Biology subjects

Ting, A. Y.

Publications and source records attributed to Ting, A. Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Cilium proteomics reveals Numb as a positive regulator of the Hedgehog signaling pathway

The transduction of Hedgehog (Hh) signaling relies on the primary cilium, a cell surface organelle serving as a signaling hub for the cell. Using proximity labeling and quantitative proteomics, we identified Numb as a new ciliary protein that positively regulates Hh signaling. Numb localizes to the ciliary pocket and acts as an endocytic adaptor to incorporate Ptch1 into clathrin-coated vesicles, thereby promoting Ptch1 exit from the cilium, a key step in Hh signaling activation. Numb loss hampers Sonic Hedgehog (Shh)-induced Ptch1 departure from the cilium, resulting in reduced activation of Hh signaling. Numb loss in spinal neural progenitors reduces Shh-induced differentiation into Nkx2.2-positive progenitors, a process reliant on high Hh signaling activity. Genetic ablation of Numb in the developing cerebellum impaired the proliferation of granule cell precursors, a Hh-dependent process, resulting in reduced cerebellar size. This study highlights Numb as a critical regulator of Ptch1 levels in the cilium during Hh signal activation and demonstrates the key role of ciliary pocket-mediated endocytosis in modulating the transduction of cell signaling.

cell biology↗

bioRxiv2022

A single-component luminescent biosensor for the SARS-CoV-2 spike protein

Many existing protein detection strategies depend on highly functionalized antibody reagents. A simpler and easier to produce class of detection reagent is highly desirable. We designed a single-component, recombinant, luminescent biosensor that can be expressed in laboratory strains of E. coli and S. cerevisiae. This biosensor is deployed in multiple homogenous and immobilized assay formats to detect recombinant SARS-CoV-2 spike antigen and cultured virus. The chemiluminescent signal generated facilitates detection by an un-augmented cell phone camera. Binding Activated Tandem split-enzyme (BAT) biosensors may serve as a useful template for diagnostics and reagents that detect SARS-CoV-2 antigens and other proteins of interest.

biochemistry↗

bioRxiv2022

Shared and distinctive neighborhoods of emerin and LBR revealed by proximity labeling and quantitative proteomics

Emerin and LBR are abundant transmembrane proteins of the nuclear envelope (NE) that are concentrated at the inner nuclear membrane (INM). Although both proteins interact with chromatin and nuclear lamins, they have distinctive biochemical and functional properties. Here we have deployed proximity labeling using the engineered biotin ligase TurboID (TbID) and quantitative proteomics to compare the neighborhoods of emerin and LBR in cultured mouse embryonic fibroblasts (MEFs). Our analysis revealed 232 high confidence proximity partners (HCPP) that interact selectively with emerin and/or LBR, 49 of which are shared by both. These included previously characterized NE-concentrated proteins, as well as a host of additional proteins not previously linked to emerin or LBR functions. Many of these are TM proteins of the ER and include two E3 ubiquitin ligases. Using the proximity ligation assay as an orthogonal approach, we validated the interactions described by proximity labeling for 11/12 proteins analyzed, supporting the robustness of our analysis. Overall, this work presents methodology that may be used for large-scale mapping of the landscape of the INM and reveals a group of new proteins with potential functional connections to emerin and LBR.

cell biology↗

bioRxiv2022

A genetic model for in vivo proximity labeling of the mammalian secretome

Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ communication. Enzyme-catalyzed proximity labeling enables the identification of proteins within a specific cellular compartment. Here, we report a BirA*G3 mouse strain that enables CRE-dependent promiscuous biotinylation of proteins trafficking through the endoplasmic reticulum. When broadly activated throughout the mouse, widespread labeling of proteins was observed within the secretory pathway. Streptavidin affinity purification and peptide mapping by quantitative mass spectrometry (MS) proteomics revealed organ-specific secretory profiles and serum trafficking. As expected, secretory proteomes were highly enriched for signal peptide-containing proteins, highlighting both conventional and non-conventional secretory processes, and ectodomain shedding. Lower-abundance proteins with hormone-like properties were recovered and validated using orthogonal approaches. Hepatocyte-specific activation of BirA*G3 highlighted liver-specific biotinylated secretome profiles. The BirA*G3 mouse model demonstrates enhanced labeling efficiency and tissue specificity over viral transduction approaches and will facilitate a deeper understanding of secretory protein interplay in development, and healthy and diseased adult states.

cell biology↗

bioRxiv2022

A light-gated transcriptional recorder for detecting cell-cell contacts

Technologies for detecting cell-cell contacts are powerful tools for studying a wide range of biological processes, from neuronal signaling to cancer-immune interactions within the tumor microenvironment. Here we report TRACC, a GPCR-based transcriptional recorder of cellular contacts, which converts contact events into stable transgene expression. TRACC is derived from our previous protein-protein interaction recorders, SPARK (Kim et al., 2017) and SPARK2 (Kim et al., 2019). TRACC incorporates light gating via the LOV domain, which provides user-defined temporal control of tool activation and reduces background. We show that TRACC detects cell-cell contacts with high specificity and sensitivity in mammalian cell culture and that it can be used to interrogate interactions between neurons and glioma, a form of brain cancer.

cell biology↗

bioRxiv2021

A dual-purpose real-time indicator and transcriptional integrator for calcium detection in living cells

Calcium is a ubiquitous second messenger in eukaryotes, correlated with neuronal activity and T-cell activation among other processes. Real-time calcium indicators such as GCaMP have recently been complemented by newer calcium integrators that convert transient calcium activity into stable gene expression. Here we introduce LuCID, a dual-purpose real-time calcium indicator and transcriptional calcium integrator that combines the benefits of both calcium detection technologies. We show that the calcium-dependent split luciferase component of LuCID provides real-time bioluminescence readout of calcium dynamics in cells, while the GI/FKF1 split GAL4 component of LuCID converts calcium-generated bioluminescence into stable gene expression. We also show that LuCIDs modular design enables it to also read out other cellular events such as protein-protein interactions. LuCID adds to the arsenal of tools for studying cells and cell populations that utilize calcium for signaling.

synthetic biology↗

bioRxiv2021

Transcription factor control of dendrite targeting via combinatorial cell-surface codes

Transcription factors specify the fate and connectivity of developing neurons. We investigate how a lineage-specific transcription factor, Acj6, controls the precise dendrite targeting of Drosophila olfactory projection neurons (PNs) by regulating the expression of cell-surface proteins. Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion molecules and proteins previously not associated with wiring, such as Piezo, whose mechanosensitive ion channel activity is dispensable for its function in PN dendrite targeting. Comprehensive genetic analyses revealed that Acj6 employs unique sets of cell-surface proteins in different PN types for dendrite targeting. Combinatorial expression of Acj6 wiring executors rescued acj6 mutant phenotypes with higher efficacy and breadth than expression of individual executors. Thus, Acj6 controls wiring specificity of different neuron types by specifying distinct combinatorial expression of cell- surface executors.

neuroscience↗

bioRxiv2021

Functional interactomes of the Ebola virus polymerase identified by proximity proteomics in the context of viral replication

Ebola virus (EBOV) critically depends on the viral polymerase to replicate and transcribe the viral RNA genome in the cytoplasm of host cells, where cellular factors can antagonize or facilitate the virus life cycle. Here we leveraged proximity proteomics and conducted an siRNA screen to define the functional interactome of EBOV polymerase. As proof-of-principle, we validated two cellular mRNA decay factors from 35 identified host factors: eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1) and up-frameshift protein 1 (UPF1). Our data suggest that EBOV can subvert restrictions of cellular mRNA decay and repurpose both GSPT1 and UPF1 to promote viral replication. Treating EBOV-infected human hepatocytes with a drug candidate that targets GSPT1 for degradation significantly reduced viral RNA load and particle production. Our work demonstrates the utility of proximity proteomics to capture the functional host-interactome of the EBOV polymerase and to illuminate host-dependent regulations of viral RNA synthesis.

microbiology↗

bioRxiv2021

Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host directed antivirals.

Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells, under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed an siRNA screen to identify those interactors that have functional roles in authentic LASV infection. As proof-of-principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet to be defined, host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses. Significance StatementLassa virus (LASV), the causative agent of Lassa fever (LF), represents an important public health problem in Western Africa. There is no FDA-approved therapeutic intervention to treat LF. Due to its limited genome coding capacity, LASV proteins are often multifunctional and orchestrate complex interactions with cellular factors to execute steps required to complete the viral life cycle. LASV polymerase is essential for replication and expression of the viral genome, and thus is an attractive target for antiviral intervention. Here we present the first host interactome of the LASV polymerase, which can guide identification of novel druggable host cellular targets for the development of cost-effective antiviral therapies for LF.

microbiology↗

bioRxiv2021

A toolbox for efficient proximity-dependent biotinylation in zebrafish embryos

Understanding how proteins are organized in compartments is essential to elucidating their function. While proximity-dependent approaches such as BioID have enabled a massive increase in information about organelles, protein complexes and other structures in cell culture, to date there have been only a few studies in living vertebrates. Here, we adapted proximity labeling for protein discovery in vivo in the vertebrate model organism, zebrafish. Using lamin A (LMNA) as bait and green fluorescent protein (GFP) as a negative control, we developed, optimized, and benchmarked in vivo TurboID and miniTurbo labeling in early zebrafish embryos. We developed both an mRNA injection protocol and a transgenic system in which transgene expression is controlled by a heat shock promoter. In both cases, biotin is provided directly in the egg water, and we demonstrate that 12 hours of labeling are sufficient for biotinylation of prey proteins, which should permit time-resolved analysis of development. After statistical scoring, we found that the proximal partners of LMNA detected in each system were enriched for nuclear envelope and nuclear membrane proteins, and included many orthologs of human proteins identified as proximity partners of lamin A in mammalian cell culture. The tools and protocols developed here will allow zebrafish researchers to complement genetic tools with powerful proteomics approaches.

systems biology↗

bioRxiv2021

An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput screens

The trafficking of specific protein cohorts to the correct subcellular location at the correct time is essential for every signaling and regulatory process in biology. Gene perturbation screens could provide a powerful approach to probe the molecular mechanisms of protein trafficking, but only if protein localization or mislocalization can be tied to a simple and robust phenotype for cell selection, such as cell proliferation or FACS. To broadly empower the study of protein trafficking processes with gene perturbation, we developed a genetically-encoded molecular tool named HiLITR. HiLITR converts protein colocalization into proteolytic release of a membrane-anchored transcription factor, which drives the expression of a chosen reporter gene. Using HiLITR in combination with FACS-based CRISPRi screening in human cell lines, we identify genes that influence the trafficking of mitochondrial and ER tail-anchored proteins. We show that loss of the SUMO E1 component SAE1 results in the mislocalization and destabilization of mitochondrial tail-anchored proteins. We also demonstrate a distinct regulatory role for EMC10 in the ER membrane complex, opposing the transmembrane-domain insertion activity of the complex. Through transcriptional integration of complex cellular functions, HiLITR expands the scope of biological processes that can be studied by genetic perturbation screening technologies.

bioengineering↗

bioRxiv2021

Proximity-labeling reveals novel host and parasite proteins at the Toxoplasma parasitophorous vacuole membrane

Toxoplasma gondii is a ubiquitous, intracellular parasite that envelopes its parasitophorous vacuole with a protein-laden membrane (PVM). The PVM is critical for interactions with the infected host cell such as nutrient transport and immune defense. Only a few parasite and host proteins have so far been identified on the host-cytosolic side of the PVM. We report here the use of human foreskin fibroblasts expressing the proximity-labeling enzyme miniTurbo, fused to a domain that targets it to this face of the PVM, in combination with quantitative proteomics to specifically identify proteins present at this crucial interface. Out of numerous human and parasite proteins with candidate PVM localization, we validate three novel parasite proteins (TGGT1_269950, TGGT1_215360, and TGGT1_217530) and four new host proteins (PDCD6IP/ALIX, PDCD6, CC2D1A, and MOSPD2) as localized to the PVM in infected human cells through immunofluorescence microscopy. These results significantly expand our knowledge of proteins present at the PVM and, given that three of the validated host proteins are components of the ESCRT machinery, they further suggest that novel biology is operating at this crucial host-pathogen interface. ImportanceToxoplasma is an intracellular pathogen which resides and replicates inside a membrane-bound vacuole in infected cells. This vacuole is modified by both parasite and host proteins which participate in a variety of host-parasite interactions at this interface, including nutrient exchange, effector transport, and immune modulation. Only a small number of parasite and host proteins present at the vacuolar membrane and exposed to the host cytosol have thus far been identified. Here we report the identification of several novel parasite and host proteins present at the vacuolar membrane using enzyme-catalyzed proximity-labeling, significantly increasing our knowledge of the molecular players present and novel biology occurring at this crucial interface.

microbiology↗

bioRxiv2020

Functional proximity mapping of RNA binding proteins uncovers a mitochondrial mRNA anchor that promotes stress recovery

Proximity labeling (PL) with genetically-targeted promiscuous enzymes has emerged as a powerful tool for unbiased proteome discovery. By combining the spatiotemporal specificity of PL with methods for functional protein enrichment, it should be possible to map specific protein subclasses within distinct compartments of living cells. Here we demonstrate this capability for RNA binding proteins (RBPs), by combining peroxidase-based PL with organic-aqueous phase separation of crosslinked protein-RNA complexes ("APEX-PS"). We validated APEX-PS by mapping nuclear RBPs, then applied it to uncover the RBPomes of two unpurifiable subcompartments - the nucleolus and the outer mitochondrial membrane (OMM). At the OMM, we discovered the RBP SYNJ2BP, which retains specific nuclear-encoded mitochondrial mRNAs during translation stress, to promote their local translation and import of protein products into the mitochondrion during stress recovery. APEX-PS is a versatile tool for compartment-specific RBP discovery and expands the scope of PL to functional protein mapping. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/387209v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@1223baforg.highwire.dtl.DTLVardef@7c87c6org.highwire.dtl.DTLVardef@1b5e7cdorg.highwire.dtl.DTLVardef@185449b_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

A molecular calcium integrator reveals a striatal cell-type driving aversion

The ability to record transient cellular events in the DNA or RNA of cells would enable precise, large-scale analysis, selection, and reprogramming of heterogeneous cell populations. Here we report a molecular technology for stable genetic tagging of cells that exhibit activity-related increases in intracellular calcium concentration (FLiCRE). We used FLiCRE to transcriptionally label activated neural ensembles in the nucleus accumbens of the mouse brain during brief stimulation of aversive inputs. Using single-cell RNA sequencing, we detected FLiCRE transcripts among the endogenous transcriptome, providing simultaneous readout of both cell-type and calcium activation history. We identified a cell-type in the nucleus accumbens activated downstream of long-range excitatory projections. Taking advantage of FLiCREs modular design, we expressed an optogenetic channel selectively in this cell-type, and showed that direct recruitment of this otherwise genetically-inaccessible population elicits behavioral aversion. The specificity and minute-resolution of FLiCRE enables molecularly-informed characterization, manipulation, and reprogramming of activated cellular ensembles.

neuroscience↗

bioRxiv2020

Proximity labeling at non-centrosomal microtubule-organizing centers reveals VAB-10B and WDR-62 as distinct microtubule regulators

Reorganization of microtubules from the centrosome to non-centrosomal subcellular sites is central to cell differentiation. To identify components of non-centrosomal microtubule organizing centers in differentiated cells of a living organism, we developed the biotin ligase-based proximity labeling approach TurboID for use in C. elegans. We identified proteins proximal to the non-centrosomal microtubule minus end protein PTRN-1/Patronin at the apical membrane of epithelial cells, focusing on two conserved proteins: spectraplakin protein VAB-10B and WDR-62, a protein we identify as homologous to vertebrate primary microcephaly disease gene WDR62. We found that WDR-62 and VAB-10B independently regulate the growth and localization of non-centrosomal microtubules and the apical targeting of microtubule minus end proteins. This regulation occurs downstream of cell polarity and in conjunction with actin. Our data suggest a division of labor where microtubule growth and anchoring are regulated by distinct complexes and uncover novel functions of spectraplakins and WDR62 family proteins.

cell biology↗

bioRxiv2020

Proteomics of protein trafficking by in vivo tissue-specific labeling

Secreted interorgan communication factors encode key regulators of homeostasis. However, long-standing questions surround their origins/destinations, mechanisms of interactions, and the number of proteins involved. Progress has been hindered by the lack of methodologies for these factors large-scale identification and characterization, as conventional approaches cannot identify low-abundance factors and the origins and destinations of secreted proteins. We established an in vivo platform to investigate secreted protein trafficking between organs proteome-wide, whereby engineered promiscuous biotin ligase BirA*G3 (a relative of TurboID) biotinylates all proteins in a subcellular compartment of one tissue, and biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Using this platform, we identified 51 putative muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, of which 60-70% have human orthologs. We demonstrate, in particular, that conserved fat body-derived novel interorgan communication factors CG31326, CG2145, and CG4332 promote muscle activity. Our results indicate that the communication network of secreted proteins is vast, and we identified systemic functions for a number of these factors. This approach is widely applicable to studies in interorgan, local and intracellular protein trafficking networks, non-conventional secretion, and to mammalian systems, under healthy or diseased states. One Sentence SummaryWe developed an in vivo platform to investigate protein trafficking between organs proteome-wide, provide a resource for interorgan communication factors, and determined conserved adipokines that affect muscles.

physiology↗

bioRxiv2020

Split-TurboID enables contact-dependent proximity labeling in cells

Proximity labeling (PL) catalyzed by promiscuous enzymes such as TurboID have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. At endoplasmic reticulum (ER)-mitochondria contact sites, reconstituted TurboID catalyzed spatially-restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to ER-mitochondria contacts. We validated eight novel candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially-specific proximity labeling in cells.

cell biology↗

bioRxiv2020

RNA-protein interaction mapping via MS2 or Cas13-based APEX targeting

RNA-protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA-protein interactions in living cells in an unbiased manner. Capitalizing on the ability of the engineered peroxidase APEX2 to identify protein interaction partners via proximity-dependent biotinylation, we used two approaches to target APEX2 to specific cellular RNAs. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were able to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured endogenous protein interaction partners of hTR, including more than a dozen proteins not previously linked to hTR. We validated the unexpected interaction between hTR and the N6-methyladenosine (m6A) demethylase ALKBH5. Further investigation showed that endogenous hTR is modified by m6A, which can be erased by ALKBH5, and that ALKBH5 influences both telomerase complex assembly and activity. These results highlight the ability of MS2- and Cas13-targeted APEX2 to identify novel RNA-protein interactions in living cells.

molecular biology↗