Uncovering the Roles and Evolved Sequence Grammar of Hypervariable Intrinsically Disordered Proteins in Bacterial Cell Division

Across all domains of life, a defining hallmark of the onset of cell division is the formation of a cytokinetic ring at the center of the cell. Cell division is a tightly controlled process that involves various regulatory factors that modulate the assembly of the cytokinetic ring. In rod-shaped bacteria, the ring is termed the Z-ring after the protein FtsZ, which is foundational to ring formation and is the bacterial homolog of tubulin. Like tubulin, FtsZ is an assembling GTPase, where GTP binding promotes the cooperative assembly into FtsZ polymers that laterally associate to form bundles. While the GTPase domain drives FtsZ polymerization, the formation of these higher-order structures requires domains outside the folded core. FtsZ has a bristled architecture, where a disordered tail, called the C-terminal tail (CTT), flanks the folded domain. The essentiality of the CTT was established through deletion experiments; however, the exact role that the CTT plays within the context of FtsZ function remained unclear. Here, we establish that the CTT, containing an intra-and intermolecular interaction motif (CTP) and a disordered linker (CTL), has a sticker-and-spacer architecture, where the CTL modulates the interactions of the CTP. We find that the modules of the CTT not only influence FtsZ assembly but also impact the catalytic efficiency of the GTPase domain. These findings add to recent findings that implicate disordered regions tethered to enzymes in auto-regulatory activities. The findings summarized above were obtained by focusing our investigations on the CTT of the FtsZ protein from B. subtilis (Bs-FtsZ). Is the stickers-and-spacers model applicable to understanding the functions of CTTs from other bacterial FtsZs? We analyzed the sequences of 1208 orthologous FtsZs, and the results show that while the CTPs and the core domains are reasonably well conserved, the CTLs are hypervariable across orthologs. The results of the sequence analysis have several implications: It might reflect a form of convergent evolution whereby different CTL sequences are interoperable with one other because different sequences serve the functionality of being spacers. Alternatively, the variation could be an example of divergent evolution, whereby changes to the CTLs engender different functionalities in different bacteria. Answering these questions will require methods to identify common sequence patterns across orthologous CTLs, and this cannot be achieved using traditional multiple sequence alignment approaches. Accordingly, we introduce computational methods that enable the quantitative analysis of conserved / distinct sequence-ensemble relationships across a family of IDRs. Additionally, we introduce a new method to uncover cryptic sequence patterns that define disordered regions as random versus non-random. These methods are shown to be applicable for high-throughput analysis of CTLs derived from different FtsZs. They are also effective in uncovering sequence patterns that are cryptic but conserved in intrinsically disordered regions (IDRs) from other bacterial proteins. Given the role of sequence-ensemble relationships and non-random motifs in IDP/IDR function, we hypothesized that these features might influence function and, therefore, might be encoded for within the amino acid sequence of the FtsZ CTL. This implies that designed CTL sequence variants that result in significant changes to these sequence features and to the ability of the CTL to function as a spacer could perturb function. To test this hypothesis, we developed scrambled sequence variants of the B. subtilis FtsZ CTL using the patterning of oppositely charged residues as a design parameter. Leveraging new methodologies, we found that the designed variants caused changes to the sequence-ensemble relationships, the non-random sequence patterning, and / or the spacer properties. Each variant was tested for complementary functions to wild type in vitro and in vivo. Indeed, deviations from wild type features had phenotypic impacts and / or influenced FtsZ assembly and activity, showing that the CTL is not a random disordered sequence but instead has specifically encoded sequence features that dictate function. As the global need to combat antibiotic-resistant infections continues to mount, studies that further understand the functions that IDRs contribute to essential bacterial processes such as cell division can be leveraged to create next-generation antibiotics

Comparison of Oxygen Flux in Hydrogel and Silicone Hydrogel Contact Lenses

The prevalence of contact lens use has been continuously growing for their convenience and for cosmetic reasons. Although contact lenses do offer many advantages over glasses, the major concern for many contact lens users is dryness that results from a lack of oxygen that goes through the contact lens to meet the demand of eye tissue. A new type of contact lens, made out of silicone hydrogel, has been introduced in the market which has garnered much attention from many contact users. The silicone hydrogel is different from the traditional hydrogel contact lens since oxygen is permeable through silicone, which was not possible through hydrogels. The hydrogel contact lenses must have high water content for oxygen delivery, silicone hydrogel contacts depends on their high oxygen diffusivity while having low water content. Night and day contact lenses are made out of silicone hydrogel whereas traditional ones for day use are often made out of hydrogel. A model was developed to validate the advantage of wearing silicone hydrogel contact lenses in both day and night conditions. By analyzing the center area of the eye around the pupil as a thin slab, the performance of these two types of contact lenses were compared by computing average oxygen concentrations in the stroma, which is the largest layer of cornea. Using COMSOL Multiphysics, the simplified geometry that included the layers of contact lens, tear, endothelium, and stroma was used as our model to find the oxygen concentration after eight hours of use either with eyes open or closed. The thickness of 80/mu m was used for both hydrogel and silicone hydrogel, the average oxygen concentration was found to be 9.100219x10-8mol/cm3 and 4.198608x10-8 mol/cm3 respectively for day setting with eyes open for eight hours and 3.536442x10-8 mol/cm3 and 2.119774x10-8 mol/cm3 respectively for night setting with eyes closed. Variations of other parameters in modeling also showed the same trend that silicone hydrogel contact lenses ended up with less oxygen in the cornea than hydrogel. Thus, the modeling showed how the silicone hydrogel did not offer any increase in oxygen delivery in both day and night settings

Information theoretic measures for quantifying sequence-ensemble relationships of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) contribute to a multitude of functions. De novo design of IDPs should open the door to modulating functions and phenotypes controlled by these systems. Recent design efforts have focused on compositional biases and specific sequence patterns as the design features. Analysis of the impact of these designs on sequence-function relationships indicates that individual sequence/compositional parameters are insufficient for describing sequence-function relationships in IDPs. To remedy this problem, we have developed information theoretic measures for sequence-ensemble relationships (SERs) of IDPs. These measures rely on prior availability of statistically robust conformational ensembles derived from all atom simulations. We show that the measures we have developed are useful for comparing sequence-ensemble relationships even when sequence is poorly conserved. Based on our results, we propose that de novo designs of IDPs, guided by knowledge of their SERs, should provide improved insights into their sequence-ensemble-function relationships

Global urban environmental change drives adaptation in white clover

Urbanization transforms environments in ways that alter biological evolution. We examined whether urban environmental change drives parallel evolution by sampling 110,019 white clover plants from 6169 populations in 160 cities globally. Plants were assayed for a Mendelian antiherbivore defense that also affects tolerance to abiotic stressors. Urban-rural gradients were associated with the evolution of clines in defense in 47% of cities throughout the world. Variation in the strength of clines was explained by environmental changes in drought stress and vegetation cover that varied among cities. Sequencing 2074 genomes from 26 cities revealed that the evolution of urban-rural clines was best explained by adaptive evolution, but the degree of parallel adaptation varied among cities. Our results demonstrate that urbanization leads to adaptation at a global scale

Role of cortactin homolog HS1 in transendothelial migration of natural killer cells.

Natural Killer (NK) cells perform many functions that depend on actin assembly, including adhesion, chemotaxis, lytic synapse assembly and cytolysis. HS1, the hematopoietic homolog of cortactin, binds to Arp2/3 complex and promotes actin assembly by helping to form and stabilize actin filament branches. We investigated the role of HS1 in transendothelial migration (TEM) by NK cells. Depletion of HS1 led to a decrease in the efficiency of TEM by NK cells, as measured by transwell assays with endothelial cell monolayers on porous filters. Transwell assays involve chemotaxis of NK cells across the filter, so to examine TEM more specifically, we imaged live-cell preparations and antibody-stained fixed preparations, with and without the chemoattractant SDF-1α. We found small to moderate effects of HS1 depletion on TEM, including whether the NK cells migrated via the transcellular or paracellular route. Expression of HS1 mutants indicated that phosphorylation of HS1 tyrosines at positions 222, 378 and 397 was required for rescue in the transwell assay, but HS1 mutations affecting interaction with Arp2/3 complex or SH3-domain ligands had no effect. The GEF Vav1, a ligand of HS1 phosphotyrosine, influenced NK cell transendothelial migration. HS1 and Vav1 also affected the speed of NK cells migrating across the surface of the endothelium. We conclude that HS1 has a role in transendothelial migration of NK cells and that HS1 tyrosine phosphorylation may signal through Vav1

Role of Vav1 in TEM by NK cells.

<p>A) Immunoblots with anti-HS1 and anti-Vav1 showing depletion of HS1 and Vav1 after 72 hrs of siRNA treatment. B) Decrease in TEM in transwell assay by NK cells treated with Vav1 siRNA, compared to control siRNA. Number of cells in the lower chamber, as a percentage of the mean of the control sample value on each day, with box and whisker plots. Boxes: 25th to 75th percentiles; whiskers: minimum and maximum values. N = 6. Asterisks indicate **P<0.005 (unpaired Student’s t-test). C) Left panel: Immunoblot with anti-HS1. The left lane shows the absence of HS1 in an anti-HS1 immunoprecipitate from a whole-cell lysate of NK cells treated with siRNA targeting HS1. The right lane shows the result for cells treated with control siRNA. Middle panel: Immunoblot with anti-Vav1. The left lane shows the presence of Vav1 protein in an anti-HS1 precipitate from a lysate of NK cells treated with control siRNA. The right lane shows the presence of Vav1 in the lysate. Right panel: Similar to the middle panel, except with a lysate from NK cells depleted for HS1.</p

Expression Rescue of HS1 Mutants in HS1-depleted NK Cells.

<p>A) Phosphorylation of HS1 Tyr397 in response to SDF-1α. Immunoblots probed with anti-Phospho-Tyr397 and anti-HS1. NK cells (5 x 10⁶) treated with SDF-1α (30 ng/mL) for the indicated times (min). B—D) Function of HS1 mutants in TEM by transwell assay. Number of cells in the lower chamber, as a percentage of the mean of the control sample value on each day, with box and whisker plots. Boxes: 25th to 75th percentiles; whiskers: minimum and maximum values. B) Mutations of phosphorylated tyrosine residues. Compared to control siRNA (blue), HS1 depletion by siRNA causes decreased TEM (red), and the defect is rescued by expression of wild-type HS1 (green) or siRNA-resistant wild-type HS1 (purple). Expression of siRNA-resistant forms of single-mutant HS1 Y378F (black), single-mutant HS1 Y397F (brown) or double-mutant HS1 Y378F Y397F (dark blue) does not rescue the defect, comparing their values to the value for siRNA-resistant wild-type (purple). Expression of siRNA-resistant HS1 Y222F (orange) rescues with a value that is slightly less, but not statistically significant, from that of siRNA-resistant wild type. Asterisks indicate *P>0.05, **P>0.005 (unpaired Student’s t-test, N = 6–9). C) Mutation of Arp2/3 complex binding site. Expression of siRNA-resistant HS1 with mutation of DDW residues to AAA (orange) rescues the defect, with no difference compared to siRNA-resistant wild-type HS1. N = 6 in each case. D) Mutation of SH3 domain at ligand-binding site. Expression of siRNA-resistant HS1 with the mutation W466K (orange) rescues the defect, with no difference compared to siRNA-resistant wild-type HS1. n = 6 in each case.</p

HS1 and TEM of NK cells in transwell assays.

<p>A) Diagram of transendothelial migration assay in a transwell device. B) Depletion of HS1 protein by siRNA, shown by immunoblot after 72 hrs. NK cells were treated with a pool of four siRNAs or one of the four. GAPDH is a loading control. C) Effects of HS1 knockdown on TEM. Plotted values are number of cells in the lower chamber, as a percentage of the mean of the control sample value on each day. Box-and-whisker plots (box: 25th to 75th percentiles, whiskers: min to max, middle line: median). Asterisks indicate statistical significance (*P<0.05. Unpaired Student’s t-test, n = 5 for each condition.) D) Fluorescence micrographs of NK cells, showing expression and co-localization of expressed HS1-tdTomato (red), F-actin (green, Alexa Fluor 488 phalloidin), and total HS1, including endogenous (blue, anti-HS1 staining). E) Fluorescence micrographs of NK cells stained with anti-HS1 to show siRNA-induced depletion of HS1 and expression of siRNA-resistant HS1 protein. F) Expression of siRNA-resistant HS1 in NK cells knocked down for HS1 with siRNA, shown by immunoblot with anti-HS1. Knockdown used a combination of HS1 siRNAs 2 and 3. G) Rescue of TEM phenotype in HS1-knockdown NK cells by expression of HS1. Cells as in panels E and F. Number of cells in the lower chamber, as a percentage of the mean of the control sample value on each day, with box-and-whisker plots as in panel C. Asterisks indicate statistical significance. (* P< 0.05, *** P < 0.0005. Unpaired Student’s t-test, N = 9–12 experiments for each condition.)</p

Migration Speeds for NK Cells.

<p>Speed (μm/min) defined as net displacement (distance start to finish) divided by time for control vs HS1-depleted cells. The distributions are not Gaussian, so the values listed are median, 95% confidence interval of the median and number of data points (N). P values from two non-parametric tests of significance are listed. Data include tracks for all cells in separate experiments on three different days.</p><p>Migration Speeds for NK Cells.</p

TEM events by NK cells on HDMVEC monolayers based on live-cell movie analysis.

<p>A) Endogenous HS1 (red) and F-actin (green) in NK cells migrating on the surface of HDMVEC monolayer observed by anti-HS1 and phalloidin fluorescence. Scale bar = 20 μm. B) DIC images from a movie (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118153#pone.0118153.s006" target="_blank">S2 Movie</a>), illustrating how the passage of the NK cell through the endothelial monolayer leaves a defect. Scale bar = 10 μm. C) Speed of cell migration, based on path length. Median and 95% confidence intervals are plotted. Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118153#pone.0118153.t004" target="_blank">Table 4-1. D</a>) Speed of cell migration, based on net displacement. Median and 95% confidence intervals are plotted. Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118153#pone.0118153.t004" target="_blank">Table 4-2. E</a>) Percentage of TEM events from movie analysis with SDF-1α. The number of TEM events as a percentage of the total number of NK cells on the surface of the endothelial monolayer in the first frame. Error bars are standard error of proportion. The difference is not statistically significant by z-test (p = 0.15) or by Fisher’s exact test for a 2 x 2 contingency table (p = 0.19). Data combined from two or three experiments per day on three days. F) Percentage of TEM events from movie analysis without SDF-1α. The number of TEM events as a percentage of the total number of NK cells on the surface of the endothelial monolayer in the first frame. Error bars are standard error of proportion. The differences between control and depleted-cell values are statistically significant with p values of 0.022, 0.024 and 0.001 for HS-depleted, Vav1-depleted and HS1+Vav1-depleted NK cells, based on chi-square tests with Yates’ correction. No other differences are statistically significant. Data combined from experiments on three days.</p