Biology subjects

Tsui, C. K.

Publications and source records attributed to Tsui, C. K..

8 recordsLinked to original sources

bioRxiv2024

Age-related remodeling of the glycocalyx drives T cell exhaustion

Cell surface glycans, termed the glycocalyx, are essential regulators of cellular signaling and thus cellular development and functions, but how aging impacts the glycocalyx remains poorly understood. Here, using immune cells as a model system for studying the relationship between aging and glycocalyx remodeling, we show that 2,6-linked sialic acid - a terminal glycan epitope typically associated with inhibitory signaling - becomes downregulated in T cells from older animals. This downregulation is tightly correlated with age-associated accumulation of effector T cells, which are decorated with little to no 2,6-linked sialic acids. T cell aging renders older individuals more vulnerable to infections and cancers. To understand the role of 2,6-linked sialic acids in T cell physiology, we generated a mouse model with T cell-specific deletion of the sialyltransferase gene St6gal1. The chronic depletion of 2,6-linked sialic acids led to naive T (TN) cells expansion in the periphery and premature T cell exhaustion. As a result, these mice were less able to control acute Listeria infection and chronic tumor growth. Blockade of the PD-1 pathway can partially restore the ability of St6gal1-deficient T cells to control tumor growth. Together, these data suggest that 2,6-linked sialic acids are critical for maintaining long-term T cell responsiveness, and the loss of 2,6-linked sialic acids may directly contribute to age-related T cell exhaustion.

immunology↗

bioRxiv2023

Combinatorial CRISPR screens and lectin microarrays identify novel glycosylation regulators

Glycans play critical roles in cellular signaling and function. Unlike proteins, glycan structures are not templated from genes but the concerted activity of many genes, making them historically challenging to study. Here, we present a strategy that utilizes pooled CRISPR screens and lectin microarrays to uncover and characterize regulators of cell surface glycosylation. We applied this approach to study the regulation of high mannose glycans - the starting structure of all asparagine(N)-linked-glycans. We used CRISPR screens to uncover the expanded network of genes controlling high mannose surface levels, followed by lectin microarrays to fully measure the complex effect of select regulators on glycosylation globally. Through this, we elucidated how two novel high mannose regulators - TM9SF3 and the CCC complex - control complex N-glycosylation via regulating Golgi morphology and function. Notably, this method allowed us to interrogate Golgi function in-depth and reveal that similar disruption to Golgi morphology can lead to drastically different glycosylation outcomes. Collectively, this work demonstrates a generalizable approach for systematically dissecting the regulatory network underlying glycosylation.

cell biology↗

bioRxiv2023

The microenvironment dictates glycocalyx construction and immune surveillance

Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with in vitro models that do not match the microenvironmental characteristics of human tissues. Using in vitro models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium. Key PointsO_LICulture medium dictates cellular mechanoresponse signatures in vitro C_LIO_LIEpithelial glycocalyx construction is mediated by Heat Shock Factor 1 (HSF1) C_LIO_LISialic acid topology dictates Natural Killer cell cytotoxicity C_LIO_LIPhysiological microenvironments reveal distinct glycobiology C_LI

cell biology↗

bioRxiv2022

Lipid homeostasis is essential for a maximal ER stress response

Changes in lipid metabolism are associated with aging and age-related diseases, including proteopathies. The endoplasmic reticulum (ER) is uniquely a major hub for protein and lipid synthesis, making its function essential for both protein and lipid homeostasis. However, it is less clear how lipid metabolism and protein quality may impact each other. Here, we identity let-767, a putative hydroxysteroid dehydrogenase, as an essential gene for both lipid and ER protein homeostasis. Knockdown of let-767 reduces lipid stores, alters ER morphology in a lipid-dependent manner, and also blocks induction of the Unfolded Protein Response of the ER (UPRER). Interestingly, a global reduction in lipogenic pathways restores UPRER induction in animals with reduced let-767. Specifically, we find that supplementation of 3-oxoacyl, the predicted metabolite directly upstream of let-767, is sufficient to block induction of the UPRER. This study highlights a novel interaction through which changes in lipid metabolism can alter a cells response to protein-induced stress.

cell biology↗

bioRxiv2022

Large-scale genetic screens identify BET-1 as a cytoskeleton regulator promoting actin health and lifespan.

The actin cytoskeleton is a three-dimensional scaffold of proteins that is a regulatory, energy-consuming network with dynamic properties to shape the structure and function of the cell. Proper actin function is required for many cellular pathways, including cell division, autophagy, chaperone function, endocytosis, and exocytosis. Deterioration of these processes manifests during aging and exposure to stress, which is in part due to the breakdown of the actin cytoskeleton. However, the regulatory mechanisms involved in preservation of cytoskeletal form and function are not well understood. Here, we performed a multi-pronged, cross-organismal screen combining a whole-genome CRISPR-Cas9 screen in human fibroblasts with in vivo C. elegans synthetic lethality screening. We identified the bromodomain protein, BET-1, as a key regulator of actin health and longevity. Overexpression of bet-1 preserves actin health at late age and promotes lifespan and healthspan in C. elegans. These beneficial effects are mediated through actin preservation by the transcriptional regulator function of BET-1. Together, our discovery assigns a key role for BET-1 in cytoskeletal health, highlighting regulatory cellular networks promoting cytoskeletal homeostasis.

cell biology↗

bioRxiv2021

In vitro and in vivo CRISPR-Cas9 screens reveal drivers of aging in neural stem cells of the brain

Aging impairs the ability of neural stem cells to transition from quiescence to activation (proliferation) in the adult mammalian brain. Neural stem cell (NSC) functional decline results in decreased production of new neurons and defective regeneration upon injury during aging1-9, and this is exacerbated in Alzheimers disease10. Many genes are upregulated with age in NSCs3, 11-13, and the knockout of some of these boosts old NSC activation and rejuvenates aspects of old brain function14-18. But systematic functional testing of genes in old NSCs - and more generally in old cells - has not been done. This has been a major limiting factor in identifying the most promising rejuvenation interventions. Here we develop in vitro and in vivo high-throughput CRISPR-Cas9 screening platforms to systematically uncover gene knockouts that boost NSC activation in old mice. Our genome-wide screening pipeline in primary cultures of young and old NSCs identifies over 300 gene knockouts that specifically restore old NSC activation. Interestingly, the top gene knockouts are involved in glucose import, cilium organization and ribonucleoprotein structures. To determine which gene knockouts have a rejuvenating effect for the aging brain, we establish a scalable CRISPR-Cas9 screening platform in vivo in old mice. Of the 50 gene knockouts we tested in vivo, 23 boost old NSC activation and production of new neurons in old brains. Notably, the knockout of Slc2a4, which encodes for the GLUT4 glucose transporter, is a top rejuvenating intervention for old NSCs. GLUT4 protein expression increases in the stem cell niche during aging, and we show that old NSCs indeed uptake [~]2-fold more glucose than their young counterparts. Transient glucose starvation increases the ability of old NSCs to activate, which is not further improved by knockout of Slc2a4/GLUT4. Together, these results indicate that a shift in glucose uptake contributes to the decline in NSC activation with age, but that it can be reversed by genetic or external interventions. Importantly, our work provides scalable platforms to systematically identify genetic interventions that boost old NSC function, including in vivo in old brains, with important implications for regenerative and cognitive decline during aging.

neuroscience↗

bioRxiv2020

Quantifying cytoskeletal heterogeneity via single-cell protein-complex fractionation

Multimeric cytoskeletal protein complexes 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 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 60 s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Co-detection of cytoskeletal protein complexes in U2OS cells treated with filamentous actin (F-actin) destabilizing LatA detects a subpopulation (~11%) exhibiting downregulated F-actin, but upregulated microtubule and intermediate filament protein complexes. Thus, some cells upregulate other cytoskeletal complexes to counteract the stress of LatA treatment. We also sought to understand the effect of non-chemical stress on cellular heterogeneity of F-actin. We find heat shock dysregulates F- and G-actin correlation. The assay overcomes selectivity limitations to biochemically quantify single-cell protein complexes perturbed with diverse stimuli.

bioengineering↗

bioRxiv2019

Systematic identification of regulators of antibody-drug conjugate toxicity using CRISPR-Cas9 screens

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 surprisingly appear to bypass the requirement of lysosomal delivery. Lastly, we show that inhibition of sialic acid biosynthesis sensitizes cells to ADC treatment by increasing the rate of ADC internalization. This sensitization was observed using several ADCs targeting different antigens in diverse cancer cell types, including the FDA-approved ADC trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Together, these results reveal novel regulators of endolysosomal trafficking, provide important insights to guide future ADC design, and identify candidate combination therapy targets as well as potential mechanisms of ADC resistance.

cancer biology↗