Cell Division Events Research Articles

Archaeal cells share common size control with bacteria despite noisier growth and division.

In nature, microorganisms exhibit different volumes spanning six orders of magnitude 1 . Despite their capability to create different sizes, a clonal population in a given environment maintains a uniform size across individual cells. Recent studies in eukaryotic and bacterial organisms showed that this homogeneity in cell size can be accomplished by growing a constant size between two cell cycle events (that is, the adder model 2-6 ). Demonstration of the adder model led to the hypothesis that this phenomenon is a consequence of convergent evolution. Given that archaeal cells share characteristics with both bacteria and eukaryotes, we investigated whether and how archaeal cells exhibit control over cell size. To this end, we developed a soft-lithography method of growing the archaeal cells to enable quantitative time-lapse imaging and single-cell analysis, which would be useful for other microorganisms. Using this method, we demonstrated that Halobacterium salinarum, a hypersaline-adapted archaeal organism, grows exponentially at the single-cell level and maintains a narrow-size distribution by adding a constant length between cell division events. Interestingly, the archaeal cells exhibited greater variability in cell division placement and exponential growth rate across individual cells in a population relative to those observed in Escherichia coli 6-9 . Here, we present a theoretical framework that explains how these larger fluctuations in archaeal cell cycle events contribute to cell size variability and control.

Cell division, endoreduplication and expansion processes: setting the cell and organ control into an integrated model of tomato fruit development

The development of a new organ is the result of coordinated events of cell division and expansion, in strong interaction with each other. This paper presents an improved version of a tomato model, which includes cells division, endoreduplication and expansion processes. The model is used to investigate the interaction among these developmental processes, in the perspective of a new cellular theory. In particular, different control schemes (either cell- or organ-based) are tested and simulation results are compared to observed data. The model shows that a pure cell-based control fails to reproduce the observed cell size distribution, and an organ-based control is needed in order to get realistic cell and fruit sizes. The model also supports the role of endoreduplication as an important modulator of the cell expansion potential.

Network-based approaches to quantify multicellular development.

Multicellularity and cellular cooperation confer novel functions on organs following a structure–function relationship. How regulated cell migration, division and differentiation events generate cellular arrangements has been investigated, providing insight into the regulation of genetically encoded patterning processes. Much less is known about the higher-order properties of cellular organization within organs, and how their functional coordination through global spatial relations shape and constrain organ function. Key questions to be addressed include: why are cells organized in the way they are? What is the significance of the patterns of cellular organization selected for by evolution? What other configurations are possible? These may be addressed through a combination of global cellular interaction mapping and network science to uncover the relationship between organ structure and function. Using this approach, global cellular organization can be discretized and analysed, providing a quantitative framework to explore developmental processes. Each of the local and global properties of integrated multicellular systems can be analysed and compared across different tissues and models in discrete terms. Advances in high-resolution microscopy and image analysis continue to make cellular interaction mapping possible in an increasing variety of biological systems and tissues, broadening the further potential application of this approach. Understanding the higher-order properties of complex cellular assemblies provides the opportunity to explore the evolution and constraints of cell organization, establishing structure–function relationships that can guide future organ design.

Gibbs free energy as a measure of complexity correlates with time within C. elegans embryonic development.

We investigate free energy behavior in the nematode Caenorhabditis elegans during embryonic development. Our approach utilizes publicly available gene expression data, which gives us a picture of developmental changes in protein concentration and, resultantly, chemical potential and free energy. Our results indicate a clear global relationship between Gibbs free energy and time spent in development and provide thermodynamic indicators of the large-scale biological events of cell division and differentiation.

Plk1 is essential for proper chromosome segregation during meiosis I/meiosis II transition in pig oocytes

BackgroundPolo-like kinase 1 (Plk1), as a characteristic regulator in meiosis, organizes multiple biological events of cell division. Although Plk1 has been implicated in various functions in somatic cell mitotic processes, considerably less is known regarding its function during the transition from metaphase I (MI) to metaphase II (MII) stage in oocyte meiotic progression.MethodsIn this study, the possible role of Plk1 during the MI-to-MII stage transition in pig oocytes was addressed. Initially, the spatiotemporal expression and subcellular localization pattern of Plk1 were revealed in pig oocytes from MI to MII stage using indirect immunofluorescence and confocal microscopy imaging techniques combined with western blot analyses. Moreover, a highly selective Plk1 inhibitor, GSK461364, was used to determine the potential role of Plk1 during this MI-to-MII transition progression.ResultsUpon expression, Plk1 exhibited a specific dynamic intracellular localization, and co-localization of Plk1 with α-tubulin was revealed in the meiotic spindle of pig oocyte during the transition from MI to MII stage. GSK461364 treatment significantly blocked the first polar body (pbI) emission in a dose-dependent manner and resulted in a failure of meiotic maturation, with a larger percentage of the GSK461364-treated oocytes arresting in the anaphase-telophase I (ATI) stage. Further subcellular structure examination results showed that inhibition of Plk1 with GSK461364 had no visible effect on spindle assembly but caused a significantly higher proportion of the treated oocytes to have obvious defects in homologous chromosome segregation at ATI stage.ConclusionsThus, these results indicate that Plk1 plays an essential role during the meiosis I/meiosis II transition in porcine oocytes, and the regulation is associated with Plk1’s effects on homologous chromosome segregation in the ATI stage.

Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation

During sporulation, the filamentous bacteria Streptomyces undergo a massive cell division event in which the synthesis of ladders of sporulation septa convert multigenomic hyphae into chains of unigenomic spores. This process requires cytokinetic Z-rings formed by the bacterial tubulin homolog FtsZ, and the stabilization of the newly formed Z-rings is crucial for completion of septum synthesis. Here we show that two dynamin-like proteins, DynA and DynB, play critical roles in this process. Dynamins are a family of large, multidomain GTPases involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. Using a cell biological approach, we show that the two Streptomyces dynamins specifically localize to sporulation septa in an FtsZ-dependent manner. Moreover, dynamin mutants have a cell division defect due to the decreased stability of sporulation-specific Z-rings, as demonstrated by kymographs derived from time-lapse images of FtsZ ladder formation. This defect causes the premature disassembly of individual Z-rings, leading to the frequent abortion of septum synthesis, which in turn results in the production of long spore-like compartments with multiple chromosomes. Two-hybrid analysis revealed that the dynamins are part of the cell division machinery and that they mediate their effects on Z-ring stability during developmentally controlled cell division via a network of protein-protein interactions involving DynA, DynB, FtsZ, SepF, SepF2, and the FtsZ-positioning protein SsgB.

Routes and machinery of primary cilium biogenesis.

Primary cilia are solitary, microtubule-based protrusions of the cell surface that play fundamental roles as photosensors, mechanosensors and biochemical sensors. Primary cilia dysfunction results in a long list of developmental and degenerative disorders that combine to give rise to a large spectrum of human diseases affecting almost any major body organ. Depending on the cell type, primary ciliogenesis is initiated intracellularly, as in fibroblasts, or at the cell surface, as in renal polarized epithelial cells. In this review, we have focused on the routes of primary ciliogenesis placing particular emphasis on the recently described pathway in renal polarized epithelial cells by which the midbody remnant resulting from a previous cell division event enables the centrosome for initiation of primary cilium assembly. The protein machinery implicated in primary cilium formation in epithelial cells, including the machinery best known for its involvement in establishing cell polarity and polarized membrane trafficking, is also discussed.

Metabolomic profiling reveals a finely tuned, starvation-induced metabolic switch in Trypanosoma cruzi epimastigotes

Trypanosoma cruzi, the etiological agent of Chagas disease, is a protozoan parasite with a complex life cycle involving a triatomine insect and mammals. Throughout its life cycle, the T. cruzi parasite faces several alternating events of cell division and cell differentiation in which exponential and stationary growth phases play key biological roles. It is well accepted that arrest of the cell division in the epimastigote stage, both in the midgut of the triatomine insect and in vitro, is required for metacyclogenesis, and it has been previously shown that the parasites change the expression profile of several proteins when entering this quiescent stage. However, little is known about the metabolic changes that epimastigotes undergo before they develop into the metacyclic trypomastigote stage. We applied targeted metabolomics to measure the metabolic intermediates in the most relevant pathways for energy metabolism and oxidative imbalance in exponentially growing and stationary growth-arrested epimastigote parasites. We show for the first time that T. cruzi epimastigotes transitioning from the exponential to the stationary phase exhibit a finely tuned adaptive metabolic mechanism that enables switching from glucose to amino acid consumption, which is more abundant in the stationary phase. This metabolic plasticity appears to be crucial for survival of the T. cruzi parasite in the myriad different environmental conditions to which it is exposed during its life cycle.

A toolkit for GFP-mediated tissue-specific protein degradation in C. elegans.

Proteins that are essential for embryo production, cell division and early embryonic events are frequently reused later in embryogenesis, during organismal development or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that uses expression of a fusion between a GFP-targeting nanobody and a SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or with rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues - the epidermis, intestine, body wall muscle, ciliated sensory neurons and touch receptor neurons - where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of C. elegans proteins.

Serine/Threonine Protein Phosphatase PstP of Mycobacterium tuberculosis Is Necessary for Accurate Cell Division and Survival of Pathogen

Protein phosphatases play vital roles in phosphorylation-mediated cellular signaling. Although there are 11 serine/threonine protein kinases in Mycobacterium tuberculosis, only one serine/threonine phosphatase, PstP, has been identified. Although PstP has been biochemically characterized and multiple in vitro substrates have been identified, its physiological role has not yet been elucidated. In this study, we have investigated the impact of PstP on cell growth and survival of the pathogen in the host. Overexpression of PstP led to elongated cells and partially compromised survival. We find that depletion of PstP is detrimental to cell survival, eventually leading to cell death. PstP depletion results in elongated multiseptate cells, suggesting a role for PstP in regulating cell division events. Complementation experiments performed with PstP deletion mutants revealed marginally compromised survival, suggesting that all of the domains, including the extracellular domain, are necessary for complete rescue. On the other hand, the catalytic activity of PstP is absolutely essential for the in vitro growth. Mice infection experiments establish a definitive role for PstP in pathogen survival within the host. Depletion of PstP from established infections causes pathogen clearance, indicating that the continued presence of PstP is necessary for pathogen survival. Taken together, our data suggest an important role for PstP in establishing and maintaining infection, possibly via the modulation of cell division events.

SuperSegger: robust image segmentation, analysis and lineage tracking of bacterial cells.

Many quantitative cell biology questions require fast yet reliable automated image segmentation to identify and link cells from frame-to-frame, and characterize the cell morphology and fluorescence. We present SuperSegger, an automated MATLAB-based image processing package well-suited to quantitative analysis of high-throughput live-cell fluorescence microscopy of bacterial cells. SuperSegger incorporates machine-learning algorithms to optimize cellular boundaries and automated error resolution to reliably link cells from frame-to-frame. Unlike existing packages, it can reliably segment microcolonies with many cells, facilitating the analysis of cell-cycle dynamics in bacteria as well as cell-contact mediated phenomena. This package has a range of built-in capabilities for characterizing bacterial cells, including the identification of cell division events, mother, daughter and neighbouring cells, and computing statistics on cellular fluorescence, the location and intensity of fluorescent foci. SuperSegger provides a variety of postprocessing data visualization tools for single cell and population level analysis, such as histograms, kymographs, frame mosaics, movies and consensus images. Finally, we demonstrate the power of the package by analyzing lag phase growth with single cell resolution.

Development of a Novel Cell Monitoring System Based on Lens-Less Imaging Toward Cultivation of Circulating Tumor Cells

Circulating tumor cells (CTCs) are defined as tumor cells circulating in the peripheral blood of patients with metastatic cancer. As CTCs could provide useful information regarding malignancy and metastatic property of primary tumor, as well as its response to anticancer agents, fundamental techniques for recovery and analysis of CTCs have been intensively studied in this decade. Although state-of-the-art microfluidic tools and whole genome amplification techniques are now available to investigate single CTCs, it is still extremely difficult to cultivate CTCs that, for example, enables examination of multiple anticancer agents toward identical CTCs. This could be due to the lack of knowledge of division and proliferation behavior of CTCs. The critical difficulty in investigation of CTC proliferation is that cell division events of CTCs are very rare, and thus conventional microscopy could hardly identify the dividing CTCs from the samples containing a number of blood cells. In this study, we propose to use a wide-field imaging system for investigation of CTC cultivation. The imaging system is composed of a light emitting diode (LED, peak wavelength: 465 nm) and a complementary metal oxide semiconductor (CMOS) imaging sensor by which an area of 6.55 × 4.92 mm2can be visualized in one shot. A cell culture dish is placed on the CMOS image sensor, and images are acquired under the LED illumination without using any lens systems (referred to as “lenss-less imaging”). The CMOS image sensor allows to develop a space-saving and inexpensive platform, so that whole system can be introduced in a general incubator, and real-time monitoring of cell growth is possible. Using this lens-less imaging system, we attempted to observe tumor cell proliferation and distinguish tumor cells from blood cells. HeLa cells were used as model tumor cells. A culture dish containing HeLa cells in 2 ml of the high glucose Dulbecco's modified Eagle's medium (DMEM, 5,000 cells/ml) was placed on the lens-less imaging system, and incubated for 18 hours at 37°C with 5% CO2. Images were captured every 15 min. For comparison, microscopic observation was also performed. As a result, HeLa cells were recorded as a spherical patterns with high intensity by a CMOS image sensor. When divided, HeLa cells were once illustrated as a dark patterns, and then two daughter cells gradually appeared with increasing the intensity. It is well known that adhesive cells temporarily go through a non-adhesive state before their division process. Indeed, we confirmed that the spherical patterns with high intensity represents the HeLa cells adhere on the bottom of the dishes, and the subsequent dark patterns do the cells in the non-adhesive state by microscopic observation. This data indicates that the lens-less imaging system can detect individual cell division events. Taking advantage of the wide field of view, it was possible to monitor the cell division events of several hundreds of cells simultaneously. Recovery of CTCs from blood samples is the first step for the cultivation and further analysis, however it is still hard to avoid blood cell contamination despite the development of sophisticated technology for CTC separation and enrichment nowadays. Consequently, it is essential to differentiate CTCs and the contaminated blood cells, otherwise accurate evaluation of CTC proliferation is impossible. To test whether tumor cells and blood cells were distinguishable based on lens-less imaging, we compared the lens-less images of HeLa cells, red blood cells and JM cells (model white blood cells). A number of differentiation parameters were extracted from the images, and computed for cluster analysis. As a result, it was successfully demonstrated to discriminate HeLa cells, red blood cells and JM cells without any labeling process, suggesting the development of a simple, inexpensive and rapid cell typing system. In summary, the lens-less imaging system could monitor cell division events of HeLa cells in a wide field of view, and differentiate HeLa cells and blood cells with high accuracy. We believe that this system could be a powerful tool to monitor the proliferation of CTCs recovered from patient blood samples.

Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes.

Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells.

Detecting cell division of Pseudomonas aeruginosa bacteria from bright-field microscopy images with hidden conditional random fields.

An approach to automatically detect bacteria division with temporal models is presented. To understand how bacteria migrate and proliferate to form complex multicellular behaviours such as biofilms, it is desirable to track individual bacteria and detect cell division events. Unlike eukaryotic cells, prokaryotic cells such as bacteria lack distinctive features, causing bacteria division difficult to detect in a single image frame. Furthermore, bacteria may detach, migrate close to other bacteria and may orientate themselves at an angle to the horizontal plane. Our system trains a hidden conditional random field (HCRF) model from tracked and aligned bacteria division sequences. The HCRF model classifies a set of image frames as division or otherwise. The performance of our HCRF model is compared with a Hidden Markov Model (HMM). The results show that a HCRF classifier outperforms a HMM classifier. From 2D bright field microscopy data, it is a challenge to separate individual bacteria and associate observations to tracks. Automatic detection of sequences with bacteria division will improve tracking accuracy.

A mechanistic stochastic framework for regulating bacterial cell division

How exponentially growing cells maintain size homeostasis is an important fundamental problem. Recent single-cell studies in prokaryotes have uncovered the adder principle, where cells add a fixed size (volume) from birth to division, irrespective of their size at birth. To mechanistically explain the adder principle, we consider a timekeeper protein that begins to get stochastically expressed after cell birth at a rate proportional to the volume. Cell-division time is formulated as the first-passage time for protein copy numbers to hit a fixed threshold. Consistent with data, the model predicts that the noise in division timing increases with size at birth. Intriguingly, our results show that the distribution of the volume added between successive cell-division events is independent of the newborn cell size. This was dramatically seen in experimental studies, where histograms of the added volume corresponding to different newborn sizes collapsed on top of each other. The model provides further insights consistent with experimental observations: the distribution of the added volume when scaled by its mean becomes invariant of the growth rate. In summary, our simple yet elegant model explains key experimental findings and suggests a mechanism for regulating both the mean and fluctuations in cell-division timing for controlling size.

Variations on a theme: Imaging cytokinetic and stable rings in situ using Caenorhabditis elegans.

Cytokinesis is an essential event in canonical cell division. In multicellular organisms, cells must divide in the context of neighboring cells in intact tissues. Recent studies have shown that tissue architecture can regulate the dynamics of and molecular requirements for cytokinesis. On the other hand, regulated cytokinesis failure occurs in, and is required for the proper function of, certain cell types and tissues including cardiomyocytes, hepatocytes, and germ lines. One way to build our understanding of cytokinesis in diverse cell types is to visualize cytokinesis in intact tissues. The nematode Caenorhabditis elegans is a powerful system for such inquiries due to the well-characterized, invariant lineage of each of its cells, the ease of genomic modifications including tagging proteins, and many more advantages. The clear cuticle of C. elegans allows for live imaging of intact tissues; however, the worm'smotility can confound imaging. Here we introduce two C. elegans tissues, an epithelial tissue and the germ line, both excellent systems for the study of cytokinesis in the context of an intact animal. Additionally, we present three protocols for overcoming the challenges of live imaging in C. elegans.

Stomagenesis versus myogenesis: Parallels in intrinsic and extrinsic regulation of transcription factor mediated specialized cell-type differentiation in plants and animals.

Although the last common unicellular ancestor of plants and animals diverged several billion years ago, and while having developed unique developmental programs that facilitate differentiation and proliferation specific to plant and animal systems, there still exists a high degree of conservation in the logic regulating these developmental processes within these two seemingly diverse kingdoms. Stomatal differentiation in plants involves a series of orchestrated cell division events mediated by a family of closely related bHLH transcription factors (TFs) to create a pair of mature guard cells. These TFs are in turn regulated by a number of upstream signaling components that ultimately function to achieve lineage specific differentiation and organized tissue patterning on the plant epidermis. The logic involved in the specification of the myogenic differentiation program in animals is intriguingly similar to stomatal differentiation in plants: Closely-related myogenic bHLHs, known as MRFs (Myogenic Regulatory Factors) provide lineage specificity essential for cell-fate determination. These MRFs, similar to the bHLHs in plants, are regulated by several upstream signaling cascades that succinctly regulate each differentiation step, leading to the production of mature muscle fibers. This review aims at providing a perspective on the emerging parallels in the logic employed by key bHLH transcription factors and their upstream signaling components that function to precisely regulate key cell-state transition events in the stomatal as well as myogenic cell lineages.

SQUAMOSA Promoter Binding Protein-like Transcription Factors: Targets for Improving Cereal Grain Yield

Grain size and shape are prime targets for crop breeders, as they affect both yield and quality of rice. In addition, breeders target plant vegetative and reproductive shoot architecture, because these traits influence grain number, and thus yield potential. Accordingly, genetic control of grain size and shape as well as shoot architecture has been extensively investigated in recent years. In a recent study, Bin Han and colleagues have used genome-wide association analysis of grain size in a diverse collection of rice varieties of worldwide origin and identified a major quantitative trait locus GLW7, which encodes a SQUAMOSA promoter binding protein-like 13 (OsSPL13), a member of the plant-specific SBP domain family of transcription factors (Si et al., 2016).

Robustness of the Process of Nucleoid Exclusion of Protein Aggregates in Escherichia coli.

Escherichia coli segregates protein aggregates to the poles by nucleoid exclusion. Combined with cell divisions, this generates heterogeneous aggregate distributions in subsequent cell generations. We studied the robustness of this process with differing medium richness and antibiotics stress, which affect nucleoid size, using multimodal, time-lapse microscopy of live cells expressing both a fluorescently tagged chaperone (IbpA), which identifies in vivo the location of aggregates, and HupA-mCherry, a fluorescent variant of a nucleoid-associated protein. We find that the relative sizes of the nucleoid's major and minor axes change widely, in a positively correlated fashion, with medium richness and antibiotic stress. The aggregate's distribution along the major cell axis also changes between conditions and in agreement with the nucleoid exclusion phenomenon. Consequently, the fraction of aggregates at the midcell region prior to cell division differs between conditions, which will affect the degree of asymmetries in the partitioning of aggregates between cells of future generations. Finally, from the location of the peak of anisotropy in the aggregate displacement distribution, the nucleoid relative size, and the spatiotemporal aggregate distribution, we find that the exclusion of detectable aggregates from midcell is most pronounced in cells with mid-sized nucleoids, which are most common under optimal conditions. We conclude that the aggregate management mechanisms of E. coli are significantly robust but are not immune to stresses due to the tangible effect that these have on nucleoid size. Escherichia coli segregates protein aggregates to the poles by nucleoid exclusion. From live single-cell microscopy studies of the robustness of this process to various stresses known to affect nucleoid size, we find that nucleoid size and aggregate preferential locations change concordantly between conditions. Also, the degree of influence of the nucleoid on aggregate positioning differs between conditions, causing aggregate numbers at midcell to differ in cell division events, which will affect the degree of asymmetries in the partitioning of aggregates between cells of future generations. Finally, we find that aggregate segregation to the cell poles is most pronounced in cells with mid-sized nucleoids. We conclude that the energy-free process of the midcell exclusion of aggregates partially loses effectiveness under stressful conditions.

Measuring Activity and Specificity of Protein Phosphatases.

Reversible protein phosphorylation plays essential roles in coordinating cell division and many other biological processes. Cell cycle regulation by opposing kinase and protein phosphatase activities is often complex and major challenges exist in identifying the direct substrates of these enzymes and the specific sites at which they act. While cell cycle kinases are known to exhibit strict substrate specificities important for coordinating the complex events of cell division, phosphatases have only recently been recognized to exert similarly precise regulatory control over cell cycle events through timely dephosphorylation of specific substrates. The molecular determinants for substrate recognition by many phosphatases that function in cell division are still poorly delineated. To understand phosphatase specificity, it is critical to employ methods that monitor the dephosphorylation of individual phosphorylation sites on physiologically relevant substrates. Here, using the cell cycle phosphatase Cdc14 as an example, we describe two methods for studying phosphatase specificity, one using synthetic phosphopeptide substrates and the other using intact phosphoprotein substrates. These methods are useful for targeted characterization of small substrate sets and are also adaptable to large-scale applications for global specificity studies.