Kimberly Tsui - Academia.edu (original) (raw)

Papers by Kimberly Tsui

Beyond the cell factory: Homeostatic regulation of and by the UPRER

Science Advances

The endoplasmic reticulum (ER) is commonly referred to as the factory of the cell, as it is respo... more The endoplasmic reticulum (ER) is commonly referred to as the factory of the cell, as it is responsible for a large amount of protein and lipid synthesis. As a membrane-bound organelle, the ER has a distinct environment that is ideal for its functions in synthesizing these primary cellular components. Many different quality control machineries exist to maintain ER stability under the stresses associated with synthesizing, folding, and modifying complex proteins and lipids. The best understood of these mechanisms is the unfolded protein response of the ER (UPRER), in which transmembrane proteins serve as sensors, which trigger a coordinated transcriptional response of genes dedicated for mitigating the stress. As the name suggests, the UPRER is most well described as a functional response to protein misfolding stress. Here, we focus on recent findings and emerging themes in additional roles of the UPRER outside of protein homeostasis, including lipid homeostasis, autophagy, apoptosis...

IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2

Commonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on dete... more Commonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection.Graphical Abstract

Quantifying cytoskeletal heterogeneity via single-cell protein-complex fractionation

Multimeric cytoskeletal protein complexes, including filamentous F-actin, orchestrate normal cell... more Multimeric cytoskeletal protein complexes, including filamentous F-actin, orchestrate normal cellular function. However, protein-complex distributions in stressed, heterogeneous cell populations remain unknown. Cell staining and proximity-based methods have limited selectivity and/or sensitivity for robust endogenous multimeric protein-complex quantification from single cells. We introduce micro-arrayed differential detergent fractionation to simultaneously detect protein complexes in 100s of individual cells. Fractionation occurs by 60s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Validating with actin-destabilizing Latrunculin A (LatA), we quantify 2.7-fold lower median F-actin complex-levels in LatA-treated single cells. Further clustering analysis of U2OS cells treated with LatA detects a subpopulation (∼11%) exhibiting downregulated F-actin, but upregulated microtubule and intermediate filament protein complexes. Thus, ...

Author response: Drosophila Fezf coordinates laminar-specific connectivity through cell-intrinsic and cell-extrinsic mechanisms

eLife

The small molecule Retro-2 prevents ricin toxicity through a poorly-defined mechanism of action (... more The small molecule Retro-2 prevents ricin toxicity through a poorly-defined mechanism of action (MOA), which involves halting retrograde vesicle transport to the endoplasmic reticulum (ER). CRISPRi genetic interaction analysis revealed Retro-2 activity resembles disruption of the transmembrane domain recognition complex (TRC) pathway, which mediates post-translational ER-targeting and insertion of tail-anchored (TA) proteins, including SNAREs required for retrograde transport. Cell-based and in vitro assays show that Retro-2 blocks delivery of newly-synthesized TA-proteins to the ER-targeting factor ASNA1 (TRC40). An ASNA1 point mutant identified using CRISPR-mediated mutagenesis abolishes both the cytoprotective effect of Retro-2 against ricin and its inhibitory effect on ASNA1-mediated ER-targeting. Together, our work explains how Retro-2 prevents retrograde trafficking of toxins by inhibiting TA-protein targeting, describes a general CRISPR strategy for predicting the MOA of smal...

HER2-overexpressing/amplified breast cancer as a testing ground for antibody-drug conjugate drug development in solid tumors

Clinical Cancer Research

CRISPR-Cas9 screens identify regulators of antibody–drug conjugate toxicity

Nature Chemical Biology

Antibody-drug conjugates (ADCs) selectively deliver highly toxic chemotherapeutic agents to targe... more Antibody-drug conjugates (ADCs) selectively deliver highly toxic chemotherapeutic agents to target antigen-expressing cells and have become an important cancer treatment in recent years. However, the molecular mechanisms by which ADCs are internalized and activated within cells remain unclear. Here we use CRISPR-Cas9 screens to identify genes that control the toxicity of ADCs. Our results demonstrate critical roles for a range of known and novel endolysosomal trafficking regulators in ADC toxicity. We identify and characterize C18orf8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of CRISPR screens with ADCs bearing a noncleavable linker versus a cleavable valine-citrulline (VC) linker, we show that a subset of late endosomal and lysosomal regulators are selectively essential for toxicity of noncleavable linker ADCs. We further show that cleavable VC linkers are rapidly processed upon internalization and therefore surprisin...

Identification of phagocytosis regulators using magnetic genome-wide CRISPR screens

Nature Genetics

Phagocytosis is required for a broad range of physiological functions, from pathogen defense to t... more Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer's disease-associated gene TM2D3 can preferentially influence uptake of amyloid-β aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.

eLife, Jan 7, 2018

Laminar arrangement of neural connections is a fundamental feature of neural circuit organization... more Laminar arrangement of neural connections is a fundamental feature of neural circuit organization. Identifying mechanisms that coordinate neural connections within correct layers is thus vital for understanding how neural circuits are assembled. In the medulla of thevisual system neurons form connections within ten parallel layers. The M3 layer receives input from two neuron types that sequentially innervate M3 during development. Here we show that M3-specific innervation by both neurons is coordinated byFezf (dFezf), a conserved transcription factor that is selectively expressed by the earlier targeting input neuron. In this cell, dFezf instructs layer specificity and activates the expression of a secreted molecule (Netrin) that regulates the layer specificity of the other input neuron. We propose that employment of transcriptional modules that cell-intrinsically target neurons to specific layers, and cell-extrinsically recruit other neurons is a general mechanism for building laye...

Hexanucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lat... more Hexanucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). The nucleotide repeat expansions are translated into dipeptide repeat (DPR) proteins, which are aggregation-prone and may contribute to neurodegeneration. Studies in model organisms, including yeast and flies have converged upon nucleocytoplasmic transport as one underlying pathogenic mechanism, but a comprehensive understanding of the molecular and cellular underpinnings of DPR toxicity in human cells is still lacking. We used the bacteria-derived clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to perform genome-wide gene knockout screens for suppressors and enhancers of C9orf72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. Our screens revealed genes involved in nucleocytoplasmic transport, reinforcing the previous findings from mo...

Finding host targets for HIV therapy

Nature genetics, Jan 31, 2017

A CRISPR screen conducted in a CD4(+) T cell leukemia line has identified host factors required f... more A CRISPR screen conducted in a CD4(+) T cell leukemia line has identified host factors required for HIV infection but dispensable for cellular survival. The results highlight sulfation on the HIV co-receptor CCR5 and cellular aggregation as potential targets for therapeutic intervention.

Neuron, 2014

Neural circuit formation relies on interactions between axons and cells within the target field. ... more Neural circuit formation relies on interactions between axons and cells within the target field. While it is well established that target-derived signals act on axons to regulate circuit assembly, the extent to which axon-derived signals control circuit formation is not known. In the Drosophila visual system, anterograde signals numerically match R1-R6 photoreceptors with their targets by controlling target proliferation and neuronal differentiation. Here we demonstrate that additional axon-derived signals selectively couple target survival with layer specificity. We show that Jelly belly (Jeb) produced by R1-R6 axons interacts with its receptor, anaplastic lymphoma kinase (Alk), on budding dendrites to control survival of L3 neurons, one of three postsynaptic targets. L3 axons then produce Netrin, which regulates the layer-specific targeting of another neuron within the same circuit. We propose that a cascade of axon-derived signals, regulating diverse cellular processes, provides a strategy for coordinating circuit assembly across different regions of the nervous system. Neuron Axon-Derived Signals Coordinate Circuit Assembly

Neuron, 2014

The study of synaptic specificity and plasticity in the CNS is limited by the inability to effici... more The study of synaptic specificity and plasticity in the CNS is limited by the inability to efficiently visualize synapses in identified neurons using light microscopy. Here, we describe synaptic tagging with recombination (STaR), a method for labeling endogenous presynaptic and postsynaptic proteins in a cell-type-specific fashion. We modified genomic loci encoding synaptic proteins within bacterial artificial chromosomes such that these proteins, expressed at endogenous levels and with normal spatiotemporal patterns, were labeled in an inducible fashion in specific neurons through targeted expression of site-specific recombinases. Within the Drosophila visual system, the number and distribution of synapses correlate with electron microscopy studies. Using two different recombination systems, presynaptic and postsynaptic specializations of synaptic pairs can be colabeled. STaR also allows synapses within the CNS to be studied in live animals noninvasively. In principle, STaR can be adapted to the mammalian nervous system. Neuron Cell-type-Specific Labeling of Synapses In Vivo

Nature communications, May 5, 2017

CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but ... more CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-th...

Beyond the cell factory: Homeostatic regulation of and by the UPRER

Science Advances

The endoplasmic reticulum (ER) is commonly referred to as the factory of the cell, as it is respo... more The endoplasmic reticulum (ER) is commonly referred to as the factory of the cell, as it is responsible for a large amount of protein and lipid synthesis. As a membrane-bound organelle, the ER has a distinct environment that is ideal for its functions in synthesizing these primary cellular components. Many different quality control machineries exist to maintain ER stability under the stresses associated with synthesizing, folding, and modifying complex proteins and lipids. The best understood of these mechanisms is the unfolded protein response of the ER (UPRER), in which transmembrane proteins serve as sensors, which trigger a coordinated transcriptional response of genes dedicated for mitigating the stress. As the name suggests, the UPRER is most well described as a functional response to protein misfolding stress. Here, we focus on recent findings and emerging themes in additional roles of the UPRER outside of protein homeostasis, including lipid homeostasis, autophagy, apoptosis...

IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2

Commonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on dete... more Commonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection.Graphical Abstract

Quantifying cytoskeletal heterogeneity via single-cell protein-complex fractionation

Multimeric cytoskeletal protein complexes, including filamentous F-actin, orchestrate normal cell... more Multimeric cytoskeletal protein complexes, including filamentous F-actin, orchestrate normal cellular function. However, protein-complex distributions in stressed, heterogeneous cell populations remain unknown. Cell staining and proximity-based methods have limited selectivity and/or sensitivity for robust endogenous multimeric protein-complex quantification from single cells. We introduce micro-arrayed differential detergent fractionation to simultaneously detect protein complexes in 100s of individual cells. Fractionation occurs by 60s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Validating with actin-destabilizing Latrunculin A (LatA), we quantify 2.7-fold lower median F-actin complex-levels in LatA-treated single cells. Further clustering analysis of U2OS cells treated with LatA detects a subpopulation (∼11%) exhibiting downregulated F-actin, but upregulated microtubule and intermediate filament protein complexes. Thus, ...

Author response: Drosophila Fezf coordinates laminar-specific connectivity through cell-intrinsic and cell-extrinsic mechanisms

eLife

The small molecule Retro-2 prevents ricin toxicity through a poorly-defined mechanism of action (... more The small molecule Retro-2 prevents ricin toxicity through a poorly-defined mechanism of action (MOA), which involves halting retrograde vesicle transport to the endoplasmic reticulum (ER). CRISPRi genetic interaction analysis revealed Retro-2 activity resembles disruption of the transmembrane domain recognition complex (TRC) pathway, which mediates post-translational ER-targeting and insertion of tail-anchored (TA) proteins, including SNAREs required for retrograde transport. Cell-based and in vitro assays show that Retro-2 blocks delivery of newly-synthesized TA-proteins to the ER-targeting factor ASNA1 (TRC40). An ASNA1 point mutant identified using CRISPR-mediated mutagenesis abolishes both the cytoprotective effect of Retro-2 against ricin and its inhibitory effect on ASNA1-mediated ER-targeting. Together, our work explains how Retro-2 prevents retrograde trafficking of toxins by inhibiting TA-protein targeting, describes a general CRISPR strategy for predicting the MOA of smal...

HER2-overexpressing/amplified breast cancer as a testing ground for antibody-drug conjugate drug development in solid tumors

Clinical Cancer Research

CRISPR-Cas9 screens identify regulators of antibody–drug conjugate toxicity

Nature Chemical Biology

Antibody-drug conjugates (ADCs) selectively deliver highly toxic chemotherapeutic agents to targe... more Antibody-drug conjugates (ADCs) selectively deliver highly toxic chemotherapeutic agents to target antigen-expressing cells and have become an important cancer treatment in recent years. However, the molecular mechanisms by which ADCs are internalized and activated within cells remain unclear. Here we use CRISPR-Cas9 screens to identify genes that control the toxicity of ADCs. Our results demonstrate critical roles for a range of known and novel endolysosomal trafficking regulators in ADC toxicity. We identify and characterize C18orf8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of CRISPR screens with ADCs bearing a noncleavable linker versus a cleavable valine-citrulline (VC) linker, we show that a subset of late endosomal and lysosomal regulators are selectively essential for toxicity of noncleavable linker ADCs. We further show that cleavable VC linkers are rapidly processed upon internalization and therefore surprisin...

Identification of phagocytosis regulators using magnetic genome-wide CRISPR screens

Nature Genetics

Phagocytosis is required for a broad range of physiological functions, from pathogen defense to t... more Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer's disease-associated gene TM2D3 can preferentially influence uptake of amyloid-β aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.

eLife, Jan 7, 2018

Laminar arrangement of neural connections is a fundamental feature of neural circuit organization... more Laminar arrangement of neural connections is a fundamental feature of neural circuit organization. Identifying mechanisms that coordinate neural connections within correct layers is thus vital for understanding how neural circuits are assembled. In the medulla of thevisual system neurons form connections within ten parallel layers. The M3 layer receives input from two neuron types that sequentially innervate M3 during development. Here we show that M3-specific innervation by both neurons is coordinated byFezf (dFezf), a conserved transcription factor that is selectively expressed by the earlier targeting input neuron. In this cell, dFezf instructs layer specificity and activates the expression of a secreted molecule (Netrin) that regulates the layer specificity of the other input neuron. We propose that employment of transcriptional modules that cell-intrinsically target neurons to specific layers, and cell-extrinsically recruit other neurons is a general mechanism for building laye...

Hexanucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lat... more Hexanucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). The nucleotide repeat expansions are translated into dipeptide repeat (DPR) proteins, which are aggregation-prone and may contribute to neurodegeneration. Studies in model organisms, including yeast and flies have converged upon nucleocytoplasmic transport as one underlying pathogenic mechanism, but a comprehensive understanding of the molecular and cellular underpinnings of DPR toxicity in human cells is still lacking. We used the bacteria-derived clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to perform genome-wide gene knockout screens for suppressors and enhancers of C9orf72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. Our screens revealed genes involved in nucleocytoplasmic transport, reinforcing the previous findings from mo...

Finding host targets for HIV therapy

Nature genetics, Jan 31, 2017

A CRISPR screen conducted in a CD4(+) T cell leukemia line has identified host factors required f... more A CRISPR screen conducted in a CD4(+) T cell leukemia line has identified host factors required for HIV infection but dispensable for cellular survival. The results highlight sulfation on the HIV co-receptor CCR5 and cellular aggregation as potential targets for therapeutic intervention.

Neuron, 2014

Neural circuit formation relies on interactions between axons and cells within the target field. ... more Neural circuit formation relies on interactions between axons and cells within the target field. While it is well established that target-derived signals act on axons to regulate circuit assembly, the extent to which axon-derived signals control circuit formation is not known. In the Drosophila visual system, anterograde signals numerically match R1-R6 photoreceptors with their targets by controlling target proliferation and neuronal differentiation. Here we demonstrate that additional axon-derived signals selectively couple target survival with layer specificity. We show that Jelly belly (Jeb) produced by R1-R6 axons interacts with its receptor, anaplastic lymphoma kinase (Alk), on budding dendrites to control survival of L3 neurons, one of three postsynaptic targets. L3 axons then produce Netrin, which regulates the layer-specific targeting of another neuron within the same circuit. We propose that a cascade of axon-derived signals, regulating diverse cellular processes, provides a strategy for coordinating circuit assembly across different regions of the nervous system. Neuron Axon-Derived Signals Coordinate Circuit Assembly

Neuron, 2014

The study of synaptic specificity and plasticity in the CNS is limited by the inability to effici... more The study of synaptic specificity and plasticity in the CNS is limited by the inability to efficiently visualize synapses in identified neurons using light microscopy. Here, we describe synaptic tagging with recombination (STaR), a method for labeling endogenous presynaptic and postsynaptic proteins in a cell-type-specific fashion. We modified genomic loci encoding synaptic proteins within bacterial artificial chromosomes such that these proteins, expressed at endogenous levels and with normal spatiotemporal patterns, were labeled in an inducible fashion in specific neurons through targeted expression of site-specific recombinases. Within the Drosophila visual system, the number and distribution of synapses correlate with electron microscopy studies. Using two different recombination systems, presynaptic and postsynaptic specializations of synaptic pairs can be colabeled. STaR also allows synapses within the CNS to be studied in live animals noninvasively. In principle, STaR can be adapted to the mammalian nervous system. Neuron Cell-type-Specific Labeling of Synapses In Vivo

Nature communications, May 5, 2017

CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but ... more CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-th...