Glycine–alanine repeats impair proper substrate unfolding by the proteasome

Proteasome ATPases unravel folded proteins. Introducing a sequence containing only glycine and alanine residues (GAr) into substrates can impair their digestion. We previously proposed that a GAr interferes with the unfolding capacity of the proteasome, leading to partial degradation of products. Here we tested that idea in several ways. Stabilizing or destabilizing a folded domain within substrate proteins changed GAr-mediated intermediate production in the way predicted by the model. A downstream folded domain determined the sites of terminal proteolysis. The spacing between a GAr and a folded domain was critical for intermediate production. Intermediates containing a GAr did not remain associated with proteasomes, excluding models whereby retained GAr-containing proteins halt further processing. The following model is supported: a GAr positioned within the ATPase ring reduces the efficiency of coupling between nucleotide hydrolysis and work performed on the substrate. If this impairment takes place when unfolding must be initiated, insertion pauses and proteolysis is limited to the portion of the substrate that has already entered the catalytic chamber of the proteasome

Bio-inspired micropatterned hydrogel to direct and deconstruct hierarchical processing of geometry-force signals by human mesenchymal stem cells during smooth muscle cell differentiation

Micropatterned biomaterial-based hydrogel platforms allow the recapitulation of in vivo-like microstructural and biochemical features that are critical physiological regulators of stem cell development. Herein, we report the use of muscle mimicking geometries patterned on polyacrylamide hydrogels as an effective strategy to induce smooth muscle cell (SMC) differentiation of human mesenchymal stem cells (hMSCs). hMSCs were systemically coerced to elongate with varying aspect ratios (AR) (that is, 1:1, 5:1, 10:1 and 15:1) at a fixed projection area of ~7000 μm2. The results showed engineered cellular anisotropy with an intermediate AR 5:1 and AR 10:1, promoting the expression of alpha smooth muscle actin (α-SMA) and enhancement of contractile output. Further mechanistic studies indicated that a threshold cell traction force of ~3.5 μN was required for SMC differentiation. Beyond the critical cytoskeleton tension, hMSCs respond to higher intracellular architectural cues such as the stress fiber (SF) alignment, SF subtype expression and diphosphorylated myosin regulatory light-chain activity to promote the expression and incorporation of α-SMA to the SF scaffold. These findings underscore the importance of exploiting biomimetic geometrical cues as an effective strategy to guide hMSC differentiation and are expected to guide the rational design of advanced tissue-engineered vascular grafts.MOE (Min. of Education, S’pore)Published versio

Spatial distribution of cell–cell and cell–ECM adhesions regulates force balance while main­taining E-cadherin molecular tension in cell pairs

Mechanical linkage between cell–cell and cell–extracellular matrix (ECM) adhesions regulates cell shape changes during embryonic development and tissue homoeostasis. We examined how the force balance between cell–cell and cell–ECM adhesions changes with cell spread area and aspect ratio in pairs of MDCK cells. We used ECM micropatterning to drive different cytoskeleton strain energy states and cell-generated traction forces and used a Förster resonance energy transfer tension biosensor to ask whether changes in forces across cell–cell junctions correlated with E-cadherin molecular tension. We found that continuous peripheral ECM adhesions resulted in increased cell–cell and cell–ECM forces with increasing spread area. In contrast, confining ECM adhesions to the distal ends of cell–cell pairs resulted in shorter junction lengths and constant cell–cell forces. Of interest, each cell within a cell pair generated higher strain energies than isolated single cells of the same spread area. Surprisingly, E-cadherin molecular tension remained constant regardless of changes in cell–cell forces and was evenly distributed along cell–cell junctions independent of cell spread area and total traction forces. Taken together, our results showed that cell pairs maintained constant E-cadherin molecular tension and regulated total forces relative to cell spread area and shape but independently of total focal adhesion area