Biology subjects

Rao, B. M.

Publications and source records attributed to Rao, B. M..

15 recordsLinked to original sources

bioRxiv2025

Glucose levels impact the morphology and cell-type composition of human cerebral organoids

Human cerebral organoids, derived from pluripotent stem cells, are powerful models for studying human brain development. The understanding of how morphogens can be used to guide patterning and differentiation has matured rapidly, however, the influence of basal media components on organoid development remains unclear. Standard organoid media frequently contain non- physiological concentrations of nutrients, including glucose, a central regulator of cellular metabolism and signaling. Here, we examine how glucose availability shapes cerebral organoid growth, morphology, and cell-type composition by comparing conventional hyperglycemic media to media with glucose levels more closely resembling physiological conditions. We find that organoids derived from multiple human pluripotent stem cell lines can grow in physiological glucose, but exhibit altered growth rates, structural features, and lineage distributions. In H9 embryonic stem cell-derived organoids, inhibition of the mTOR pathway under physiological glucose restores neurodevelopmental cell types otherwise diminished in these conditions. These findings highlight glucose as a key determinant of organoid lineage specification and cellular signaling. Importantly, however, glucose modulation does not reduce variability across organoids or cell lines, underscoring the need to better understand and control sources of heterogeneity to improve organoid models.

cell biology↗

bioRxiv2024

Derivation of human trophoblast stem cells from placentas at birth

Human trophoblast stem cells (hTSCs) have emerged as a powerful tool for modeling the placental cytotrophoblast (CTB) in vitro. hTSCs were originally derived from CTBs of the first trimester placenta or blastocyst-stage embryos in trophoblast stem cell medium (TSCM) that contains epidermal growth factor (EGF), the glycogen synthase kinase-beta (GSK3{beta}) inhibitor CHIR99021, the transforming growth factor-beta (TGF{beta}) inhibitors A83-01 and SB431542, valproic acid (VPA), and the Rho-associated protein kinase (ROCK) inhibitor Y-27632. Here we show that hTSCs can be derived from CTBs isolated from the term placenta, using TSCM supplemented with a low concentration of mitochondrial pyruvate uptake inhibitor UK5099 and lipid-rich albumin (TUA medium). Notably, hTSCs could not be derived from term CTBs using TSCM alone, or in the absence of either UK5099 or lipid-rich albumin. Strikingly, hTSCs cultured in TUA medium for a few passages could be transitioned into TSCM and cultured thereafter in TSCM. hTSCs from term CTBs cultured in TUA medium as well as those transitioned into and cultured in TSCM thereafter could be differentiated to the extravillous trophoblast and syncytiotrophoblast lineages and exhibited high transcriptome similarity with hTSCs derived from first trimester CTBs. We anticipate that these results will enable facile derivation of hTSCs from normal and pathological placentas at birth with diverse genetic backgrounds and facilitate in vitro mechanistic studies in trophoblast biology. Significance statementHuman trophoblast stem cells (hTSCs) derived from early pregnancy have emerged as a powerful tool for modeling early human placental development in vitro. However, restrictions on derivation of cell lines from fetal tissues limit the availability and genetic diversity of hTSC lines. Also, the associated pregnancy outcome of hTSCs derived from early gestation is unknown. Here we describe the derivation and characterization of hTSCs from placentas obtained at birth. Our results will enable derivation of genetically diverse hTSCs associated with normal and pathological pregnancies, enabling research in placental biology.

developmental biology↗

bioRxiv2022

Experimental and analytical framework for ''mix-and-read'' assays based on split luciferase

The use of immunodetection assays including the widely used enzyme-linked immunosorbent assay (ELISA) in applications such as point-of-care detection is often limited by the need for protein immobilization and multiple binding and washing steps. Here, we describe an experimental and analytical framework for the development of simple and modular "mix-and-read" enzymatic complementation assays based on split luciferase that enable sensitive detection and quantification of analytes in solution. In this assay, two engineered protein binders targeting non-overlapping epitopes on the target analyte were each fused to non-active fragments of luciferase to create biosensor probes. Binding proteins to two model targets, lysozyme and Sso6904, were isolated from a combinatorial library of Sso7d mutants using yeast surface display. In the presence of the analyte, probes were brought into close proximity, reconstituting enzymatic activity of luciferase and enabling detection of low picomolar concentrations of the analyte by chemiluminescence. Subsequently, we constructed an equilibrium binding model that relates binding affinities of the binding proteins for the target, assay parameters such as the concentrations of probes used, and assay performance (limit of detection and concentration range over which the target can be quantified). Overall, our experimental and analytical framework provide the foundation for the development of split luciferase assays for detection and quantification of various targets.

bioengineering↗

bioRxiv2021

Identifying challenges and opportunities for histone peptides in protein engineering platforms

Histone post-translational modifications are small chemical changes to histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET based systems. High throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet published studies on their use with histones have been noticeably sparse. Here we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding.

bioengineering↗

bioRxiv2021

Laminin switches terminal differentiation fate of human trophoblast stem cells under chemically defined culture conditions.

Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we do not utilize transforming growth factor-beta inhibitors or a passage step for EVT differentiation, or forskolin for STB formation. Strikingly, under these conditions, presence of a single additional extracellular cue - lam-inin-1 - switched the terminal differentiation of hTSCs from STB to the EVT lineage. Activation of the sphingosine-1 receptor 3 receptor (S1PR3) using a chemical agonist could drive EVT differentiation of hTSCs in the absence of exogenous laminin, albeit less efficiently. To illustrate the utility of a chemically defined culture system for mechanistic studies, we examined the role of protein kinase C (PKC) signaling during hTSC differentiation to the EVT lineage. Inhibition of PKC/{beta} signaling significantly reduced HLA-G expression and the formation of HLA-G+ mesen-chymal EVTs during hTSC differentiation mediated by laminin exposure; however, it did not prevent commitment to the EVT lineage or STB differentiation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation, and will enable mechanistic studies in vitro. SignificanceDespite its importance to a healthy pregnancy, early human placental development remains poorly understood. Mechanistic studies are impeded by restrictions on research with human embryos and fetal tissues, and significant differences in placentation between humans and commonly used animal models. In this context, human trophoblast stem cells (hTSCs) have emerged as attractive in vitro models for the epithelial cytotrophoblast of the early gestation human placenta. Here we describe chemically defined culture conditions for differentiation of hTSCs to the two major differentiated cell types - extravillous trophoblast and syncytiotrophoblast. These culture conditions enable in vitro studies to reveal molecular mechanisms regulating hTSC differentiation.

developmental biology↗

bioRxiv2021

REMY: A platform for the rapid interrogation of epigenome modifications on yeast

Histone proteins are decorated with a combinatorially and numerically diverse set of biochemical modifications. Here we describe a versatile and scalable platform termed Rapid interrogation of Epigenome Modifications using Yeast surface display (REMY), which enables efficient characterization of histone modifications without the need for recombinant protein production. As proof-of-concept, we first used REMY to rapidly profile the histone H3 and H4 residue writing specificities of the human histone acetyltransferase, p300. Subsequently, we used REMY to screen a large panel of commercially available anti-acetylation antibodies for their specificities, identifying many suitable and unsuitable reagents. Further, use of REMY enabled efficient mapping of the large binary crosstalk space between acetylated residues on histones H3 and H4, and uncovered previously unreported residue interdependencies affecting p300 activity. Our results show that REMY is a useful tool that can advance our understanding of chromatin biology by enabling efficient interrogation of the complexity of epigenome modifications.

synthetic biology↗

bioRxiv2020

Use of target-displaying magnetized yeast in screening mRNA display peptide libraries to identify ligands

This work presents the first use of yeast-displayed protein targets for screening mRNA display libraries of cyclic and linear peptides. The WW domains of Yes-Associated Protein 1 (WW-YAP) and mitochondrial import receptor subunit TOM22 were adopted as protein targets. Yeast cells displaying WW-YAP or TOM22 were magnetized with iron oxide nanoparticles to enable the isolation of target-binding mRNA-peptide fusions. Equilibrium adsorption studies were conducted to estimate the binding affinity (KD) of selected WW-YAP-binding peptides: KD values of 37 M and 4 M were obtained for cyclo[M-AFRLC-K] and its linear cognate, and 40 M and 3 M for cyclo[M-LDFVNHRSRG-K] and its linear cognate, respectively. TOM22-binding peptide cyclo[M-PELNRAI-K] was conjugated to magnetic beads and incubated with yeast cells expressing TOM22 and luciferase. A luciferase-based assay showed a 4.5-fold higher binding of TOM22+ yeast compared to control cells. This work demonstrates that integrating mRNA and yeast display accelerates the discovery of biospecific peptides.

biochemistry↗

bioRxiv2020

Quantitative yeast-yeast two hybrid for discovery and binding affinity estimation of protein-protein interactions

Quantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well as characterization of binding proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two hybrid (qYY2H) system that not only enables discovery of specific protein-protein interactions, but also efficient, quantitative estimation of their binding affinities (KD). In qYY2H, the bait and prey proteins are expressed as yeast cell surface fusions using yeast surface display. We developed a semi-empirical framework for estimating the KD of monovalent bait-prey interactions, using measurements of the apparent KD of yeast-yeast binding, which is mediated by multivalent interactions between yeast-displayed bait and prey. Using qYY2H, we identified interaction partners of SMAD3 and the tandem WW domains of YAP from a cDNA library and characterized their binding affinities. Finally, we showed that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein.

biochemistry↗

bioRxiv2020

Screening of yeast display libraries of enzymatically-cyclized peptides to discover macrocyclic peptide ligands

We present the construction and screening of yeast display libraries of cyclic peptides wherein site-selective enzymatic cyclization of linear peptides is achieved using bacterial transglu-taminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve cyclization in the endoplasmic reticulum prior to yeast surface display. The cyclization yield was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-cyclized peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified cyclic peptide cyclo[E-LYLAYPAH-K] featured a KD of 1.67 {micro}M for YAP and 0.84 {micro}M for WW as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of yeast surface display for screening transglutaminase-cyclized peptide libraries, and efficient identification of cyclic peptide ligands.

biochemistry↗

bioRxiv2020

Discovery of membrane-permeating cyclic peptides via mRNA display

Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and non-disruptively controlling intracellular or intraembryonic processes.

biochemistry↗

bioRxiv2020

Isolation of Chemically Cyclized Peptide Binders Using Yeast Surface Display

Cyclic peptides with engineered protein-binding activity have gained increasing attention for use in therapeutic and biotechnology applications. We describe the efficient isolation and characterization of cyclic peptide binders from genetically encoded combinatorial libraries using yeast surface display. Here, peptide cyclization is achieved by disuccinimidyl glutarate-mediated crosslinking of amine groups within a linear peptide sequence that is expressed as a yeast cell surface fusion. Using this approach, we first screened a library of cyclic heptapeptides by magnetic selection and fluorescence activated cell sorting (FACS), to isolate binders for a model target (lysozyme) with low micromolar binding affinity (KD ~ 1.2 - 3.7 {micro}M). The isolated peptides bound lysozyme selectively, and only when cyclized. Importantly, we showed that yeast surface displayed cyclic peptides could be used to efficiently obtain quantitative estimates of binding affinity, without chemical synthesis of the selected peptides. Subsequently, to demonstrate broader applicability of our approach, we isolated cyclic heptapeptides that bind human interleukin-17 (IL-17) using yeast-displayed IL-17 as a target for magnetic selection, followed by FACS using recombinant IL-17. Molecular docking simulations and follow-up experimental analyses identified a candidate cyclic peptide that binds IL-17 in its receptor binding region with moderate affinity (KD ~ 300 nM). Taken together, our results show that yeast surface display can be used to efficiently isolate and characterize cyclic peptides generated by chemical modification from combinatorial libraries.

synthetic biology↗

bioRxiv2020

Genetically encoded live cell sensor for tyrosinated microtubules

Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin post-translational modification (PTMs). These PTMs are known to affect microtubule stability, dynamics and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present there exist no tool that can specifically mark tubulin PTMs in live cells, thus severely limiting our understanding of tubulin PTMs. Using yeast display library, we identified a binder against terminal tyrosine of alpha tubulin, a unique PTM site. Extensive characterization validates the robustness and non-perturbing nature of our binder as tyrosination sensor, a live cell tubulin nanobody specific towards tyrosinated or unmodified microtubules. Using which, in real time we followed nocodazole, colchicine and vincristine induced depolymerization events of unmodified microtubules, and found each distinctly perturb microtubule polymer. Together, our work describes the tyrosination sensor and potential applications to study microtubule and PTM processes in living cells.

cell biology↗

bioRxiv2019

Two distinct trophectoderm lineage stem cells from human pluripotent stem cells

Trophoblasts are the principal cell type of the placenta. The use of human trophoblast stem cells (hTSCs) as a model for studies of early placental development is hampered by limited genetic diversity of existing hTSC lines, and constraints on using human fetal tissue or embryos needed to generate additional cell lines. Here we report the derivation of two distinct stem cells of the trophectoderm lineage from human pluripotent stem cells. The first is a CDX2- stem cell equivalent to primary hTSCs - they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2+ putative human trophectoderm stem cell (hTESC) with distinct cell culture requirements and differences in gene expression and differentiation relative to hTSCs. Derivation of hTSCs and hTESCs from pluripotent stem cells significantly enables construction of models for normal and pathological placental development.

developmental biology↗

bioRxiv2019

Screening yeast display libraries against magnetized yeast cell targets enables efficient isolation of membrane protein binders.

When isolating binders from yeast displayed combinatorial libraries, a soluble, recombinantly expressed form of the target protein is typically utilized. As an alternative, we describe the use of target proteins displayed as surface fusions on magnetized yeast cells. In our strategy, the target protein is co-expressed on the yeast surface with an iron oxide binding protein; incubation of these yeast cells with iron oxide nanoparticles results in their magnetization. Subsequently, binder cells that interact with the magnetized target cells can be isolated using a magnet. Using a known binder-target pair with modest binding affinity (KD ~ 400 nM), we showed that a binder present at low frequency (1 in 105) could be enriched >100-fold, in a single round of screening, suggesting feasibility of screening combinatorial libraries. Subsequently, we screened yeast display libraries of Sso7d and nanobody variants against yeast displayed targets to isolate binders specific to the cytosolic domain of the mitochondrial membrane protein TOM22 (KD ~ 271-2009 nM) and the extracellular domain of the c-Kit receptor (KD ~ 131 to KD >2000 nM). Additional studies showed that the TOM22 binders identified using this approach could be used for the enrichment of mitochondria from cell lysates, thereby confirming binding to the native mitochondrial protein. The ease of expressing a membrane protein or a domain thereof as a yeast cell surface fusion - in contrast to recombinant soluble expression - makes the use of yeast-displayed targets particularly attractive. Therefore, we expect the use of magnetized yeast cell targets will enable efficient isolation of binders to membrane proteins.\n\nO_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

bioengineering↗

bioRxiv2019

A Fyn - specific biosensor reveals localized, pulsatile kinase activity and spatially regulated signaling crosstalk

Cell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor (FynSensor) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. FynSensor imaging uncovers spatially-organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate.

cell biology↗