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

Autoinhibition regulates cellular localization and actin assembly activity of the diaphanous-related formins FRLα and mDia1

Diaphanous-related formins (DRFs) are key regulators of actin cytoskeletal dynamics whose in vitro actin assembly activities are thought to be regulated by autoinhibition. However, the in vivo consequences of autoinhibition and the involvement of DRFs in specific biological processes are not well understood. In this study, we show that in the DRFs FRLα (formin-related gene in leukocytes α) and mouse diaphanous 1, autoinhibition regulates a novel membrane localization activity in vivo as well as actin assembly activity in vitro. In FRLα, the Rho family guanosine triphosphatase Cdc42 relieves the autoinhibition of both membrane localization and biochemical actin assembly activities. FRLα is required for efficient Fc-γ receptor–mediated phagocytosis and is recruited to the phagocytic cup by Cdc42. These results suggest that mutual autoinhibition of biochemical activity and cellular localization may be a general regulatory principle for DRFs and demonstrate a novel role for formins in immune function

Ideals Generated by Principal Minors

A minor is principal means it is defined by the same row and column indices. Let $X$ be a square generic matrix, $K[X]$ the polynomial ring in entries of $X$, over an algebraically closed field, $K$. For fixed $t\leq n$, let $\mathfrak P_t$ denote the ideal generated by the size $t$ principal minors of $X$. When $t=2$ the resulting quotient ring $K[X]/\mathfrak P_2$ is a normal complete intersection domain. When $t>2$ we break the problem into cases depending on a fixed rank, $r$, of $X$. We show when $r=n$ for any $t$, the respective images of $\mathfrak P_t$ and $\mathfrak P_{n-t}$ in the localized polynomial ring, where we invert $\det X$, are isomorphic. From that we show the algebraic set given by $\mathfrak P_{n-1}$ has a codimension $n$ component, plus a codimension 4 component defined by the determinantal ideal (which is given by all the submaximal minors of $X$). When $n=4$ the two components are linked, and we prove some consequences

A novel mechanism of actin filament processive capping by formin: solution of the rotation paradox

The FH2 domains of formin family proteins act as processive cappers of actin filaments. Previously suggested stair-stepping mechanisms of processive capping imply that a formin cap rotates persistently in one direction with respect to the filament. This challenges the formin-mediated mechanism of intracellular cable formation. We suggest a novel scenario of processive capping that is driven by developing and relaxing torsion elastic stresses. Based on the recently discovered crystal structure of an FH2–actin complex, we propose a second mode of processive capping—the screw mode. Within the screw mode, the formin dimer rotates with respect to the actin filament in the direction opposite to that generated by the stair-stepping mode so that a combination of the two modes prevents persistent torsion strain accumulation. We determine an optimal regime of processive capping, whose essence is a periodic switch between the stair-stepping and screw modes. In this regime, elastic energy does not exceed feasible values, and supercoiling of actin filaments is prevented

Statics and dynamics of single DNA molecules confined in nanochannels

The successful design of nanofluidic devices for the manipulation of biopolymers requires an understanding of how the predictions of soft condensed matter physics scale with device dimensions. Here we present measurements of DNA extended in nanochannels and show that below a critical width roughly twice the persistence length there is a crossover in the polymer physics

Regulation of Early Adipose Commitment by Zfp521

While there has been significant progress in determining the transcriptional cascade involved in terminal adipocyte differentiation, less is known about early events leading to lineage commitment and cell fate choice. It has been recently discovered that zinc finger protein 423 (Zfp423) is an early actor in adipose determination. Here, we show that a close paralog of Zfp423, Zfp521, acts as a key regulator of adipose commitment and differentiation in vitro and in vivo. Zfp521 exerts its actions by binding to early B cell factor 1 (Ebf1), a transcription factor required for the generation of adipocyte progenitors, and inhibiting the expression of Zfp423. Overexpression of Zfp521 in cells greatly inhibits adipogenic potential, whereas RNAi-mediated knock-down or genetic ablation of Zfp521 enhances differentiation. In addition, $Zfp521^{−/−}$ embryos exhibit increased mass of interscapular brown adipose tissue and subcutaneous white adipocytes, a cell autonomous effect. Finally, Ebf1 participates in a negative feedback loop to repress Zfp521 as differentiation proceeds. Because Zfp521 is known to promote bone development, our results suggest that it acts as a critical switch in the commitment decision between the adipogenic and osteogenic lineages

A quantitative inventory of yeast P body proteins reveals principles of composition and specificity

P bodies are archetypal biomolecular condensates that concentrate proteins and RNA without a surrounding membrane. While dozens of P body proteins are known, the concentrations of components in the compartment have not been measured. We used live cell imaging to generate a quantitative inventory of the major proteins in yeast P bodies. Only seven proteins are highly concentrated in P bodies (5.1&ndash;15&micro;M); the 24 others examined are appreciably lower (most &le; 2.6&micro;M). P body concentration correlates inversely with cytoplasmic exchange rate. Sequence elements driving Dcp2 concentration into P bodies are distributed across the protein and act synergistically. Our data indicate that P bodies, and probably other condensates, are compositionally simpler than suggested by proteomic analyses, with implications for specificity, reconstitution and evolution.</p