Fan Tu

Panorama of ancient metazoan macromolecular complexes.

Macromolecular complexes are essential to conserved biological processes, but their prevalence across animals is unclear. By combining extensive biochemical fractionation with quantitative mass spectrometry, here we directly examined the composition of soluble multiprotein complexes among diverse metazoan models. Using an integrative approach, we generated a draft conservation map consisting of more than one million putative high-confidence co-complex interactions for species with fully sequenced genomes that encompasses functional modules present broadly across all extant animals. Clustering reveals a spectrum of conservation, ranging from ancient eukaryotic assemblies that have probably served cellular housekeeping roles for at least one billion years, ancestral complexes that have accrued contemporary components, and rarer metazoan innovations linked to multicellularity. We validated these projections by independent co-fractionation experiments in evolutionarily distant species, affinity purification and functional analyses. The comprehensiveness, centrality and modularity of these reconstructed interactomes reflect their fundamental mechanistic importance and adaptive value to animal cell systems.

Coordinated genomic control of ciliogenesis and cell movement by RFX2

The mechanisms linking systems-level programs of gene expression to discrete cell biological processes in vivo remain poorly understood. In this study, we have defined such a program for multi-ciliated epithelial cells (MCCs), a cell type critical for proper development and homeostasis of the airway, brain and reproductive tracts. Starting from genomic analysis of the cilia-associated transcription factor Rfx2, we used bioinformatics and in vivo cell biological approaches to gain insights into the molecular basis of cilia assembly and function. Moreover, we discovered a previously un-recognized role for an Rfx factor in cell movement, finding that Rfx2 cell-autonomously controls apical surface expansion in nascent MCCs. Thus, Rfx2 coordinates multiple, distinct gene expression programs in MCCs, regulating genes that control cell movement, ciliogenesis, and cilia function. As such, the work serves as a paradigm for understanding genomic control of cell biological processes that span from early cell morphogenetic events to terminally differentiated cellular functions. DOI: http://dx.doi.org/10.7554/eLife.01439.001

Integration of over 9,000 mass spectrometry experiments builds a global map of human protein complexes

Macromolecular protein complexes carry out many of the essential functions of cells, and many genetic diseases arise from disrupting the functions of such complexes. Currently, there is great interest in defining the complete set of human protein complexes, but recent published maps lack comprehensive coverage. Here, through the synthesis of over 9,000 published mass spectrometry experiments, we present hu.MAP, the most comprehensive and accurate human protein complex map to date, containing > 4,600 total complexes, > 7,700 proteins, and > 56,000 unique interactions, including thousands of confident protein interactions not identified by the original publications. hu.MAP accurately recapitulates known complexes withheld from the learning procedure, which was optimized with the aid of a new quantitative metric (k‐cliques) for comparing sets of sets. The vast majority of complexes in our map are significantly enriched with literature annotations, and the map overall shows improved coverage of many disease‐associated proteins, as we describe in detail for ciliopathies. Using hu.MAP, we predicted and experimentally validated candidate ciliopathy disease genes in vivo in a model vertebrate, discovering CCDC138, WDR90, and KIAA1328 to be new cilia basal body/centriolar satellite proteins, and identifying ANKRD55 as a novel member of the intraflagellar transport machinery. By offering significant improvements to the accuracy and coverage of human protein complexes, hu.MAP (http://proteincomplexes.org) serves as a valuable resource for better understanding the core cellular functions of human proteins and helping to determine mechanistic foundations of human disease.

Proteome-wide dataset supporting the study of ancient metazoan macromolecular complexes.

Our analysis examines the conservation of multiprotein complexes among metazoa through use of high resolution biochemical fractionation and precision mass spectrometry applied to soluble cell extracts from 5 representative model organisms Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, Strongylocentrotus purpuratus, and Homo sapiens. The interaction network obtained from the data was validated globally in 4 distant species (Xenopus laevis, Nematostella vectensis, Dictyostelium discoideum, Saccharomyces cerevisiae) and locally by targeted affinity-purification experiments. Here we provide details of our massive set of supporting biochemical fractionation data available via ProteomeXchange (PXD002319-PXD002328), PPIs via BioGRID (185267); and interaction network projections via (http://metazoa.med.utoronto.ca) made fully accessible to allow further exploration. The datasets here are related to the research article on metazoan macromolecular complexes in Nature [1].

RhoA regulates actin network dynamics during apical surface emergence in multiciliated epithelial cells

ABSTRACT Homeostatic replacement of epithelial cells from basal precursors is a multistep process involving progenitor cell specification, radial intercalation and, finally, apical surface emergence. Recent data demonstrate that actin-based pushing under the control of the formin protein Fmn1 drives apical emergence in nascent multiciliated epithelial cells (MCCs), but little else is known about this actin network or the control of Fmn1. Here, we explore the role of the small GTPase RhoA in MCC apical emergence. Disruption of RhoA function reduced the rate of apical surface expansion and decreased the final size of the apical domain. Analysis of cell shapes suggests that RhoA alters the balance of forces exerted on the MCC apical surface. Finally, quantitative time-lapse imaging and fluorescence recovery after photobleaching studies argue that RhoA works in concert with Fmn1 to control assembly of the specialized apical actin network in MCCs. These data provide new molecular insights into epithelial apical surface assembly and could also shed light on mechanisms of apical lumen formation. Summary: The role of the small GTPase RhoA in the assembly of the apical surface in nascent multiciliated cells is explored, revealing that RhoA and formin 1 control forces acting on the apical surface to regulate apical emergence.

Protein localization screening in vivo reveals novel regulators of multiciliated cell development and function

ABSTRACT Multiciliated cells (MCCs) drive fluid flow in diverse tubular organs and are essential for the development and homeostasis of the vertebrate central nervous system, airway and reproductive tracts. These cells are characterized by dozens or hundreds of motile cilia that beat in a coordinated and polarized manner. In recent years, genomic studies have not only elucidated the transcriptional hierarchy for MCC specification but also identified myriad new proteins that govern MCC ciliogenesis, cilia beating and cilia polarization. Interestingly, this burst of genomic data has also highlighted that proteins with no obvious role in cilia do, in fact, have important ciliary functions. Understanding the function of proteins with little prior history of study presents a special challenge, especially when faced with large numbers of such proteins. Here, we define the subcellular localization in MCCs of ∼200 proteins not previously implicated in cilia biology. Functional analyses arising from the screen provide novel links between actin cytoskeleton and MCC ciliogenesis. Summary: An unbiased screen reveals the localization of ∼200 proteins in multiciliated cell. Functional analyses arising from the screen provide novel links between actin cytoskeleton and MCC ciliogenesis.

A phase separated organelle at the root of motile ciliopathy

Hundreds of different cell types emerge in the developing embryo, each of which must compartmentalize cell type specific biochemical processes in a crowded intracellular environment. To study cell type specific compartmentalization, we examined motile ciliated cells, which must assemble vast numbers of dynein motors to drive ciliary beating, as mutation of dyneins or their assembly factors causes motile ciliopathy. We show that dyneins, their assembly factors, and chaperones all concentrate together in Dynein Assembly Particles (DynAPs). These phase-separated organelles are specific to ciliated cells but share machinery with stress granules. Our data suggest that a common framework underlies ubiquitous and cell-type specific phase separated organelles and that one such organelle is defective in a human genetic disease.

A synthesis of over 9,000 mass spectrometry experiments reveals the core set of human protein complexes

Macromolecular protein complexes carry out many of the essential functions of cells, and many genetic diseases arise from disrupting the functions of such complexes. Currently there is great interest in defining the complete set of human protein complexes, but recent published maps lack comprehensive coverage. Here, through the synthesis of over 9,000 published mass spectrometry experiments, we present hu.MAP, the most comprehensive and accurate human protein complex map to date, containing >4,600 total complexes, >7,700 proteins and >56,000 unique interactions, including thousands of confident protein interactions not identified by the original publications. hu.MAP accurately recapitulates known complexes withheld from the learning procedure, which was optimized with the aid of a new quantitative metric (k-cliques) for comparing sets of sets. The vast majority of complexes in our map are significantly enriched with literature annotations and the map overall shows improved coverage of many disease-associated proteins, as we describe in detail for ciliopathies. Using hu.MAP, we predicted and experimentally validated candidate ciliopathy disease genes in vivo in a model vertebrate, discovering CCDC138, WDR90, and KIAA1328 to be new cilia basal body/centriolar satellite proteins, and identifying ANKRD55 as a novel member of the intraflagellar transport machinery. By offering significant improvements to the accuracy and coverage of human protein complexes, hu.MAP (http://proteincomplexes.org) serves as a valuable resource for better understanding the core cellular functions of human proteins and helping to determine mechanistic foundations of human disease.

High-content protein localization screening in vivo reveals novel regulators of multiciliated cell development and function

Multiciliated cells (MCCs) drive fluid flow in diverse tubular organs and are essential for development and homeostasis of the vertebrate central nervous system, airway, and reproductive tracts. These cells are characterized by dozens or hundreds of long, motile cilia that beat in a coordinated and polarized manner (Brooks and Wallingford, 2014). In recent years, genomic studies have not only elucidated the transcriptional hierarchy for MCC specification, but also identified myriad new proteins that govern MCC ciliogenesis, cilia beating, or cilia polarization (e.g. (Choksi et al., 2014b; Chung et al., 2014; Hoh et al., 2012; Ma et al., 2014; Treutlein et al., 2014)). Interestingly, this burst of genomic data has also highlighted the obvious importance of the “ignorome,” that large fraction of vertebrate genes that remain only poorly characterized (Pandey et al., 2014). Understanding the function of novel proteins with little prior history of study presents a special challenge, especially when faced with large numbers of such proteins. Here, we explored the MCC ignorome by defining the subcellular localization of 260 poorly defined proteins in vertebrate MCCs in vivo. Moreover, functional analyses that arose from results of the screen provide novel insights into the mechanisms by which the actin cytoskeleton simultaneously influences diverse aspects of MCC biology, including basal body docking, and ciliogenesis.