Prediction of MET Overexpression in Lung Adenocarcinoma from Hematoxylin and Eosin Images.

Mesenchymal epithelial transition (MET) protein overexpression is a targetable event in non-small cell lung cancer and is the subject of active drug development. Challenges in identifying patients for these therapies include lack of access to validated testing, such as standardized immunohistochemistry assessment, and consumption of valuable tissue for a single gene/protein assay. Development of prescreening algorithms using routinely available digitized hematoxylin and eosin (H&E)-stained slides to predict MET overexpression could promote testing for those who will benefit most. Recent literature reports a positive correlation between MET protein overexpression and RNA expression. In this work, a large database of matched H&E slides and RNA expression data were leveraged to train a weakly supervised model to predict MET RNA overexpression directly from H&E images. This model was evaluated on an independent holdout test set of 300 overexpressed and 289 normal patients, demonstrating a receiver operating characteristic area under curve of 0.70 (95th percentile interval: 0.66 to 0.74) with stable performance characteristics across different patient clinical variables and robust to synthetic noise on the test set. These results suggest that H&E-based predictive models could be useful to prioritize patients for confirmatory testing of MET protein or MET gene expression status.

Validation of an Expanded Carrier Screen that Optimizes Sensitivity via Full-Exon Sequencing and Panel-wide Copy Number Variant Identification.

BACKGROUND: By identifying pathogenic variants across hundreds of genes, expanded carrier screening (ECS) enables prospective parents to assess the risk of transmitting an autosomal recessive or X-linked condition. Detection of at-risk couples depends on the number of conditions tested, the prevalence of the respective diseases, and the screen's analytical sensitivity for identifying disease-causing variants. Disease-level analytical sensitivity is often <100% in ECS tests because copy number variants (CNVs) are typically not interrogated because of their technical complexity. METHODS: We present an analytical validation and preliminary clinical characterization of a 235-gene sequencing-based ECS with full coverage across coding regions, targeted assessment of pathogenic noncoding variants, panel-wide CNV calling, and specialized assays for technically challenging genes. Next-generation sequencing, customized bioinformatics, and expert manual call review were used to identify single-nucleotide variants, short insertions and deletions, and CNVs for all genes except FMR1 and those whose low disease incidence or high technical complexity precluded novel variant identification or interpretation. RESULTS: Screening of 36859 patients' blood or saliva samples revealed the substantial impact on fetal disease-risk detection attributable to novel CNVs (9.19% of risk) and technically challenging conditions (20.2% of risk), such as congenital adrenal hyperplasia. Of the 7498 couples screened, 335 were identified as at risk for an affected pregnancy, underscoring the clinical importance of the test. Validation of our ECS demonstrated >99% analytical sensitivity and >99% analytical specificity. CONCLUSIONS: Validated high-fidelity identification of different variant types-especially for diseases with complicated molecular genetics-maximizes at-risk couple detection.

Machine-Learning-Based Analysis in Genome-Edited Cells Reveals the Efficiency of Clathrin-Mediated Endocytosis.

Cells internalize various molecules through clathrin-mediated endocytosis (CME). Previous live-cell imaging studies suggested that CME is inefficient, with about half of the events terminated. These CME efficiency estimates may have been confounded by overexpression of fluorescently tagged proteins and inability to filter out false CME sites. Here, we employed genome editing and machine learning to identify and analyze authentic CME sites. We examined CME dynamics in cells that express fluorescent fusions of two defining CME proteins, AP2 and clathrin. Support vector machine classifiers were built to identify and analyze authentic CME sites. From inception until disappearance, authentic CME sites contain both AP2 and clathrin, have the same degree of limited mobility, continue to accumulate AP2 and clathrin over lifetimes >∼20 s, and almost always form vesicles as assessed by dynamin2 recruitment. Sites that contain only clathrin or AP2 show distinct dynamics, suggesting they are not part of the CME pathway.

Centrosomes. Regulated assembly of a supramolecular centrosome scaffold in vitro.

The centrosome organizes microtubule arrays within animal cells and comprises two centrioles surrounded by an amorphous protein mass called the pericentriolar material (PCM). Despite the importance of centrosomes as microtubule-organizing centers, the mechanism and regulation of PCM assembly are not well understood. In Caenorhabditis elegans, PCM assembly requires the coiled-coil protein SPD-5. We found that recombinant SPD-5 could polymerize to form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Only the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other PCM proteins. Thus, PCM size and binding capacity emerge from the regulated polymerization of one coiled-coil protein to form a porous network.

Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) involves the recruitment of numerous proteins to sites on the plasma membrane with prescribed timing to mediate specific stages of the process. However, how choreographed recruitment and function of specific proteins during CME is achieved remains unclear. Using genome editing to express fluorescent fusion proteins at native levels and live-cell imaging with single-molecule sensitivity, we explored dynamin2 stoichiometry, dynamics, and functional interdependency with actin. Our quantitative analyses revealed heterogeneity in the timing of the early phase of CME, with transient recruitment of 2-4 molecules of dynamin2. In contrast, considerable regularity characterized the final 20 s of CME, during which ∼26 molecules of dynamin2, sufficient to make one ring around the vesicle neck, were typically recruited. Actin assembly generally preceded dynamin2 recruitment during the late phases of CME, and promoted dynamin recruitment. Collectively, our results demonstrate precise temporal and quantitative regulation of the dynamin2 recruitment influenced by actin polymerization.

Caulobacter chromosome in vivo configuration matches model predictions for a supercoiled polymer in a cell-like confinement.

We measured the distance between fluorescent-labeled DNA loci of various interloci contour lengths in Caulobacter crescentus swarmer cells to determine the in vivo configuration of the chromosome. For DNA segments less than about 300 kb, the mean interloci distances, , scale as n(0.22), where n is the contour length, and cell-to-cell distribution of the interloci distance r is a universal function of r/n(0.22) with broad cell-to-cell variability. For DNA segments greater than about 300 kb, the mean interloci distances scale as n, in agreement with previous observations. The 0.22 value of the scaling exponent for short DNA segments is consistent with theoretical predictions for a branched DNA polymer structure. Predictions from Brownian dynamics simulations of the packing of supercoiled DNA polymers in an elongated cell-like confinement are also consistent with a branched DNA structure, and simulated interloci distance distributions predict that confinement leads to "freezing" of the supercoiled configuration. Lateral positions of labeled loci at comparable positions along the length of the cell are strongly correlated when the longitudinal locus positions differ by <0.16 µm. We conclude that the chromosome structure is supercoiled locally and elongated at large length scales and that substantial cell-to-cell variability in the interloci distances indicates that in vivo crowding prevents the chromosome from reaching an equilibrium arrangement. We suggest that the force causing rapid transport of loci remote from the parS centromere to the distal cell pole may arise from the release at the polar region of potential energy within the supercoiled DNA.

Compaction and transport properties of newly replicated Caulobacter crescentus DNA.

Upon initiating replication of the Caulobacter chromosome, one copy of the parS centromere remains at the stalked pole; the other moves to the distal pole. We identified the segregation dynamics and compaction characteristics of newly replicated Caulobacter DNA during transport (highly variable from cell to cell) using time-lapse fluorescence microscopy. The parS centromere and a length (also highly variable) of parS proximal DNA on each arm of the chromosome are segregated with the same relatively slow transport pattern as the parS locus. Newly replicated DNA further than about 100 kb from parS segregates with a different and faster pattern, while loci at 48 kb from parS segregate with the slow pattern in some cells and the fast pattern in others. The observed parS-proximal DNA compaction characteristics have scaling properties that suggest the DNA is branched. HU2-deletion strains exhibited a reduced compaction phenotype except near the parS site where only the ΔHU1ΔHU2 double mutant had a compaction phenotype. The chromosome shows speed-dependent extension during translocation suggesting the DNA polymer is under tension. While DNA segregation is highly reliable and succeeds in virtually all wild-type cells, the high degree of cell to cell variation in the segregation process is noteworthy.

The three-dimensional architecture of a bacterial genome and its alteration by genetic perturbation.

We have determined the three-dimensional (3D) architecture of the Caulobacter crescentus genome by combining genome-wide chromatin interaction detection, live-cell imaging, and computational modeling. Using chromosome conformation capture carbon copy (5C), we derive ~13 kb resolution 3D models of the Caulobacter genome. The resulting models illustrate that the genome is ellipsoidal with periodically arranged arms. The parS sites, a pair of short contiguous sequence elements known to be involved in chromosome segregation, are positioned at one pole, where they anchor the chromosome to the cell and contribute to the formation of a compact chromatin conformation. Repositioning these elements resulted in rotations of the chromosome that changed the subcellular positions of most genes. Such rotations did not lead to large-scale changes in gene expression, indicating that genome folding does not strongly affect gene regulation. Collectively, our data suggest that genome folding is globally dictated by the parS sites and chromosome segregation.

Assembly of the Caulobacter cell division machine.

Cytokinesis in Gram-negative bacteria is mediated by a multiprotein machine (the divisome) that invaginates and remodels the inner membrane, peptidoglycan and outer membrane. Understanding the order of divisome assembly would inform models of the interactions among its components and their respective functions. We leveraged the ability to isolate synchronous populations of Caulobacter crescentus cells to investigate assembly of the divisome and place the arrival of each component into functional context. Additionally, we investigated the genetic dependence of localization among divisome proteins and the cell cycle regulation of their transcript and protein levels to gain insight into the control mechanisms underlying their assembly. Our results revealed a picture of divisome assembly with unprecedented temporal resolution. Specifically, we observed (i) initial establishment of the division site, (ii) recruitment of early FtsZ-binding proteins, (iii) arrival of proteins involved in peptidoglycan remodelling, (iv) arrival of FtsA, (v) assembly of core divisome components, (vi) initiation of envelope invagination, (vii) recruitment of polar markers and cytoplasmic compartmentalization and (viii) cell separation. Our analysis revealed differences in divisome assembly among Caulobacter and other bacteria that establish a framework for identifying aspects of bacterial cytokinesis that are widely conserved from those that are more variable.

High-throughput identification of protein localization dependency networks.

Bacterial cells are highly organized with many protein complexes and DNA loci dynamically positioned to distinct subcellular sites over the course of a cell cycle. Such dynamic protein localization is essential for polar organelle development, establishment of asymmetry, and chromosome replication during the Caulobacter crescentus cell cycle. We used a fluorescence microscopy screen optimized for high-throughput to find strains with anomalous temporal or spatial protein localization patterns in transposon-generated mutant libraries. Automated image acquisition and analysis allowed us to identify genes that affect the localization of two polar cell cycle histidine kinases, PleC and DivJ, and the pole-specific pili protein CpaE, each tagged with a different fluorescent marker in a single strain. Four metrics characterizing the observed localization patterns of each of the three labeled proteins were extracted for hundreds of cell images from each of 854 mapped mutant strains. Using cluster analysis of the resulting set of 12-element vectors for each of these strains, we identified 52 strains with mutations that affected the localization pattern of the three tagged proteins. This information, combined with quantitative localization data from epitasis experiments, also identified all previously known proteins affecting such localization. These studies provide insights into factors affecting the PleC/DivJ localization network and into regulatory links between the localization of the pili assembly protein CpaE and the kinase localization pathway. Our high-throughput screening methodology can be adapted readily to any sequenced bacterial species, opening the potential for databases of localization regulatory networks across species, and investigation of localization network phylogenies.

Caulobacter PopZ forms a polar subdomain dictating sequential changes in pole composition and function.

The bacterium Caulobacter crescentus has morphologically and functionally distinct cell poles that undergo sequential changes during the cell cycle. We show that the PopZ oligomeric network forms polar ribosome exclusion zones that change function during cell cycle progression. The parS/ParB chromosomal centromere is tethered to PopZ at one pole prior to the initiation of DNA replication. During polar maturation, the PopZ-centromere tether is broken, and the PopZ zone at that pole then switches function to act as a recruitment factor for the ordered addition of multiple proteins that promote the transformation of the flagellated pole into a stalked pole. Stalked pole assembly, in turn, triggers the initiation of chromosome replication, which signals the formation of a new PopZ zone at the opposite cell pole, where it functions to anchor the newly duplicated centromere that has traversed the long axis of the cell. We propose that pole-specific control of PopZ function co-ordinates polar development and cell cycle progression by enabling independent assembly and tethering activities at the two cell poles.

Caulobacter requires a dedicated mechanism to initiate chromosome segregation.

Chromosome segregation in bacteria is rapid and directed, but the mechanisms responsible for this movement are still unclear. We show that Caulobacter crescentus makes use of and requires a dedicated mechanism to initiate chromosome segregation. Caulobacter has a single circular chromosome whose origin of replication is positioned at one cell pole. Upon initiation of replication, an 8-kb region of the chromosome containing both the origin and parS moves rapidly to the opposite pole. This movement requires the highly conserved ParABS locus that is essential in Caulobacter. We use chromosomal inversions and in vivo time-lapse imaging to show that parS is the Caulobacter site of force exertion, independent of its position in the chromosome. When parS is moved farther from the origin, the cell waits for parS to be replicated before segregation can begin. Also, a mutation in the ATPase domain of ParA halts segregation without affecting replication initiation. Chromosome segregation in Caulobacter cannot occur unless a dedicated parS guiding mechanism initiates movement.