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Actin in vitro reconstitution

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https://www.readbyqxmd.com/read/29042512/in-vitro-reconstitution-of-t-cell-receptor-mediated-segregation-of-the-cd45-phosphatase
#1
Catherine B Carbone, Nadja Kern, Ricardo A Fernandes, Enfu Hui, Xiaolei Su, K Christopher Garcia, Ronald D Vale
T cell signaling initiates upon the binding of peptide-loaded MHC (pMHC) on an antigen-presenting cell to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR-pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase-phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR-pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells...
October 31, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29017047/cell-biology-capturing-formin-s-mechano-inhibition
#2
Dimitrios Vavylonis, Brandon G Horan
Formins polymerize actin filaments for the cytokinetic contractile ring. Using in vitro reconstitution of fission yeast contractile ring precursor nodes containing formins and myosin, a new study shows that formin-mediated polymerization is strongly inhibited upon the capture and pulling of actin filaments by myosin, a result that has broad implications for cellular mechanosensing.
October 9, 2017: Current Biology: CB
https://www.readbyqxmd.com/read/28951543/mechanoregulated-inhibition-of-formin-facilitates-contractile-actomyosin-ring-assembly
#3
Dennis Zimmermann, Kaitlin E Homa, Glen M Hocky, Luther W Pollard, Enrique M De La Cruz, Gregory A Voth, Kathleen M Trybus, David R Kovar
Cytokinesis physically separates dividing cells by forming a contractile actomyosin ring. The fission yeast contractile ring has been proposed to assemble by Search-Capture-Pull-Release from cytokinesis precursor nodes that include the molecular motor type-II myosin Myo2 and the actin assembly factor formin Cdc12. By successfully reconstituting Search-Capture-Pull in vitro, we discovered that formin Cdc12 is a mechanosensor, whereby myosin pulling on formin-bound actin filaments inhibits Cdc12-mediated actin assembly...
September 26, 2017: Nature Communications
https://www.readbyqxmd.com/read/28935896/network-heterogeneity-regulates-steering-in-actin-based-motility
#4
Rajaa Boujemaa-Paterski, Cristian Suarez, Tobias Klar, Jie Zhu, Christophe Guérin, Alex Mogilner, Manuel Théry, Laurent Blanchoin
The growth of branched actin networks powers cell-edge protrusions and motility. A heterogeneous density of actin, which yields to a tunable cellular response, characterizes these dynamic structures. We study how actin organization controls both the rate and the steering during lamellipodium growth. We use a high-resolution surface structuration assay combined with mathematical modeling to describe the growth of a reconstituted lamellipodium. We demonstrate that local monomer depletion at the site of assembly negatively impacts the network growth rate...
September 21, 2017: Nature Communications
https://www.readbyqxmd.com/read/28872124/in-vitro-polymerization-of-f-actin-on-early-endosomes
#5
Olivia Muriel, Cameron Christopher Scott, Jorge Larios, Vicent Mercier, Jean Gruenberg
Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto the endosomal membrane. We have established a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to genetic and biochemical manipulations. Endosomal fractions are prepared by floatation in sucrose gradients from cells expressing the early endosomal protein GFP-RAB5...
August 28, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28867286/load-adaptation-of-lamellipodial-actin-networks
#6
Jan Mueller, Gregory Szep, Maria Nemethova, Ingrid de Vries, Arnon D Lieber, Christoph Winkler, Karsten Kruse, J Victor Small, Christian Schmeiser, Kinneret Keren, Robert Hauschild, Michael Sixt
Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane...
September 21, 2017: Cell
https://www.readbyqxmd.com/read/28854360/spatiotemporal-control-of-lipid-conversion-actin-based-mechanical-forces-and-curvature-sensors-during-clathrin-ap-1-coated-vesicle-biogenesis
#7
Mihaela Anitei, Christoph Stange, Cornelia Czupalla, Christian Niehage, Kai Schuhmann, Pia Sala, Aleksander Czogalla, Theresia Pursche, Ünal Coskun, Andrej Shevchenko, Bernard Hoflack
Clathrin/adaptor protein-1-coated carriers connect the secretory and the endocytic pathways. Carrier biogenesis relies on distinct protein networks changing membrane shape at the trans-Golgi network, each regulating coat assembly, F-actin-based mechanical forces, or the biophysical properties of lipid bilayers. How these different hubs are spatiotemporally coordinated remains largely unknown. Using in vitro reconstitution systems, quantitative proteomics, and lipidomics, as well as in vivo cell-based assays, we characterize the protein networks controlling membrane lipid composition, membrane shape, and carrier scission...
August 29, 2017: Cell Reports
https://www.readbyqxmd.com/read/28754385/alignment-of-actin-filament-streams-driven-by-myosin-motors-in-crowded-environments
#8
Takahiro Iwase, Yasuhiko Sasaki, Kuniyuki Hatori
BACKGROUND: Cellular dynamics depend on cytoskeletal filaments and motor proteins. Collective movements of filaments driven by motor proteins are observed in the presence of dense filaments in in vitro systems. As multiple macromolecules exist within cells and the physiological ionic conditions affect their interactions, crowding might contribute to ordered cytoskeletal architecture because of collective behavior. METHODS: Using an in vitro reconstituted system, we observed the emergence of stripe patterns resulting from collective actin filament streaming driven by myosin motors in the presence of the crowding agent, methylcellulose (MC)...
November 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28282023/competition-between-tropomyosin-fimbrin-and-adf-cofilin-drives-their-sorting-to-distinct-actin-filament-networks
#9
Jenna R Christensen, Glen M Hocky, Kaitlin E Homa, Alisha N Morganthaler, Sarah E Hitchcock-DeGregori, Gregory A Voth, David R Kovar
The fission yeast actin cytoskeleton is an ideal, simplified system to investigate fundamental mechanisms behind cellular self-organization. By focusing on the stabilizing protein tropomyosin Cdc8, bundling protein fimbrin Fim1, and severing protein coffin Adf1, we examined how their pairwise and collective interactions with actin filaments regulate their activity and segregation to functionally diverse F-actin networks. Utilizing multi-color TIRF microscopy of in vitro reconstituted F-actin networks, we observed and characterized two distinct Cdc8 cables loading and spreading cooperatively on individual actin filaments...
March 10, 2017: ELife
https://www.readbyqxmd.com/read/28255696/reconstitution-of-tcr-signaling-using-supported-lipid-bilayers
#10
Xiaolei Su, Jonathon A Ditlev, Michael K Rosen, Ronald D Vale
Biochemical reconstitution has served as an important tool for understanding the mechanisms of many cellular processes including DNA replication, transcription, translation, vesicle trafficking, and ubiquitin-mediated proteolysis. Here, we demonstrate that biochemical reconstitution can be applied to studying a complex signaling pathway involving as many as 12 proteins or protein complexes acting at the surface of model membranes. We show that a temporal sequence of events in activated T cells beginning with phosphorylation of the T cell receptor and culminating in the activation of actin polymerization can be replicated in vitro...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27983818/structural-and-functional-effects-of-cardiomyopathy-causing-mutations-in-the-troponin-t-binding-region-of-cardiac-tropomyosin
#11
Alexander M Matyushenko, Daniil V Shchepkin, Galina V Kopylova, Katerina E Popruga, Natalya V Artemova, Anastasia V Pivovarova, Sergey Y Bershitsky, Dmitrii I Levitsky
Hypertrophic cardiomyopathy (HCM) is a severe heart disease caused by missense mutations in genes encoding sarcomeric proteins of cardiac muscle. Many of these mutations are identified in the gene encoding the cardiac isoform of tropomyosin (Tpm), an α-helical coiled-coil actin-binding protein that plays a key role in Ca(2+)-regulated contraction of cardiac muscle. We employed various methods to characterize structural and functional features of recombinant human Tpm species carrying HCM mutations that lie either within the troponin T-binding region in the C-terminal part of Tpm (E180G, E180V, and L185R) or near this region (I172T)...
January 10, 2017: Biochemistry
https://www.readbyqxmd.com/read/27698406/triggering-signaling-pathways-using-f-actin-self-organization
#12
A Colin, L Bonnemay, C Gayrard, J Gautier, Z Gueroui
The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers...
October 4, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27693515/how-cellular-membrane-properties-are-affected-by-the-actin-cytoskeleton
#13
REVIEW
J Lemière, F Valentino, C Campillo, C Sykes
Lipid membranes define the boundaries of living cells and intracellular compartments. The dynamic remodelling of these membranes by the cytoskeleton, a very dynamic structure made of active biopolymers, is crucial in many biological processes such as motility or division. In this review, we present some aspects of cellular membranes and how they are affected by the presence of the actin cytoskeleton. We show that, in parallel with the direct study of membranes and cytoskeleton in vivo, biomimetic in vitro systems allow reconstitution of biological processes in a controlled environment...
November 2016: Biochimie
https://www.readbyqxmd.com/read/27666967/fascin-and-%C3%AE-actinin-bundled-networks-contain-intrinsic-structural-features-that-drive-protein-sorting
#14
Jonathan D Winkelman, Cristian Suarez, Glen M Hocky, Alyssa J Harker, Alisha N Morganthaler, Jenna R Christensen, Gregory A Voth, James R Bartles, David R Kovar
Cells assemble and maintain functionally distinct actin cytoskeleton networks with various actin filament organizations and dynamics through the coordinated action of different sets of actin-binding proteins. The biochemical and functional properties of diverse actin-binding proteins, both alone and in combination, have been increasingly well studied. Conversely, how different sets of actin-binding proteins properly sort to distinct actin filament networks in the first place is not nearly as well understood...
October 24, 2016: Current Biology: CB
https://www.readbyqxmd.com/read/27551056/septins-promote-macropinosome-maturation-and-traffic-to-the-lysosome-by-facilitating-membrane-fusion
#15
Lee Dolat, Elias T Spiliotis
Macropinocytosis, the internalization of extracellular fluid and material by plasma membrane ruffles, is critical for antigen presentation, cell metabolism, and signaling. Macropinosomes mature through homotypic and heterotypic fusion with endosomes and ultimately merge with lysosomes. The molecular underpinnings of this clathrin-independent endocytic pathway are largely unknown. Here, we show that the filamentous septin GTPases associate preferentially with maturing macropinosomes in a phosphatidylinositol 3,5-bisphosphate-dependent manner and localize to their contact/fusion sites with macropinosomes/endosomes...
August 29, 2016: Journal of Cell Biology
https://www.readbyqxmd.com/read/27473903/in-vitro-reconstitution-of-septin-assemblies-on-supported-lipid-bilayers
#16
A A Bridges, A S Gladfelter
Septins are polymerizing eukaryotic proteins that play conserved roles in cell cortex organization and are essential in many cell types. How septin dynamics and protein-protein interactions determine their function at the plasma membrane remains a mystery. Here, we present a method for recapitulating septin polymerization and lipid interaction utilizing supported lipid bilayers to mimic the eukaryotic plasma membrane. Septins on supported lipid bilayers can be visualized with single-molecule sensitivity using total internal reflective fluorescence microscopy...
2016: Methods in Cell Biology
https://www.readbyqxmd.com/read/27378946/molecular-and-functional-effects-of-a-splice-site-mutation-in-the-myl2-gene-associated-with-cardioskeletal-myopathy-and-early-cardiac-death-in-infants
#17
Zhiqun Zhou, Wenrui Huang, Jingsheng Liang, Danuta Szczesna-Cordary
The homozygous appearance of the intronic mutation (IVS6-1) in the MYL2 gene encoding for myosin ventricular/slow-twitch skeletal regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy and early cardiac death. The IVS6-1 (c403-1G>C) mutation resulted from a cryptic splice site in MYL2 causing a frameshift and replacement of the last 32 codons by 19 different amino acids in the RLC mutant protein. Infants who were IVS6-1(+∕+)-positive died between 4 and 6 months of age due to cardiomyopathy and heart failure...
2016: Frontiers in Physiology
https://www.readbyqxmd.com/read/27310470/dynamic-filament-formation-by-a-divergent-bacterial-actin-like-parm-protein
#18
Anthony J Brzoska, Slade O Jensen, Deborah A Barton, Danielle S Davies, Robyn L Overall, Ronald A Skurray, Neville Firth
Actin-like proteins (Alps) are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E...
2016: PloS One
https://www.readbyqxmd.com/read/27126922/permeabilization-activated-reduction-in-fluorescence-a-novel-method-to-measure-kinetics-of-protein-interactions-with-intracellular-structures
#19
Pali P Singh, Jenci L Hawthorne, Christie A Davis, Omar A Quintero
Understanding kinetic information is fundamental in understanding biological function. Advanced imaging technologies have fostered the development of kinetic analyses in cells. We have developed Permeabilization Activated Reduction in Fluorescence (PARF) analysis for determination of apparent t1/2 and immobile fraction, describing the dissociation of a protein of interest from intracellular structures. To create conditions where dissociation events are observable, cells expressing a fluorescently-tagged protein are permeabilized with digitonin, diluting the unbound protein into the extracellular media...
June 2016: Cytoskeleton
https://www.readbyqxmd.com/read/27119641/regulation-of-pi3k-by-pkc-and-marcks-single-molecule-analysis-of-a-reconstituted-signaling-pathway
#20
Brian P Ziemba, John E Burke, Glenn Masson, Roger L Williams, Joseph J Falke
In chemotaxing ameboid cells, a complex leading-edge signaling circuit forms on the cytoplasmic leaflet of the plasma membrane and directs both actin and membrane remodeling to propel the leading edge up an attractant gradient. This leading-edge circuit includes a putative amplification module in which Ca(2+)-protein kinase C (Ca(2+)-PKC) is hypothesized to phosphorylate myristoylated alanine-rich C kinase substrate (MARCKS) and release phosphatidylinositol-4,5-bisphosphate (PIP2), thereby stimulating production of the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3) by the lipid kinase phosphoinositide-3-kinase (PI3K)...
April 26, 2016: Biophysical Journal
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