Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton

Abstract

Intermediate filaments (IF) are a major component of the metazoan cytoskeleton and are essential for normal cell morphology, motility, and signal transduction. Dysregulation of IFs causes a wide range of human diseases, including skin disorders, cardiomyopathies, lipodystrophy, and neuropathy. Despite this pathophysiological significance, how cells regulate IF structure, dynamics, and function remains poorly understood. Here, we show that site-specific modification of the prototypical IF protein vimentin with O-linked β-N-acetylglucosamine (O-GlcNAc) mediates its homotypic protein-protein interactions and is required in human cells for IF morphology and cell migration. In addition, we show that the intracellular pathogen Chlamydia trachomatis, which remodels the host IF cytoskeleton during infection, requires specific vimentin glycosylation sites and O-GlcNAc transferase activity to maintain its replicative niche. Our results provide new insight into the biochemical and cell biological functions of vimentin O-GlcNAcylation, and may have broad implications for our understanding of the regulation of IF proteins in general.

Combined Atomic Force Microscope and Volumetric Light Sheet System for Mechanobiology

“…2a). We anticipate that the ability to couple force measurements with highresolution, volumetric optical imaging will be a powerful tool for studying the mechanobiology and regulation of the IF cytoskeleton, which remain poorly understood 27,37 . For example, vimentin filaments organize around the nucleus (Fig.…”

“…Our results also set the stage for new discoveries on IF cytoskeleton regulation. All IF proteins are decorated with post-translational modifications (PTMs), including phosphorylation and O-glycosylation, which may alter IF function 37,40 . We previously demonstrated that the site-specific O-glycosylation of vimentin is required for IF assembly and function in human cells 37 , but the real-time effects of these PTMs on IF network dynamics and mechanical properties remain elusive.…”

“…All IF proteins are decorated with post-translational modifications (PTMs), including phosphorylation and O-glycosylation, which may alter IF function 37,40 . We previously demonstrated that the site-specific O-glycosylation of vimentin is required for IF assembly and function in human cells 37 , but the real-time effects of these PTMs on IF network dynamics and mechanical properties remain elusive. Using simultaneous AFM-LS, we are poised to characterize the 4D effects of chemical or genetic inhibition of PTMs (e.g., phosphorylation, O-glycosylation) on the vimentin cytoskeleton, addressing a longstanding question in the IF field.…”

“…Cell culture for vimentin, actin and lysosome 3D imaging. Vimentin-/-HeLa cells stably expressing vimentin-mEmerald were generated as described 37 . In brief, the endogenous vimentin gene was deleted via CRISPR-Cas9 methods and a verified vimentin-/-clone was stably transduced with a lentiviral vimentin-mEmerald vector.…”

Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis

“…Our observation that silencing GFPT expression increases bacterial 403 diameter strongly supports the hypothesis that UDP-GlcNAc, or an intermediate along the 404 HBP, is hijacked into the inclusion to fuel bacterial division, possibly by feeding peptidoglycan 405 synthesis. One recent publication showed that UDP-GlcNAc is also used during infection to 406 post-translationally modify the intermediate filament vimentin and this also could contribute 407 to significant UDP-GlcNAc consumption in C. trachomatis infection(Tarbet, Dolat et al, 2018). 408…”