Biology subjects

Willis, L. M.

Publications and source records attributed to Willis, L. M..

5 recordsLinked to original sources

bioRxiv2025

Polysialic acid is a versatile marker for retinal Müller glia in common vertebrate model organisms and systems

Muller glia are retinal support cells that play crucial roles in tissue structure, waste management, and repair. A challenge for the field has been to find Muller glia markers that detect non-reactive cells or work well in non-mammalian models. We introduce two novel markers for identifying reactive and non-reactive Muller glia in vertebrate retinas: a modified enzyme lectin (GFP-EndoNDM) and a monoclonal antibody (mAb735). These markers recognize polysialic acid (polySia), which is a highly conserved glycosylation modification in humans and vertebrates. In the retina, polySia is present on Muller glia predominantly in the form of polySia-NCAM. We used GFP-EndoNDM and mAb735 to investigate polySia distribution in Muller glia of fish, amphibians, reptiles, birds, rodents, retinal organoids, and humans. In adult retinas of most species, polySia was localized to the Muller glia and spanned outer to inner retina, with inner plexiform layer (IPL) sublaminae ramifications. Gliosis was also detectable in degenerating murine retinas. Notable species differences were that only outer retinal regions of Muller glia were labelled in adult zebrafish, whereas the outer Muller glia body up to the first IPL sublamina was labelled in adult turquoise killifish. There was no significant retinal polySia labeling in larval zebrafish, but it was present in the brain. Larval turquoise killifish have polySia throughout the retina, similar to other adult vertebrates. Labelling polySia expands the scientific toolbox for Muller glia markers, and offers a versatile way to visualize and monitor structural changes in non-reactive and reactive Muller glia across most vertebrate species. HighlightsO_LIPolySia is a highly conserved Muller glia marker in vertebrate retina and retinal organoids C_LIO_LILabeling Muller glia with markers of polySia, facilitates the analysis of fine Muller glia processes in the IPL sublaminae, whole Muller glia morphology, and degenerative gliosis C_LIO_LIPolySia can be labeled with an engineered lectin conjugate (GFP-EndoNDM) and a commercially-available monoclonal antibody C_LI

cell biology↗

bioRxiv2025

Polysialylation is a general feature of immune activation

Sialic acids are critical regulators of immune responses and the sialic acid-Siglec axis has receive much attention recently for it role as a new immune checkpoint. While 2,3- and 2,6-linked sialosides have been well studied, much less is known about the 2,8-linked polysialic acid (polySia), especially in the adaptive immune system. We demonstrate here that polySia is actually found in all major classes of circulating immune cells, including B and T cells, where it is strongly associated with prior antigen exposure. Cell surface expression of polySia could be observed within 24 h of activation of naive T cells but did not correlate with expression of the immune-specific polysialyltransferase ST8Sia4. Finally, we assessed the effects of smoking on polySia levels, and noted substantial increased levels of polySia, particularly on effector T cells and preferentially in males. Significance StatementThe immune system is central to human health and holds the key to treating many chronic diseases. We show for the first time that the immunomodulatory glycan polysialic acid is widespread in the human immune system and associated with immune cell activation. Moreover, sex-dependent increases in polysialic acid could be observed on key populations of immune effector cells in smokers. This study offers insight into how glycans might contribute to sex differences in development and progression of chronic diseases.

immunology↗

bioRxiv2022

A new strategy for identifying polysialylated proteins reveals they are secreted from cancer cells as soluble proteins and as part of extracellular vesicles

Polysialic acid (polySia) is a long homopolymer consisting of 2,8-linked sialic acid with tightly regulated expression in humans. In healthy adults, it occurs on cell surface glycoproteins in neuronal, reproductive, and immune tissues; however, it is aberrantly present in many cancers and its overexpression correlates with significantly increased metastasis and poor prognosis. Prompted by the observation that the MCF-7 breast cancer cell line contains only intracellular polySia, we investigated the secretion of polySia from MCF-7 cells. PolySia was found predominantly on soluble proteins in MCF-7 conditioned media, but also on extracellular vesicles (EVs), secreted from the cells. Since MCF-7 cells do not express known polysialylated proteins, we developed a robust method for purifying polysialylated proteins that uses a metabolic labelling strategy to introduce a bioorthogonal functionality into polySia. Using this method we identified three previously unknown polysialylated proteins, and found that two of these proteins - AGR2 and QSOX2 - were secreted from MCF-7 cells. We confirmed that QSOX2 found in EV-depleted MCF-7 cell conditioned media was polysialylated. Herein we report the secretion of polysialic acid on both soluble and EV-associated proteins from MCF-7 cancer cells and introduce a new method to efficiently identify polysialylated proteins. These findings have exciting implications for understanding the roles of polySia in cancer progression and metastasis and for identifying new cancer biomarkers.

biochemistry↗

bioRxiv2022

Attenuation of polysialic acid biosynthesis in cells by the small molecule inhibitor 8-keto-sialic acid

Sialic acids are key mediators of cell function, particularly with regards to cellular interactions with the surrounding environment. Reagents that modulate the display of specific sialyl glycoforms at the cell surface would be useful biochemical tools and potentially allow for therapeutic intervention in numerous challenging chronic diseases. While multiple strategies are being explored for the control of cell surface sialosides, none that shows high selectivity between sialyltransferases or that targets a specific sialyl glycoform has yet to emerge. Here we describe a strategy to block the formation of 2,8-linked sialic acid chains (oligo- and polysialic acid) through the use of 8-keto-sialic acid as a chain-terminating metabolic inhibitor that, if incorporated, can not be elongated. 8-Keto-sialic acid is non-toxic at effective concentrations and serves to block polysialic acid synthesis in cancer cell lines and primary immune cells, with minimal effects on other sialyl glycoforms.

biochemistry↗

bioRxiv2021

Large differences in carbohydrate degradation and transport potential in the genomes of lichen fungal symbionts

Lichen symbioses are generally thought to be stabilized by the transfer of fixed carbon compounds from a photosynthesizing unicellular symbiont to a fungus. In other fungal symbioses, carbohydrate subsidies correlate with genomic reductions in the number of genes for plant cell wall-degrading enzymes (PCWDEs), but whether this is the case with lichen fungal symbionts (LFSs) is unknown. We predicted genes encoding carbohydrate-active enzymes (CAZymes) and sugar transporters in 17 existing and 29 newly sequenced genomes from across the class Lecanoromycetes, the largest extant clade of LFSs. Despite possessing lower mean numbers of PCWDE genes compared to non-symbiont Ascomycota, all LFS genomes possessed a robust suite of predicted PCWDEs. The largest CAZyme gene numbers, on par with model species such as Penicillium, were retained in genomes from the subclass Ostropomycetidae, which are found in crust lichens with highly specific ecologies. The lowest numbers were in the subclass Lecanoromycetidae, which are symbionts of many generalist macrolichens. Our results suggest that association with phototroph symbionts does not in itself result in functional loss of PCWDEs and that PCWDE losses may have been driven by adaptive processes within the evolution of specific LFS lineages. The inferred capability of some LFSs to access a wide range of carbohydrates suggests that some lichen symbioses may augment fixed CO2 with carbon from external sources. SignificanceLichen symbioses are considered self-contained autotrophic systems in which the total carbon economy is the sum of phototroph-fixed CO2, supplied to a fungus as sugars. In other fungal-plant symbioses, such as mycorrhizae, plant-derived sugar subsidies are associated with loss of plant cell wall-degrading enzymes (PCWDEs). We compared PCWDE inventories in 46 genomes from the largest group of lichen fungal symbionts (LFSs) with non-symbionts from across Ascomycota. We found that despite lower overall gene numbers, all LFSs retain PCWDEs, and some possess gene numbers and functional diversity on par with non-symbionts. Our results suggest that association with a phototroph does not necessarily result in PCWDE loss, and some lichens may obtain carbon from sources other than CO2 fixation.

genomics↗