Phosphorylation of nephrin induces phase separated domains that move through actomyosin contraction

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Kim, S., Kalappurakkal, J. M., Mayor, S., & Rosen, M. K. Phosphorylation of nephrin induces phase separated domains that move through actomyosin contraction. Molecular Biology of the Cell, 30(24), (2019): 2996–3012, doi:10.1091/mbc.E18-12-0823.The plasma membrane of eukaryotic cells is organized into lipid and protein microdomains, whose assembly mechanisms and functions are incompletely understood. We demonstrate that proteins in the nephrin/Nck/N-WASP actin-regulatory pathway cluster into micron-scale domains at the basal plasma membrane upon triggered phosphorylation of transmembrane protein nephrin. The domains are persistent but readily exchange components with their surroundings, and their formation is dependent on the number of Nck SH3 domains, suggesting they are phase separated polymers assembled through multivalent interactions among the three proteins. The domains form independent of the actin cytoskeleton, but acto-myosin contractility induces their rapid lateral movement. Nephrin phosphorylation induces larger clusters at the cell periphery, which are associated with extensive actin assembly and dense filopodia. Our studies illustrate how multivalent interactions between proteins at the plasma membrane can produce micron-scale organization of signaling molecules, and how the resulting clusters can both respond to and control the actin cytoskeleton.We thank Hongtao Yu (University of Texas Southwestern Medical Center [UTSW]) for providing the HeLa cell line used in this work; Dan Billadeau and Timothy Gomez (Mayo Clinic) for providing antibodies; Nico Stuurman (University of California, San Francisco) for assistance with STORM imaging; Kate Luby-Phelps and Abhijit Bugde (UTSW Live Cell Imaging Core Facility) for their assistance in epifluorescence and spinning disk confocal experiments; Sudeep Banjade for advice on designing the S3, S2, S1 constructs; Khuloud Jaqaman (UTSW) for advice on cluster motility analysis; Salman Banani and Jonathan Ditlev (UTSW) for critical reading of the manuscript; and members of the Rosen lab and participants in the MBL/HHMI Summer Institutes for advice and helpful discussions. This work was supported by a Howard Hughes Medical Institute Collaborative Innovation Award; the Welch Foundation (I-1544 to M.K.R.); a J.C. Bose Fellowship from the Department of Science and Technology, government of India (to S.M.); a Margadarshi Fellowship from the Wellcome Trust—Department of Biotechnology, India Alliance (IA/M/15/1/502018 to S.M.). Research in the Rosen lab is supported by the Howard Hughes Medical Institute

Population distribution analyses reveal a hierarchy of molecular players underlying parallel endocytic pathways.

Single-cell-resolved measurements reveal heterogeneous distributions of clathrin-dependent (CD) and -independent (CLIC/GEEC: CG) endocytic activity in Drosophila cell populations. dsRNA-mediated knockdown of core versus peripheral endocytic machinery induces strong changes in the mean, or subtle changes in the shapes of these distributions, respectively. By quantifying these subtle shape changes for 27 single-cell features which report on endocytic activity and cell morphology, we organize 1072 Drosophila genes into a tree-like hierarchy. We find that tree nodes contain gene sets enriched in functional classes and protein complexes, providing a portrait of core and peripheral control of CD and CG endocytosis. For 470 genes we obtain additional features from separate assays and classify them into early- or late-acting genes of the endocytic pathways. Detailed analyses of specific genes at intermediate levels of the tree suggest that Vacuolar ATPase and lysosomal genes involved in vacuolar biogenesis play an evolutionarily conserved role in CG endocytosis

Population distribution analyses reveal a hierarchy of molecular players underlying parallel endocytic pathways.

Single-cell-resolved measurements reveal heterogeneous distributions of clathrin-dependent (CD) and -independent (CLIC/GEEC: CG) endocytic activity in Drosophila cell populations. dsRNA-mediated knockdown of core versus peripheral endocytic machinery induces strong changes in the mean, or subtle changes in the shapes of these distributions, respectively. By quantifying these subtle shape changes for 27 single-cell features which report on endocytic activity and cell morphology, we organize 1072 Drosophila genes into a tree-like hierarchy. We find that tree nodes contain gene sets enriched in functional classes and protein complexes, providing a portrait of core and peripheral control of CD and CG endocytosis. For 470 genes we obtain additional features from separate assays and classify them into early- or late-acting genes of the endocytic pathways. Detailed analyses of specific genes at intermediate levels of the tree suggest that Vacuolar ATPase and lysosomal genes involved in vacuolar biogenesis play an evolutionarily conserved role in CG endocytosis

Parody as positive dissent in Hindi theatre

Parody (etymologically a voice alongside another voice) involves imitation, but what is crucial is the co-presence of these two voices, the parodying and the parodied. It is the dialogue between two enunciative spheres, two utterances, hence its preeminent position in the Bakhtinian concept of dialogism. The two points of view, set against each other dialogically, represent two utterances, speakers, styles, languages, and axiological systems, even if they issue from a single speaker. As a reflexive device and critical manipulation of canonized forms, parody has often been considered as the epitome of postmodernism in European and North American literature and artistic expression. The paper aims to show that, in Hindi theatre, parody is politically significant. The article focuses on Bhartendu Hariścandra (1850—1885) and Habīb Tanvīr (1923—2009). It argues that the use of the quotes of Nazīr Akbarābādīin Tanvīr’s most famous play Āgrā Bāzār, a poet who himself parodies the traditional poetical canons, enhances a literary reflexivity that is one of the deepest creative devices of Indian culture

Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions

Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven “retrograde flow” of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues

Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration

Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, μm-scale measurements

Endocytic phenotypes in mutant primary hemocytes from <i>Drosophila</i>.

<p>(A–D) dsRNA treated S2R+ cells phenocopy corresponding allelic mutants in primary hemocyte cultures in a secondary assay. Scatter plots (A, B) show normalized fold change in fluorescence intensity of dextran that was pulsed (A) or chased (B) in S2R+ cells treated with different dsRNAs (y axis) or in hemocytes (x axis) from the corresponding mutant flies. In all cases, representative values were normalized to those from negative controls (CS hemocytes or zeo dsRNA treated S2R+ cells) and are plotted as mean± SEM. (n>30 for hemocyte assays, n>200 for S2R+ assays in all cases). For the chase assay in (B), we utilized <i>dor⁴</i> and <i>car¹</i> mutant hemocytes as positive controls (shown in light blue; Sriram et al., 2003). (C) Representative micrographs of hemocyte cultures from flies carrying hypomorphic alleles of <i>vps35</i>, <i>epac</i>, <i>α-cop</i> and <i>CG1418</i> assayed as in (B). (D) Summary of the experiment in (A–B) displaying statistically significant (Student's T-test, p<0.05) changes in uptake/retention of mutant hemocytes or gene-depleted S2R+ cells as colour coded maps. Scale bar in (C) = 5 µm.</p

Quantitative profiling of two endocytic routes at single cell resolution.

<p>(A) Experimental workflow outline for cell seeding, transfection and multiplex endocytic assays to obtain multifeature data across 7131 gene depletions. The entire procedure was performed on a cell array (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100554#pone.0100554.s001" target="_blank">Figure S1A</a>; details in SOM) and the positions of negative and positive (dsRNA against <i>sec23</i>, <i>arf1</i>, <i>shi</i>) controls are highlighted in their respective colours. (B) Table grouping the 27 quantitative features into categories. The top half of the table contains direct measurements of intensity, while the lower half contains geometric parameters of the cell, endosomes and nucleus. Various measurements are made from each fluorescent channel, including those utilizing different pixel radii for local background subtraction while detecting endosomes. (C) Representative brightfield (bf) and fluorescent micrographs of a field of view of individual cells (zoomed in insets) labeled with four different fluorescent probes: Hoechst; FITC-Dextran (Fdex); Alexa568-Tf (Tf); Alexa647-αOkt9 (Okt9); (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100554#pone.0100554.s018" target="_blank">Methods S1</a> for details). The psuedocolour merge image is a composite of the Fdex (green), TfR (red) and Okt9 (blue) channels. Scale bar = 15 µm; inset = 3×. (D) Grayscale heatmap representing the fraction of four control genes (<i>arf1</i> (<i>arf79f</i>); <i>shi</i>; <i>sec23</i>; <i>chc</i>) picked up as hits (above a Z-score threshold of 3) across all 27 features in the entire dataset. Higher values on the grayscale bar denote higher pick-up rates. The features with higher pick-up rates correspond to the known endocytic roles of these genes.</p

Primary hits validated in a secondary classification assay.

<p>(A–B) Schema (A) and positional patterning (B) on cell arrays of secondary endocytic classification assays carried out for all CG features (upper schema) or a subset of CD features (lower schema).All the test genes were surrounded with local positive controls, and negative controls (see legend in (B)). With this patterning, each gene was tested in triplicate, with three local positive controls and six local negative controls. (C) Heatmap representing raw mean fluorescence intensities (in the pulse channel) across a test cell array used to validate the CG secondary endocytic assay described in (A). Only the means of control wells are shown in the top panel and the inter-control variation in means is representative of a typical experiment. For comparison, the lower panel depicts the mean fluorescence intensities of test genes. (D) The green bars show the fraction of genes predicted as hits for each feature in the primary screen that were also picked up as hits for that feature in the secondary. The gray bars show the fraction of genes not predicted as a hit for each feature in the primary screen that were nevertheless picked up as hits for that feature in the secondary. With a single exception (Tnum) we find that the green bars exceed the gray (p-value 5×10⁻⁶ for 22 fair coin flips) demonstrating the selectivity and reproducibility of our primary assay. (E) Psuedocoloured fluorescence micrographs of representative control and <i>drab5</i>- and <i>dvps4</i>- dsRNA treated populations of cells that were subjected to the CG pulse-chase assay from (<b>A</b>). Both Drab5 and Dvps4 depleted cells were affected in the chase (with Fdex, green) portion of the assay, while the pulse portion (with Rdex, red) was unaffected (see quantitation in bar graphs on the right, normalized to control). Scale bar = 10 µm.</p

Role of lysosomal genes.

<p>(A) Network map depicting known and predicted interactions (green lines: genetic; blue lines: physical; brown lines: predicted based on conserved data) between the ‘Granule group’ set of eye colour mutants (pink) and selected hits (gray). In this network, genes encoding Carnation (<i>car</i>; the fly homolog of VPS33), Deep orange (<i>dor</i>), Carmine (<i>cm</i>) and Rab7 were identified with roles in CG endocytosis in this study (denoted by black asterisks), while White (<i>w</i>) depletion affected at least one Tf pathway feature (white asterisk). (B) Localization of Carnation on early fluid endosomes. <i>Drosophila</i> S2R+ cells were pulsed with TMR-Dextran for two minutes and fixed and labeled with antibodies to Carnation (αCar). Micrographs show a representative cell imaged in two channels and a pseudo colour merge image (labeled TMRdex and αCar), in red, green and merge respectively). Carnation (green) is seen enriched on peripheral, small, early fluid endosomes (red). Three examples of such endosomes (white arrows in merge panel) are shown in the magnified inset. (C) Fluorescent micrographs depict the levels of fluid uptake in representative S2R+ cells treated with dsRNA against <i>car</i> (first lower panel) or <i>syx1A</i> (last lower panel) or in hemocytes from <i>car¹</i> mutant flies (middle lower panel), with their respective controls (upper panels). Bar graph represents mean and SD of normalized fluorescent integrated intensity per cell from 2–3 experiments, with 100–150 cells per treatment (S2R+ cells) or 40 cells per genotype (hemocytes). (D) Representative fluorescent micrographs depict fluid uptake measured in hemocytes as in (C), in flies that were: homozygous for a mutant allele of <i>car</i> (<i>car¹</i>); a hetero-allelic combination of <i>car¹</i>/+;<i>syx1</i>/+;or wild type (CS). Also tested were flies heterozygous for <i>syx1</i>/+ and <i>car¹</i>/+. Bar graph represents mean and SD of normalized fluorescent integrated intensity per hemocyte from 2–3 experiments with 40 cells per genotype. (E) Representative micrographs show human AGS cells treated with control siRNA or siRNA to hSYX1A and hVPS33A/B and pulsed with FITC-Dextran for 5 min. Right panel - Bar graphs show population averaged mean fluorescence intensity uptake per cell (representative experiment with n>50 cells per replicate, 2 replicates). Scale bar in (B–E) main panel = 5 µm, inset = 1 µm. Double asterisks denote significance <i>p</i> values lower than 0.01 with the Student's T-Test.</p