Biology subjects

Raghunathan, V.

Publications and source records attributed to Raghunathan, V..

3 recordsLinked to original sources

bioRxiv2021

Extracellular matrix stiffness and TGFβ2 regulate YAP/TAZ activity in human trabecular meshwork cells

Primary open-angle glaucoma progression is associated with increased human trabecular meshwork (HTM) stiffness and elevated transforming growth factor beta 2 (TGF{beta}2) levels in aqueous humor. Increased transcriptional activity of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), central players in mechanotransduction, are implicated in glaucomatous HTM (GTM) cell dysfunction. Yet, the detailed mechanisms underlying YAP/TAZ modulation in HTM cells in response to alterations in extracellular matrix (ECM) stiffness and TGF{beta}2 levels are not well understood. Using biomimetic ECM hydrogels with tunable stiffness, here we show that increased ECM stiffness elevates YAP/TAZ transcriptional activity potentially through modulating focal adhesions and cytoskeletal rearrangement. Furthermore, TGF{beta}2 increased YAP/TAZ nuclear localization in both normal and glaucomatous HTM cells, which was prevented by inhibiting extracellular-signal-regulated kinase and Rho-associated kinase signaling pathways. Filamentous (F)-actin depolymerization reversed TGF{beta}2-induced YAP/TAZ nuclear localization. YAP/TAZ depletion using siRNA or verteporfin decreased focal adhesions, ECM remodeling and cell contractile properties. Similarly, YAP/TAZ inactivation with verteporfin partially blocked TGF{beta}2-induced HTM/GTM hydrogel contraction and stiffening. Collectively, our data provide evidence for a pathologic role of aberrant YAP/TAZ signaling in glaucomatous HTM cell dysfunction, and may help inform strategies for the development of novel multifactorial approaches to prevent progressive ocular hypertension in glaucoma.

cell biology↗

bioRxiv2020

Macrophage aggresome-like induced structures are flexible organizing platforms for immune signaling

Macrophages adopt a pro-inflammatory phenotype in response to environmental challenges in a process that often coincides with the formation of transient cytosolic p62/SQSTM1 inclusions containing ubiquitinated proteins in structures known as aggresome-like induced structures (ALIS). Although described as stress-induced inclusions that accumulate aggregated proteins, little direct evidence supports their hypothesized structural role in the context of immune stimulation. Here, we showed that these structures in primary macrophages are induced by multiple microbialbased ligands, including exposure to cytosolic double-stranded DNA. Rather than accumulating aggregated proteins, we observed that ubiquitinated proteins form a ring-shaped structure around the perimeter of these circular foci. We identified that different microbial stimuli induced the formation of ubiquitin-positive foci with distinct characteristics and we observed selective recruitment of context-dependent immune regulators. Our findings are consistent with a model where these ubiquitin-containing structures act as adaptable organizing centers for innate immune signaling. SUMMARYCharbonneau et al. demonstrate that ubiquitin- and p62-containing cytosolic ring-shaped structures induced by bacterial infections, microbial ligands and cytosolic double-stranded DNA contain context-dependent immune regulators, revealing an important insight on the cellular architecture required to coordinate signal transduction in macrophage.

immunology↗

bioRxiv2020

Differential levels of dermatan sulfate generate distinct Collagen I gel architectures

Collagen I is the most abundant extracellular matrix (ECM) protein in vertebrate tissues. As an endogenously synthesized fibrillar biopolymer or as a synthetic hydrogel, it provides mechanical durability to tissue microenvironments and regulates cell function. Predominant regulators of its fibrillogenesis are dermatan sulfate proteoglycans (DSPGs), proteins conjugated with iduronic acid containing DS glycosaminoglycans (GAGs). Although DS is known to regulate tissue function through its modulation of Collagen I architecture, a precise quantifiable understanding of the latter remains elusive. We investigate this problem by visualizing the pattern of structural elements within fixed Collagen I gels polymerized in the presence of varying concentrations of DS (50-, 200- and 400- g/mL) using second harmonic generation microscopy (SHG). Measuring four independent imaging parameters: fibril density, mean SHG signal (which estimates the ordering of the fibrils), surface occupancy (which estimates the space occupied by fibrils), and the fibril width allows us to construct an informative model of the effects of DS on Collagen I element architecture. Supported by confocal microscopy, our observations indicate that the effect on collagen fibril pattern of DS is contextual upon its concentrations. Lower levels of DS result in more numerous disorganized fibrils; higher levels restore organization, but at lower fibril densities. Such Collagen I gel pattern-tuning of DS is likely of relevance for understanding its functions in disease progression and biomaterial applications.

physiology↗