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1.  Dynamics of TGF-β/Smad Signaling 
FEBS letters  2012;586(14):1921-1928.
The physiological responses to TGF-β stimulation are diverse and vary amongst different cell types and environmental conditions. Even though the principal molecular components of the canonical and the noncanonical TGF-β signaling pathways have been largely identified, the mechanism that underlies the well-established context dependent physiological responses remains a mystery. Understanding how the components of TGF-β signaling function as a system and how this system functions in the context of the global cellular regulatory network requires a more quantitative and systematic approach. Here, we review the recent progress in understanding TGF-β biology using integration of mathematical modeling and quantitative experimental analysis. These studies reveal many interesting dynamics of TGF-β signaling and how cells quantitatively decode variable doses of TGF-β stimulation.
doi:10.1016/j.febslet.2012.03.063
PMCID: PMC4127320  PMID: 22710166
TGF-β; Smad; mathematical model; dose response; switch-like response
2.  Leader cell positioning drives wound-directed collective migration in TGFβ-stimulated epithelial sheets 
Molecular Biology of the Cell  2014;25(10):1586-1593.
Motility analysis of collectively migrating epithelial sheets reveals the interplay between cellular density and leader cell positioning throughout a collective group, as well as the functional role of leader cell constraint in directing a migrating group of cells.
During wound healing and cancer metastasis, cells are frequently observed to migrate in collective groups. This mode of migration relies on the cooperative guidance of leader and follower cells throughout the collective group. The upstream determinants and molecular mechanisms behind such cellular guidance remain poorly understood. We use live-cell imaging to track the behavior of epithelial sheets of keratinocytes in response to transforming growth factor β (TGFβ), which stimulates collective migration primarily through extracellular regulated kinase 1/2 (Erk1/2) activation. TGFβ-treated sheets display a spatial pattern of Erk1/2 activation in which the highest levels of Erk1/2 activity are concentrated toward the leading edge of a sheet. We show that Erk1/2 activity is modulated by cellular density and that this functional relationship drives the formation of patterns of Erk1/2 activity throughout sheets. In addition, we determine that a spatially constrained pattern of Erk1/2 activity results in collective migration that is primarily wound directed. Conversely, global elevation of Erk1/2 throughout sheets leads to stochastically directed collective migration throughout sheets. Our study highlights how the spatial patterning of leader cells (cells with elevated Erk1/2 activity) can influence the guidance of a collective group of cells during wound healing.
doi:10.1091/mbc.E14-01-0697
PMCID: PMC4019490  PMID: 24623725
3.  Analysis of Ligand Dependent Nuclear Accumulation of Smads in TGF-beta Signaling 
The growth inhibition of dividing cells and most of the transcriptional responses upon TGF-beta treatment depend on the Smad2, Smad3, and Smad4 transcription factors. These proteins shuttle continuously between the cytoplasm and the nucleus, transmitting the ligand status of the TGF-beta receptors to the nuclear transcription machinery. In the absence of TGF-beta ligand, Smads 2/3/4 reside predominantly in the cytoplasm. Following ligand binding to the TGF-beta receptors, the dynamic equilibrium of shuttling Smads 2/3/4 shifts towards a predominantly nuclear state, where a high concentration of these transcription factors drives transcriptional activation and repression of genes required for proper cellular response. Here, we describe live-cell imaging and immunofluorescence microscopy methods for tracking Smads subcellular localization in response to TGF-beta and leptomycin B treatment. In addition, a method of fractionating nuclear and cytoplasmic proteins used to confirm the imaging results was presented. Our results support the notion that the R-Smad shuttling mechanism is distinct from Co-Smad.
doi:10.1007/978-1-60761-738-9_5
PMCID: PMC4083701  PMID: 20694662
TGF-beta; SMAD4; SMAD2; Nuclear Accumulation; Cellular Fractionation; Immunofluorescence; Leptomycin B
4.  The Development of a Novel High Throughput Computational Tool for Studying Individual and Collective Cellular Migration 
PLoS ONE  2013;8(12):e82444.
Understanding how cells migrate individually and collectively during development and cancer metastasis can be significantly aided by a computation tool to accurately measure not only cellular migration speed, but also migration direction and changes in migration direction in a temporal and spatial manner. We have developed such a tool for cell migration researchers, named Pathfinder, which is capable of simultaneously measuring the migration speed, migration direction, and changes in migration directions of thousands of cells both instantaneously and over long periods of time from fluorescence microscopy data. Additionally, we demonstrate how the Pathfinder software can be used to quantify collective cell migration. The novel capability of the Pathfinder software to measure the changes in migration direction of large populations of cells in a spatiotemporal manner will aid cellular migration research by providing a robust method for determining the mechanisms of cellular guidance during individual and collective cell migration.
doi:10.1371/journal.pone.0082444
PMCID: PMC3873918  PMID: 24386097
5.  Identification and Mechanistic Studies of a Novel Ubiquitin E1 Inhibitor 
Journal of Biomolecular Screening  2012;17(4):421-434.
Protein degradation via the ubiquitin-proteasome pathway is important for a diverse number of cellular processes ranging from cell signaling to development. Disruption of the ubiquitin pathway occurs in a variety of human diseases, including several cancers and neurological disorders. Excessive proteolysis of tumor suppressor proteins, such as p27, occurs in numerous aggressive human tumors. To discover small-molecule inhibitors that potentially prevent p27 degradation, we developed a series of screening assays, including a cell-based screen of a small-molecule compound library and two novel nucleotide exchange assays. Several small-molecule inhibitors, including NSC624206, were identified and subsequently verified to prevent p27 ubiquitination in vitro. The mechanism of NSC624206 inhibition of p27 ubiquitination was further unraveled using the nucleotide exchange assays and shown to be due to antagonizing ubiquitin activating enzyme (E1). We determined that NSC624206 and PYR-41, a recently reported inhibitor of ubiquitin E1, specifically block ubiquitin-thioester formation but have no effect on ubiquitin adenylation. These studies reveal a novel E1 inhibitor that targets a specific step of the E1 activation reaction. NSC624206 could, therefore, be potentially useful for the control of excessive ubiquitin-mediated proteolysis in vivo.
doi:10.1177/1087057111433843
PMCID: PMC3339042  PMID: 22274912
ubiquitin E1; inhibitor; p27kip1; ubiquitin; proteolysis
6.  Partners in crime: the TGFβ and MAPK pathways in cancer progression 
Cell & Bioscience  2011;1:42.
The TGFβ and Ras-MAPK pathways play critical roles in cell development and cell cycle regulation, as well as in tumor formation and metastasis. In the absence of cellular transformation, these pathways operate in opposition to one another, where TGFβ maintains an undifferentiated cell state and suppresses proliferation, while Ras-MAPK pathways promote proliferation, survival and differentiation. However, in colorectal and pancreatic cancers, the opposing pathways' mechanisms are simultaneously activated in order to promote cancer progression and metastasis. Here, we highlight the roles of the TGFβ and Ras-MAPK pathways in normal and malignant states, and provide an explanation for how the concomitant activation of these pathways drives tumor biology. Finally, we survey potential therapeutic targets in these pathways.
doi:10.1186/2045-3701-1-42
PMCID: PMC3275500  PMID: 22204556
TGFβ; Metastasis; ERK; MEK; Ras; Crosstalk; Signal Transduction
7.  Quantitative analysis of transient and sustained transforming growth factor-β signaling dynamics 
Mathematical modeling and experimental analyses reveal that TGF-β ligand depletion has an important role in converting short-term graded signaling responses to long-term switch-like responses.
Cells respond in real time to the absolute number of TGF-β molecules in their environment.A single pulse of TGF-β stimulation results in transient SMAD activation whereas repeated short pulses of stimulation result in sustained SMAD activation.Ligand-induced short-term TGF-β/SMAD signaling activation is graded while long-term signaling response is switch-like or ultrasensitive.TGF-β ligand depletion is a major cause of conversion from graded short-term responses to ultrasensitive long-term responses.
The transforming growth factor-β (TGF-β) pathway is a prominent signaling pathway that regulates diverse aspects of cellular homeostasis, including proliferation, differentiation, migration, and death (Massague, 1998). Remarkably, the pleiotropic biological effects of TGF-β are mediated by a relatively simple signaling module (Clarke and Liu, 2008). An interesting question is how such an apparently straightforward and simple cascade can generate a wide array of biological responses depending on the cellular context.
Members of the TGF-β superfamily are frequently used as morphogens in early embryo development (Green, 2002). The best-studied examples include Dpp in Drosophila and Activin in Xenopus (Gurdon and Bourillot, 2001; Lander, 2007). In the developmental context, cells can respond to a graded ligand concentration and produce discrete biological responses (e.g., transcription of certain genes, proliferation, or differentiation; Green, 2002). To convert continuous morphogen stimulation into discrete responses, mechanisms must exist to provide a threshold for the cellular response. How variable TGF-β ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remains poorly understood.
We have previously reported that ligand molecules per cell is the input variable to which the cells respond, and ligand number per cell is the best predictor of signaling responses (Zi and Klipp, 2007a; Clarke et al, 2009). Here, we developed an improved mathematical model to predict TGF-β signaling responses by calibrating the model with various experimental data sets from different TGF-β stimulations. Using a combined experimental and mathematical modeling approach, we showed that TGF-β pulse stimulation results in transient activation of the pathway while repeated short pulses at short time intervals lead to a sustained activation similar to persistent ligand exposure.
We next investigate the system response to variable doses of TGF-β in HaCaT cells. Our mathematical model predicts that the short-term Smad2 phosphorylation (after 45 min of TGF-β stimulation) is a graded response, while long-term Smad2 activation (after 24 h of TGF-β stimulation) is a switch-like response (Figure 5A and B). As shown in Figure 5A–D, both short- and long-term Smad2 phosphorylation can be saturated but doses of TGF-β that cause maximum response are quite different. Additionally, the shapes of response curves were different. The short-term Smad2 activation was a graded (Michaelis–Menten-like) response with a very low apparent Hill coefficient of about 0.8 (Figure 5A and C) while the long-term Smad2 activation (P-Smad2 at 24 h) yielded a switch-like response with an apparent Hill coefficient of about 4.5 (Figure 5B and D). Thus, the Smad2 response is initially graded and sharpens over time to become ultrasensitive. To address whether TGF-β-inducible gene expression responses are graded or switch-like in the short and long term, we measured mRNA levels of Smad7, an early responsive gene of TGF-β and protein levels of p21 and PAI-1 whose inductions are delayed and late, respectively. The experimental data show that Smad7 induction exhibits a graded response with corresponding Hill coefficients of about 1.3 (Figure 5E), which is consistent with the graded P-Smad2 response at 45 min (Figure 5A and C). PAI-1 induction in response to variable doses of TGF-β for 24 h is highly ultrasensitive with an apparent Hill coefficient of ∼5.3. Compared with Smad7 and PAI-1, p21 induction is only modest ultrasensitive (nHill≈2) (Figure 5G). These results suggest short-term gene induction by TGF-β appears to be graded while long-term targets are more switch-like. Finally, we measured the growth inhibitory response of HaCaT cells to variable doses of TGF-β. The level of BrdU incorporation is also ultrasensitive with an apparent Hill coefficient of about 4.3 (Figure 5H). Therefore, the long-term TGF-β growth inhibitory response also shows a switch-like behavior. Finally, we show that TGF-β depletion affects long-term Smad phosphorylation and switch-like response of TGF-β signaling system. These findings shed new light on how continuous ligand doses are translated into discontinuous cell fate decisions in biological systems.
In summary, we have shown that the dose and time course of TGF-β stimulation have profound effects on Smad signaling dynamics. The rate of ligand depletion controls the duration of Smad2 phosphorylation. Cells can respond to a short pulse of TGF-β stimulation, and periodic short ligand exposures are sufficient to generate long-term signaling responses. Short-term TGF-β stimulation causes only transient pathway activation and can be terminated by ligand depletion. TGF-β-induced Smad2 phosphorylation is graded in the short-term but ultrasensitive (switch-like) in the long-term (Figure 7). Additionally, cell growth arrest in response to TGF-β shows switch-like rather than graded behavior. Our modeling and experimental analyses suggest that ligand depletion is likely to be involved in sharpening a graded response into a switch-like response.
Mammalian cells can decode the concentration of extracellular transforming growth factor-β (TGF-β) and transduce this cue into appropriate cell fate decisions. How variable TGF-β ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remain poorly understood. Using a combined experimental and mathematical modeling approach, we discovered that cells respond differently to continuous and pulsating TGF-β stimulation. The TGF-β pathway elicits a transient signaling response to a single pulse of TGF-β stimulation, whereas it is capable of integrating repeated pulses of ligand stimulation at short time interval, resulting in sustained phospho-Smad2 and transcriptional responses. Additionally, the TGF-β pathway displays different sensitivities to ligand doses at different time scales. While ligand-induced short-term Smad2 phosphorylation is graded, long-term Smad2 phosphorylation is switch-like to a small change in TGF-β levels. Correspondingly, the short-term Smad7 gene expression is graded, while long-term PAI-1 gene expression is switch-like, as is the long-term growth inhibitory response. Our results suggest that long-term switch-like signaling responses in the TGF-β pathway might be critical for cell fate determination.
doi:10.1038/msb.2011.22
PMCID: PMC3130555  PMID: 21613981
mathematical model; Smad; TGF-β; ultrasensitivity
8.  Allosteric Site Variants of Haemophilus influenzae β-Carbonic Anhdyrase†, ‡ 
Biochemistry  2009;48(26):6146-6156.
Haemophilus influenzae β-carbonic anhydrase (HICA) is hypothesized to be an allosteric protein that is regulated by the binding of bicarbonate ion to a non-catalytic (inhibitory) site that controls the ligation of Asp44 to the catalytically essential zinc ion. We report here the X-ray crystallographic structures of two variants (W39F and Y181F) involved in the binding of bicarbonate ion in the non-catalytic site and an active site variant (D44N) that is incapable of forming a strong zinc ligand. The alteration of Trp39 to Phe increases the apparent Ki for bicarbonate inhibition by 4.8-fold. While the structures of W39F and Y181F are very similar to the wild-type enzyme, the X-ray crystal structure of the D44N variant reveals that it has adopted an active site conformation nearly identical to that of non-allosteric β-carbonic anhydrases. We propose that the structure of the D44N variant is likely to be representative of the active conformation of the enzyme. These results lend additional support to the hypothesis that HICA is an allosteric enzyme that can adopt active and inactive conformations, the latter of which is stabilized by bicarbonate ion binding to a non-catalytic site.
doi:10.1021/bi900663h
PMCID: PMC2740983  PMID: 19459702
beta carbonic anhydrase; Haemophilus influenzae; allosteric site; bicarbonate; zinc metalloenzyme

Results 1-8 (8)