TRPV4 channel regulates both TGFbeta1 and matrix stiffness-induced normal human dermal myofibroblast differentiation

Presenter: Shweta Sharma                          Status: Faculty

Authors: S. Sharma, R. Goswami, M. Merth, K. Y. Lei, S. O. Rahaman


Abstract: TRPV4 channel regulates both TGFbeta1 and matrix stiffness-induced normal human dermal myofibroblast differentiation     S. Sharma1, R. Goswami1, M. Merth1, K. Y. Lei1, S. O. Rahaman1*    1University of Maryland, Department of Nutrition and Food Science, College Park, MD 20742    *Corresponding author     Scleroderma is a multisystem connective tissue disease with no effective medical treatment. Myofibroblasts are critical to the fibrogenic tissue repair process in the skin and many internal organs. Emerging data support a role for both a mechanical signal, e.g., matrix stiffness, and a biochemical signal, e.g., transforming growth factor beta1 (TGFbeta1), in myofibroblast differentiation. However, the mechanisms by which crosstalk between mechanical and biochemical signal promotes fibrogenesis are still elusive. Transient receptor potential vanilloid 4 (TRPV4) is a stretch-activated calcium channel activated by both mechanical and biochemical stimuli. We investigated if TRPV4-initiated signals play a role in TGFbeta1 or matrix stiffness-induced differentiation of normal human dermal fibroblasts (HDF). We demonstrate that TRPV4 channels are expressed and functional in HDF. Importantly, TRPV4 activity (agonist-induced calcium influx) is induced 2 fold in myofibroblasts, under condition of increasing matrix stiffness in the pathophysiological range (1-25 kPa), or in HDF pretreated with TGbeta1. Furthermore, TGFbeta1 induces expression of TRPV4 proteins in a dose-dependent manner. Importantly, TRPV4 antagonism by a selective small molecule antagonist abrogates calcium influx and both TGFbeta1 and matrix stiffness-induced myofibroblast differentiation as assessed by i) alpha-smooth muscle actin (SMA) expression/incorporation into stress fibers, and ii) generation of polymerized actin (F-actin). Mechanistically, we demonstrate that TRPV4 channel inhibition abrogates TGFbeta1-induced activation of transcription factors, Smad2/3. Taken altogether, these data identify a novel reciprocal functional link between TRPV4 activation and TGFbeta1 signals regulating dermal myofibroblast differentiation. Successful manipulation of the TRPV4 activity may be a targeted therapeutic approach to the treatment of scleroderma.     Funding: AHA (13SDG17310007), Startup and MAES grant from University of Maryland 


Collective cell migration over long time scales reveals distinct phenotypes

Presenter: Rachel Lee                                   Status: Graduate Student

Authors: Rachel M. Lee, Christina H. Stuelten, Carole A. Parent, and Wolfgang Losert


Abstract: Migratory phenotypes of metastasizing tumor cells include single and collective cell migration. While it is commonly acknowledged that migration of tumor cells differs from that of normal epithelial cells, our understanding of collective cell migration, as well as the connection between individual cell properties and the collective behavior of cell groups, is incomplete.  We investigate migration in the context of a cancer progression model to determine how perturbation of cell-cell adhesions, specifically reduced E-cadherin expression, affects the collective migration phenotype.  Time lapse imaging of epithelial sheets and particle image velocimetry (PIV) are used to quantitatively study the dynamics of cell motion over ten hours.  We find that non-malignant MCF10A cells are distinguished from malignant MCF10CA1a cells by both their short term and long term dynamics. Short termdynamics distinguish non-malignant E-cadherin knockdown cells from the control, butlong term dynamics such as increasing spatial correlations remain unchanged.  Collective behavior in epithelial sheets includes long term dynamics that cannot be captured by short term metrics such as instantaneous speed or directionality. The use of metrics incorporating migration data over long times allows us to distinguish features of collective migration that are and are not effected by E-cadherin, a clinically relevant adhesion molecule. These tools will allow future studies to investigate which adhesion components and time scales are most predictive of malignant behavior, which may lead to improved cancer diagnosis.


Mechanisms of T Lymphocyte Activation Revealed by Super Resolution Microscopy

Presenter: Leonard Campanello                             Status: Graduate Student

Authors: Leonard Campanello, Wolfgang Losert


Abstract: Tight regulatory control of lymphocyte activation is necessary to avoid the deleterious consequences of an uncontrolled immune response. An intricate web of positive and negative regulation governs a key activation pathway in lymphocytes, the antigen-receptor-to-NF-κB pathway, particularly at a crucial signal transduction step mediated by a complex intracellular signaling structure called the POLKADOTS signalosome. However, the interplay between positive and negative control in this structure is currently poorly understood. We have utilized cutting-edge super-resolution imaging technologies combined with quantitative imaging analysis to examine the spatial organization of the POLKADOTS signalosome. Our preliminary results suggest that autophagosomes, small degradative organelles, localize preferentially to the ends of the filamentous POLKADOTS structure. Quantitative analyses of fixed cells images lend support to these initial imaging observations. Together, these results provide new insights into the complex regulatory processes which govern T lymphocyte activation.


Structural and Dynamic Analysis of Disordered H4 Histone Tail by Modified AWSEM-MD

Presenter: Hao                                  Status: Graduate Student

Authors: Hao Wu, Garegin Papoian


Abstract: DNA compaction in eukaryotic cells is mediated by positively charged histone protein octamers. The latter consists of well folded core segments, that come together to form a central cylinder, and flexible tails, protruding out from the surrounding DNA. Despite being disordered, histone tails play an important role in bridging interactions between neighboring nucleosomes, regulating chromatin folding structure and dynamics. Hence, it is desirable to develop coarse-grained, yet accurate models of histone tails, such that subsequent nucleosomal and polynucleosomal simulation time scales could be physiologically significant. To achieve this goal, we added new interactions to the coarse-grained force field AWSEM-MD, which is typically used for folding of globular proteins or binding studies. 



Characterization of hinge mutants in E. coli mechanosensitive channel of small conductance (MscS)

Presenter: Stephanie Sansbury                              Status: Undergraduate Student

Authors: Stephanie Sansbury, Ugur Cetiner, Ian Rowe, Abigail Cember, Sergei Sukharev


Abstract: Mechanosensitive channels (MS) allow E. coli and non-model bacterial species to survive otherwise lethal changes in osmolarity. The rapid influx of water associated with hypo-osmotic conditions leads to elevated turgor pressure complicit with increased tension across the membrane. The subsequent mechanical stress opens the MS channels embedded in the membrane, relieving tension and preventing cell lysis.  However, the behavior of E. coli MscS (mechanosensitive channel of small conductance) and other mechanosensitive channels is more dynamic than existing in either the closed or open state: electrophysiological characterization reveals complex behaviors including inactivation and recovery profiles, allowing resolution of channel function with greater granularity.  To elaborate on the interplay of channel structure and function, mutants of the homoheptamer MscS were generated and analyzed in E. coli spheroplasts via patch-clamp experiments. Each of these mutants exhibits substitutions along the transmembrane alpha-helix lining the channel pore that effectively relocate the hinges responsible for opening and inactivation. These studies provide insight into the precise residues responsible for elements of channel behavior and suggest the significance of sequence homology between MS channels at those loci. 


Characterizing the Effects of Phosphorylation on the Structural Dynamics of Ubiquitin

Presenter: Yaniv Kazansky                          Status: Undergraduate Student

Authors: Yaniv Kazansky, David Fushman


Abstract: Ubiquitination, the covalent attachment of ubiquitin to a substrate protein, is a key post-translational modification that, among other outcomes, marks eukaryotic proteins for degradation. The ubiquitin ligase parkin has been implicated in mitochondrial quality control, with a role in tagging many outer mitochondrial substrates for degradation. Parkin is autoinhibited in its native state, and phosphorylation of both parkin and ubiquitin by PINK1 is necessary for parkin activation. Defects in the PINK1-parkin pathway have been linked to forms of early-onset Parkinson’s Disease. Recent findings have revealed ubiquitin phosphorylation by PINK1 to result in significant conformational changes to ubiquitin; however the nature and full implications of those changes are not yet clear. In particular, ubiquitin phosphorylation appears to result in a novel conformation that is in slow exchange with the native conformation. Using nuclear magnetic resonance (NMR) spectroscopy, we have observed striking dynamic effects in phospho-ubiquitin.  We tested the ability of phospho-ubiquitin to bind to common ubiquitin-binding proteins. We found that phospho-ubiquitin binds the UBA domain of ubiquilin-1 with an affinity comparable to that of wild-type ubiquitin. However, binding to a ligand appears to lock phospho-ubiquitin into its native conformation, suggesting that the novel conformation is binding-incompetent. We also found that lowering the pH of the sample buffer resulted in a disappearance of the NMR signals from the novel conformation. These results provide structural insights into how the novel conformation of ubiquitin arises, as the charge of the attached phosphate group may regulate the two conformations. Next, we examined the effects of phosphorylation on Lys48-linked poly-ubiquitin chains. We found that phosphorylation of a single ubiquitin unit in di-ubiquitin results in significant chemical shift perturbations and even novel signals in the non-phosphorylated ubiquitin unit, suggesting a possibility that phospho-ubiquitin impacts the conformational states and dynamics of the chain or of its chain partner. 



Structural Characterization of K6/K63-linked Non-Canonical Ubiquitin Chains Using NMR

Presenter: Meredith Miller                          Status: Undergraduate Student

Authors: Meredith Miller, David Fushman


Abstract: The small protein ubiquitin (Ub) plays a vital role in all eukaryotic cells as a post-translational modifier of proteins. Understanding the structure and recognition mechanisms of Ub chains of different linkages and lengths is necessary in order to elucidate their diverse range of functions. Research has shown that Ub chains linked through lysine residues K6 and K63 bind to the BRCA1 DNA repair complex via ubiquitin interacting motifs (UIM). Experimental data indicate that Ub chains containing K6 and K63 linkages are necessary for co-localization of the BRCA1 complex at sites of DNA damage; however, the structural features and molecular recognition mechanisms of these signals are poorly understood. In order to address this deficiency in our knowledge, a series of non-canonical Ub trimers containing these specific linkages were assembled enzymatically, and their structural properties were examined using nuclear magnetic resonance (NMR) spectroscopy. The results showed no significant changes to the interdomain interfaces within the mixed-linkage unbranched Ub trimers in comparison to the respective K6-linked and K63-linked Ub dimers. Branched Ub trimers containing K6 and K63 linkages are being assembled, and any resulting structural variations will be evaluated. Future research will focus on receptor-recognition properties of these mixed-linkage Ub trimers.