Effect of heterogeneous substrate adhesivity of follower cells on speed and tension profile of leader cells in primary keratocyte collective cell migration

In single keratocyte motility, membrane tension is reported to be high at cell-fronts and believed to establish front coherence. To understand role of membrane mechanics in collective cell migration, we study membrane height fluctuations in cell sheets from fish scales using interference reflection microscopy (IRM). We report the monolayer to have cells lacking substrate adhesion and show that such ‘non-sticky’ cells can form bridges between leader cells and far-away follower cells. Do such interactions alter motility and membrane mechanics in such leaders? We find non-significant, but reduced speed for leaders with ‘non-sticky’ followers in comparison to other leaders. Cells show high phenotypic variability in their membrane fluctuation tension profiles. On average, this tension is found to be lower at cell fronts than the mid-section. However, leaders with non-sticky followers are more prone to display higher tension at their front and have a negative correlation between cell speed and front-mid tension difference. Thus, we conclude that intracellular tension gradients are heterogeneous in cell sheets and substrate adhesivity of followers can control the coupling of the gradient to cell speed

Collaborative Enhancement of Antibody Binding to Distinct PECAM-1 Epitopes Modulates Endothelial Targeting

Antibodies to platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitate targeted drug delivery to endothelial cells by “vascular immunotargeting.” To define the targeting quantitatively, we investigated the endothelial binding of monoclonal antibodies (mAbs) to extracellular epitopes of PECAM-1. Surprisingly, we have found in human and mouse cell culture models that the endothelial binding of PECAM-directed mAbs and scFv therapeutic fusion protein is increased by co-administration of a paired mAb directed to an adjacent, yet distinct PECAM-1 epitope. This results in significant enhancement of functional activity of a PECAM-1-targeted scFv-thrombomodulin fusion protein generating therapeutic activated Protein C. The “collaborative enhancement” of mAb binding is affirmed in vivo, as manifested by enhanced pulmonary accumulation of intravenously administered radiolabeled PECAM-1 mAb when co-injected with an unlabeled paired mAb in mice. This is the first demonstration of a positive modulatory effect of endothelial binding and vascular immunotargeting provided by the simultaneous binding a paired mAb to adjacent distinct epitopes. The “collaborative enhancement” phenomenon provides a novel paradigm for optimizing the endothelial-targeted delivery of therapeutic agents

SPE-44 Implements Sperm Cell Fate

The sperm/oocyte decision in the hermaphrodite germline of Caenorhabditis elegans provides a powerful model for the characterization of stem cell fate specification and differentiation. The germline sex determination program that governs gamete fate has been well studied, but direct mediators of cell-type-specific transcription are largely unknown. We report the identification of spe-44 as a critical regulator of sperm gene expression. Deletion of spe-44 causes sperm-specific defects in cytokinesis, cell cycle progression, and organelle assembly resulting in sterility. Expression of spe-44 correlates precisely with spermatogenesis and is regulated by the germline sex determination pathway. spe-44 is required for the appropriate expression of several hundred sperm-enriched genes. The SPE-44 protein is restricted to the sperm-producing germline, where it localizes to the autosomes (which contain sperm genes) but is excluded from the transcriptionally silent X chromosome (which does not). The orthologous gene in other Caenorhabditis species is similarly expressed in a sex-biased manner, and the protein likewise exhibits autosome-specific localization in developing sperm, strongly suggestive of an evolutionarily conserved role in sperm gene expression. Our analysis represents the first identification of a transcriptional regulator whose primary function is the control of gamete-type-specific transcription in this system

Detection of microorganisms using biosensors-a smarter way towards detection techniques.

Along with useful microorganisms, there are some that cause potential damage to the animals and plants. Detection and identification of these harmful organisms in a cost and time effective way is a challenge for the researchers. The future of detection methods for microorganisms shall be guided by biosensor, which has already contributed enormously in sensing and detection technology. Here, we aim to review the use of various biosensors, developed by integrating the biological and physicochemical/mechanical properties (of tranducers), which can have enormous implication in healthcare, food, agriculture and biodefence. We have also highlighted the ways to improve the functioning of the biosensor

Monoclonal antibody (mAb) ligands recognizing distinct extracellular epitopes of PECAM-1.

<p>(<b>A</b>) MAbs investigated in this study to probe the affinity and accessibility to distinct epitopes of human PECAM-1 (huPECAM-1; mAbs 62 and 37) and mouse PECAM-1 (muPECAM-1; mAbs 390 and MEC13.3). Listed is the effect of various anti-PECAM-1 mAbs on PECAM-1-dependent homophilic adhesion, as defined by the aggregation of L-cells fibroblast transfectants expressing PECAM-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#pone.0034958-Nakada1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#pone.0034958-Yan3" target="_blank">[50]</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#pone.0034958-Yan1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#pone.0034958-Nakada1" target="_blank">[22]</a>. (<b>B</b>–<b>C</b>) Diagram of immunoreactive regions within PECAM-1 domains 1 and 2. (<b>B</b>) Amino acid (AA) location of distinct non-overlapping epitopes for binding of mAbs 62 and 37 on Ig-domain 1 (IgD1) of huPECAM-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#pone.0034958-Nakada1" target="_blank">[22]</a>. (<b>C</b>) AA location of epitopes for mAbs 390 and MEC13.3 on Ig-domain 2 (IgD2) of muPECAM-1 (H. DeLisser, unpublished results). Peptide sequence recognized by mAbs are colored in red.</p

<i>In vitro</i> binding properties of mAb to live cells expressing PECAM-1.

<p>Cell surface binding of mAbs to PECAM-1 was determined by ELISA-based method with (<b>A</b>) HUVECs, (<b>B</b>) REN-muP cells. Proteins were added to confluent cellular monolayers at the indicated dilutions and incubated for 2 h at 4°C. The results shown are from a representative experiment. Non-targeted IgG or non-PECAM-1 expressing cells were used as negative control. Representative plots for mAb binding to MS1 cells are available in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#pone.0034958.s002" target="_blank">Figure S2</a></b>. (<b>C</b>) Analysis of the relative binding affinity of anti-PECAM-1 mAbs, when binding to cells is half-maximal (IC₅₀). Data points were fit as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#s4" target="_blank">Methods</a>.” The IC₅₀ is reported as the mean IC₅₀ value ± SD of three independent experiments performed in triplicate.</p

Binding parameters of anti-PECAM-1 [¹²⁵I]-mAbs to live cells expressing PECAM-1.

<p>Cell surface binding parameters (K_d and B_max) of [¹²⁵I]-mAbs to PECAM-1 was determined by RIA-based method with (<b>A</b>) native huPECAM-1 on HUVECs, and (<b>B</b>) recombinant muPECAM-1 on REN-muP cells. Serial dilutions of [¹²⁵I]-mAbs were added to confluent cellular monolayers and incubated for 2 h at 4°C. The results shown are from a representative experiment, with the inset showing Scatchard plot of binding data. Note that total binding was corrected for NSB using 100−fold excess of unlabeled mAb for HUVECs or using parent REN cells for REN-muP binding. (<b>C</b>–<b>D</b>) K_d and B_max Binding parameters are for [¹²⁵I]-mAbs to huPECAM-1 and muPECAM-1 are listed. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#s2" target="_blank">Results</a> were determined by three independent RIA experiments performed in quadruplicate, with data expressed as mean ± SD.</p

In vitro enhancement of binding, accessibility and therapeutic output of anti-PECAM-1 390 scFv-TM fusion protein <i>via</i> dual epitope-engagement of muPECAM-1.

<p>(<b>A</b>) Cell surface binding of the therapeutic fusion protein 390 scFv-TM to REN-muP cells was assessed in the presence of 200 nM self-paired parental mAb 390 or paired mAb MEC13.3 by ELISA. The curves shown are representative ELISA. Only binding to REN-muP cells shown; there was no significant binding detected using control REN cells lacking muPECAM-1. Binding affinity of 390 scFv-TM, reflected by IC₅₀, increases 3.8−fold when paired with MEC13.3. The IC₅₀ is reported as the mean IC₅₀ value ± SD of three independent experiments performed in triplicate. (<b>B</b>) Generation of activated protein c (APC), a cell-protective species, on the surface of REN-muP cells is initiated by targeted binding of 390 scFv-TM (+thrombin). APC generation is augmented up to 5−fold when 390 scFv-TM binding is enhanced with paired mAb MEC13.3 compared to 390 scFv-TM alone. (<b>C</b>) Co-IP of the MEC13.3/muPECAM-1/390 scFv-TM-FLAG complex in REN-muP cells. REN-muP cells were treated with muPECAM-1 targeted rat anti-mouse IgG MEC13.3 and anti-mouse 390 scFv-TM-FLAG combinations. Cell lysates were immunoprecipitated with Protein G agarose beads to MEC13.3 and analyzed by SDS-PAGE and immunoblotting (IB) using anti-muPECAM-1, anti-FLAG, and rat polyclonal anti-mouse antibodies, as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#s4" target="_blank">Methods</a>.” For controls, REN-muP cells ±390 scFv-TM FLAG were incubated with Protein G beads alone (lanes 1 and 5, 3 and 7). 390 scFv-TM-FLAG was only detected in the IP for REN-muP cells co-treated with MEC13.3 and 390 scFv-TM-FLAG (lane 6), indicating an interaction between MEC13.3 and 390 scFv-TM through muPECAM-1. Data are representative of two independent experiments.</p

Anti-PECAM-1 [¹²⁵I]-mAb binding in live cells is enhanced by paired mAb directed to adjacent PECAM-1 epitope.

<p>The modulation of PECAM-1 binding was determined by co-incubation of [¹²⁵I]-mAb with indicated concentrations of unlabeled self-paired mAb or paired mAb with cells for 2 h at 4°C. Binding data were plotted as [¹²⁵I]-mAb molecules bound per cell (mAb/cell) and data points were fit as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#s4" target="_blank">Methods</a>.” (<b>A</b> and <b>B</b>) Unlabeled mAb 62 competitively inhibits binding of [¹²⁵I]-mAb 62 to huPECAM-1 in HUVEC. However, mAb 37 enhances [¹²⁵I]-mAb 62 binding to huPECAM-1 in HUVEC by 1.5−fold over binding of [¹²⁵I]-mAb 62 alone. Interestingly, mAb 62 does not enhance the binding of [¹²⁵I]-mAb 37 (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034958#pone.0034958.s003" target="_blank">Figure S3</a></b>). (<b>C</b>–<b>D</b>) Collaborative binding studies of mAbs 390 and MEC13.3 with REN-muP cells as described in panel A. Unlabeled self-paired mAb 390 and mAb MEC13.3 competitively inhibit binding of [¹²⁵I]-mAb390 and [¹²⁵I]-mAb MEC13.3 to REN-muP cells, respectively. In contrast, mAb pairs [¹²⁵I]-mAb 390/MEC13.3 and [¹²⁵I]-mAb MEC13.3/390 enhance binding by ∼1.5−fold and ∼2.7−fold, respectively, over [¹²⁵I]-mAb alone (***, P<0.001, <i>n</i> = 3–4).</p

Collaborative Enhancement of Endothelial Targeting of Nanocarriers by Modulating Platelet-Endothelial Cell Adhesion Molecule-1/CD31 Epitope Engagement

Nanocarriers (NCs) coated with antibodies (Abs) to extracellular epitopes of the transmembrane glycoprotein PECAM (platelet endothelial cell adhesion molecule-1/CD31) enable targeted drug delivery to vascular endothelial cells. Recent studies revealed that paired Abs directed to adjacent, yet distinct epitopes of PECAM stimulate each other’s binding to endothelial cells <i>in vitro</i> and <i>in vivo</i> (“collaborative enhancement”). This phenomenon improves targeting of therapeutic fusion proteins, yet its potential role in targeting multivalent NCs has not been addressed. Herein, we studied the effects of Ab-mediated collaborative enhancement on multivalent NC spheres coated with PECAM Abs (Ab/NC, ∼180 nm diameter). We found that PECAM Abs do mutually enhance endothelial cell binding of Ab/NC coated by paired, but not “self” Ab. <i>In vitro</i>, collaborative enhancement of endothelial binding of Ab/NC by paired Abs is modulated by Ab/NC avidity, epitope selection, and flow. Cell fixation, but not blocking of endocytosis, obliterated collaborative enhancement of Ab/NC binding, indicating that the effect is mediated by molecular reorganization of PECAM molecules in the endothelial plasmalemma. The collaborative enhancement of Ab/NC binding was affirmed <i>in vivo</i>. Intravascular injection of paired Abs enhanced targeting of Ab/NC to pulmonary vasculature in mice by an order of magnitude. This stimulatory effect greatly exceeded enhancement of Ab targeting by paired Abs, indicating that ‘“collaborative enhancement”’ effect is even more pronounced for relatively large multivalent carriers <i>versus</i> free Abs, likely due to more profound consequences of positive alteration of epitope accessibility. This phenomenon provides a potential paradigm for optimizing the endothelial-targeted nanocarrier delivery of therapeutic agents