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Actin in vitro reconstitution

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
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...
September 28, 2016: Biochimie
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...
September 12, 2016: Current Biology: CB
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
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
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
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
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
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
Xiaolei Su, Jonathon A Ditlev, Enfu Hui, Wenmin Xing, Sudeep Banjade, Julia Okrut, David S King, Jack Taunton, Michael K Rosen, Ronald D Vale
Activation of various cell surface receptors triggers the reorganization of downstream signaling molecules into micrometer- or submicrometer-sized clusters. However, the functional consequences of such clustering have been unclear. We biochemically reconstituted a 12-component signaling pathway on model membranes, beginning with T cell receptor (TCR) activation and ending with actin assembly. When TCR phosphorylation was triggered, downstream signaling proteins spontaneously separated into liquid-like clusters that promoted signaling outputs both in vitro and in human Jurkat T cells...
April 29, 2016: Science
Andrew E Messer, Christopher R Bayliss, Mohammed El-Mezgueldi, Charles S Redwood, Douglas G Ward, Man-Ching Leung, Maria Papadaki, Cristobal Dos Remedios, Steven B Marston
We investigated the effect of 7 Hypertrophic Cardiomyopathy (HCM)-causing mutations in troponin T (TnT) on troponin function in thin filaments reconstituted with actin and human cardiac tropomyosin. We used the quantitative in vitro motility assay to study Ca(2+)-regulation of unloaded movement and its modulation by troponin I phosphorylation. Troponin from a patient with the K280N TnT mutation showed no difference in Ca(2+)-sensitivity when compared with donor heart troponin and the Ca(2+)-sensitivity was also independent of the troponin I phosphorylation level (uncoupled)...
July 1, 2016: Archives of Biochemistry and Biophysics
Darius Vasco Köster, Kabir Husain, Elda Iljazi, Abrar Bhat, Peter Bieling, R Dyche Mullins, Madan Rao, Satyajit Mayor
The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin. We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors...
March 22, 2016: Proceedings of the National Academy of Sciences of the United States of America
Haruko Ueda, Etsuo Yokota, Keiko Kuwata, Natsumaro Kutsuna, Shoji Mano, Tomoo Shimada, Kentaro Tamura, Giovanni Stefano, Yoichiro Fukao, Federica Brandizzi, Teruo Shimmen, Mikio Nishimura, Ikuko Hara-Nishimura
The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In animals and yeast, homotypic ER membrane fusion is mediated by fusogens (atlastin and Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis (Arabidopsis thaliana), another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as an ER membrane fusogen, but direct evidence is lacking. Here, we show that RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its C terminus...
February 2016: Plant Physiology
Sandra Donkervoort, Maria Papadaki, Josine M de Winter, Matthew B Neu, Janbernd Kirschner, Véronique Bolduc, Michele L Yang, Melissa A Gibbons, Ying Hu, Jahannaz Dastgir, Meganne E Leach, Anne Rutkowski, A Reghan Foley, Marcus Krüger, Eric P Wartchow, Elyshia McNamara, Royston Ong, Kristen J Nowak, Nigel G Laing, Nigel F Clarke, Coen A C Ottenheijm, Steven B Marston, Carsten G Bönnemann
OBJECTIVE: Mutations in TPM3, encoding Tpm3.12, cause a clinically and histopathologically diverse group of myopathies characterized by muscle weakness. We report two patients with novel de novo Tpm3.12 single glutamic acid deletions at positions ΔE218 and ΔE224, resulting in a significant hypercontractile phenotype with congenital muscle stiffness, rather than weakness, and respiratory failure in one patient. METHODS: The effect of the Tpm3.12 deletions on the contractile properties in dissected patient myofibers was measured...
December 2015: Annals of Neurology
Kabir H Biswas, Kevin L Hartman, Cheng-han Yu, Oliver J Harrison, Hang Song, Adam W Smith, William Y C Huang, Wan-Chen Lin, Zhenhuan Guo, Anup Padmanabhan, Sergey M Troyanovsky, Michael L Dustin, Lawrence Shapiro, Barry Honig, Ronen Zaidel-Bar, Jay T Groves
Epithelial (E)-cadherin-mediated cell-cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin (E-cad-ECD) in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility...
September 1, 2015: Proceedings of the National Academy of Sciences of the United States of America
Rodrigo Cáceres, Majdouline Abou-Ghali, Julie Plastino
Actin filament dynamics have been studied for decades in pure protein solutions or in cell extracts, but a breakthrough in the field occurred at the turn of the century when it became possible to reconstitute networks of actin filaments, growing in a controlled but physiological manner on surfaces, mimicking the actin assembly that occurs at the plasma membrane during cell protrusion and cell shape changes. The story begins with the bacteria Listeria monocytogenes, the study of which led to the reconstitution of cellular actin polymerization on a variety of supports including plastic beads...
November 2015: Biochimica et Biophysica Acta
Anastasia Karabina, Katarzyna Kazmierczak, Danuta Szczesna-Cordary, Jeffrey R Moore
Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin's ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010)...
August 15, 2015: Archives of Biochemistry and Biophysics
Renu Mohan, Annie John
The cytoskeletal polymers--actin, microtubules, and intermediate filaments--are interlinked by coordinated protein interactions to form a complex three-dimensional cytoskeletal network. Association of actin filaments with microtubules is important for various cellular processes such as cell division, migration, vesicle and organelle transport, and axonal growth. Several proteins including signaling molecules, motor proteins, and proteins directly or indirectly associated with microtubules and actin are involved in bridging the cytoskeletal components...
June 2015: IUBMB Life
Astrid Walrant, Daniel S Saxton, Guilherme Pereira Correia, Jennifer L Gallop
Xenopus egg extracts are a powerful tool to reconstitute complex cell biological processes using a cell-free strategy. When used in conjunction with liposomes and supported lipid bilayers, they can recapitulate the biochemical activities occurring at the cytosol/plasma membrane interface of the cell that underlie remodeling of the actin cytoskeleton. We use these in vitro systems to elucidate how membranes and proteins collaborate to make the appropriate actin structure at a given time and place. We have recently broadened the types of membrane substrate used, and also optimized protocols for preparation of Xenopus egg extracts for actin assembly assays from membranes...
2015: Methods in Cell Biology
José Alvarado, Gijsje H Koenderink
The actin-myosin cytoskeleton allows cells to move, change shape, and exert forces. These fascinating functions involve active contraction of cross-linked networks of actin filaments by myosin II motor proteins. Unlike muscle cells, where actin and myosin form ordered bundles that contract homogeneously, nonmuscle cells have a variety of more disordered types of actin-myosin meshworks. Active gels reconstituted from purified actin and myosin proteins offer a useful in vitro model system to systematically and quantitatively investigate the mechanisms of contraction and the role of physical parameters like motor activity and network connectivity...
2015: Methods in Cell Biology
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