The influence of molecular reach and diffusivity on the efficacy of membrane-confined reactions

Signaling by surface receptors often relies on tethered reactions whereby an enzyme bound to the cytoplasmic tail of a receptor catalyzes reactions on substrates within reach. The overall length and stiffness of the receptor tail, the enzyme, and the substrate determine a biophysical parameter termed the molecular reach of the reaction. This parameter determines the probability that the receptor-tethered enzyme will contact the substrate in the volume proximal to the membrane when separated by different distances within the membrane plane. In this work, we develop particle-based stochastic reaction-diffusion models to study the interplay between molecular reach and diffusion. We find that increasing the molecular reach can increase reaction efficacy for slowly diffusing receptors, whereas for rapidly diffusing receptors, increasing molecular reach reduces reaction efficacy. In contrast, if reactions are forced to take place within the two-dimensional plasma membrane instead of the three-dimensional volume proximal to it or if molecules diffuse in three dimensions, increasing molecular reach increases reaction efficacy for all diffusivities. We show results in the context of immune checkpoint receptors (PD-1 dephosphorylating CD28), a standard opposing kinase-phosphatase reaction, and a minimal two-particle model. The work highlights the importance of the three-dimensional nature of many two-dimensional membrane-confined interactions, illustrating a role for molecular reach in control-ling biochemical reactions.Published versio

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Biophysical assay for tethered signaling reactions reveals tether-controlled activity for the phosphatase SHP-1

Tethered enzymatic reactions are ubiquitous in signaling networks but are poorly understood. A previously unreported mathematical analysis is established for tethered signaling reactions in surface plasmon resonance (SPR). Applying the method to the phosphatase SHP-1 interacting with a phosphorylated tether corresponding to an immune receptor cytoplasmic tail provides five biophysical/biochemical constants from a single SPR experiment: two binding rates, two catalytic rates, and a reach parameter. Tether binding increases the activity of SHP-1 by 900-fold through a binding-induced allosteric activation (20-fold) and a more significant increase in local substrate concentration (45-fold). The reach parameter indicates that this local substrate concentration is exquisitely sensitive to receptor clustering. We further show that truncation of the tether leads not only to a lower reach but also to lower binding and catalysis. This work establishes a new framework for studying tethered signaling processes and highlights the tether as a control parameter in clustered receptor signaling

Integration of CHO cell culture process improvements with continued process verification

Continued verification of the manufacturing process is a key component of the process validation lifecycle. It provides assurance that the process remains in a state of control during commercial manufacturing through ongoing process and product monitoring and review of quality system elements, such as change control, deviation management. Continuous improvement of cell culture process and operations is an important aspect of routine commercial manufacturing. This is driven by factors such as increased health authority requirements, advanced technology, emerging knowledge on mammalian cell culture process and product quality, and cost benefit. Most operational improvements, when implemented, are assumed not to impact culture performance and/or critical quality attributes. However, unintended consequences have been encountered during the continuous improvement of CHO cell culture processes in Vacaville facility. Through the continued process verification (CPV), the impact to process performance and product quality is able to be detected and corrected when needed. A few examples shared in this poster will focus on the areas easily overlooked during implementation of a change, and the cumulative impact of multiple changes through life cycle of the products. Cell culture process A uses depth filter as part of medium filtration, and it has been known that depth filter can impact the glycosylation pattern as part of the effort to evaluate depth filter removal during lab scale. Due to a vendor initiated change (VIC) for the depth filter manufacturing process, intensive studies were conducted in lab scale to understand the potential impact to product quality. However, when utilizing the post VIC change filter in large scale, an unexpected shift in glycosylation profile was observed. Troubleshooting studies were performed to better understand the change, and further process changes were implemented to revert the product quality back to pre VIC level. A second case study will discuss the impact pH control related changes implemented to cell culture process B, and their impact to culture performance and product quality. The result from the interaction of those changes will be discussed. This poster will shed some light on the need to integrate CPV into the implementation of ongoing process and operational improvements

Improving glycosylation profiles and cell culture performance with a sensitive cell line in commercial manufacturing

A history of sporadically poor culture performance in the inoculum train and subsequent production cultures has been observed in a CHO cell line expressing a recombinant mAb X, resulting in insufficient titer for forward processing downstream. Through data analysis and experimentation in controlled bioreactors, this poor performance has been linked to seed train culture conditions- primarily seed train culture pH. Standard manufacturing practices for pH monitoring and control were modified to more precisely control culture pH to desired set point. Non-standard metrics (such as cell size and oxygen uptake rate) have been used to monitor seed train cultures. Evidence of the poor growth phenotype can also be found in elevated levels of binding immunoglobin protein (BiP) in the seed train culture cells. With seed train pH control improvements and enhanced monitoring in place, the poor culture performance has not been observed in recent commercial campaigns. In addition to reduced titer, product quality is also impacted when slower growth is observed in production culture, specifically out of specification high afucosylated species is observed. For one case of poor production performance that resulted in a high out of specification result for the afucosylated species, a review of equipment, cell age, raw material, adventitious agent testing, mechanical and process change records, and production media composition did not identify anything that could be linked to the slow growth observed. However, trend analysis revealed an abnormally high ammonium level in the early inoculum train stage. This case showed that minor deviations in the inoculum train cultures could result in poor performance in production cultures later, ultimately resulting in product quality failures. In order to reduce impact to product quality using this sensitive cell line, efforts to move the process into a more robust zone have been explored. Modified pH control strategies and impact to culture performance and product quality control will be discussed

Comprehensive analysis of T cell leukemia signals reveals heterogeneity in the PI3 kinase-Akt pathway and limitations of PI3 kinase inhibitors as monotherapy.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer. Poly-chemotherapy with cytotoxic and genotoxic drugs causes substantial toxicity and more specific therapies targeting the underlying molecular lesions are highly desired. Perturbed Ras signaling is prevalent in T-ALL and occurs via oncogenic RAS mutations or through overexpression of the Ras activator RasGRP1 in ~65% of T-ALL patients. Effective small molecule inhibitors for either target do not currently exist. Genetic and biochemical evidence link phosphoinositide 3-kinase (PI3K) signals to T-ALL, PI3Ks are activated by Ras-dependent and Ras-independent mechanisms, and potent PI3K inhibitors exist. Here we performed comprehensive analyses of PI3K-Akt signaling in T-ALL with a focus on class I PI3K. We developed a multiplex, multiparameter flow cytometry platform with pan- and isoform-specific PI3K inhibitors. We find that pan-PI3K and PI3K γ-specific inhibitors effectively block basal and cytokine-induced PI3K-Akt signals. Despite such inhibition, GDC0941 (pan-PI3K) or AS-605240 (PI3Kγ-specific) as single agents did not efficiently induce death in T-ALL cell lines. Combination of GDC0941 with AS-605240, maximally targeting all p110 isoforms, exhibited potent synergistic activity for clonal T-ALL lines in vitro, which motivated us to perform preclinical trials in mice. In contrast to clonal T-ALL lines, we used a T-ALL cancer model that recapitulates the multi-step pathogenesis and inter- and intra-tumoral genetic heterogeneity, a hallmark of advanced human cancers. We found that the combination of GDC0941 with AS-605240 fails in such trials. Our results reveal that PI3K inhibitors are a promising avenue for molecular therapy in T-ALL, but predict the requirement for methods that can resolve biochemical signals in heterogeneous cell populations so that combination therapy can be designed in a rational manner

Essential role of membrane cholesterol in accelerated BCR internalization and uncoupling from NF-κB in B cell clonal anergy

Divergent hypotheses exist to explain how signaling by the B cell receptor (BCR) is initiated after antigen binding and how it is qualitatively altered in anergic B cells to selectively uncouple from nuclear factor κB and c-Jun N-terminal kinase pathways while continuing to activate extracellular signal–regulated kinase and calcium–nuclear factor of activated T cell pathways. Here we find that BCRs on anergic cells are endocytosed at a very enhanced rate upon binding antigen, resulting in a large steady-state pool of intracellularly sequestered receptors that appear to be continuously cycling between surface and intracellular compartments. This endocytic mechanism is exquisitely sensitive to the lowering of plasma membrane cholesterol by methyl-β-cyclodextrin, and, when blocked in this way, the sequestered BCRs return to the cell surface and RelA nuclear accumulation is stimulated. In contrast, when plasma membrane cholesterol is lowered and GM1 sphingolipid markers of membrane rafts are depleted in naive B cells, this does not diminish BCR signaling to calcium or RelA. These results provide a possible explanation for the signaling changes in clonal anergy and indicate that a chief function of membrane cholesterol in B cells is not to initiate BCR signaling, but instead to terminate a subset of signals by rapid receptor internalization

Satellite Content and Halo Mass of Galaxy Clusters: Comparison between Red-Sequence and Halo-based Optical Cluster Finders

Cluster cosmology depends critically on how the optical clusters are selected from imaging surveys. We compare the conditional luminosity function (CLF) and weak lensing halo masses between two different cluster samples at fixed richness, detected within the same volume ($0.1{<}z{<}0.34$) using the red-sequence and halo-based methods. After calibrating our CLF deprojection method against mock galaxy samples, we measure the 3D CLFs by cross-correlating clusters with SDSS photometric galaxies. As expected, the CLFs of the red-sequence and halo-based finders exhibit redder and bluer populations, respectively. We also find significant shape discrepancies between the two CLFs at the faint end, where the red-sequence clusters show a strong deficit of faint galaxies but a bump at $M_r{\sim}-20.5$, while the halo-based clusters host an increasing number of blue satellites. By comparing the subsamples of clusters that have a match between the two catalogues to those without matches, we discover that the CLF shape depends sensitively on the cluster centroiding. However, the average weak lensing halo mass between the matched and non-matched clusters are roughly consistent with each other in either cluster sample. Since the colour preferences of the two cluster finders are almost orthogonal, such a consistency indicates that the scatter in the mass-richness relation of either cluster sample is close to random. Therefore, while the choice of how optical clusters are identified impacts the satellite content, our result suggests that it should not introduce strong systematics biases in cluster cosmology.Comment: 17 pages, 12 figures, 6 table

Bayesian Inference of Spatial Organizations of Chromosomes

Knowledge of spatial chromosomal organizations is critical for the study of transcriptional regulation and other nuclear processes in the cell. Recently, chromosome conformation capture (3C) based technologies, such as Hi-C and TCC, have been developed to provide a genome-wide, three-dimensional (3D) view of chromatin organization. Appropriate methods for analyzing these data and fully characterizing the 3D chromosomal structure and its structural variations are still under development. Here we describe a novel Bayesian probabilistic approach, denoted as “Bayesian 3D constructor for Hi-C data” (BACH), to infer the consensus 3D chromosomal structure. In addition, we describe a variant algorithm BACH-MIX to study the structural variations of chromatin in a cell population. Applying BACH and BACH-MIX to a high resolution Hi-C dataset generated from mouse embryonic stem cells, we found that most local genomic regions exhibit homogeneous 3D chromosomal structures. We further constructed a model for the spatial arrangement of chromatin, which reveals structural properties associated with euchromatic and heterochromatic regions in the genome. We observed strong associations between structural properties and several genomic and epigenetic features of the chromosome. Using BACH-MIX, we further found that the structural variations of chromatin are correlated with these genomic and epigenetic features. Our results demonstrate that BACH and BACH-MIX have the potential to provide new insights into the chromosomal architecture of mammalian cells.Statistic

Prevalent and Dynamic Binding of the Cell Cycle Checkpoint Kinase Rad53 to Gene Promoters

Replication of the genome must be coordinated with gene transcription and cellular metabolism, especially following replication stress in the presence of limiting deoxyribonucleotides. The Saccharomyces cerevisiae Rad53 (CHEK2 in mammals) checkpoint kinase plays a major role in cellular responses to DNA replication stress. Cell cycle regulated, genome-wide binding of Rad53 to chromatin was examined. Under replication stress, the kinase bound to sites of active DNA replication initiation and fork progression, but unexpectedly to the promoters of about 20% of genes encoding proteins involved in multiple cellular functions. Rad53 promoter binding correlated with changes in expression of a subset of genes. Rad53 promoter binding to certain genes was influenced by sequence-specific transcription factors and less by checkpoint signaling. However, in checkpoint mutants, untimely activation of late-replicating origins reduces the transcription of nearby genes, with concomitant localization of Rad53 to their gene bodies. We suggest that the Rad53 checkpoint kinase coordinates genome-wide replication and transcription under replication stress conditions