Like the body's skeleton, the cytoskeleton gives shape and structure to the inside of a cell. Yet, unlike a skeleton, the cytoskeleton is ever changing. The cytoskeleton consists of many fibers each made from chains of protein molecules. One of these proteins is called vimentin and it forms intermediate filaments in the cytoskeleton. Many different types of cells contain vimentin and a lot of it is found in cancer cells that have spread beyond their original location to other sites in the body.

Cells use chemical modifications to regulate cytoskeleton proteins. For example, through a process called glycosylation, cells can reversibly attach a sugar modification called O-GlcNAc to vimentin. O-GlcNAc can be attached to several different parts of vimentin and each location may have a different effect. It is not currently clear how cells control their vimentin filaments or what role O-GlcNAc plays in this process.

Using genetic engineering, Tarbet et al. produced human cells in the laboratory with modified vimentin proteins. These altered proteins lacked some of the sites for O-GlcNAc attachment. The goal was to see whether the loss of O-GlcNAc at a specific location would affect fiber formation and cell behavior. The results showed one site where vimentin needs O-GlcNAc to form fibers. Without O-GlcNAc at this site, cells could not migrate towards chemical signals. In addition, in normal human cells, Chlamydia bacteria hijack vimentin and rearrange the filaments to form a cage around themselves for protection. However, the cells lacking O-GlcNAc on vimentin were resistant to infection by Chlamydia bacteria.

These findings highlight the importance of O-GlcNAc on vimentin in healthy cells and during infection. Vimentin’s contribution to cell migration may also help to explain its role in the spread of cancer. The importance of O-GlcNAc suggests it could be a new target for therapies. Yet, it also highlights the need for caution due to the delicate balance between the activity of vimentin in healthy and diseased cells. In addition, human cells produce about 70 other vimentin-like proteins and further work will examine if they are also affected by O-GlcNAc.

Intermediate filaments (IF) are a major component of the metazoan cytoskeleton, distinct from the actin and microtubule systems (Lowery et al., 2015; Herrmann and Aebi, 2016; Chernyatina et al., 2015; Köster et al., 2015; Leduc and Etienne-Manneville, 2015). Humans express over 70 IF proteins, including both cytoplasmic (e.g., vimentin, keratins, neurofilaments) and nuclear (lamins) members, many with tissue-specific functions (Szeverenyi et al., 2008). All IF proteins comprise a central, conserved α-helical rod domain, as well as amino-terminal head and carboxy-terminal tail domains of varying lengths (Lowery et al., 2015; Herrmann and Aebi, 2016; Chernyatina et al., 2015; Köster et al., 2015; Leduc and Etienne-Manneville, 2015). IF proteins homo- or heterodimerize through the parallel association of their rod domains into coiled coils, forming an elongated dimer of ~45–48 nm for cytoplasmic IFs and ~50–52 nm for nuclear lamins (Quinlan et al., 1986; Aebi et al., 1986). These dimers laterally associate in antiparallel fashion to form tetramers, which in turn assemble into ~65 nm unit-length filaments (ULFs) composed of eight tetramers (Herrmann and Aebi, 2016; Chernyatina et al., 2015; Herrmann et al., 1996). Finally, ULFs associate end-to-end to assemble mature IFs, measuring ~10 nm across (Lowery et al., 2015; Herrmann and Aebi, 2016; Chernyatina et al., 2015).

Unlike actin- or microtubule-based structures, IFs are nonpolar and do not serve as tracks for molecular motors. Instead, IFs contribute to the mechanical integrity of the cell through their unique viscoelastic properties (Lowery et al., 2015; Herrmann and Aebi, 2016; Chernyatina et al., 2015; Köster et al., 2015; Leduc and Etienne-Manneville, 2015). In general, the IF network is flexible under low strain but stiffens and resists breakage under an applied force (Janmey et al., 1991; Fudge et al., 2003; Guzmán et al., 2006; Kreplak et al., 2005). Remarkably, individual IFs can be stretched up to 3.6-fold before rupture, demonstrating their elastic nature, as compared to actin cables or microtubules (Kreplak et al., 2005). The IF network is also highly dynamic in vivo, with IF subunits (likely tetramers) exchanging rapidly at many points along mature filaments (Mendez et al., 2010; Goldman et al., 2012; Miller et al., 1991; Vikstrom et al., 1989; Ho et al., 1998; Martys et al., 1999; Vikstrom et al., 1992; Nöding et al., 2014). Similarly, the IF cytoskeleton quickly reorganizes in response to numerous physiological cues, including cell cycle progression, migration, spreading, and growth factor stimulation (Lowery et al., 2015; Herrmann and Aebi, 2016; Chernyatina et al., 2015; Köster et al., 2015; Leduc and Etienne-Manneville, 2015; Yoon et al., 1998; Helfand et al., 2003).

IFs participate in many cellular processes, including maintenance of cell shape, organelle anchoring, cell motility, and signal transduction (Helfand et al., 2011; Ben-Ze'ev, 1984). For example, vimentin, among the most widely studied IF proteins, is required for mesenchymal cell adhesion, migration, chemotaxis, and wound healing in both cell culture and animal models (Ivaska et al., 2007; Yamaguchi et al., 2005; Eckes et al., 2000; Rogel et al., 2011; Menko et al., 2014). Vimentin IFs also contribute to the mechanical properties of the cytoplasm, and stabilize and localize mitochondria (Nekrasova et al., 2011; Buehler, 2013; Guo et al., 2013; Eckes et al., 1998). Importantly, genetic lesions that dysregulate the IF cytoskeleton cause a wide range of human diseases, including skin and nail disorders (keratins), cardiomyopathies (desmin), lipodystrophy, muscular dystrophy and progeria (lamins), giant axonal neuropathy and Charcot-Marie-Tooth disease (neurofilaments), and cataracts (vimentin) (Omary, 2009; Omary et al., 2004). In addition, the ectopic expression of wild type (WT) vimentin is a hallmark of the epithelial-to-mesenchymal transition, and is widely observed in human metastatic cancers (Satelli and Li, 2011; Nieto, 2011; De Craene and Berx, 2013). IFs are likely functionally important in this context, because vimentin expression levels correlate with the invasive phenotypes of breast, prostate, and other epithelial cancers (Satelli and Li, 2011; Wei et al., 2008; Zhu et al., 2011; Vuoriluoto et al., 2011). Finally, the IF cytoskeleton is also intimately involved in host-microbe interactions (Geisler and Leube, 2016; Mak and Brüggemann, 2016). For example, changes in vimentin IFs are implicated in the adhesion, invasion, and replication of a wide range of bacteria, including such important pathogens as Chlamydia trachomatis (Kumar and Valdivia, 2008; Jorgensen et al., 2011; Snavely et al., 2014; Bednar et al., 2011), Mycobacterium tuberculosis (Garg et al., 2006; Mahesh et al., 2016), Streptococcus pyogenes (Bryant et al., 2006; Icenogle et al., 2012; Lin et al., 2015) and Salmonella enterica (Murli et al., 2001; Guignot and Servin, 2008).

Despite this broad pathophysiological significance, the regulation of IF cytoskeleton morphology, dynamics, and signaling functions remains incompletely understood. Several recent lines of evidence indicate that post-translational modifications (PTMs) are an important mode of IF regulation, and indeed all IFs are subject to extensive PTMs, including phosphorylation, ubiquitination, sumoylation, acetylation, farnesylation, and glycosylation (Snider and Omary, 2014). However, in most cases, the functional impact of these PTMs on IF structure and function is poorly characterized.

To better understand the dynamic regulation of the IF cytoskeleton, we focused on O-linked β-N-acetylglucosamine (O-GlcNAc), an intracellular form of protein glycosylation that reversibly decorates serine and threonine residues on thousands of nuclear, cytoplasmic, and mitochondrial proteins. In mammals, O-GlcNAc is added by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA) (Figure 1A) (Hanover et al., 2010; Hart et al., 2011; Hart, 2014). O-GlcNAc cycling controls many processes, including nutrient sensing, cell cycle progression, and apoptosis (Hanover et al., 2010; Hart et al., 2011), and is essential, as genetic ablation of OGT or OGA is lethal in mice (Shafi et al., 2000; Keembiyehetty et al., 2015; Yang et al., 2012). In addition, aberrant O-GlcNAc cycling is implicated in numerous human diseases, including cancer (Hart et al., 2011; Ma and Vosseller, 2013; Yi et al., 2012; Singh et al., 2015), diabetes (Vaidyanathan and Wells, 2014; Hardivillé and Hart, 2014; Ma and Hart, 2013), cardiac dysfunction (Erickson et al., 2013; Erickson, 2014; Dassanayaka and Jones, 2014; Darley-Usmar et al., 2012), and neurodegeneration (Yuzwa et al., 2012; Vaidyanathan et al., 2014; Yuzwa and Vocadlo, 2014; Zhu et al., 2014).

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Proteomic analysis of vimentin GlcNDAz crosslinks.

Full proteomics results are available for download as a Scaffold (.sf3) file on the eLife web site. Free software to view Scaffold files is available at http://www.proteomesoftware.com/products/scaffold/download/.

https://doi.org/10.7554/eLife.31807.007

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Interestingly, numerous IF proteins are O-GlcNAcylated (King and Hounsell, 1989; Chou et al., 1992). For example, keratin-18 glycosylation is required for the recruitment and activation of the pro-survival kinase Akt, and mice expressing an unglycosylatable keratin-18 mutant are sensitized to chemical injury of the liver and pancreas (Ku et al., 2010; Ku et al., 1996). Several neurofilament proteins are also glycosylated, especially in axons, and neurofilament-M O-GlcNAcylation is reduced in both human Alzheimer’s disease patients and a rat model of amyotrophic lateral sclerosis, suggesting a potential role for dysregulated IF glycosylation in neurodegeneration (Dong et al., 1996; Dong et al., 1993; Lüdemann et al., 2005; Deng et al., 2008; Cheung and Hart, 2008). Finally, vimentin is O-GlcNAcylated on several sites, primarily in its head domain (Slawson et al., 2008; Wang et al., 2007). Changes in vimentin glycosylation have been observed in models of differentiating adipocytes (Ishihara et al., 2010) and neurons (Farach and Galileo, 2008), and a recent study observed a correlation between vimentin O-GlcNAcylation and the invasive potential of cholangiocarcinoma (Phoomak et al., 2017), implicating glycosylation changes in both the physiological and pathological functions of vimentin. These studies suggest that O-GlcNAcylation is a prominent mode of IF regulation in homeostasis and disease alike. Nevertheless, the mechanistic and functional impacts of O-GlcNAcylation on IF proteins remain largely unexplored.

Here, we report that vimentin O-GlcNAcylation is required for the structure and function of IFs in human cells. We demonstrate that site-specific glycosylation of vimentin mediates its self-association, and is required in human cells for both IF morphology and its facilitating role in cell migration. In addition, we provide evidence that vimentin glycosylation is co-opted by an intracellular bacterial pathogen to produce a replicative niche, revealing a new connection between IF O-GlcNAcylation and microbial pathogenesis. Our results provide new insight into the physiological role of vimentin glycosylation in particular, and may have broad implications for our understanding of the dynamic regulation of the IF cytoskeleton in general.

Vimentin is O-GlcNAcylated at multiple sites in its head domain (Slawson et al., 2008; Wang et al., 2007), but the functional consequences of this modification are poorly understood. Like other intracellular PTMs, O-GlcNAc can exert a wide range of biochemical effects on its substrates, including conformational change, relocalization or destruction (Hanover et al., 2010; Hart et al., 2011; Shafi et al., 2000; Keembiyehetty et al., 2015; Mondoux et al., 2011; Bond and Hanover, 2013). Because IF proteins self-associate into homo-oligomeric complexes, we hypothesized that vimentin O-GlcNAcylation might influence its protein-protein interactions. Indeed, O-GlcNAc regulates protein-protein interactions in other contexts, including chromatin remodeling, nutrient sensing, and the interaction between keratins and Akt (Ku et al., 2010; Fujiki et al., 2011; Tarbet et al., 2018; Dentin et al., 2008). However, physiological O-GlcNAc-mediated interactions are often low-affinity, sub-stoichiometric, and transient, presenting a technical barrier to studying them (Hanover et al., 2010; Hart et al., 2011; Shafi et al., 2000; Keembiyehetty et al., 2015; Mondoux et al., 2011; Bond and Hanover, 2013; Tarbet et al., 2018).

To address these challenges, we used a chemical biology method to capture and characterize O-GlcNAc-mediated protein-protein interactions in living cells (Yu et al., 2012). In this strategy, cells are metabolically labeled with a GlcNAc analog bearing a diazirine photocrosslinking moiety, termed ‘GlcNDAz' (Yu et al., 2012). GlcNDAz is accepted by the GlcNAc salvage pathway, converted to the nucleotide-sugar UDP-GlcNDAz, and used by OGT to decorate its native substrates (Yu et al., 2012). Brief treatment of GlcNDAz-labeled live cells with UV light triggers the covalent crosslinking of O-GlcNDAz moieties to any binding partner proteins within ~2–4 Å of the sugar (Yu et al., 2012). Because of this short radius, GlcNDAz crosslinking occurs exclusively at sites where the glycan contributes to the interaction interface, without crosslinking to distant or nonspecific proteins (Yu et al., 2012). Therefore, GlcNDAz is a powerful tool for identifying direct, glycosylation-mediated interactions between endogenous proteins in live cells (Yu et al., 2012).

To determine whether vimentin engages in O-GlcNAc-mediated protein-protein interactions, we treated human cells with GlcNDAz and UV, and analyzed lysates by immunoblot (IB). Endogenous vimentin crosslinked into distinct, high molecular weight bands that were diazirine- and UV-dependent (Figure 1B), indicating that O-GlcNAc mediates protein-protein interactions between vimentin and one or more binding partners. Overexpression of OGT caused an increase in vimentin crosslinks (Figure 1C), whereas overexpression of OGA (Figure 1C) or treatment with a small molecule inhibitor of OGT, abbreviated 5SGlcNAc (Gloster et al., 2011) (Figure 1—figure supplement 1A), reduced crosslinking, confirming that this assay reports on authentic O-GlcNAc-mediated interactions, and not a nonspecific action of the GlcNDAz probe.

We next sought to identify the O-GlcNAc-mediated binding partner(s) of vimentin detected in our crosslinking assay. We created a myc-6xHis-tagged human vimentin construct and confirmed that it crosslinked via GlcNDAz similarly to endogenous vimentin (Figure 1—figure supplement 1B). Then, we purified preparative amounts of vimentin crosslinks from transfected cells via tandem immunoprecipitation (IP) and immobilized metal affinity chromatography, and analyzed the crosslinks by mass spectrometry (MS)-based proteomics (Figure 1—figure supplement 1C, Figure 1—figure supplement 2D,E and Figure 1—source data 1). We obtained 80% tryptic peptide coverage of vimentin in these samples, without significant enrichment of other proteins, beyond common contaminants (Figure 1—figure supplement 2 and Figure 1—source data 1). This result suggested that the vimentin GlcNDAz crosslinks represent homotypic, O-GlcNAc-mediated vimentin-vimentin interactions.

In vivo, vimentin exists in a range of assembly states, from soluble tetramers and ULFs, to relatively insoluble mature IFs (Lowery et al., 2015; Herrmann and Aebi, 2016; Chernyatina et al., 2015; Köster et al., 2015; Leduc and Etienne-Manneville, 2015). To determine the assembly state of the crosslinked vimentin species, we performed a well-established differential extraction procedure (Ridge et al., 2016) on GlcNDAz-crosslinked cells. The GlcNDAz crosslinks of endogenous vimentin extracted into a denaturing buffer but not low or high ionic strength non-denaturing buffers, indicating that the crosslinks occur within the highly assembled filament population (Figure 1D) (Ridge et al., 2016). Importantly, extracted crosslinks remained soluble when exchanged from denaturing into non-denaturing buffers, demonstrating that GlcNDAz crosslinking per se does not reduce vimentin solubility (Figure 1—figure supplement 2F). To further confirm that crosslinks occur within assembled IFs, we treated cells with β,β’-iminodipropionitrile (IDPN), which blocks vimentin assembly beyond the ULF state (Durham, 1986). Consistent with previous reports, IDPN treatment abrogated vimentin IFs and caused the accumulation of vimentin aggregates (Figure 1—figure supplement 2G) (Durham, 1986). In the crosslinking assay, IDPN also suppressed the formation of GlcNDAz-dependent adducts, indicating that the crosslinks occur in assembly states beyond ULFs (Figure 1E). Using quantitative IBs, we detected ~10% of endogenous vimentin crosslinked into GlcNDAz-dependent adducts (Figure 1—figure supplement 2H), though this measurement likely significantly underestimates the fraction of vimentin that engages in O-GlcNAc-mediated interactions, since several steps in the GlcNDAz crosslinking protocol are less than 100% efficient (Yu et al., 2012; Rodriguez et al., 2015). Based on these results, we concluded that O-GlcNAc-mediated vimentin-vimentin interactions are prevalent in cells, and occur primarily within assembled IFs, but not in soluble pools of lower-order assembly states.

The Hart lab previously mapped several glycosylation sites on vimentin’s flexible head domain (Slawson et al., 2008; Wang et al., 2007). We used these results to identify vimentin O-GlcNAc sites that contribute to its homotypic, glycosylation-mediated interactions. We mutated each reported O-GlcNAcylation site to alanine and screened these constructs in the GlcNDAz crosslinking assay (Figure 2). Mutation of several individual residues, including T33, S34, and S39, reduced vimentin crosslinking, while mutation of S49 abolished all detectable crosslinks. Because of the dramatic reduction of crosslinking in the S49A mutant, we measured the fraction of total vimentin glycosylation occurring at S49. We transfected cells with vector, WT or S49A vimentin and then labeled them with GalNAz, an azide-bearing unnatural monosaccharide that we have described previously as a metabolic reporter of O-GlcNAcylation (Boyce et al., 2011; Palaniappan et al., 2013; Chen et al., 2017). S49A mutant vimentin exhibited ~80% the GalNAz signal of WT (Figure 2—figure supplement 1), suggesting that approximately one-fifth of vimentin glycosylation occurs on S49 under these conditions. Together, these results suggest that robust and site-specific O-GlcNAcylation in the vimentin head domain mediates homotypic protein-protein interactions within IFs.

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We next tested whether O-GlcNAc-mediated interactions influence vimentin IF structure or function in live human cells. We used CRISPR/Cas9 methods to delete endogenous vimentin from two different human cell lines, selected single cell-derived clones, and confirmed the absence of vimentin mRNA and protein (Figure 3—figure supplement 1A,B). Then, we stably reconstituted individual vimentin^−/− clones with WT or unglycosylatable point-mutant versions of a well-characterized vimentin-mEmerald fusion protein (Mendez et al., 2010; Yoon et al., 1998; Helfand et al., 2011; Hookway et al., 2015) to permit live-cell visualization of IFs. We verified that vimentin-mEmerald was expressed at uniform levels comparable to endogenous vimentin (Figure 3—figure supplement 1C,D), and that the mEmerald signal precisely coincided with anti-vimentin immunofluorescence in cells expressing both endogenous vimentin and vimentin-mEmerald (Figure 3—figure supplement 2E). These results confirmed that the vimentin-mEmerald construct is a faithful proxy for the untagged protein in this system. In our panels of vimentin-mEmerald-reconstituted cells, we included Y117L, a point-mutation in the rod domain that blocks vimentin assembly beyond the ULF stage (Meier et al., 2009), as a positive control for IF disruption. We used fluorescence-activated cell sorting, IB and fluorescence microscopy to ensure equal expression of the various WT and mutant vimentin-mEmerald transgenes across reconstituted vimentin^−/− cell lines (Figure 3A, Figure 3—figure supplements 1D,3A).

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Cells reconstituted with WT vimentin-mEmerald exhibited canonical IF morphology, whereas the Y117L-expressing cells lacked assembled filaments and instead displayed punctate structures consistent with ULFs (Figure 3A,B, Figure 3—figure supplement 3A,B). These results indicate that our reconstituted cell systems recapitulate the previously reported characteristics of WT and Y117L vimentin (Meier et al., 2009; Helfand et al., 2011; Robert et al., 2014). We also observed dramatic alterations in vimentin IF organization in several unglycosylatable point-mutants. For example, the S34A mutant, which had an intermediate phenotype in our crosslinking assay (Figure 2), displayed a partial defect in IF formation, with both punctate and filamentous fluorescence detected (Figure 3A, Figure 3—figure supplement 3A). Interestingly, the S49A mutant, which lacked detectable O-GlcNAc-mediated interactions in the GlcNDAz assay (Figure 2), exhibited a significant reduction of assembled IFs in live cells, displaying a higher proportion of punctate vimentin fluorescence as compared to WT (Figure 3A,B, Figure 3—figure supplement 3A,B). Importantly, the S49A mutation had no impact on vimentin stability (Figure 3—figure supplement 2), further ruling out an effect of vimentin expression level in this system.

These results suggested that O-GlcNAc on specific residues of vimentin, particularly S49, may be required for its homotypic assembly into mature IFs in live cells. However, S/T→A mutations eliminate all O-linked PTMs at the mutated site, including both O-GlcNAcylation and phosphorylation. Therefore, phenotypes arising from S/T→A mutations may be due to loss of phosphorylation, glycosylation, or both. Vimentin S49 is a known glycosylation site (Slawson et al., 2008; Wang et al., 2007) but not a reported phosphorylation site, suggesting that the phenotypes we observed in the S49A mutant (Figures 2 and 3, Figure 3—figure supplement 3) are due to the loss of O-GlcNAc, and not the loss of phosphorylation. To further test this hypothesis, we reconstituted vimentin^−/− cells with a phosphomimetic S49E mutant. Vimentin-S49E-expressing cells displayed a loss of filament morphology and a proportion of puncta indistinguishable from the S49A mutant (Figure 3 and Figure 3—figure supplement 3). These results suggest that the abnormal vimentin IF structures observed with the S49A mutant in live cells are due to the loss of glycosylation, not loss of phosphorylation, at S49.

We next investigated whether vimentin O-GlcNAcylation is required for known functions of IFs. The IF cytoskeleton facilitates cell migration, and vimentin^−/− cells and tissues exhibit migration defects (Ivaska et al., 2007; Yamaguchi et al., 2005; Eckes et al., 2000; Rogel et al., 2011; Menko et al., 2014). We used a well-characterized Transwell assay (Justus et al., 2014) to measure cell migration by vimentin-reconstituted cell lines across a collagen matrix. Consistent with prior reports in other systems (Ivaska et al., 2007; Yamaguchi et al., 2005; Eckes et al., 2000; Rogel et al., 2011; Menko et al., 2014), vimentin^−/− cells stably transduced with empty vector or the Y117L mutant construct were impaired in serum-stimulated migration, relative to cells expressing WT vimentin (Figure 4, Figure 4—source data 1). Interestingly, vimentin-S49A-expressing cells also exhibited migration defects compared to WT (Figure 4, Figure 4—source data 1). This result suggested that O-GlcNAc on vimentin may be required for optimal cell migration. To further confirm this hypothesis, we assayed the serum-induced migration of vimentin-reconstituted cells upon treatment with 5SGlcNAc or Thiamet-G, a specific small molecule inhibitor of OGA (Yuzwa et al., 2008) (Figure 4, Figure 4—source data 1). 5SGlcNAc or Thiamet-G treatment each significantly inhibited the serum-induced migration of cells expressing WT vimentin, but not cells lacking vimentin or expressing the S49A mutant (Figure 4, Figure 4—source data 1). Taken together, these results indicate that O-GlcNAcylation of vimentin, especially on S49, is required for optimal serum-stimulated cell migration.

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Full statistical analyses of cell migration data.

(A) Migration data were first compared by analysis of variance (ANOVA) using JMP software. The results of the ANOVA, as well as the effect test, are displayed. (B) Pairwise t-tests for all combinations of data points were performed. Level and –Level indicate the sample and the sample it is being compared to, respectively. The calculated difference, standard error, and upper and lower confidence levels (CL) are shown, as well as the p value for each t-test. (C) Comprehensive comparison of all samples and conditions. Any two samples that do not share a letter in column two are statistically significantly different from each other.

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Several intracellular pathogens co-opt the IF cytoskeleton to stabilize the large membrane-bound vacuoles in which they replicate, presumably by providing structural scaffolds (Geisler and Leube, 2016; Mak and Brüggemann, 2016). For example, we previously showed that the obligate intracellular pathogen Chlamydia trachomatis recruits a meshwork of vimentin IFs to its vacuolar ‘inclusion’ compartment, stabilizing its replicative niche and shielding bacterial components from the host’s cytoplasmic innate immune surveillance machinery (Kumar and Valdivia, 2008; Jorgensen et al., 2011; Snavely et al., 2014; Bednar et al., 2011). The molecular mechanisms of Chlamydia-induced vimentin recruitment and reorganization are incompletely understood. To examine the potential role of vimentin O-GlcNAcylation in these processes, we treated HeLa cells reconstituted with WT vimentin-mEmerald with vehicle control, 5SGlcNAc or Thiamet-G, infected them with Chlamydia, and visualized pathogen-containing inclusions and vimentin IFs by fluorescence microscopy (Figure 5A, Figure 5—figure supplement 1). We found that 5SGlcNAc treatment inhibited the formation of a vimentin meshwork around the inclusion, consistent with a requirement for vimentin glycosylation in IF remodeling during Chlamydia infection (Figure 5A). 5SGlcNAc treatment reduced the average size of the inclusions in infected cells and increased the number of extra-inclusion bacteria (Figure 5B), demonstrating that OGT activity is required for inclusion expansion and integrity. Thiamet-G treatment had little effect in these assays, likely because basal levels of O-GlcNAcylation are already sufficient for optimal Chlamydia replication (Figure 5, Figure 5—figure supplement 1).

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We next tested whether O-GlcNAcylation of vimentin itself is required for inclusion integrity. We infected vimentin-reconstituted HeLa cell lines with Chlamydia and visualized vimentin IFs and bacteria by fluorescence microscopy. Compared to WT vimentin-expressing cells, cells expressing either the S49A or Y117L mutant vimentin displayed reduced IF recruitment to the inclusions, smaller average inclusion size, and larger numbers of bacteria escaping into the cytoplasm (Figure 5C,D). These data suggest that the site-specific glycosylation of vimentin itself is required for IF remodeling during Chlamydia infection. To further test this hypothesis, we infected empty vector-reconstituted vimentin^−/− cells with Chlamydia in the presence or absence of 5SGlcNAc. In contrast to our observations with cells expressing WT vimentin (Figure 5A,B), 5SGlcNAc had no impact on Chlamydia inclusion size or extra-inclusion bacteria in cells lacking vimentin, indicating that vimentin, but not other host- or pathogen-encoded targets, is required for the effects of 5SGlcNAc in this context (Figure 5E). We concluded that both OGT activity and vimentin glycosylation sites are required for IF reorganization during Chlamydia infection, and are co-opted by this pathogen to promote inclusion integrity and growth.

The IF cytoskeleton plays critical roles in both physiological and pathological processes, but how cells regulate IF structure and function remains poorly understood. We provide evidence that site-specific glycosylation of the vimentin head domain regulates its homotypic association in human cells, and is required for both IF morphology and cell migration (Figure 6). In addition, site-specific O-GlcNAcylation of vimentin is exploited by an intracellular pathogen to promote its own replication, underlining the importance of IF dynamics in disease states. Because many IF proteins are O-GlcNAc-modified (King and Hounsell, 1989; Chou et al., 1992; Ku et al., 2010; Dong et al., 1996; Dong et al., 1993; Lüdemann et al., 2005; Deng et al., 2008; Cheung and Hart, 2008; Slawson et al., 2008; Wang et al., 2007; Srikanth et al., 2010; Kakade et al., 2016; Tao et al., 2006), our results may provide new insight into the regulation of both vimentin in particular and the IF cytoskeleton in general.

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Early evidence for vimentin glycosylation was reported nearly twenty-five years ago (Shikhman et al., 1993) and confirmed with sophisticated MS methods thirteen years later (Slawson et al., 2008; Wang et al., 2007). However, the biochemical and cellular effects of this modification have remained largely uncharacterized. Through GlcNDAz crosslinking (Figure 2) and live-cell imaging (Figure 3, Figure 3—figure supplement 3), we determined that the vimentin glycosylation sites S34, S39 and especially S49 are required for normal homotypic vimentin-vimentin interactions and for IF morphology in live human cells. These O-GlcNAc-mediated interactions likely occur within assembled IFs and not smaller oligomeric states (Figure 1D,E). It is well established that portions of the vimentin head domain are required for filament assembly (Eriksson et al., 2004), and that head domain PTMs (especially phosphorylation) govern IF dynamics in vivo (Helfand et al., 2011; Eriksson et al., 2004; Chou et al., 1990; Eriksson et al., 1992; Sihag et al., 2007; Goto et al., 2002; Chan et al., 2002). Our results are consistent with this general model of regulated filament assembly/disassembly through head domain PTMs (Figure 6). Interestingly, however, internal deletion mutations within the head domain demonstrate that S49 itself is not required for vimentin filament formation in vitro or in vivo (Shoeman et al., 2002). Therefore, we propose that the phenotypes observed in the S49A vimentin mutant are caused by a loss of PTM regulation, rather than simply a requirement for serine at that site.

Given the very short crosslinking radius of the GlcNDAz reagent (~2–4 Å) (Yu et al., 2012) and our MS proteomics analysis of the crosslinked complexes (Figure 1—figure supplement 1C and Figure 1—figure supplement 2D,E), the O-GlcNAc-mediated interactions we observe are likely homotypic dimeric or trimeric vimentin adducts. The precise molecular nature of these crosslinks, and why we observe multiple discrete crosslinked complexes, remain uncertain. The high MS proteomic coverage we obtained of vimentin and the lack of other proteins in the crosslinks (Figure 1—figure supplement 2 and Figure 1—source data 1) suggest that the complexes do not contain other binding partner proteins or known protein-based PTMs of vimentin, such as SUMO (Kaminsky et al., 2009; Wang et al., 2010). Because vimentin is multiply phosphorylated (Helfand et al., 2011; Eriksson et al., 2004; Chou et al., 1990; Eriksson et al., 1992; Sihag et al., 2007; Goto et al., 2002; Chan et al., 2002), the crosslinks of distinct molecular weight (Figures 1B and 2) could in principle represent different phospho-forms of vimentin. However, several commercially available anti-phospho-vimentin antibodies failed to recognize the crosslinked complexes by IB (not shown), and phosphatase treatment of crosslinked adducts had no effect on their SDS-PAGE migration (Figure 1—figure supplement 3), arguing against differential phosphorylation as an explanation.

Instead, we postulate that the discrete bands in the GlcNDAz-induced vimentin complexes represent different crosslinking geometries of vimentin multimers. Because the O-GlcNAc-mediated interactions we detect occur primarily in assembled IFs (Figure 1D,E), an individual glycan on the vimentin head domain (e.g., at residue S49) may contact the head domain of its dimeric partner, and/or the rod domain of a vimentin molecule in an adjacent dimer, and/or another vimentin molecule within the same ULF. These different crosslinking geometries might produce migration differences in our GlcNDAz/SDS-PAGE assay (Figure 1B).

Irrespective of the molecular identities of the individual adducts, the fact that we observe a highly reproducible crosslinking pattern suggests that O-GlcNAc mediates specific contacts among vimentin molecules, and not a large variety of nonspecific interactions, which would produce variable or smeared crosslinking (Yu et al., 2012). This evidence of specific contacts is perhaps surprising, given the widely accepted model that the head domain of vimentin is intrinsically disordered (Guharoy et al., 2013). It is possible that, in vivo, O-GlcNAcylation mediates site-specific contacts between glycans on one vimentin molecule and discrete glycan recognition sites or domains on adjacent vimentin molecules within assembled filaments. Interestingly, several prior reports identified a GlcNAc-binding property of vimentin on the cell surface (Ise et al., 2010; Ise et al., 2011; Kim et al., 2012; Komura et al., 2012). Although the physiological relevance of extracellular vimentin is controversial, these biochemical observations are reminiscent of our own, in that vimentin is reported to bind to GlcNAc residues in both cases. However, two mutations in vimentin that abolish extracellular GlcNAc binding, E382A and E396A (Komura et al., 2012), do not reduce intracellular GlcNDAz crosslinking (Figure 2—figure supplement 2). Therefore, our observations are distinct from the reported glycan-binding property of cell surface vimentin (Ise et al., 2010; Ise et al., 2011; Kim et al., 2012; Komura et al., 2012). The vimentin site(s) that bind O-GlcNAc moieties are not readily discernible from our MS studies, due to the unpredictable masses and fragmentation spectra of crosslinked adducts. However, the identification of these sites will be an important priority for future studies, because this information may elucidate the biophysical regulation of IF assembly and dynamics by O-GlcNAc.

At the cellular level, specific glycosylation sites of vimentin, including S34, S39 and especially S49, are essential for IF morphology and for facilitating serum-stimulated migration (Figures 3 and 4, Figure 3—figure supplement 3). We propose that O-GlcNAcylation at one or more of these sites influences the assembly and/or disassembly of vimentin filaments, as has been described for other PTMs. Indeed, phosphorylation of several residues in the vimentin head domain promotes IF disassembly in response to multiple stimuli (Helfand et al., 2011; Eriksson et al., 2004; Chou et al., 1990; Eriksson et al., 1992; Sihag et al., 2007; Goto et al., 2002; Chan et al., 2002). O-GlcNAcylation and phosphorylation often compete for nearby or identical residues on specific protein substrates, giving rise to a complex functional interplay between these PTMs (Hart et al., 2011; Butkinaree et al., 2010). Moreover, specific O-GlcNAc sites required for in vivo IF assembly in our work, such as S34 and S39 (Figure 3, Figure 3—figure supplement 3), can also be phosphorylated (Helfand et al., 2011; Eriksson et al., 2004; Chou et al., 1990; Eriksson et al., 1992; Sihag et al., 2007; Goto et al., 2002; Chan et al., 2002). Therefore, reciprocal PTMs at certain sites may have antagonistic effects, with glycosylation of S34 or S39 promoting IF assembly, while phosphorylation at those sites induces disassembly.

In contrast, vimentin S49 is required for normal IF assembly in human cells (Figures 2 and 3, Figure 3—figure supplement 3) and is a known glycosylation site (Slawson et al., 2008; Wang et al., 2007) but is not a reported phosphorylation site. These observations suggest a functional role for O-GlcNAc, but not O-phosphate, at this residue. In support of this hypothesis, a phosphomimetic S49E mutant phenocopies an S49A mutant in IF morphology (Figure 3), and an OGT inhibitor impacts on cell migration and Chlamydia inclusion size only in the presence of WT vimentin, but not in cells expressing S49A mutant vimentin or lacking vimentin (Figures 4 and 5E). Taken together, these results strongly indicate that (de)glycosylation of S49 is required for WT levels of cell migration and pathogen-induced IF remodeling. Although we cannot strictly rule out the possibility that S49 is phosphorylated under as-yet untested conditions, we suggest that S49 O-GlcNAcylation is required for homotypic vimentin-vimentin interactions (Figure 2), filament assembly (Figure 3), and downstream behaviors of the vimentin IF cytoskeleton in normal and pathological contexts (Figures 4 and 5).

Finally, our results indicate that O-GlcNAc-mediated vimentin IF assembly is required for Chlamydia inclusion expansion and integrity, because we observed smaller inclusions and more cytoplasmic bacteria in Chlamydia-infected cells expressing Y117L or S49A mutants of vimentin (Figure 5C,D). Chlamydia remodels the IF cytoskeleton to stabilize the inclusion during its replication phase, and deploys the CPAF protease to dismantle the IF cytoskeleton and promote bacterial release at late stages of infection (Kumar and Valdivia, 2008; Jorgensen et al., 2011; Snavely et al., 2014; Bednar et al., 2011). Our current data extend these results by demonstrating that vimentin filament remodeling by Chlamydia depends on both OGT activity (Figure 5A,B) and on the site-specific glycosylation of vimentin itself (Figure 5C–E). We detected WT levels of CPAF-mediated vimentin cleavage with the S49A and Y117L mutants (Figure 5—figure supplement 2), indicating that the phenotypes we observe are not due to differential vimentin cleavage during infection. The molecular mechanisms by which Chlamydia co-opts OGT activity and vimentin glycosylation sites (e.g., S49) to reshape the IF cytoskeleton remain to be determined, and will be an interesting focus of future studies.

In conclusion, we have shown that site-specific glycosylation of vimentin mediates its homotypic protein-protein interactions, and is required in human cells for IF morphology and cell migration. In addition, Chlamydia relies on OGT activity and particular vimentin glycosylation sites to remodel the IF cytoskeleton and promote inclusion expansion and integrity. Our results provide new insight into the biochemical and cell biological functions of vimentin O-GlcNAcylation. In addition, our work may have broad implications for other IF proteins. Numerous IF proteins are dynamically glycosylated in vivo (King and Hounsell, 1989; Chou et al., 1992; Ku et al., 2010; Dong et al., 1996; Dong et al., 1993; Lüdemann et al., 2005; Deng et al., 2008; Cheung and Hart, 2008; Slawson et al., 2008; Wang et al., 2007; Srikanth et al., 2010; Kakade et al., 2016; Tao et al., 2006), and the S49 residue of vimentin is conserved both among vertebrate vimentin orthologs and in human desmin, another type III IF protein (Figure 6—figure supplement 1). Therefore, site-specific O-GlcNAcylation may be a general mode of regulating IF dynamics and function. In the future, pharmacological modulation of O-GlcNAcylation may provide a way to manipulate filament form and function for therapeutic benefit in diseases of dysregulated IFs, ranging from neurodegeneration and cardiomyopathy to cancer.

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Author details

1. Heather J Tarbet

   Department of Biochemistry, Duke University School of Medicine, Durham, United States

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

2. Lee Dolat

   1. Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, United States
   2. Center for Host-Microbial Interactions, Duke University School of Medicine, Durham, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

3. Timothy J Smith

   Department of Biochemistry, Duke University School of Medicine, Durham, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

4. Brett M Condon

   Department of Biochemistry, Duke University School of Medicine, Durham, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

5. E Timothy O'Brien III

   1. Department of Biochemistry, Duke University School of Medicine, Durham, United States
   2. Department of Physics and Astronomy, University of North Carolina, Chapel Hill, United States

   Contribution

   Software, Formal analysis, Validation

   Competing interests

   No competing interests declared

6. Raphael H Valdivia

   1. Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, United States
   2. Center for Host-Microbial Interactions, Duke University School of Medicine, Durham, United States

   Contribution

   Resources, Software, Formal analysis, Writing—review and editing

   Competing interests

   No competing interests declared

7. Michael Boyce

   1. Department of Biochemistry, Duke University School of Medicine, Durham, United States
   2. Center for Host-Microbial Interactions, Duke University School of Medicine, Durham, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Project administration, Writing—review and editing

   For correspondence

   michael.boyce@duke.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2729-4876

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