The distribution of RhoA and actomyosin at the epithelial zonula adherens.

<p>i. Scheme of coherent epithelial cells and the distribution of the active RhoA zone (green) at the apical adherens junctions (zonula adherens, ZA). ii. Diagram of the actomyosin cortex located on the cytoplasmic side of a cell. The ZA corresponds to a cortical zone enriched in NMIIA and RhoA. Note that RhoA is activated at the ZA, but this lipid-anchored molecule can potentially diffuse horizontally onto the apical surface of the cells as well as vertically down into the lateral cell-cell junctions. iii. Scheme of the top view of the RhoA zone from a group of cells (left) and its comparison with a spinning disk confocal image taken at the apical region of the cells expressing the GTP-RhoA reporter, GFP-AHPH. The inset in the right image corresponds to the same field of view seen by differential interference contrast microscopy (DIC) and shows that the analyzed field belongs to a confluent epithelial monolayer. Note also that the optical (X-Y) plane of the imaging captures the apical surface of the epithelium.</p

Space-time plot of the response to an external perturbation at <i>t</i> = 40, that decreases the stability of <i>NMIIA</i> by a 2-fold increase of its decay rate and at the same time the contractility of <i>NMIIA</i> is switched off (i.e. <i>Pe</i> = 0).

<p>The initial parameters are: <i>Pe</i> = 8, α = 0.02, κ₁ = κ₂ = 0.2, <i>K</i> = 1.</p

Space-time plots of the concentration field <i>RhoA(x</i>,<i>t)</i> and flow field <i>v(x</i>,<i>t)</i>: top row: <i>Pe</i> = 10, the front propagates with constant speed; bottom row: <i>Pe</i> = 12, stationary <i>RhoA</i> zone; the other parameters are: <i>α</i> = 1, <i>κ</i>_<i>1</i> = <i>κ</i>_<i>2</i> = 0.2, <i>K</i> = 1

<p>Space-time plots of the concentration field <i>RhoA(x</i>,<i>t)</i> and flow field <i>v(x</i>,<i>t)</i>: top row: <i>Pe</i> = 10, the front propagates with constant speed; bottom row: <i>Pe</i> = 12, stationary <i>RhoA</i> zone; the other parameters are: <i>α</i> = 1, <i>κ</i>_<i>1</i> = <i>κ</i>_<i>2</i> = 0.2, <i>K</i> = 1</p

Phase-portrait of the bistable spatially uniform system described by Eq (1).

<p>The steady states are at the intersections of the nullclines (red and yellow curves), the light green/blue curves show the stable/unstable manifolds of the unstable steady state, and the arrows show the direction and rate of change at different locations on the <i>RhoA-NMIIA</i> phase-plane. The parameters are: <i>α</i> = 1, <i>κ</i>_<i>1</i> = <i>κ</i>_<i>2</i> = 0.4, <i>n</i> = 4. For this choice of parameters the location of the unstable saddle point is closer to the low fixed point at origin.</p

The width of the contractile zone (with higher concentrations of <i>RhoA</i> and <i>NMIIA</i>), defined as the integral of the <i>RhoA</i> concentration over the whole domain, as a function of time, starting from a localized initial condition for a range of different contraction strengths (<i>Pe = 2</i> to 16).

<p>Note, that i) the slope (i.e. propagation speed) decreases as <i>Pe</i> is increased and there is a transition to a stationary contractile zone when <i>Pe</i> > 10. (<i>α</i> = 1, <i>κ</i>_<i>1</i> = <i>κ</i>_<i>2</i> = 0.2, <i>K</i> = 1); ii) the curves corresponding to Pe = 0–10 propagate with constant speed until they reach the end of the computational domain.</p

Characterization of protein isoform diversity in human umbilical vein endothelial cells via long-read proteogenomics

Endothelial cells (ECs) comprise the lumenal lining of all blood vessels and are critical for the functioning of the cardiovascular system. Their phenotypes can be modulated by alternative splicing of RNA to produce distinct protein isoforms. To characterize the RNA and protein isoform landscape within ECs, we applied a long read proteogenomics approach to analyse human umbilical vein endothelial cells (HUVECs). Transcripts delineated from PacBio sequencing serve as the basis for a sample-specific protein database used for downstream mass-spectrometry (MS) analysis to infer protein isoform expression. We detected 53,863 transcript isoforms from 10,426 genes, with 22,195 of those transcripts being novel. Furthermore, the predominant isoform in HUVECs does not correspond with the accepted “reference isoform” 25% of the time, with vascular pathway-related genes among this group. We found 2,597 protein isoforms supported through unique peptides, with an additional 2,280 isoforms nominated upon incorporation of long-read transcript evidence. We characterized a novel alternative acceptor for endothelial-related gene CDH5, suggesting potential changes in its associated signalling pathways. Finally, we identified novel protein isoforms arising from a diversity of RNA splicing mechanisms supported by uniquely mapped novel peptides. Our results represent a high-resolution atlas of known and novel isoforms of potential relevance to endothelial phenotypes and function.</p