Elucidating the wiring diagram of the human cell is a central goal of the postgenomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry, and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery. We found that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions, and that RNA binding proteins form a specific subgroup defined by unique interaction and localization properties. Paired with a fully interactive website (opencell.czbiohub.org), our work constitutes a resource for the quantitative cartography of human cellular organization.

Supporting Information: Figure S1 Figure. S1 -Endogenous tagging of endolysosomal compartments (A) Representative fluorescence microscopy images of CRISPRi-HEK293T cells with RAB7A-mNG 11 endogenously labeled with the split-mNeonGreen system. Cells were treated with AF555-tau fibrils for 22 hours. (B) Representative fluorescence microscopy images of HEK293T cells with mNG 11 -RAB7A (top) or LAMP1-mNG 11 (bottom) endogenously labeled with the split-mNeonGreen system.

Elucidating the wiring diagram of the human cell is one of the central goals of the post-genomic era. Here, we integrate genome engineering, confocal imaging, mass spectrometry and data science to systematically map protein localization in live cells and protein interactions under endogenous expression conditions. For this, we generated a library of 1,311 CRISPR-edited cell lines harboring fluorescent tags that also serve as handles for affinity capture, and applied a new machine learning framework to encode the interaction and localization profiles of each protein. Our approach provides a data-driven description of the molecular and spatial networks that organize the human proteome. We show that unsupervised clustering of these networks delineates functional groups and facilitates biological discovery, while hierarchical analyses uncover the core features that template cellular architecture. Furthermore, we discover that localization signatures are remarkably predictive of protein function, and often contain enough information to identify molecular interactions. Paired with a fully interactive website (opencell.czbiohub.org), OpenCell is a resource for the quantitative cartography of human cellular organization.

To control their movement, cells need to coordinate actin assembly with the geometric features of their substrate. Here, we uncover a role for the actin regulator WASP in the 3D migration of neutrophils. We show that WASP responds to substrate topology by enriching to sites of inward, substrate-induced membrane deformation. Superresolution imaging reveals that WASP preferentially enriches to the necks of these substrate-induced invaginations, a distribution that could support substrate pinching. WASP facilitates recruitment of the Arp2/3 complex to these sites, stimulating local actin assembly that couples substrate features with the cytoskeleton. Surprisingly, WASP only enriches to membrane deformations in the front half of the cell, within a permissive zone set by WASP’s front-biased regulator Cdc42. While WASP KO cells exhibit relatively normal migration on flat substrates, they are defective at topology-directed migration. Our data suggest that WASP integrates substrate topology with cell polarity by selectively polymerizing actin around substrate-induced membrane deformations in the front half of the cell.

Intercellular propagation of protein aggregation is emerging as a key mechanism in the progression of several neurodegenerative diseases, including Alzheimer's Disease and frontotemporal dementia. However, we lack a systematic understanding of the cellular pathways controlling prion-like propagation. To uncover such pathways, we performed CRISPR interference (CRISPRi) screens in a human cellbased model of propagation of tau aggregation. Our screens uncovered that knockdown of several components of the ESCRT machinery, including CHMP6, or CHMP2A in combination with CHMP2B (a gene linked to familial frontotemporal dementia), promote propagation of tau aggregation. We found that knockdown of these genes caused damage to endolysosomal membranes, consistent with a role for the ESCRT

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Diarrhoeagenic E. coli (DEC) is one of the most common causes of diarrhoeal death in children less than five years globally. It is responsible for 30%–40% of all diarrhoeal episodes in developing countries. It is estimated that 0.12 million children died of diarrhoea caused by DEC in 2011 globally. There is no baseline data on the occurrence of DEC diarrhoea in Andaman Islands, the remote islands of India. The study is particularly important as these strains are the emerging enteric pathogen in both developed and developing countries. DEC was screened from E. coli isolates obtained from diarrhoeal stool samples by multiplex PCR with specific primers using stasndard protocols. During the study period, among the 1394 stool samples collected, 95 (6.82%) patients were found infected with DEC. Of the 97 isolates from 95 patients, 68 (70.1%) were EAEC, 19 (19.6%) were EPEC and 10 (10.3%) were ETEC. Of the 19 EPEC isolates, 63.2% were atypical EPEC which is the emerging enteric pathogen among the children in developing as well as developed countries. More than 80% of the patients had watery diarrhoea and 6% of them had invasive diarrhoea. Persistent diarrhoea was also found in three infected children. This study documents the occurrence and type of DEC diarrhoea in Andaman Islands first time and highlights the significant proportions of E. coli diarrhoea being caused by EAEC and atypical EPEC strains.

Bacterial biofilm infections, particularly those of Pseudomonas aeruginosa (PA), have high rates of antimicrobial tolerance and are commonly found in chronic wound and cystic fibrosis lung infections. Combination therapeutics that act synergistically can overcome antimicrobial tolerance; however, the delivery of multiple therapeutics at relevant dosages remains a challenge. We therefore developed a nanoscale drug carrier for antimicrobial codelivery by combining approaches from polyelectrolyte nanocomplex (NC) formation and layer-by-layer electrostatic self-assembly. This strategy led to NC drug carriers loaded with tobramycin antibiotics and antimicrobial silver nanoparticles (AgTob-NCs). AgTob-NCs displayed synergistic enhancements in antimicrobial activity against both planktonic and biofilm PA cultures, with positively charged NCs outperforming negatively charged formulations. NCs were evaluated in mouse models of lung infection, leading to reduced bacterial burden and improved survival outcomes. This approach therefore shows promise for nanoscale therapeutic codelivery to treat recalcitrant bacterial infections.