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.

The COPII coat complex, which mediates secretory cargo trafficking from the endoplasmic reticulum, is a key control point for subcellular protein targeting. Because misdirected proteins cannot function, protein sorting by COPII is critical for establishing and maintaining normal cell and tissue homeostasis. Indeed, mutations in COPII genes cause a range of human pathologies, including cranio-lenticulo-sutural dysplasia (CLSD), which is characterized by collagen trafficking defects, craniofacial abnormalities, and skeletal dysmorphology. Detailed knowledge of the COPII pathway is required to understand its role in normal cell physiology and to devise new treatments for disorders in which it is disrupted. However, little is known about how vertebrates dynamically regulate COPII activity in response to developmental, metabolic, or pathological cues. Several COPII proteins are modified by O-linked β-N-acetylglucosamine (O-GlcNAc), a dynamic form of intracellular protein glycosylation, but the biochemical and functional effects of these modifications remain unclear. Here, we use a combination of chemical, biochemical, cellular, and genetic approaches to demonstrate that site-specific O-GlcNAcylation of COPII proteins mediates their protein-protein interactions and modulates cargo secretion. In particular, we show that individual O-GlcNAcylation sites of SEC23A, an essential COPII component, are required for its function in human cells and vertebrate development, because mutation of these sites impairs SEC23A-dependent in vivo collagen trafficking and skeletogenesis in a zebrafish model of CLSD. Our results indicate that O-GlcNAc is a conserved and critical regulatory modification in the vertebrate COPII-dependent trafficking pathway.

5'-AMP-activated protein kinase (AMPK), by way of its inhibition of acetyl-CoA carboxylase (ACC), plays an important role in regulating malonyl-CoA levels and the rate of fatty acid oxidation in skeletal and cardiac muscle. In these tissues, LKB1 is the major AMPK kinase and is therefore critical for AMPK activation. The purpose of this study was to determine how the lack of muscle LKB1 would affect malonyl-CoA levels and/or fatty-acid oxidation. Comparing wild-type (WT) and skeletal/cardiac muscle-specific LKB1 knockout (KO) mice, we found that the 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR)-stimulated decrease in malonyl-CoA levels in WT heart and quadriceps muscles was entirely dependent on the presence of LKB1, as was the AICAR-induced increase in fatty-acid oxidation in EDL muscles in vitro, since these responses were not observed in KO mice. Likewise, the decrease in malonyl-CoA levels after muscle contraction was attenuated in KO gastrocnemius muscles, suggesting that LKB1 plays an important role in promoting the inhibition of ACC, likely by activation of AMPK. However, since ACC phosphorylation still increased and malonyl-CoA levels decreased in KO muscles (albeit not to the levels observed in WT mice), whereas AMPK phosphorylation was entirely unresponsive, LKB1/AMPK signaling cannot be considered the sole mechanism for inhibiting ACC during and after muscle activity. Regardless, our results suggest that LKB1 is an important regulator of malonyl-CoA levels and fatty acid oxidation in skeletal muscle.

Branvold DJ, Allred DR, Beckstead DJ, Kim HJ, Fillmore N, Condon BM, Brown JD, Sudweeks SN, Thomson DM, Winder WW. Thyroid hormone effects on LKB1, MO25, phospho-AMPK, phospho-CREB, and PGC-1␣ in rat muscle. J Appl Physiol 105: 1218-1227, 2008. First published July 31, 2008 doi:10.1152/japplphysiol.00997.2007.-Expression of all of the isoforms of the subunits of AMP-activated protein kinase (AMPK) and AMPK activity is increased in skeletal muscle of hyperthyroid rats. Activity of AMPK in skeletal muscle is regulated principally by the upstream kinase, LKB1. This experiment was designed to determine whether the increase in AMPK activity is accompanied by increased expression of the LKB1, along with binding partner proteins. LKB1, MO25, and downstream targets were determined in muscle extracts in control rats, in rats given 3 mg of thyroxine and 1 mg of triiodothyronine per kilogram chow for 4 wk, and in rats given 0.01% propylthiouracil (PTU; an inhibitor of thyroid hormone synthesis) in drinking water for 4 wk (hypothyroid group). LKB1 and MO25 increased in the soleus of thyroid hormone-treated rats vs. the controls. In other muscle types, LKB1 responses were variable, but MO25 increased in all. In soleus, MO25 mRNA increased with thyroid hormone treatment, and STRAD mRNA increased with PTU treatment. Phospho-AMPK and phospho-ACC were elevated in soleus and gastrocnemius of hyperthyroid rats. Thyroid hormone treatment also increased the amount of phosphocAMP response element binding protein (CREB) in the soleus, heart, and red quadriceps. Four proteins having CREB response elements (CRE) in promoter regions of their genes (peroxisome proliferatoractivated receptor-␥ coactivator-1␣, uncoupling protein 3, cytochrome c, and hexokinase II) were all increased in soleus in response to thyroid hormones. These data provide evidence that thyroid hormones increase soleus muscle LKB1 and MO25 content with subsequent activation of AMPK, phosphorylation of CREB, and expression of mitochondrial protein genes having CRE in their promoters.adenosine 5Ј-monophosphate-activated protein kinase; acetyl-coenzyme A carboxylase; muscle mitochondria EXPERIMENTAL HYPERTHYROIDISM has previously been demonstrated to increase mitochondrial enzyme content of rat skeletal muscle and other tissues (48, 49). Rat soleus muscle, consisting predominantly of type I, slow-twitch fibers, is much more sensitive than other muscles to this action of thyroid hormones (3, 48), possibly due to increased thyroid hormone receptor expression (3). This model has been used to study some of the basic molecular signaling pathways responsible for this effect of thyroid hormone excess on mitochondrial biogenesis (18,19,33). Hyperthyroidism is one of relatively few conditions that increases metabolic rate and increases expression of mitochondrial proteins in skeletal muscle. Previous studies suggest that the LKB1/AMP-activated protein kinase (AMPK) signaling pathway is important in mitochondrial biogenesis (47,53). In this present study, the adaptation of the LKB1...

the central goals of mechanobiology are to understand how cells generate force and how they respond to environmental mechanical stimuli. A full picture of these processes requires high-resolution, volumetric imaging with time-correlated force measurements. Here we present an instrument that combines an open-top, single-objective light sheet fluorescence microscope with an atomic force microscope (AfM), providing simultaneous volumetric imaging with high spatiotemporal resolution and high dynamic range force capability (10 pN-100 nN). With this system we have captured lysosome trafficking, vimentin nuclear caging, and actin dynamics on the order of one second per single-cell volume. to showcase the unique advantages of combining Line Bessel light sheet imaging with AfM, we measured the forces exerted by a macrophage during fcɣR-mediated phagocytosis while performing both sequential two-color, fixed plane and volumetric imaging of F-actin. This unique instrument allows for a myriad of novel studies investigating the coupling of cellular dynamics and mechanical forces. Cells interact mechanically with their environment by generating and responding to forces. A focus on the mechanical dynamics of cell phenomena such as motility, division and phagocytosis is essential for understanding stem cell fate 1 , cancer progression 2 and innate immunity 3. These mechanical processes are inherently three-dimensional (3D) and are regulated both by very local (nm) interactions as well as whole-cell scale (μm) biochemical and mechanical signaling. Obtaining a more complete picture of a cell's mechanical interaction with its environment requires monitoring local and global structures in 3D while simultaneously measuring associated forces. These goals necessitate integrating high spatial and temporal resolution volumetric imaging methods with high resolution force acquisition. Light sheet florescence microscopy (LSFM) enables acquisition of volumetric, multicolor time series at a high resolution and frame rate with low background fluorescence and low phototoxicity 4-6. Among single cell force methods 7 , Atomic Force Microscopy (AFM) 8 is unique in combining a large force range (10-11-10-6 N) that enables molecular-scale to tissue-level mechanics measurements, with high bandwidth temporal resolution (μs) and sub-nanometer spatial control. LSFM provides sufficient spatiotemporal resolution for studying cell-wide protein specific dynamics that AFM imaging cannot. Therefore, by using the AFM for force spectroscopy and LSFM for imaging we have exploited the strengths of each technique resulting in a system that is greater than its parts. Combining LSFM with AFM (AFM-LS) imposes significant geometrical constraints to the optical system design, and demands low vibration operation to accommodate sensitive force measurements. To address this challenge, we used a single-objective selective plane microscopy (soSPIM) technique integrated with, and time-synchronized to, an AFM. As described in a report on our first generation system 9 , we gene...