Genetically inspired in vitro reconstitution of<i>Saccharomyces cerevisiae</i>actin cables from seven purified proteins

Pollard, Luther W.; Garabedian, Mikael V.; Alioto, Salvatore L.; Shekhar, Shashank; Goode, Bruce L.

doi:10.1091/mbc.e19-10-0576

Cited by 12 publications

(11 citation statements)

References 71 publications

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“…Those studies demonstrated that robust and tunable actin flows are regulated by two main components: the actin turnover rate and the network geometry. In parallel, reconstitution of dynamic networks with a balance between assembly and disassembly mediated by actin polymerization and disassembly by ADF/cofilin (Michelot et al , 2007; Akin & Mullins, 2008; Reymann et al , 2011; Manhart et al , 2019; Pollard et al , 2020) has allowed to better understand the role of the different molecular actors in the establishment of a dynamic steady state. However, those experiments were performed either with cell extracts where precise protein content is unknown or with purified proteins in unlimited volumes (therefore masking some crucial steps existing when a limited amount of component is available).…”

Section: Introductionmentioning

confidence: 99%

Recycling limits the lifetime of actin turnover

Blanchoin

Théry

Colin

et al. 2022

Preprint

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Intracellular organization is largely mediated by the actin turnover. Cellular actin networks consume matter and energy to sustain their dynamics, while maintaining their appearance. This behavior, called 'dynamic steady state', enables cells to sense and adapt to their environment. However, how structural stability can be maintained during the constant turnover of a limited actin monomer pool is poorly understood. To answer this question, we developed an experimental system using actin bead motility in a cell-sized compartment with a limited amount of monomer. We used the speed and the size of the actin comet tails to evaluate the system's monomer consumption and its lifetime. We established the relative contribution of actin assembly, disassembly and recycling for a bead movement over tens of hours. Recycling mediated by cyclase-associated proteins is the key step in allowing the reuse of monomers for multiple assembly cycles. Energy supply and protein aging are also factors that limit the lifetime of actin turnover. This work reveals the balancing mechanism for long-term network assembly with a limited amount of building blocks.

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Section: Introductionmentioning

confidence: 99%

Recycling limits the lifetime of actin turnover

Blanchoin

Théry

Colin

et al. 2022

Preprint

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“…Assays can be further modified to use cell extracts that may add the “missing” unknown key factors required to recapitulate cell-like phenomena. For example, TIRF-based assays employing yeast or Xenopus extracts have reconstituted contractile actomyosin rings 37 , mitotic spindles 26 , 38 , components of actin or microtubule assembly 39 , 40 , and even dynamics at the centrosome and kinetochores 36 , 41 , 42 , 43 . Moreover, such systems may pave the way toward artificial cell systems that have lipids or signaling factors present 44 , 45 , 46 .…”

Section: Discussionmentioning

confidence: 99%

Visualizing Actin and Microtubule Coupling Dynamics <em>In Vitro</em> by Total Internal Reflection Fluorescence (TIRF) Microscopy

Henty-Ridilla¹

2022

JoVE

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Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actinmicrotubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actinmicrotubule dynamics at a resolution of single filaments or higher-order complexes.

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“…Elegant in vitro studies confirm at the level of single actin filaments that tropomyosin excludes other ABPs, such as fimbrin or ADF/cofilin, therefore preventing filaments from disassembly [129,134,148,149]. Interestingly, tropomyosin is not required per se to assemble cables in vitro in the absence of disassembling factors but it becomes necessary to maintain cable assembly in biomimetic assays where treadmilling has been reconstituted [136,150]. Tropomyosins are hence major biochemical regulators that define the identity of actin filaments and regulate the binding of many families of ABPs, thereby leading to the segregation of these proteins to different actin networks.…”

Section: Tropomyosins and The Biogenesis Of New Actin Substratesmentioning

confidence: 99%

Diversity from similarity: cellular strategies for assigning particular identities to actin filaments and networks

2020

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The actin cytoskeleton has the particularity of being assembled into many functionally distinct filamentous networks from a common reservoir of monomeric actin. Each of these networks has its own geometrical, dynamical and mechanical properties, because they are capable of recruiting specific families of actin-binding proteins (ABPs), while excluding the others. This review discusses our current understanding of the underlying molecular mechanisms that cells have developed over the course of evolution to segregate ABPs to appropriate actin networks. Segregation of ABPs requires the ability to distinguish actin networks as different substrates for ABPs, which is regulated in three different ways: (1) by the geometrical organization of actin filaments within networks, which promotes or inhibits the accumulation of ABPs; (2) by the identity of the networks' filaments, which results from the decoration of actin filaments with additional proteins such as tropomyosin, from the use of different actin isoforms or from covalent modifications of actin; (3) by the existence of collaborative or competitive binding to actin filaments between two or multiple ABPs. This review highlights that all these effects need to be taken into account to understand the proper localization of ABPs in cells, and discusses what remains to be understood in this field of research.

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Genetically inspired in vitro reconstitution ofSaccharomyces cerevisiaeactin cables from seven purified proteins

Abstract: Yeast actin cables are reconstituted from seven purified proteins, providing a powerful demonstration of how a minimal set of components can self-organize into a micron-scale structure that has many of the same features of actin cables found in vivo.

Cited by 12 publications

References 71 publications

Recycling limits the lifetime of actin turnover

Recycling limits the lifetime of actin turnover

Visualizing Actin and Microtubule Coupling Dynamics <em>In Vitro</em> by Total Internal Reflection Fluorescence (TIRF) Microscopy

Diversity from similarity: cellular strategies for assigning particular identities to actin filaments and networks

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