Mary Teruel is an Assistant Professor of Chemical and Systems Biology and, by Courtesy, of Bioengineering at Stanford University. She received a B.S. degree in Mechanical Engineering from the University of Pennsylvania and an M.S. and Ph.D. in Aeronautical Engineering from Stanford. She has been working at the interface of engineering, medicine, and biology for over 15 years - designing, building, and implementing new quantitative microscopy, proteomic, and computational tools to understand fundamental principles in cell differentiation and tissue regeneration, especially in the context of obesity, diabetes, cardiovascular disease, and cancer. Her current research focuses on understanding how timed application and synergy of hormones, particular in the context of circadian rhythms, can be harnessed to dynamically control tissue regeneration and to restore healthy tissue function in vivo. The Teruel Lab uses mesenchymal stem cell differentiation into the fat lineage (stem cell differentiation, adipogenesis) as model systems.
Ph.D., Stanford University, Aeronautical Engineering
M.S., Stanford University, Aeronautical Engineering
B.S., University of Pennsylvannia, Mechanical Engineering
Current Research and Scholarly Interests
The Teruel Lab uses a combination of engineering and biological approaches including high-throughput screening of RNAi and DNA construct libraries, CRISPR libraries, targeted mass spectrometry, live-cell fluorescence microscopy, and bioinformatics to investigate the systems biology of cell differentiation and tissue renegeneration, with a particular focus on uncovering the molecular mechanisms underlying insulin resistance, diabetes, and obesity.
- Research Seminar
CSB 270 (Aut, Win, Spr)
Independent Studies (8)
- Directed Reading in Biophysics
BIOPHYS 399 (Win, Spr)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Sum)
- Directed Reading in Chemical and Systems Biology
CSB 299 (Aut, Win, Spr, Sum)
- Graduate Research
BIOPHYS 300 (Win, Spr)
- Graduate Research
CBIO 399 (Aut, Sum)
- Graduate Research
CSB 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
CSB 370 (Aut, Win, Spr, Sum)
- Undergraduate Research
CSB 199 (Aut, Win, Spr, Sum)
- Directed Reading in Biophysics
Prior Year Courses
- Research Seminar
CSB 270 (Aut, Win, Spr)
- Chemical and Systems Biology Bootcamp
CSB 201 (Aut)
- Imaging: Biological Light Microscopy
BIO 152, CSB 222, MCP 222 (Spr)
- Research Seminar
CSB 270 (Aut, Win, Spr)
- Research Seminar
Loss of adipocyte identity through synergistic repression of PPARγ by TGF-β and mechanical stress
View details for DOI 10.1101/604231
A terminal cell differentiation decision based on a tug-of-war between differentiation and mitogenic signals during an extended G1 period
View details for DOI 10.1101/632570
A Transcriptional Circuit Filters Oscillating Circadian Hormonal Inputs to Regulate Fat Cell Differentiation.
2018; 27 (4): 854–68.e8
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. In striking contrast, equally strong persistent signals trigger maximal differentiation. We identify the mechanism of how hormone oscillations are filtered as a combination of slow and fast positive feedback centered on PPARG. Furthermore, we confirm in mice that flattening of daily glucocorticoid oscillations significantly increases the mass of subcutaneous and visceral fat pads. Together, our study provides a molecular mechanism for why stress, Cushing's disease, and other conditions for which glucocorticoid secretion loses its pulsatility may lead to obesity.
View details for PubMedID 29617644
Expression variation and covariation impair analog and enable binary signaling control.
Molecular systems biology
2018; 14 (5): e7997
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.
View details for PubMedID 29759982
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. 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.
View details for PubMedID 28625553
Using SRM-MS to quantify nuclear protein abundance differences between adipose tissue depots of insulin-resistant mice
JOURNAL OF LIPID RESEARCH
2015; 56 (5): 1068-1078
Insulin resistance underlies metabolic disease. Visceral, but not subcutaneous, white adipose tissue (WAT) has been linked to the development of insulin resistance, potentially due to differences in regulatory protein abundance. Here we investigate how protein levels are changed in insulin resistance 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 (SVC) was impaired. In an in vitro approach to understand the cause of this impaired differentiation, we compared insulin-resistant visceral SVC 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.
View details for DOI 10.1194/jlr.D056317
View details for Web of Science ID 000353767200012
View details for PubMedID 25840986
Controlling low rates of cell differentiation through noise and ultrahigh feedback.
2014; 344 (6190): 1384-1389
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.
View details for DOI 10.1126/science.1252079
View details for PubMedID 24948735
View details for PubMedCentralID PMC4733388
Consecutive Positive Feedback Loops Create a Bistable Switch that Controls Preadipocyte-to-Adipocyte Conversion
2012; 2 (4): 976-990
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.
View details for DOI 10.1016/j.celrep.2012.08.038
View details for Web of Science ID 000314455600027
View details for PubMedID 23063366
Parallel adaptive feedback enhances reliability of the Ca2+ signaling system
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (35): 14485-14490
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.
View details for DOI 10.1073/pnas.1018266108
View details for Web of Science ID 000294425900029
View details for PubMedID 21844332
View details for PubMedCentralID PMC3167526
Matrix stiffness induces a tumorigenic phenotype in mammary epithelium through changes in chromatin accessibility.
Nature biomedical engineering
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.
View details for DOI 10.1038/s41551-019-0420-5
View details for PubMedID 31285581
- A dynamic picture of protein behavior in cells. Nature biotechnology 2015; 33 (4): 356-357
Measuring Gli2 Phosphorylation by Selected Reaction Monitoring Mass Spectrometry.
Methods in molecular biology (Clifton, N.J.)
2015; 1322: 105-123
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.
View details for DOI 10.1007/978-1-4939-2772-2_10
View details for PubMedID 26179043
Gli protein activity is controlled by multisite phosphorylation in vertebrate hedgehog signaling.
2014; 6 (1): 168-181
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.
View details for DOI 10.1016/j.celrep.2013.12.003
View details for PubMedID 24373970
View details for PubMedCentralID PMC3915062
- The proteome of cholesteryl-ester-enriched versus triacylglycerol-enriched lipid droplets. PloS one 2014; 9 (8)
The E3 ubiquitin ligase UBE3C enhances proteasome processivity by ubiquitinating partially proteolyzed substrates.
journal of biological chemistry
2013; 288 (48): 34575-34587
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.
View details for DOI 10.1074/jbc.M113.499350
View details for PubMedID 24158444
View details for PubMedCentralID PMC3843071
Neuropilins are positive regulators of Hedgehog signal transduction
GENES & DEVELOPMENT
2011; 25 (22): 2333-2346
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.
View details for DOI 10.1101/gad.173054.111
View details for PubMedID 22051878
Comprehensive identification of PIP3-regulated PH domains from C elegans to H sapiens by model prediction and live imaging
2008; 30 (3): 381-392
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.
View details for DOI 10.1016/j.molcel.2008.04.008
View details for Web of Science ID 000255761200015
View details for PubMedID 18471983
View details for PubMedCentralID PMC3523718
Rab10, a target of the AS160 rab GAP, is required for insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane
2007; 5 (4): 293-303
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.
View details for DOI 10.1016/j.cmet.2007.03.001
View details for Web of Science ID 000245484100008
View details for PubMedID 17403373
siRNA screen of the human signaling proteome identifies the PtdIns(3,4,5) P-3-mTOR signaling pathway as a primary regulator of transferrin uptake
2007; 8 (7)
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.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.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.
View details for DOI 10.1186/gb-2007-8-7-r142
View details for Web of Science ID 000249416400022
View details for PubMedID 17640392
View details for PubMedCentralID PMC2323231
Single cell imaging of PI3K activity and glucose transporter insertion into the plasma membrane by dual color evanescent wave microscopy.
Science's STKE : signal transduction knowledge environment
2003; 2003 (169): PL4-?
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.
View details for PubMedID 12582202
Fluorescence imaging of signaling networks
TRENDS IN CELL BIOLOGY
2003; 13 (2): 101-106
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*.
View details for Web of Science ID 000180957300008
View details for PubMedID 12559761
Parallel single-cell monitoring of receptor-triggered membrane translocation of a calcium-sensing protein module
2002; 295 (5561): 1910-1912
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.
View details for Web of Science ID 000174299500045
View details for PubMedID 11884760
Control of astrocyte Ca2+ oscillations and waves by oscillating translocation and activation of protein kinase C
2001; 11 (14): 1089-1097
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.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.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.
View details for Web of Science ID 000170093700018
View details for PubMedID 11509231
Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis
JOURNAL OF CELL BIOLOGY
2000; 151 (7): 1353-1367
Phagocytosis requires localized and transient remodeling of actin filaments. Phosphoinositide signaling is believed to play an important role in cytoskeletal organization, but it is unclear whether lipids, which can diffuse along the membrane, can mediate the focal actin assembly required for phagocytosis. We used imaging of fluorescent chimeras of pleckstrin homology and C1 domains in live macrophages to monitor the distribution of phosphatidylinositol-4,5-bisphosphate (4,5-PIP(2)) and diacylglycerol, respectively, during phagocytosis. Our results reveal a sequence of exquisitely localized, coordinated steps in phospholipid metabolism: a focal, rapid accumulation of 4,5-PIP(2) accompanied by recruitment of type Ialpha phosphatidylinositol phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals. Loss of 4,5-PIP(2) correlated with mobilization of phospholipase Cgamma (PLCgamma) and with the localized formation of diacylglycerol. The presence of 4, 5-PIP(2) and active PLCgamma at the phagosome was shown to be essential for effective particle ingestion. The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP(2) is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.
View details for Web of Science ID 000166237200001
View details for PubMedID 11134066
Spatial sensing in fibroblasts mediated by 3 ' phosphoinositides
JOURNAL OF CELL BIOLOGY
2000; 151 (6): 1269-1279
The directed movement of fibroblasts towards locally released platelet-derived growth factor (PDGF) is a critical event in wound healing. Although recent studies have implicated polarized activation of phosphoinositide (PI) 3-kinase in G protein-mediated chemotaxis, the role of 3' PI lipids in tyrosine kinase-triggered chemotaxis is not well understood. Using evanescent wave microscopy and green fluorescent protein-tagged Akt pleckstrin homology domain (GFP-AktPH) as a molecular sensor, we show that application of a shallow PDGF gradient triggers a markedly steeper gradient in 3' PI lipids in the adhesion zone of fibroblasts. Polar GFP-AktPH gradients, as well as a new type of radial gradient, were measured from front to rear and from the periphery to the center of the adhesion zone, respectively. A strong spatial correlation between polarized 3' PI production and rapid membrane spreading implicates 3' PI lipids as a direct mediator of polarized migration. Analysis of the temporal changes of 3' PI gradients in the adhesion zone revealed a fast diffusion coefficient (0.5 microm(2)/s) and short lifetime of 3' PIs of <1 min. Together, this study suggests that the tyrosine kinase-coupled directional movement of fibroblasts and their radial membrane activity are controlled by local generation and rapid degradation of 3' PI second messengers.
View details for Web of Science ID 000165851300016
View details for PubMedID 11121441
- Translocation and reversible localization of signaling proteins: A dynamic future for signal transduction CELL 2000; 103 (2): 181-184
Molecular memory by reversible translocation of calcium/calmodulin-dependent protein kinase II
2000; 3 (9): 881-886
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.
View details for Web of Science ID 000167177400014
View details for PubMedID 10966618
Differential codes for free Ca2+-calmodulin signals in nucleus and cytosol
2000; 10 (2): 86-94
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.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.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.
View details for Web of Science ID 000085042100019
View details for PubMedID 10662666
A versatile microporation technique for the transfection of cultured CNS neurons
JOURNAL OF NEUROSCIENCE METHODS
1999; 93 (1): 37-48
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.
View details for Web of Science ID 000084033900005
View details for PubMedID 10598863
CaMKII beta functions as an F-actin targeting module that localizes CaMKII alpha/beta heterooligomers to dendritic spines
1998; 21 (3): 593-606
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.
View details for Web of Science ID 000076196400015
View details for PubMedID 9768845
Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells
JOURNAL OF CELL BIOLOGY
1998; 140 (3): 485-498
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.
View details for Web of Science ID 000072026300003
View details for PubMedID 9456311
Electroporation-induced formation of individual calcium entry sites in the cell body and processes of adherent cells
1997; 73 (4): 1785-1796
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.
View details for Web of Science ID A1997XY95500008
View details for PubMedID 9336174