Molecular Determinants of Tubulin’s C‑Terminal Tail Conformational Ensemble

Tubulin is important for a wide variety of cellular processes including cell division, ciliogenesis, and intracellular trafficking. To perform these diverse functions, tubulin is regulated by post-translational modifications (PTM), primarily at the C-terminal tails of both the α- and β-tubulin heterodimer subunits. The tubulin C-terminal tails are disordered segments that are predicted to extend from the ordered tubulin body and may regulate both intrinsic properties of microtubules and the binding of microtubule associated proteins (MAP). It is not understood how either interactions with the ordered tubulin body or PTM affect tubulin’s C-terminal tails. To probe these questions, we developed a method to isotopically label tubulin for C-terminal tail structural studies by NMR. The conformational changes of the tubulin tails as a result of both proximity to the ordered tubulin body and modification by mono- and polyglycine PTM were determined. The C-terminal tails of the tubulin dimer are fully disordered and, in contrast with prior simulation predictions, exhibit a propensity for β-sheet conformations. The C-terminal tails display significant chemical shift differences as compared to isolated peptides of the same sequence, indicating that the tubulin C-terminal tails interact with the ordered tubulin body. Although mono- and polyglycylation affect the chemical shift of adjacent residues, the conformation of the C-terminal tail appears insensitive to the length of polyglycine chains. Our studies provide important insights into how the essential disordered domains of tubulin function

Extracellular Vesicles Secreted from Cancer Cell Lines Stimulate Secretion of MMP-9, IL-6, TGF-β1 and EMMPRIN

<div><p>Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-β1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles.</p></div

IL-6 is secreted upon stimulation with EVs in THP-1 cells.

<p>IL-6 secretion in THP-1 cells was measured with an ELISA assay upon stimulation with EV^MCF-7, EV^MDA, EV^U937 and EV^L.6pL. The EVs secrete low levels of IL-6 that did not contribute significantly to the overall observed secreted IL-6 levels.</p

Mass Spectrometry identification of EV proteins.

*<p>Protein identification was performed using 99% confidence threshold.</p>†<p>Protein identification required at least two unique peptides.</p

EMMPRIN is secreted upon stimulation with EVs.

<p>Western blot analysis of THP-1 cells shows the change in EMMPRIN secretion with and without stimulation with EV^MCF-7 and EV^MDA.</p

TGF-β1 is secreted upon stimulation with EVs in THP-1 cells.

<p>TGF-β1 secretion in THP-1 cells was measured with an ELISA assay upon stimulation with EV^MCF-7, EV^MDA, EV^U937 and EV^L.6pL. Most purified EVs secrete low levels of TGF-β1 that did not contribute significantly to the overall observed secreted TGF-β1 levels. Interestingly, EV^U937 alone comprised high levels of TGF-β1 that were reduced upon incubation with THP-1 cells, potentially suggesting internalization of TGF-β1 that is well known <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071225#pone.0071225-Zwaagstra1" target="_blank">[36]</a>.</p

MMP-9 is secreted upon stimulation with EVs in THP-1 cells.

<p>MMP-9 secretion in THP-1 cells was measured with an ELISA assay upon stimulation with EV^MCF-7, EV^MDA, EV^U937 and EV^L.6pL. The EVs themselves secrete low levels of MMP-9 that did not contribute significantly to the overall observed secreted MMP-9 levels.</p

EVs are internalized into THP-1 cells.

<p>Fluorescence microscopy images of THP-1 cells treated with Texas red stained EVs. Left panel - THP-1 cell stained with EMMPRIN FITC and DAPI. The red signal is the auto-fluorescence signal of the cell. Middle panel - THP-1 cell treated with the Texas Red stain alone. The signal observed is the same as in the left image and corresponds to the Texas red background and cell auto-fluorescence signal. Right panel - THP-1 cells treated with Texas Red stained EVs and visualized after a 5-minute incubation period. The cell membrane is stained with EMMPRIN-FITC antibody, the EVs are shown in red and the DAPI stained nucleus is shown in blue. The reference bar is 10 µm.</p

Purification of secreted EVs.

<p>A) Size exclusion chromatography elution profile of the purification of conditioned media to isolate EVs. The red box corresponds to the elution peak for the purified EVs. B) EMMPRIN is secreted via EVs in all cell lines tested as shown by the Western blot probed for EMMPRIN in the vesicle fractions purified using size exclusion chromatography. C) NanoParticle Tracking analysis detects EVs in the purified sample and validates our size exclusion method to purify EVs from conditioned media. D) Electron microscopy was used to visualize the secreted EVs. Data shown is for EV^MDA.</p

Supplemental Material for Baschal et al., 2018

<p>Idiopathic scoliosis (IS) is a complex genetic disease of unknown etiology. We completed exome sequencing for five IS families and performed GO term enrichment analyses on the resulting variant lists. Overall, we identified enriched categories in our affected families that include stereocilia and other actin-based cellular projections, cilia and other microtubule-based cellular projections, and the extracellular matrix (ECM). Our results suggest that there are multiple paths to IS and provide a foundation for future studies of IS pathogenesis.</p><p></p