Biology subjects

Wang, M. S.

Publications and source records attributed to Wang, M. S..

5 recordsLinked to original sources

bioRxiv2026

MAFA drives pancreatic islet maturation through coordination of cell growth, metabolic maturation, and resilience to stress.

Stem cell-derived islets (SC-islets) are a promising source for replacing the loss of insulin-secreting {beta} cells in insulin-dependent diabetes patients, but compared to their primary human counter parts, SC-islets are considered functionally immature and do not immediately regulate blood glucose after transplantation. To better understand the molecular underpinnings of {beta} cell maturation, we genetically engineered human pluripotent stem cells (hPSCs) to control the induction of the {beta} cell-maturing transcription factor MAFA or a MAFA S64F mutant variant associated with premature {beta} cell aging and senescence in SC-islets. We employ a combination of in vitro assays, multi-omics analyses, and murine in vivo transplantation studies to demonstrate that MAFA remodeling of the SC-islet transcriptional landscape promotes SC-islet biogenesis and metabolic maturation giving rise to greater SC-islet resilience and insulin secretory capacity. Parallel MAFA S64F studies highlight the importance for compensatory mechanisms for sustaining accelerated oxidative metabolism and enhanced insulin secretion without inducing intracellular stress responses and islet dysfunction. Overall, these results reveal MAFA-governed transcriptional changes that drive SC-islet functional maturation, which provides the foundation for maturing SC-islets in vitro, preserving {beta} cell integrity, and improving SC-islet cell replacement therapies.

cell biology↗

bioRxiv2023

Topographical analysis of immune cell interactions reveals a biomechanical signature for immune cytolysis

Immune cells live intensely physical lifestyles characterized by structural plasticity, mechanosensitivity, and force exertion. Whether specific immune functions require stereotyped patterns of mechanical output, however, is largely unknown. To address this question, we used super-resolution traction force microscopy to compare cytotoxic T cell immune synapses with contacts formed by other T cell subsets and macrophages. T cell synapses were globally and locally protrusive, which was fundamentally different from the coupled pinching and pulling of macrophage phagocytosis. By spectrally decomposing the force exertion patterns of each cell type, we associated cytotoxicity with compressive strength, local protrusiveness, and the induction of complex, asymmetric interfacial topographies. These features were further validated as cytotoxic drivers by genetic disruption of cytoskeletal regulators, direct imaging of synaptic secretory events, and in silico analysis of interfacial distortion. We conclude that T cell-mediated killing and, by implication, other effector responses are supported by specialized patterns of efferent force.

immunology↗

bioRxiv2023

Selection of a Promiscuous Minimalist cAMP Phosphodiesterase from a Library of De Novo Designed Proteins

The ability of unevolved amino acid sequences to become biological catalysts was key to the emergence of life on Earth. However, billions of years of evolution separate complex modern enzymes from their simpler early ancestors. To study how unevolved sequences can develop new functions, we screened for enzymatic activity in a collection of > 1 million novel sequences based on a de novo 4-helix bundle library of semi-random sequences. To mirror evolutionary selection for biological function, we screened the collection using ultrahigh-throughput droplet microfluidics to identify features that yield phosphoesterase activity. Characterization of active hits demonstrated that acquiring new function required a large jump in sequence space: screening enriched for truncations that removed > 40% of the protein chain and introduced a catalytically important cysteine. The truncated protein dimerized into a dynamic -helical structure, consistent with the idea that gain of function was accompanied by an increase in structural dynamics relative to the parental 4-helix bundle. The purified protein catalyzes the hydrolysis of a range of phosphodiesters, with the greatest activity toward the biological second messenger cyclic AMP (cAMP). The novel cAMPase is a manganese-dependent metalloenzyme and catalyzes cAMP hydrolysis with a rate acceleration on the order of 109 and catalytic proficiency on the order of 1014 M-1, comparable to large enzymes shaped by billions of years of evolution. These findings suggest that fragmentation to modular primordial peptides can be a fertile avenue for introducing structural and functional diversity into proteins. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/528392v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@180e004org.highwire.dtl.DTLVardef@1f32a75org.highwire.dtl.DTLVardef@16876aforg.highwire.dtl.DTLVardef@147ffc9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Mechanically active integrins direct cytotoxic secretion at the immune synapse

The secretory output of cell-cell interfaces must be tightly controlled in space and time to ensure functional efficacy. This is particularly true for the cytotoxic immune synapse (IS), the stereotyped junction formed between a cytotoxic lymphocyte and the infected or transformed target cell it aims to destroy1. Cytotoxic lymphocytes kill their targets by channeling a mixture of granzyme proteases and the pore forming protein perforin directly into the IS2, 3. The synaptic secretion of these toxic molecules constrains their deleterious effects to the target cell alone, thereby protecting innocent bystander cells in the surrounding tissue from collateral damage. Despite the importance of this process for immune specificity, the molecular and cellular mechanisms that establish secretory sites within the IS remain poorly understood. Here, we identified an essential role for integrin mechanotransduction in cytotoxic secretion using a combination of single cell biophysical measurements, ligand micropatterning, and functional assays. Upon ligand-binding, the L{beta}2 integrin LFA-1 functioned as a spatial cue, attracting lytic granules containing perforin and granzyme and inducing their fusion at closely adjacent sites within the synaptic membrane. LFA-1 molecules were subjected to pulling forces within these secretory domains, and genetic or pharmacological suppression of these forces abrogated cytotoxicity. We conclude that lymphocytes employ an integrin-dependent mechanical checkpoint to enhance both the potency and the security of their cytotoxic output.

immunology↗

bioRxiv2018

Interfacial actin protrusions mechanically potentiate killing by cytotoxic T cells

Cytotoxic T lymphocytes (CTLs) kill by forming immunological synapses with target cells and secreting toxic proteases and the pore forming protein perforin into the intercellular space. Immunological synapses are highly dynamic structures that potentiate perforin activity by applying mechanical force against the target cell. Here, we employed high-resolution imaging and microfabrication to investigate how CTLs exert synaptic forces and coordinate their mechanical output with perforin secretion. Using micropatterned stimulatory substrates that enable synapse growth in three dimensions, we found that perforin release occurs at the base of actin-rich protrusions that extend from central and intermediate locations within the synapse. These protrusions, which depended on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, were required for synaptic force exertion and efficient killing. They also mediated physical distortion of the target cell surface during CTL-target cell interactions. Our results reveal the mechanical basis of cellular cytotoxicity and highlight the functional importance of dynamic, three-dimensional architecture in immune cell-cell interfaces.\n\nOne sentence summaryCytotoxic T lymphocytes use F-actin-rich protrusions at the immunological synapse to potentiate perforin-and granzyme-mediated target cell killing.

immunology↗