Pooled protein tagging,cellular imaging,and in situ sequencing for monitoring drug action in real time

The levels and subcellular localizations of proteins regulate critical aspects of many cellular processes and can become targets of therapeutic intervention. However, high-throughput methods for the discovery of proteins that change localization either by shuttling between compartments, by binding larger complexes, or by localizing to distinct membraneless organelles are not available. Here we describe a scalable strategy to characterize effects on protein localizations and levels in response to different perturbations. We use CRISPR-Cas9-based intron tagging to generate cell pools expressing hundreds of GFP-fusion proteins from their endogenous promoters and monitor localization changes by time-lapse microscopy followed by clone identification using in situ sequencing. We show that this strategy can characterize cellular responses to drug treatment and thus identify nonclassical effects such as modulation of protein–protein interactions, condensate formation, and chemical degradation.

Currently available mass-spectrometry methods (Rix and Superti-Furga 2009; Martinez Molina et al. 2013; Savitski et al. 2014; Huber et al. 2015; Drewes and Knapp 2018) for monitoring the effects of cellular perturbations on proteomes cannot be scaled efficiently to monitor time-dependent effects in high throughput. A different approach to study drug action is live-cell imaging of protein dynamics in cells expressing a protein of interest fused to a fluorescent tag. Traditionally, such reporter cells are generated either by overexpression to nonphysiologic levels, by oligonucleotide-directed homologous recombination in yeast, or by using CRISPR-Cas9 and homology-directed repair (HDR) to endogenously tag proteins in human cells (Ghaemmaghami et al. 2003; Huh et al. 2003; Chong et al. 2015; Leonetti et al. 2016). In addition to those targeted approaches, “gene trapping” or “CD-tagging” strategies, which rely on the random, viral integration of fluorescent tags as synthetic exons, have been used for analyzing dynamic changes in response to drugs (Jarvik et al. 1996; Morin et al. 2001; Cohen et al. 2008; Kang et al. 2016), but they are limited by integration site biases and require the isolation and characterization of clones before using them in an arrayed format. Recently, a strategy combining genome engineering and gene trapping using homology-independent CRISPR-Cas9 editing to place a fluorescent tag as a synthetic exon into introns of individual target genes has been described (Serebrenik et al. 2019). The strategy relies on a generic sgRNA excising a fluorescent tag flanked by splice acceptor and donor sites from a generic donor plasmid, which is coexpressed with a gene-specific intron-targeting sgRNA specifying the integration site. Here we show the scalability of that strategy to enable pooled protein tagging of more than 900 metabolic enzymes and epigenetic modifiers. Exposing the GFP-tagged cells to compounds allows us to monitor drug effects on the localization and levels of hundreds of proteins in real time in a pooled format, followed by identification of responding clones by in situ sequencing of the expressed intron-targeting sgRNA that corresponds to the tagged protein (Fig. 1A).Open in a separate windowFigure 1.Pooled GFP intron-tagging of metabolic enzymes. (A) Schematic outline of the approach. (B) Identification of targetable introns within metabolic genes. (C) FACS sorting of clones with successful GFP-tagging by signal enrichment over background mCherry intensity used as control for autofluorescence. (D) Representative image of sorted GFP-tagged cell pool. Scale bar, 25 µm. (E) Comparison of RNA-seq expression in HAP1 cells between genes for which GFP-tagged cells could be isolated and genes that were targeted in the sgRNA library but did not result in successful clone isolation.     

Kinetics and mechanism of formation and properties of polymers composed of paired macromolecules

The influence of formation conditions on structure and properties of reaction products from two different macromolecules (paired polymers) such as polystyrene and poly(1,1,2-trichlorobutadiene) or polystyrene and poly(vinyl chloride) is investigated. Mechanical properties, molecular mobility, heat resistance, thermostability, and fire resistance are shown to be regulated over a wide range by changing the molecular weight of the initial polymers, their ratio in the reaction mixture, etc. The interaction of different macromolecules in solution to form paired polymers is analyzed theoretically and experimentally. An analysis of structure and properties of the resulting products by refractometry, viscometry, sedimentation velocity, statistical analysis, and others shows that paired polymers are systems of the “coil-in-coil” type held together by chemical bonds in the zones of mutual penetration.     

Multiple mechanisms govern the dynamics of depression at neocortical synapses of young rats

Synaptic transmission between pairs of excitatory neurones in layers V ( N = 38) or IV ( N = 6) of somatosensory cortex was examined in a parasagittal slice preparation obtained from young Wistar rats (14–18 days old). A combined experimental and theoretical approach reveals two characteristics of short-term synaptic depression. Firstly, as well as a release-dependent depression, there is a release-independent component that is evident in smaller postsynaptic responses even following failure to release transmitter. Secondly, recovery from depression is activity dependent and is faster at higher input frequencies. Frequency-dependent recovery is a Ca²⁺-dependent process and does not reflect an underlying augmentation. Frequency-dependent recovery and release-independent depression are correlated, such that at those connections with a large amount of release-independent depression, recovery from depression is faster. In addition, both are more pronounced in experiments performed at physiological temperatures. Simulations demonstrate that these homeostatic properties allow the transfer of rate information at all frequencies, essentially linearizing synaptic responses at high input frequencies.     

Modeling the presentation of C3d-coated antigen by B lymphocytes: enhancement by CR1/2-BCR co-ligation is selective for the co-ligating antigen

We have used a set of single-chain variable fragment antibodies (sc) genetically fused with an influenza hemagglutinin-derived peptide as a means to investigate the role of CR1 and CR2 in antigen presentation by B cells. When incubated with the B cell lymphoma 2PK3, peptide-containing sc specific for either CR1 or CR1/2 mediated activation of the hemagglutinin peptide-specific T cell line IP-12-7, as assessed by IL-2 production. Efficient presentation was dependent on the binding of the constructs to CR1/2, implying that receptor-mediated endocytosis is responsible for the effect. Cross-linkage of CR1/2 or CD19 by mAb did not increase the extent of T cell activation. However, when CR1/2 was co-ligated with the BCR--using either polyclonal goat anti-mouse IgG or recombinant protein LA--the antigen concentration required to activate T cells decreased by two orders of magnitude. Moreover, this enhancement was selective for the antigen included in these complexes and did not affect the presentation of a free peptide or of antigen bound to CR1/2 excluded from the complexes. These results suggest that B cells may bind various C3d-coated antigens at a time, but only the one which reacts with the BCR will be processed with high efficiency. This mechanism may ensure the specificity of cognate T cell help.     

Alteration of the LRP1B gene region is associated with high grade of urothelial cancer

We have delineated regions of interest at chromosome 2q21.2, 2q36.3, and 2q37.1 by deletion mapping of 114 urothelial cancers (UC). Altogether, 17%, 18%, and 63% of the G1, G2, and G3 tumors displayed loss of heterozygosity at chromosome 2q, respectively, The region at 2q21.2 was narrowed down to the LRP1B gene (NT_005129.6). Hemi- and homozygous deletion at the LRP1B gene region was seen in 31 of 114 UCs. Only 8% of the UCs with G1 and none with G2 tumors showed loss of heterozygosity at the LRP1B gene, whereas 49% of the G3 UCs had allelic loss at this region. RT-PCR analysis of the LRP1B gene showed the lack of expression of several exons in 2 of 9 cases analyzed. Our analysis suggests that the LRP1B gene is a candidate tumor suppressor gene in UCs.     

Metastatic potential of human CX-1 colon adenocarcinoma cells is dependent on the expression of sialosyl Le a antigen

Several lines of evidence indicate that sialosyl Le a , tumor-associated carbohydrate antigen present on human colon carcinoma cells, is involved in formation of metastases. To study the role of this carbohydrate structure in development of metastases, we have used the clone of human colon carcinoma CX-1 cells transfected with antisense expression vector containing fragment of cDNA for a1,3/4-fucosyltransferase (FT III), which is involved in synthesis of sialosyl Le a tetrasaccharide. It has been reported previously that, in contrast to the parental cells, the antisense-transfected CX-1.1AS5 cells do not express sialosyl Le a and do not adhere to E-selectin-expressing CHO cells. In the present work we have studied the formation of liver metastases by CX-1.1AS5 cells after their orthotopic or intrasplenic implantation into athymic nu/nu mice. After orthotopic implantation of sialosyl Le a -negative colon carcinoma CX-1.1AS5 cells, the number of mice with liver metas-tases was markedly lower (21% of mice) in comparison with their number after implantation of the parental CX-1.1 cells (86% of mice). However, no differences in ability to form colonies in liver were observed between parental CX-1.1 cells and antisense-transfected CX-1.1AS5 cells after intrasplenic inoculation. The liver metastases were formed in 89% and 84% of mice, respectively. Our data support the thesis on the importance of sialosyl Le a antigen expression in the development of liver metastases by colon cancer cells, and indicate the role of transplantation route and primary tumor localization in formation of metastases.© Kluwer Academic Publishers 1998     

Two-color FISH characterization of i(1q) and der(1;16) in human breast cancer cells

Two-color fluorescent in situ hybridizations using probes for alphoid (α) and classical satellite (CS) DNAs from chromosomes 1 and 16 were performed to characterize i(1q), der(1;16), and complex rearrangements observed in breast cancer cells from fresh tumors and established cell lines. Six of seven i(1q) occurred after breakage in the α1 containing region and one of seven was dicentric, with breakage in 1p11.2. The five der(1;16)(q10;p10) studied appeared to result from a variety of breakpoints involving α1, α16, CS1, and CS16 DNAs. All had conserved α16 DNA, suggesting a segregation of the der(1;16) leading to a loss of 16q and a gain of 1q in most cases. One complex rearrangement of chromosome 1 also appeared to involve chromosome 16, suggesting that a der(1;16) occurred first, followed by another rearrangement. Both the apparent preferential involvement of constitutive heterochromatin harboring α and CS DNAs and the variety of breakpoints spanning along heterochromatin suggest that the important consequence of the rearrangement is not the breakage per se but the resulting imbalance. © 1993 Wiley-Liss, Inc.     

Tissue zinc levels in precancerous tissue in the gastrointestinal tract of azoxymethane (AOM)-treated rats

Alterations in tissue zinc levels have been documented in patients with gastrointestinal tract malignancies and more frequently, in those with colonic cancer. However, the precise role of tissue zinc in carcinogenesis is not well elucidated. This study, using a well-established colon cancer model in rats, was designed to investigate the relationship of tissue zinc to the carcinogenic process. The aim was to examine tissue zinc levels in the preneoplastic tissues and to study the changes that occur during transition of mucosa from normal to preneoplastic state. Six-week old rats were given a single dose subcutaneous injection of azoxymethane (AOM) (30mg/kg body weight) and sacrificed after 1, 2, 5, and 9 months of the treatment. Plasma zinc levels showed a significant decrease (p<0.05) at 9 months compared with controls. Tissue zinc levels showed a significant decrease in the large intestine at 1 and 2 months (p<0.05) and at 5 and 9 months (p<0.01), in the small intestine at 2, 5, and 9 months (p<0.05), and in the stomach at 5 and 9 months (p<0.05). The maximum percent decrease (45%) in tissue zinc was observed in the large intestine at 9 months. Tissue copper zinc super oxide dismutase (CuZnSOD) activity was assessed in the body of the stomach, small intestine, and large intestine and compared with the control group. There was a significant fall in CuZnSOD levels in the small intestine at 9 months (p<0.05) and in the large intestine at 5 and 9 months (p<0.01). Two of these six rats showed histological evidence of precancerous lesions in the mucosa of the colon. This study suggests that the decrease in plasma zinc, tissue zinc and activity of CuZnSOD is associated with development of preneoplastic lesions in the colonic mucosa.     

Involvement of MMPs in delayed neuronal death after global ischemia

Spatial and temporal relations between metalloproteinase (MMP-2 and MMP-9) activation and laminin degradation in gerbil hippocampus after transient cerebral ischemia has been studied. Activity of MMPs was determined by gelatin zymography in homogenates from dorsal (DP, an equivalent of CA1 sector) and abdominal (AbP, containing CA2-4 and gyrus dentatus) parts of hippocampus. A significant activation of both investigated metalloproteinases was found at 72 h of recovery. Whereas MMP-2 up-regulation did not show any spatial preferences, the increase of MMP-9 activity was observed exclusively in DP. Activation of MMP-9 at this time point correlated spatially with degradation of laminin-protein of extracellular matrix. These results show that MMP pathway may function as a component of delayed neuronal death cascade in the apoptogenic CA1 sector after transient global ischemia.     

Morphometric study of the ductus arteriosus during human development

During prenatal life, the ductus arteriosus connects the left pulmonary artery and the descending aorta. Morphometric features (length, external diameter, volume) of the ductus arteriosus in 131 human fetuses (65 males, 66 females) were studied by means of anatomical, digital and statistical methods. Regression analysis was used to investigate the growth of the ductus arteriosus during gestation. The values of the length of the ductus arteriosus ranged from 3.95 mm for the 15 week gestational group to 12.20 mm for the 34th week of gestation. The length of the ductus arteriosus related to fetal age (x) increased according to the linear function y = -3.0726 + 0.4381x. The mean values of the diameter of the ductus arteriosus ranged from 1.34 to 3.49mm for the 15 and 34 week gestational groups, respectively. The growth of the ductus arteriosus diameter followed in accordance with the linear function y = 0.2072 + 0.0935x. The mean values of the ductus arteriosus volume ranged from 5.08 mm3 for the 15 week group to 117.30 mm3 of the 34 week gestation group. The volume growth increased according to the function y = 0.0007x3.3782. Positive correlation coefficients between arterial parameters and fetal age were statistically significant (P < or = 0.01) and reached the following values: r1 = 0.98 for Length, r2 = 0.90 for diameter and r3 = 0.94 for volume. Despite the increase in absolute diameter, the relative diameter of the ductus arteriosus (ductus arteriosus-to-aortic bulb diameter ratio) decreased from 0.80 to 0.48.