population whereby individual preadipocytes commit to differentiate primarily during a 12-h window each day, corresponding to the time of rest. Daily gating occurs because cells irreversibly commit to differentiate within only a few hours, which is much faster than the rest phase and the overall multiday differentiation process. The daily bursts in differentiation commitment result from a differentiation-stimulus driven variable and slow increase in expression of PPARG, the master regulator of adipogenesis, overlaid with circadian boosts in PPARG expression driven by fast, clock-driven PPARG regulators such as CEBPA. Our finding of daily bursts in cell differentiation only during the circadian cycle phase corresponding to evening in humans is broadly relevant, given that most differentiating somatic cells are regulated by the circadian clock. Having a restricted time each day when differentiation occurs may open therapeutic strategies to use timed treatment relative to the clock to promote tissue regeneration.

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. Transcriptomic and Cd36-knockout mouse analyses show that hyperinsulinemia-mediated de novo fatty acid synthesis and Cd36-mediated fatty acid uptake drive fat mass increases. Intriguingly, this mechanism by which glucocorticoid flattening causes acute hyperinsulinemia and adipocyte hypertrophy is unexpectedly beneficial in preventing high levels of circulating fatty acids and glucose for weeks, thus serving as a protective response to preserve metabolic health during chronic stress., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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. Using live, single-cell imaging of cell cycle progression and differentiation commitment during adipogenesis, we show that a rapid switch mechanism engages exclusively in G1 to trigger differentiation commitment simultaneously with permanent exit from the cell cycle. We identify a molecular competition in G1 between when the differentiation switch is triggered and when the proliferative window closes that allows mitogen and differentiation stimuli to control the balance between terminally differentiating cells produced and progenitor cells kept in reserve, a parameter of critical importance for enabling proper development of tissue domains and organs., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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. We found that increased stiffness yielded cells with more wrinkled nuclei and with increased lamina-associated chromatin, that cells cultured in stiff matrices displayed more accessible chromatin sites, which exhibited footprints of Sp1 binding, and that this transcription factor acts along with the histone deacetylases 3 and 8 to regulate the induction of stiffness-mediated tumorigenicity. Just as cell culture on soft environments or in them rather than on tissue-culture plastic better recapitulates the acinar morphology observed in mammary epithelium in vivo, mammary epithelial cells cultured on soft microenvironments or in them also more closely replicate the in vivo chromatin state. Our results emphasize the importance of culture conditions for epigenomic studies, and reveal that chromatin state is a critical mediator of mechanotransduction.

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 Xenopus laevis 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. Focusing on bimodal ERK signaling, we show that variation and covariation in MEK and ERK expression improves controllability of the percentage of activated cells, demonstrating how variation and covariation in expression enables population-level control of binary cell-fate decisions. Together, our study argues for a control principle whereby low expression variation enables accurate control of analog single-cell signaling, while increased variation, covariation, and numbers of pathway components are required to widen the stimulus range over which external inputs regulate binary cell activation to enable precise control of the fraction of activated cells in a population., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

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. We find that heterogeneity in RP composition endows ribosomes with differential selectivity for translating subpools of transcripts, including those controlling metabolism, cell cycle, and development. As an example, mRNAs enriched in binding to RPL10A/uL1-containing ribosomes are shown to require RPL10A/uL1 for their efficient translation. Within several of these transcripts, this level of regulation is mediated, at least in part, by internal ribosome entry sites. Together, these results reveal a critical functional link between ribosome heterogeneity and the post-transcriptional circuitry of gene expression., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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 most differentially expressed proteins were important in adipogenesis, as confirmed by siRNA-mediated depletion experiments, suggesting a defect in adipogenesis in visceral, but not subcutaneous, insulin-resistant WAT. Furthermore, differentiation of visceral, but not subcutaneous, insulin-resistant stromal vascular cells (SVCs) was impaired. In an in vitro approach to understand the cause of this impaired differentiation, we compared insulin-resistant visceral SVCs to preadipocyte cell culture models made insulin resistant by different stimuli. The insulin-resistant visceral SVC protein abundance profile correlated most with preadipocyte cell culture cells treated with both palmitate and TNFα. Together, our study introduces a method to simultaneously measure and quantitatively compare nuclear protein expression patterns in primary adipose tissue and adipocyte cell cultures, which we show can reveal relationships between differentiation and disease states of different adipocyte tissue types.

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. Here, we describe a method of quantitatively monitoring the phosphorylation of Gli proteins by triple quadrupole mass spectrometry of Gli2 immunoprecipitated from cell lysates. This method is broadly applicable to the monitoring of phosphorylation changes of immunoprecipitated Gli proteins when the putative phosphosites are known.

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. LD proteins have been found to be quite diverse, from structural proteins to metabolic enzymes, proteins involved in vesicular transport, and proteins that may play a role in LD formation. Previous proteomics analyses have focused on TAG-enriched LDs, whereas CE-enriched LDs have been largely ignored. Our study has compared the LD proteins from CE-enriched LDs to TAG-enriched LDs in steroidogenic cells. In primary rat granulosa cells loaded with either HDL to produce CE-enriched LDs or fatty acids to produce TAG-enriched LDs, 61 proteins were found to be elevated in CE-enriched LDs and 40 proteins elevated in TAG-enriched LDs with 278 proteins in similar amounts. Protein expression was further validated by selected reaction monitoring (SRM) mass spectrometry (MS). SRM verified expression of 25 of 27 peptides that were previously detected by tandem mass tagging MS. Several proteins were confirmed to be elevated in CE-enriched LDs by SRM including the intermediate filament vimentin. This study is the first to compare the proteins found on CE-enriched LDs with TAG-enriched LDs and constitutes the first step in creating a better understanding of the proteins found on CE-enriched LDs in steroidogenic cells.

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. This reconciles two fundamental opposing requirements: High cell-to-cell signal variability is needed to generate very low differentiation rates, whereas low signal variability is needed to prevent differentiated cells from de-differentiating. Higher eukaryotes can thus control low rates of near irreversible cell fate decisions through a balancing act between noise and ultrahigh feedback connectivity., (Copyright © 2014, American Association for the Advancement of Science.)

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. Activation of signaling leads to a global remodeling of the Gli phosphorylation landscape: the PKA target sites become dephosphorylated, while a second cluster of sites undergoes phosphorylation. The pattern of Gli phosphorylation can regulate Gli transcriptional activity in a graded fashion, suggesting a phosphorylation-based mechanism for how a gradient of Hh signaling in a morphogenetic field can be converted into a gradient of transcriptional activity., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

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. Upon knockdown of UBE3C, an E3 ubiquitin ligase, a reporter protein consisting of a destabilizing domain fused to GFP is degraded more slowly and incompletely by the proteasome. Partial proteolysis is also observed when UBE3C is present but cannot ubiquitinate substrates because its active site has been mutated, it is unable to bind to the proteasome, or the substrate lacks lysine residues. UBE3C knockdown also results in less substrate polyubiquitination. Finally, knockdown renders cells more susceptible to the Hsp90 inhibitor 17-AAG, suggesting that UBE3C protects against the harmful accumulation of protein fragments arising from incompletely degraded proteasome substrates.

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. Conversion begins by glucocorticoid and cAMP signals raising C/EBPβ levels above a critical threshold, triggering three consecutive positive feedback loops: from PPARγ to C/EBPα, then to C/EBPβ, and last to the insulin receptor. Experiments and modeling show that these feedbacks create a robust, irreversible transition to a terminally differentiated state by rejecting short- and low-amplitude stimuli. After the differentiation switch is triggered, insulin controls fat accumulation in a graded fashion. Altogether, our study introduces a regulatory motif that locks cells in a differentiated state by engaging a sequence of positive feedback loops., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

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. Using cell lines lacking key Hh pathway components, we show that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish, as we show that morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance our knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental 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). Model calculations show that this combined parallel regulation in protein expression levels effectively stabilizes basal cytosolic and ER Ca(2+) levels and preserves receptor signaling. Our results demonstrate that, rather than directly controlling the relative level of signaling proteins in a forward regulation strategy, cells prevent transmission failure by sensing the state of the signaling pathway and using multiple parallel adaptive feedbacks.

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. We developed a recursive-learning algorithm to predict PIP3 regulation of 1200 PH domains from different eukaryotes and validated that it accurately predicts PIP3 regulation. Strikingly, this algorithm showed that PIP3 regulation is specified by amino acids across the PH domain, not just the PIP3-binding pocket, and must have evolved several times independently from PIP3-insensitive ancestral PH domains. Finally, our algorithm and live-cell experiments provide a functional survey of PH domains in different species, showing that PI3K regulation increased from approximately two C. elegans and four Drosophila to 40 vertebrate proteins.

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. Overexpression of a mutant of Rab10 defective for GTP hydrolysis increased GLUT4 on the surface of basal adipocytes. Rab10 knockdown resulted in an attenuation of insulin-induced GLUT4 redistribution to the plasma membrane and a concomitant 2-fold decrease in GLUT4 exocytosis rate. Re-expression of a wild-type Rab10 restored normal GLUT4 translocation. The basal increase in plasma-membrane GLUT4 due to AS160 knockdown was partially blocked by knocking down Rab10 in the same cells, further indicating that Rab10 is a target of AS160 and a positive regulator of GLUT4 trafficking to the cell surface upon insulin stimulation.

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. In addition to known transport proteins, we identified 11 signaling proteins that included a striking signature set for the phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3)-target of rapamycin (mTOR) signaling pathway. We show that the PI3K-mTOR signaling pathway is a positive regulator of transferrin uptake that increases the number of transferrin receptors per endocytic vesicle without affecting endocytosis or recycling rates., Conclusion: Our study identifies the PtdIns(3,4,5)P3-mTOR signaling pathway as a new regulator of iron-transferrin uptake and serves as a proof-of-concept that targeted RNA interference screens of the signaling proteome provide a powerful and unbiased approach to discover or rank signaling pathways that regulate a particular cell function.

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. In the microscope design we have developed, the beams from helium-cadmium (442 nm) and argon (514 nm) lasers are merged and focused through a dove prism at an angle that yields total internal reflection. In this configuration, evanescent wave-excited fluorescence at the glass-water interface can be detected with either high or low magnification, to allow for high-resolution imaging or the study of many cells in parallel. We applied this technique to make parallel measurements of the time-course of insulin-triggered activation of phosphatidylinositol 3-kinase (PI3K) and GLUT4 glucose transporter insertion into the plasma membrane of individual differentiated 3T3L1 adipocytes using a phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P(3)]-binding pleckstrin homology domain fused to CFP, and GLUT4 conjugated to YFP. The technique should have wide applicability to various cell types and diverse signaling processes.

Receptor-triggered signaling processes exhibit complex cross-talk and feedback interactions, with many signaling proteins and second messengers acting locally within the cell. The flow of information in this input-output system can only be understood by tracking where and when local signaling activities are induced. Systematic strategies are therefore needed to measure the localization and translocation of all signaling proteins, and to develop fluorescent biosensors that can track local signaling activities in individual cells. Such a biosensor tool chest can be based on two types of green fluorescent protein constructs that either translocate or undergo fluorescence-resonance-energy transfer when local signaling occurs. Broad strategies to measure quantitative, dynamic parameters in signaling networks, together with perturbation approaches, are needed to develop comprehensive models of signaling networks*.

Time courses of translocation of fluorescently conjugated proteins to the plasma membrane were simultaneously measured in thousands of individual rat basophilic leukemia cells. We found that the C2 domain---a calcium-sensing, lipid-binding protein module that is an essential regulator of protein kinase C and numerous other proteins---targeted proteins to the plasma membrane transiently if calcium was released from internal stores, and persistently in response to entry of extracellular calcium across the plasma membrane. The C2 domain translocation time courses of stimulated cells clustered into only two primary modes. Hence, the reversible recruitment of families of signaling proteins from one cellular compartment to another is a rapid bifurcation mechanism for inducing discrete states of cellular signaling networks.

Background: Glutamate-induced Ca2+ oscillations and waves coordinate astrocyte signaling responses, which in turn regulate neuronal excitability. Recent studies have suggested that the generation of these Ca2+ oscillations requires a negative feedback that involves the activation of conventional protein kinase C (cPKC). Here, we use total internal reflection fluorescence (TIRF) microscopy to investigate if and how periodic plasma membrane translocation of cPKC is used to generate Ca2+ oscillations and waves., Results: Glutamate stimulation of astrocytes triggered highly localized GFP-PKCgamma plasma membrane translocation events, induced rapid oscillations in GFP-PKCgamma translocation, and generated GFP-PKCgamma translocation waves that propagated across and between cells. These translocation responses were primarily mediated by the Ca2+-sensitive C2 domains of PKCgamma and were driven by localized Ca2+ spikes, by oscillations in Ca2+ concentration, and by propagating Ca(2+) waves, respectively. Interestingly, GFP-conjugated C1 domains from PKCgamma or PKCdelta that have been shown to bind diacylglycerol (DAG) also oscillated between the cytosol and the plasma membrane after glutamate stimulation, suggesting that PKC is repetitively activated by combined oscillating increases in Ca(2+) and DAG concentrations. The expression of C1 domains, which increases the DAG buffering capacity and thereby delays changes in DAG concentrations, led to a marked prolongation of Ca(2+) spikes, suggesting that PKC activation is involved in terminating individual Ca(2+) spikes and waves and in defining the time period between Ca(2+) spikes., Conclusions: Our study suggests that cPKCs have a negative feedback role on Ca(2+) oscillations and waves that is mediated by their repetitive activation by oscillating DAG and Ca(2+) concentrations. Periodic translocation and activation of cPKC can be a rapid and markedly localized signaling event that can limit the duration of individual Ca(2+) spikes and waves and can define the Ca(2+) spike and wave frequencies.

Synaptic plasticity is thought to be a key process for learning, memory and other cognitive functions of the nervous system. The initial events of plasticity require the conversion of brief electrical signals into alterations of the biochemical properties of synapses that last for much longer than the initial stimuli. Here we show that a regulator of synaptic plasticity, calcium/calmodulin-dependent protein kinase IIalpha (CaMKII), sequentially translocates to postsynaptic sites, undergoes autophosphorylation and gets trapped for several minutes until its dissociation is induced by secondary autophosphorylation and phosphatase 1 action. Once dissociated, CaMKII shows facilitated translocation for several minutes. This suggests that trapping of CaMKII by its targets and priming of CaMKII translocation may function as biochemical memory mechanisms that change the signaling capacity of synapses.

Background: Many targets of calcium signaling pathways are activated or inhibited by binding the Ca(2+)-liganded form of calmodulin (Ca(2+)-CaM). Here, we test the hypothesis that local Ca(2+)-CaM-regulated signaling processes can be selectively activated by local intracellular differences in free Ca(2+)-CaM concentration., Results: Energy-transfer confocal microscopy of a fluorescent biosensor was used to measure the difference in the concentration of free Ca(2+)-CaM between nucleus and cytoplasm. Strikingly, short receptor-induced calcium spikes produced transient increases in free Ca(2+)-CaM concentration that were of markedly higher amplitude in the cytosol than in the nucleus. In contrast, prolonged increases in calcium led to equalization of the nuclear and cytosolic free Ca(2+)-CaM concentrations over a period of minutes. Photobleaching recovery and translocation measurements with fluorescently labeled CaM showed that equalization is likely to be the result of a diffusion-mediated net translocation of CaM into the nucleus. The driving force for equalization is a higher Ca(2+)-CaM-buffering capacity in the nucleus compared with the cytosol, as the direction of the free Ca(2+)-CaM concentration gradient and of CaM translocation could be reversed by expressing a Ca(2+)-CaM-binding protein at high concentration in the cytosol., Conclusions: Subcellular differences in the distribution of Ca(2+)-CaM-binding proteins can produce gradients of free Ca(2+)-CaM concentration that result in a net translocation of CaM. This provides a mechanism for dynamically regulating local free Ca(2+)-CaM concentrations, and thus the local activity of Ca(2+)-CaM targets. Free Ca(2+)-CaM signals in the nucleus remain low during brief or low-frequency calcium spikes, whereas high-frequency spikes or persistent increases in calcium cause translocation of CaM from the cytoplasm to the nucleus, resulting in similar concentrations of nuclear and cytosolic free Ca(2+)-CaM.

The application of molecular techniques to cultured central nervous system (CNS) neurons has been limited by a lack of simple and efficient methods to introduce macromolecules into their cytosol. We have developed an electroporation technique that efficiently transfers RNA, DNA and other large membrane-impermeant molecules into adherent hippocampal neurons. Microporation allowed the use of either in vitro transcribed RNA or cDNA to transfect neurons. While RNA transfection yielded a higher percentage of transfected neurons and produced quantitative co-expression of two proteins, DNA transfection yielded higher levels of protein expression. Dextran-based calcium indicators also were efficiently delivered into the cytosol. Microporated neurons appear to survive poration quite well, as indicated by their morphological integrity, electrical excitability, ability to produce action potential-evoked calcium signals, and intact synaptic transmission. Furthermore, green fluorescent protein (GFP)-tagged marker proteins were expressed and correctly localized to the cytosol, plasma membrane, or endoplasmic reticulum. The microporation method is efficient, convenient, and inexpensive: macromolecules can be introduced into most adherent neurons in a 3 mm2 surface area while requiring as little as 1 microl of the material to be introduced. We conclude that the microporation of macromolecules is a versatile approach to investigate signaling, secretion, and other processes in CNS neurons.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine protein kinase that regulates long-term potentiation and other forms of neuronal plasticity. Functional differences between the neuronal CaMKIIalpha and CaMKIIbeta isoforms are not yet known. Here, we use green fluorescent protein-tagged (GFP-tagged) CaMKII isoforms and show that CaMKIIbeta is bound to F-actin in dendritic spines and cell cortex while CaMKIIalpha is largely a cytosolic enzyme. When expressed together, the two isoforms form large heterooligomers, and a small fraction of CaMKIIbeta is sufficient to dock the predominant CaMKIIalpha to the actin cytoskeleton. Thus, CaMKIIbeta functions as a targeting module that localizes a much larger number of CaMKIIalpha isozymes to synaptic and cytoskeletal sites of action.

Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.

Electroporation is a widely used method for introducing macromolecules into cells. We developed an electroporation device that requires only 1 microl of sample to load adherent cells in a 10-mm2 surface area while retaining greater than 90% cell survivability. To better understand this device, field-induced permeabilization of adherent rat basophilic leukemia and neocortical neuroblastoma cells was investigated by using fluorescent calcium and voltage indicators. Rectangular field pulses led to the formation of only a few calcium entry sites, preferentially in the hyperpolarized parts of the cell body and processes. Individual entry sites were formed at the same locations when field pulses were repeated. Before calcium entry, a partial breakdown of the membrane potential was observed in both polar regions. Based on our results, a model is proposed for the formation and closure of macromolecule entry sites in adherent cells. First, the rapid formation of a large number of small pores leads to a partial membrane potential breakdown in both polar regions of the cell. Second, over tens of milliseconds, a few entry sites for macromolecules are formed, preferentially in the hyperpolarized part of cell body and processes, at locations defined by the local membrane structure. These entry sites reseal on a time scale of 50 ms to several seconds, with residual small pores remaining open for several minutes.