scholarly journals Stoichiometry controls activity of phase-separated clusters of actin signaling proteins

Science ◽  
2019 ◽  
Vol 363 (6431) ◽  
pp. 1093-1097 ◽  
Author(s):  
Lindsay B. Case ◽  
Xu Zhang ◽  
Jonathon A. Ditlev ◽  
Michael K. Rosen
Keyword(s):  
Lipid Bilayers ◽  
Dwell Time ◽  
Specific Activity ◽  
Signaling Proteins ◽  
Actin Assembly ◽  
Protein Activity ◽  
Signaling Systems ◽  
Multivalent Interactions ◽  
Actin Regulatory Proteins ◽  
Surrounding Membrane

Biomolecular condensates concentrate macromolecules into foci without a surrounding membrane. Many condensates appear to form through multivalent interactions that drive liquid-liquid phase separation (LLPS). LLPS increases the specific activity of actin regulatory proteins toward actin assembly by the Arp2/3 complex. We show that this increase occurs because LLPS of the Nephrin–Nck–N-WASP signaling pathway on lipid bilayers increases membrane dwell time of N-WASP and Arp2/3 complex, consequently increasing actin assembly. Dwell time varies with relative stoichiometry of the signaling proteins in the phase-separated clusters, rendering N-WASP and Arp2/3 activity stoichiometry dependent. This mechanism of controlling protein activity is enabled by the stoichiometrically undefined nature of biomolecular condensates. Such regulation should be a general feature of signaling systems that assemble through multivalent interactions and drive nonequilibrium outputs.

scholarly journals The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly.

1993 ◽  
Vol 120 (4) ◽  
pp. 909-922 ◽  
Author(s):  
C P Chia ◽  
A Shariff ◽  
S A Savage ◽  
E J Luna
Keyword(s):  
Membrane Protein ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Lipid Vesicles ◽  
Integral Membrane Protein ◽  
Actin Binding ◽  
Actin Assembly ◽  
Nucleation Activity

Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F-actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.

scholarly journals Direct measurement of DNA-mediated adhesion between lipid bilayers

2015 ◽  
Vol 17 (24) ◽  
pp. 15615-15628 ◽  
Author(s):  
S. F. Shimobayashi ◽  
B. M. Mognetti ◽  
L. Parolini ◽  
D. Orsi ◽  
P. Cicuta ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Direct Measurement ◽  
Living Cells ◽  
Solid Substrates ◽  
Multivalent Interactions

Multivalent interactions between deformable mesoscopic units are ubiquitous in biology, where membrane macromolecules mediate the interactions between neighbouring living cells and between cells and solid substrates.

scholarly journals Synthetic protein condensates that recruit and release protein activity in living cells

2020 ◽  
Author(s):  
Tatsuyuki Yoshii ◽  
Masaru Yoshikawa ◽  
Masahiro Ikuta ◽  
Shinya Tsukiji
Keyword(s):  
Mammalian Cells ◽  
Chemical Biology ◽  
Cell Engineering ◽  
Signaling Proteins ◽  
Protein Activity ◽  
Cellular Processes ◽  
On Demand ◽  
Synthetic Protein ◽  
Intracellular Proteins ◽  
Condensate System

AbstractCompartmentation of proteins into biomolecular condensates or membraneless organelles formed by phase separation is an emerging principle for the regulation of cellular processes. Creating synthetic condensates that accommodate specific intracellular proteins on demand would have various applications in chemical biology, cell engineering and synthetic biology. Here, we report the construction of synthetic protein condensates capable of recruiting and/or releasing proteins of interest in living mammalian cells in response to a small molecule or light. We first present chemogenetic protein-recruiting and -releasing condensates, which rapidly inhibited and activated signaling proteins, respectively. An optogenetic condensate system was successfully constructed that enables reversible release and sequestration of protein activity using light. This proof-of-principle work provides a new platform for chemogenetic and optogenetic control of protein activity in mammalian cells and represents a step towards tailor-made engineering of synthetic protein condensates with various functionalities.

scholarly journals Phosphorylation of Nephrin induces phase separated domains that move through actomyosin contraction

2019 ◽  
Author(s):  
Soyeon Kim ◽  
Joseph M. Kalappurakkal ◽  
Satyajit Mayor ◽  
Michael K. Rosen
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Regulatory Pathway ◽  
Basal Plasma Membrane ◽  
Basal Plasma ◽  
Multivalent Interactions ◽  
And Control ◽  
Actomyosin Contraction

AbstractThe 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 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.

scholarly journals Collateral fitness effects of mutations

2020 ◽  
Vol 117 (21) ◽  
pp. 11597-11607 ◽  
Author(s):  
Jacob D. Mehlhoff ◽  
Frank W. Stearns ◽  
Dahlia Rohm ◽  
Buheng Wang ◽  
Erh-Yeh Tsou ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Protein Evolution ◽  
Physiological Function ◽  
Specific Activity ◽  
Antibiotic Resistance Gene ◽  
Cell Phenotype ◽  
Missense Mutations ◽  
Protein Activity ◽  
Fitness Effects ◽  
As Effects

The distribution of fitness effects of mutation plays a central role in constraining protein evolution. The underlying mechanisms by which mutations lead to fitness effects are typically attributed to changes in protein specific activity or abundance. Here, we reveal the importance of a mutation’s collateral fitness effects, which we define as effects that do not derive from changes in the protein’s ability to perform its physiological function. We comprehensively measured the collateral fitness effects of missense mutations in theEscherichia coli TEM-1β-lactamase antibiotic resistance gene using growth competition experiments in the absence of antibiotic. At least 42% of missense mutations inTEM-1were deleterious, indicating that for some proteins collateral fitness effects occur as frequently as effects on protein activity and abundance. Deleterious mutations caused improper posttranslational processing, incorrect disulfide-bond formation, protein aggregation, changes in gene expression, and pleiotropic effects on cell phenotype. Deleterious collateral fitness effects occurred more frequently inTEM-1than deleterious effects on antibiotic resistance in environments with low concentrations of the antibiotic. The surprising prevalence of deleterious collateral fitness effects suggests they may play a role in constraining protein evolution, particularly for highly expressed proteins, for proteins under intermittent selection for their physiological function, and for proteins whose contribution to fitness is buffered against deleterious effects on protein activity and protein abundance.

scholarly journals Distinct Chemotaxis Protein Paralogs Assemble into Chemoreceptor Signaling Arrays To Coordinate Signaling Output

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Lindsey O’Neal ◽  
Jessica M. Gullett ◽  
Anastasia Aksenova ◽  
Adam Hubler ◽  
Ariane Briegel ◽  
...  
Keyword(s):  
Signaling Proteins ◽  
Signal Integration ◽  
Content Type ◽  
Signaling Systems ◽  
Chemotaxis Proteins ◽  
Membrane Bound ◽  
Chemotaxis Receptors ◽  
Genome Analyses ◽  
Response Output ◽  
Motile Bacteria

ABSTRACT Most chemotactic motile bacteria possess multiple chemotaxis signaling systems, the functions of which are not well characterized. Chemotaxis signaling is initiated by chemoreceptors that assemble as large arrays, together with chemotaxis coupling proteins (CheW) and histidine kinase proteins (CheA), which form a baseplate with the cytoplasmic tips of receptors. These cell pole-localized arrays mediate sensing, signaling, and signal amplification during chemotaxis responses. Membrane-bound chemoreceptors with different cytoplasmic domain lengths segregate into distinct arrays. Here, we show that a bacterium, Azospirillum brasilense, which utilizes two chemotaxis signaling systems controlling distinct motility parameters, coordinates its chemotactic responses through the production of two separate membrane-bound chemoreceptor arrays by mixing paralogs within chemotaxis baseplates. The polar localization of chemoreceptors of different length classes is maintained in strains that had baseplate signaling proteins from either chemotaxis system but was lost when both systems were deleted. Chemotaxis proteins (CheA and CheW) from each of the chemotaxis signaling systems (Che1 and Che4) could physically interact with one another, and chemoreceptors from both classes present in A. brasilense could interact with Che1 and Che4 proteins. The assembly of paralogs from distinct chemotaxis pathways into baseplates provides a straightforward mechanism for coordinating signaling from distinct pathways, which we predict is not unique to this system given the propensity of chemotaxis systems for horizontal gene transfer. IMPORTANCE The assembly of chemotaxis receptors and signaling proteins into polar arrays is universal in motile chemotactic bacteria. Comparative genome analyses indicate that most motile bacteria possess multiple chemotaxis signaling systems, and experimental evidence suggests that signaling from distinct chemotaxis systems is integrated. Here, we identify one such mechanism. We show that paralogs from two chemotaxis systems assemble together into chemoreceptor arrays, forming baseplates comprised of proteins from both chemotaxis systems. These mixed arrays provide a straightforward mechanism for signal integration and coordinated response output from distinct chemotaxis systems. Given that most chemotactic bacteria encode multiple chemotaxis systems and the propensity for these systems to be laterally transferred, this mechanism may be common to ensure chemotaxis signal integration occurs.

scholarly journals Transient assembly of F-actin on the outer mitochondrial membrane contributes to mitochondrial fission

2014 ◽  
Vol 208 (1) ◽  
pp. 109-123 ◽  
Author(s):  
Sunan Li ◽  
Shan Xu ◽  
Brian A. Roelofs ◽  
Liron Boyman ◽  
W. Jonathan Lederer ◽  
...  
Keyword(s):  
De Novo ◽  
Mitochondrial Fission ◽  
Regulatory Proteins ◽  
Actin Assembly ◽  
Membrane Remodeling ◽  
Mitochondrial Fragmentation ◽  
Down Regulation ◽  
Dynamic Assembly ◽  
Actin Regulatory Proteins ◽  
Underlying Mechanisms

In addition to established membrane remodeling roles in various cellular locations, actin has recently emerged as a participant in mitochondrial fission. However, the underlying mechanisms of its participation remain largely unknown. We report that transient de novo F-actin assembly on the mitochondria occurs upon induction of mitochondrial fission and F-actin accumulates on the mitochondria without forming detectable submitochondrial foci. Impairing mitochondrial division through Drp1 knockout or inhibition prolonged the time of mitochondrial accumulation of F-actin and also led to abnormal mitochondrial accumulation of the actin regulatory factors cortactin, cofilin, and Arp2/3 complexes, suggesting that disassembly of mitochondrial F-actin depends on Drp1 activity. Furthermore, down-regulation of actin regulatory proteins led to elongation of mitochondria, associated with mitochondrial accumulation of Drp1. In addition, depletion of cortactin inhibited Mfn2 down-regulation– or FCCP-induced mitochondrial fragmentation. These data indicate that the dynamic assembly and disassembly of F-actin on the mitochondria participates in Drp1-mediated mitochondrial fission.

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

2019 ◽  
Vol 30 (24) ◽  
pp. 2996-3012 ◽  
Author(s):  
Soyeon Kim ◽  
Joseph M. Kalappurakkal ◽  
Satyajit Mayor ◽  
Michael K. Rosen
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Transmembrane Protein ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Basal Plasma Membrane ◽  
Basal Plasma ◽  
Multivalent Interactions ◽  
And Control ◽  
Actomyosin Contraction

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.

scholarly journals Arf6 Can Trigger Wave Regulatory Complex-Dependent Actin Assembly Independent of Arno

2020 ◽  
Vol 21 (7) ◽  
pp. 2457 ◽  
Author(s):  
Vikash Singh ◽  
Anthony C. Davidson ◽  
Peter J. Hume ◽  
Vassilis Koronakis
Keyword(s):  
Lipid Bilayers ◽  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Small Gtpase ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Nucleotide Exchange Factor ◽  
Regulatory Complex

The small GTPase ADP-ribosylation factor 6 (Arf6) anchors at the plasma membrane to orchestrate key functions, such as membrane trafficking and regulating cortical actin cytoskeleton rearrangement. A number of studies have identified key players that interact with Arf6 to regulate actin dynamics in diverse cell processes, yet it is still unknown whether Arf6 can directly signal to the wave regulatory complex to mediate actin assembly. By reconstituting actin dynamics on supported lipid bilayers, we found that Arf6 in co-ordination with Rac1(Ras-related C3 botulinum toxin substrate 1) can directly trigger actin polymerization by recruiting wave regulatory complex components. Interestingly, we demonstrated that Arf6 triggers actin assembly at the membrane directly without recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO (ARF nucleotide-binding site opener), which is able to activate Arf1 to enable WRC-dependent actin assembly. Furthermore, using labelled E. coli, we demonstrated that actin assembly by Arf6 also contributes towards efficient phagocytosis in THP-1 macrophages. Taken together, this study reveals a mechanism for Arf6-driven actin polymerization.

scholarly journals A major role for noncoding regulatory mutations in the evolution of enzyme activity

2019 ◽  
Vol 116 (25) ◽  
pp. 12383-12389 ◽  
Author(s):  
David W. Loehlin ◽  
Jesse R. Ames ◽  
Kathy Vaccaro ◽  
Sean B. Carroll
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Functional Divergence ◽  
Primary Source ◽  
Protein Activity ◽  
Genetic Changes ◽  
Gene Enhancer ◽  
Ethanol Toxicity ◽  
Regulatory Mutations

The quantitative evolution of protein activity is a common phenomenon, yet we know little about any general mechanistic tendencies that underlie it. For example, an increase (or decrease) in enzyme activity may evolve from changes in protein sequence that alter specific activity, or from changes in gene expression that alter the amount of protein produced. The latter in turn could arise via mutations that affect gene transcription, posttranscriptional processes, or copy number. Here, to determine the types of genetic changes underlying the quantitative evolution of protein activity, we dissected the basis of ecologically relevant differences in Alcohol dehydrogenase (Adh) enzyme activity between and within several Drosophila species. By using recombinant Adh transgenes to map the functional divergence of ADH enzyme activity in vivo, we find that amino acid substitutions explain only a minority (0 to 25%) of between- and within-species differences in enzyme activity. Instead, noncoding substitutions that occur across many parts of the gene (enhancer, promoter, and 5′ and 3′ untranslated regions) account for the majority of activity differences. Surprisingly, one substitution in a transcriptional Initiator element has occurred in parallel in two species, indicating that core promoters can be an important natural source of the tuning of gene activity. Furthermore, we show that both regulatory and coding substitutions contribute to fitness (resistance to ethanol toxicity). Although qualitative changes in protein specificity necessarily derive from coding mutations, these results suggest that regulatory mutations may be the primary source of quantitative changes in protein activity, a possibility overlooked in most analyses of protein evolution.

Sign in / Sign up

Export Citation Format

Share Document