keyword
https://read.qxmd.com/read/38493594/arp2-3-complex-and-formin-mediated-actin-cytoskeleton-networks-facilitate-actin-binding-protein-sorting-in-fission-yeast
#1
JOURNAL ARTICLE
Kaitlin E Homa, Glen M Hocky, Cristian Suarez, David R Kovar
While it is well-established that F-actin networks with specific organizations and dynamics are tightly regulated by distinct sets of associated actin-binding proteins (ABPs), how ABPs self-sort to particular F-actin networks remains largely unclear. We report that actin assembly factors Arp2/3 complex and formin Cdc12 tune the association of ABPs fimbrin Fim1 and tropomyosin Cdc8 to different F-actin networks in fission yeast. Genetic and pharmacological disruption of F-actin networks revealed that Fim1 is preferentially directed to Arp2/3-complex mediated actin patches, whereas Cdc8 is preferentially targeted to formin Cdc12-mediated filaments in the contractile ring...
March 16, 2024: European Journal of Cell Biology
https://read.qxmd.com/read/38086388/ckap5-enables-formation-of-persistent-actin-bundles-templated-by-dynamically-instable-microtubules
#2
JOURNAL ARTICLE
Jan Sabo, Michaela Dujava Zdimalova, Paula G Slater, Vojtech Dostal, Stepan Herynek, Lenka Libusova, Laura A Lowery, Marcus Braun, Zdenek Lansky
Cytoskeletal rearrangements and crosstalk between microtubules and actin filaments are vital for living organisms. Recently, an abundantly present microtubule polymerase, CKAP5 (XMAP215 homolog), has been reported to play a role in mediating crosstalk between microtubules and actin filaments in the neuronal growth cones. However, the molecular mechanism of this process is unknown. Here, we demonstrate, in a reconstituted system, that CKAP5 enables the formation of persistent actin bundles templated by dynamically instable microtubules...
December 6, 2023: Current Biology: CB
https://read.qxmd.com/read/38038849/motility-assay-to-probe-the-calcium-sensitivity-of-myosin-and-regulated-thin-filaments
#3
JOURNAL ARTICLE
Chao Liu, Kathleen M Ruppel, James A Spudich
Calcium-dependent activation of the thin filament mediated by the troponin-tropomyosin complex is key in the regulation of actin-myosin based muscle contraction. Perturbations to this system, either physiological (e.g., phosphorylation of myosin light chains) or pathological (e.g., mutations that cause familial cardiomyopathies), can alter calcium sensitivity and thus have important implications in human health and disease. The in vitro motility assay provides a quantitative and precise method to study the calcium sensitivity of the reconstituted myosin-thin filament motile system...
2024: Methods in Molecular Biology
https://read.qxmd.com/read/38003336/a-novel-variant-in-tpm3-causing-muscle-weakness-and-concomitant-hypercontractile-phenotype
#4
JOURNAL ARTICLE
Katarzyna Robaszkiewicz, Małgorzata Siatkowska, Renske I Wadman, Erik-Jan Kamsteeg, Zhiyong Chen, Ashirwad Merve, Matthew Parton, Enrico Bugiardini, Charlotte de Bie, Joanna Moraczewska
A novel variant of unknown significance c.8A > G (p.Glu3Gly) in TPM3 was detected in two unrelated families. TPM3 encodes the transcript variant Tpm3.12 (NM_152263.4), the tropomyosin isoform specifically expressed in slow skeletal muscle fibers. The patients presented with slowly progressive muscle weakness associated with Achilles tendon contractures of early childhood onset. Histopathology revealed features consistent with a nemaline rod myopathy. Biochemical in vitro assays performed with reconstituted thin filaments revealed defects in the assembly of the thin filament and regulation of actin-myosin interactions...
November 9, 2023: International Journal of Molecular Sciences
https://read.qxmd.com/read/37922812/reconstitution-of-actin-based-cellular-processes-why-encapsulation-changes-the-rules
#5
JOURNAL ARTICLE
Fabina Binth Kandiyoth, Alphée Michelot
While in vitro reconstitution of cellular processes is progressing rapidly, the encapsulation of biomimetic systems to reproduce the cellular environment is a major challenge. Here we review the difficulties, using reconstitution of processes dependent on actin polymerization as an example. Some of the problems are purely technical, due to the need for engineering strategies to encapsulate concentrated solutions in micrometer-sized compartments. However, other significant issues arise from the reduction of experimental volumes, which alters the chemical evolution of these non-equilibrium systems...
October 30, 2023: European Journal of Cell Biology
https://read.qxmd.com/read/37909673/spatial-control-of-arp2-3-induced-actin-polymerization-on-phase-separated-giant-unilamellar-vesicles
#6
JOURNAL ARTICLE
Rogério Lopes Dos Santos, Michel Malo, Clément Campillo
Deciphering the physical mechanisms underlying cell shape changes, while avoiding the cellular interior's complexity, involves the development of controlled basic biomimetic systems that imitate cell functions. In particular, the reconstruction of cytoskeletal dynamics on cell-sized giant unilamellar vesicles (GUVs) has allowed for the reconstituting of some cell-like processes in vitro . In fact, such a bottom-up strategy could be the basis for forming protocells able to reorganize or even move autonomously...
November 1, 2023: ACS Synthetic Biology
https://read.qxmd.com/read/37890285/reconstitution-of-the-transition-from-a-lamellipodia-to-filopodia-like-actin-network-with-purified-proteins
#7
JOURNAL ARTICLE
Cristian Suarez, Jonathan D Winkelman, Alyssa J Harker, Hannah J Ye, Patrick M McCall, Alisha N Morganthaler, Margaret L Gardel, David R Kovar
How cells utilize complex mixtures of actin binding proteins to assemble and maintain functionally diverse actin filament networks with distinct architectures and dynamics within a common cytoplasm is a longstanding question in cell biology. A compelling example of complex and specialized actin structures in cells are filopodia which sense extracellular chemical and mechanical signals to help steer motile cells. Filopodia have distinct actin architecture, composed of long, parallel actin filaments bundled by fascin, which form finger-like membrane protrusions...
October 20, 2023: European Journal of Cell Biology
https://read.qxmd.com/read/37889477/split-luciferase-fragment-reconstitution-for-unveiling-rna-localization-and-dynamics-in-live-cells
#8
JOURNAL ARTICLE
Masatoshi Eguchi, Hideaki Yoshimura, Yoshibumi Ueda, Takeaki Ozawa
The intracellular distribution and dynamics of RNAs play pivotal roles in various physiological phenomena. The ability to monitor the amount and localization of endogenous RNAs in living cells allows for elucidating the mechanisms of various intracellular events. Protein-based fluorescent RNA probes are now widely used to visualize and analyze RNAs in living cells. However, continuously monitoring the temporal changes in RNA localization and dynamics in living cells is challenging. In this study, we developed a bioluminescent probe for spatiotemporal monitoring of RNAs in living cells by using a split-luciferase reconstitution technique...
October 27, 2023: ACS Sensors
https://read.qxmd.com/read/37870863/myosin-and-tropomyosin-troponin-complementarily-regulate-thermal-activation-of-muscles
#9
JOURNAL ARTICLE
Shuya Ishii, Kotaro Oyama, Fuyu Kobirumaki-Shimozawa, Tomohiro Nakanishi, Naoya Nakahara, Madoka Suzuki, Shin'ichi Ishiwata, Norio Fukuda
Contraction of striated muscles is initiated by an increase in cytosolic Ca2+ concentration, which is regulated by tropomyosin and troponin acting on actin filaments at the sarcomere level. Namely, Ca2+-binding to troponin C shifts the "on-off" equilibrium of the thin filament state toward the "on" state, promoting actomyosin interaction; likewise, an increase in temperature to within the body temperature range shifts the equilibrium to the on state, even in the absence of Ca2+. Here, we investigated the temperature dependence of sarcomere shortening along isolated fast skeletal myofibrils using optical heating microscopy...
December 4, 2023: Journal of General Physiology
https://read.qxmd.com/read/37870087/microtubules-under-mechanical-pressure-can-breach-dense-actin-networks
#10
JOURNAL ARTICLE
Matthieu Gélin, Alexandre Schaeffer, Jérémie Gaillard, Christophe Guérin, Benoit Vianay, Magali Orhant-Prioux, Marcus Braun, Christophe Leterrier, Laurent Blanchoin, Manuel Théry
The crosstalk between the actin network and microtubules is essential for cell polarity. It orchestrates microtubule organization within the cell, driven by the asymmetry of actin architecture along the cell periphery. The physical intertwining of these networks regulates spatial organization and force distribution in the microtubule network. While their biochemical interactions are becoming clearer, the mechanical aspects remain less understood. To explore this mechanical interplay, we developed an in vitro reconstitution assay to investigate how dynamic microtubules interact with various actin filament structures...
October 23, 2023: Journal of Cell Science
https://read.qxmd.com/read/37838354/direct-observation-of-oriented-behavior-of-actin-filaments-interacting-with-desmin-intermediate-filaments
#11
JOURNAL ARTICLE
Takumi Ishizaka, Kuniyuki Hatori
BACKGROUND: Associations between actin filaments (AFs) and intermediate filaments (IFs) are frequently observed in living cells. The crosstalk between these cytoskeletal components underpins cellular organization and dynamics; however, the molecular basis of filamentous interactions is not fully understood. Here, we describe the mode of interaction between AFs and desmin IFs (DIFs) in a reconstituted in vitro system. METHODS: AFs (rabbit skeletal muscle) and DIFs (chicken gizzard) were labeled with fluorescent dyes...
October 12, 2023: Biochimica et Biophysica Acta. General Subjects
https://read.qxmd.com/read/37790449/synthetic-control-of-actin-polymerization-and-symmetry-breaking-in-active-protocells
#12
Shiva Razavi, Felix Wong, Bedri Abubaker-Sharif, Hideaki T Matsubayashi, Hideki Nakamura, Eduardo Sandoval, Douglas N Robinson, Baoyu Chen, Jian Liu, Pablo A Iglesias, Takanari Inoue
Non-linear biomolecular interactions on the membranes drive membrane remodeling that underlies fundamental biological processes including chemotaxis, cytokinesis, and endocytosis. The multitude of biomolecules, the redundancy in their interactions, and the importance of spatiotemporal context in membrane organization hampers understanding the physical principles governing membrane mechanics. A minimal, in vitro system that models the functional interactions between molecular signaling and membrane remodeling, while remaining faithful to cellular physiology and geometry is powerful yet remains unachieved...
September 23, 2023: bioRxiv
https://read.qxmd.com/read/37778219/purification-of-modified-mammalian-actin-isoforms-for-in-vitro-reconstitution-assays
#13
JOURNAL ARTICLE
David J Kast, Silvia Jansen
In vitro reconstitution assays using purified actin have greatly improved our understanding of cytoskeletal dynamics and their regulation by actin-binding proteins. However, early purification methods consisted of harsh conditions to obtain pure actin and often did not include correct maturation and obligate modification of the isolated actin monomers. Novel insights into the folding requirements and N-terminal processing of actin as well as a better understanding of the interaction of actin with monomer sequestering proteins such as DNaseI, profilin and gelsolin, led to the development of more gentle approaches to obtain pure recombinant actin isoforms with known obligate modifications...
September 28, 2023: European Journal of Cell Biology
https://read.qxmd.com/read/37749275/molecular-mechanisms-of-inorganic-phosphate-release-from-the-core-and-barbed-end-of-actin-filaments
#14
JOURNAL ARTICLE
Wout Oosterheert, Florian E C Blanc, Ankit Roy, Alexander Belyy, Micaela Boiero Sanders, Oliver Hofnagel, Gerhard Hummer, Peter Bieling, Stefan Raunser
The release of inorganic phosphate (Pi ) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. The mechanisms underlying Pi release from the core and barbed end of actin filaments remain unclear. Here, using human and bovine actin isoforms, we combine cryo-EM with molecular-dynamics simulations and in vitro reconstitution to demonstrate how actin releases Pi through a 'molecular backdoor'. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and opens only transiently through concerted amino acid rearrangements...
September 25, 2023: Nature Structural & Molecular Biology
https://read.qxmd.com/read/37725653/friction-patterns-guide-actin-network-contraction
#15
JOURNAL ARTICLE
Alexandra Colin, Magali Orhant-Prioux, Christophe Guérin, Mariya Savinov, Wenxiang Cao, Benoit Vianay, Ilaria Scarfone, Aurélien Roux, Enrique M De La Cruz, Alex Mogilner, Manuel Théry, Laurent Blanchoin
The shape of cells is the outcome of the balance of inner forces produced by the actomyosin network and the resistive forces produced by cell adhesion to their environment. The specific contributions of contractile, anchoring and friction forces to network deformation rate and orientation are difficult to disentangle in living cells where they influence each other. Here, we reconstituted contractile actomyosin networks in vitro to study specifically the role of the friction forces between the network and its anchoring substrate...
September 26, 2023: Proceedings of the National Academy of Sciences of the United States of America
https://read.qxmd.com/read/37645911/a-platform-for-dissecting-force-sensitivity-and-multivalency-in-actin-networks
#16
Joseph T Levin, Ariel Pan, Michael T Barrett, Gregory M Alushin
The physical structure and dynamics of cells are supported by micron-scale actin networks with diverse geometries, protein compositions, and mechanical properties. These networks are composed of actin filaments and numerous actin binding proteins (ABPs), many of which engage multiple filaments simultaneously to crosslink them into specific functional architectures. Mechanical force has been shown to modulate the interactions between several ABPs and individual actin filaments, but it is unclear how this phenomenon contributes to the emergent force-responsive functional dynamics of actin networks...
August 16, 2023: bioRxiv
https://read.qxmd.com/read/37406300/filopodia-in-vitro-and-in-vivo
#17
REVIEW
Thomas C A Blake, Jennifer L Gallop
Filopodia are dynamic cell surface protrusions used for cell motility, pathogen infection, and tissue development. The molecular mechanisms determining how and where filopodia grow and retract need to integrate mechanical forces and membrane curvature with extracellular signaling and the broader state of the cytoskeleton. The involved actin regulatory machinery nucleates, elongates, and bundles actin filaments separately from the underlying actin cortex. The refined membrane and actin geometry of filopodia, importance of tissue context, high spatiotemporal resolution required, and high degree of redundancy all limit current models...
July 5, 2023: Annual Review of Cell and Developmental Biology
https://read.qxmd.com/read/37318128/phase-separation-mediated-actin-bundling-by-the-postsynaptic-density-condensates
#18
JOURNAL ARTICLE
Xudong Chen, Bowen Jia, Shihan Zhu, Mingjie Zhang
The volume and the electric strength of an excitatory synapse is near linearly correlated with the area of its postsynaptic density (PSD). Extensive research in the past has revealed that the PSD assembly directly communicates with actin cytoskeleton in the spine to coordinate activity-induced spine volume enlargement as well as long-term stable spine structure maintenance. However, the molecular mechanism underlying the communication between the PSD assembly and spine actin cytoskeleton is poorly understood...
June 15, 2023: ELife
https://read.qxmd.com/read/37113335/in-vitro-reconstitution-of-phase-separated-p62-bodies-on-the-arp2-3-derived-actin-network
#19
JOURNAL ARTICLE
Tong Liu, Mengbo Xu, Na Mi
In cells, p62/SQSTM1 undergoes liquid-liquid phase separation (LLPS) with poly-ubiquitin chains to form p62 bodies that work as a hub for various cellular events, including selective autophagy. Cytoskeleton components such as Arp2/3-derived branched actin network and motor protein myosin 1D have been shown to actively participate in the formation of phase-separated p62 bodies. Here, we describe a detailed protocol on the purification of p62 and other proteins, the assembly of the branched actin network, and the reconstitution of p62 bodies along with cytoskeletal structures in vitro...
April 20, 2023: Bio-protocol
https://read.qxmd.com/read/36971445/the-mechanics-of-poro-elastic-contractile-actomyosin-networks-as-a-model-system-of-the-cell-cytoskeleton
#20
JOURNAL ARTICLE
Sakshi Choudhary, Gefen Livne, Shachar Gat, Anne Bernheim-Groswasser
Cells can actively change their shapes and become motile, a property that depends on their ability to actively reorganize their internal structure. This feature is attributed to the mechanical and dynamic properties of the cell cytoskeleton, notably, the actomyosin cytoskeleton, which is an active gel of polar actin filaments, myosin motors, and accessory proteins that exhibit intrinsic contraction properties. The usually accepted view is that the cytoskeleton behaves as a viscoelastic material. However, this model cannot always explain the experimental results, which are more consistent with a picture describing the cytoskeleton as a poroelastic active material-an elastic network embedded with cytosol...
March 10, 2023: Journal of Visualized Experiments: JoVE
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