Regulators of actin filament barbed ends at a glance

Shekhar, Shashank; Pernier, Julien; Carlier, Marie-France

doi:10.1242/jcs.179994

Cited by 82 publications

(79 citation statements)

References 71 publications

(86 reference statements)

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“…The turnover dynamics of actin filaments occur preferentially at filaments ends, with polymerization occurring at uncapped barbed ends and depolymerization at pointed ends [1,6]. To map the distribution of uncapped filaments ends we permeabilize fragments, following a brief fixation step, and introduce fluorescent actin monomers that polymerize onto the uncapped filament ends and label them [35,36].…”

Section: Resultsmentioning

confidence: 99%

“…As new actin assembles at the front, older filaments are pushed away from the leading edge and eventually disassemble. Despite substantial progress in studying the regulation of lamellipodial actin dynamics [2,5,6], its complexity still defies quantitative understanding [7]. …”

Section: Introductionmentioning

confidence: 99%

“…Since a detailed molecular picture of all the reactions involved in actin turnover in motile cells is still beyond reach [6,28], our aim here is to characterize actin turnover and transport in a coarse-grained, yet quantitative manner. We seek to measure the spatial distributions of the main lamellipodial actin subpopulations and the transitions between them.…”

Section: Introductionmentioning

confidence: 99%

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Actin Turnover in Lamellipodial Fragments

et al. 2017

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Summary Actin turnover is the central driving force underlying lamellipodial motility. The molecular components involved are largely known, and their properties have been studied extensively in vitro. However, a comprehensive picture of actin turnover in vivo is still missing. We focus on fragments from fish epithelial keratocytes, which are essentially stand-alone motile lamellipodia. The geometric simplicity of fragments and the absence of additional actin structures allow us to characterize the spatiotemporal lamellipodial actin organization with unprecedented detail. We use fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and extraction experiments to show that about two thirds of the lamellipodial actin diffuses in the cytoplasm with nearly uniform density, while the rest forms the treadmilling polymer network. Roughly a quarter of the diffusible actin pool is in filamentous form as diffusing oligomers, indicating that severing and debranching are important steps in the disassembly process generating oligomers as intermediates. The remaining diffusible actin concentration is orders of magnitude higher than the in vitro actin monomer concentration required to support the observed polymerization rates, implying that the majority of monomers are transiently kept in a nonpolymerizable ‘reserve’ pool. The actin network disassembles and reassembles throughout the lamellipodium within seconds, so the lamellipodial network turnover is local. The diffusible actin transport, on the other hand, is global: actin subunits typically diffuse across the entire lamellipodium before reassembling into the network. This combination of local network turnover and global transport of dissociated subunits through the cytoplasm makes actin transport robust, yet rapidly adaptable and amenable to regulation.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Actin Turnover in Lamellipodial Fragments

et al. 2017

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“…Actin filaments grow and shrink by addition and loss, respectively, of actin subunits at the ends of filaments. The barbed (plus) end of the filament is favored over the pointed (minus) end for assembly, both thermodynamically and kinetically (Pollard, 2016), and cells control their shape and migration by regulating barbed-end filament assembly spatially and temporally (Shekhar et al , 2016). Examples of such regulation are numerous and affect a wide range of processes, including development and differentiation (Harris et al , 2009), immunity and inflammation (Marcos-Ramiro et al , 2014), and cancer cell invasion and metastasis (Kumar and Weaver, 2009; Mierke, 2013).…”

Section: Introductionmentioning

confidence: 99%

CARMIL family proteins as multidomain regulators of actin-based motility

Stark

Lanier

Cooper

2017

MBoC

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CARMILs are large multidomain proteins that regulate the actin-binding activity of capping protein (CP), a major capper of actin filament barbed ends in cells. CARMILs bind directly to CP and induce a conformational change that allosterically decreases but does not abolish its actin-capping activity. The CP-binding domain of CARMIL consists of the CP-interaction (CPI) and CARMIL-specific interaction (CSI) motifs, which are arranged in tandem. Many cellular functions of CARMILs require the interaction with CP; however, a more surprising result is that the cellular function of CP in cells appears to require binding to a CARMIL or another protein with a CPI motif, suggesting that CPI-motif proteins target CP and modulate its actin-capping activity. Vertebrates have three highly conserved genes and expressed isoforms of CARMIL with distinct and overlapping localizations and functions in cells. Various domains of these CARMIL isoforms interact with plasma membranes, vimentin intermediate filaments, SH3-containing class I myosins, the dual-GEF Trio, and other adaptors and signaling molecules. These biochemical properties suggest that CARMILs play a variety of membrane-associated functions related to actin assembly and signaling. CARMIL mutations and variants have been implicated in several human diseases. We focus on roles for CARMILs in signaling in addition to their function as regulators of CP and actin.

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“…Continued generation of the monomer pool by filament disassembly is therefore crucial. Disassemblers like actin depolymerizing factor (ADF)/cofilin and filament cappers like capping protein (CP) are essential agonists of motility [3, 4, 5, 6, 7, 8], but the exact molecular mechanisms by which they accelerate actin polymerization at the leading edge and filament turnover has been debated for over two decades [9, 10, 11, 12]. Whereas filament fragmentation by ADF/cofilin has long been demonstrated by total internal reflection fluorescence (TIRF) [13, 14], filament depolymerization was only inferred from bulk solution assays [15].…”

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confidence: 99%

Enhanced Depolymerization of Actin Filaments by ADF/Cofilin and Monomer Funneling by Capping Protein Cooperate to Accelerate Barbed-End Growth

Shekhar

Carlier

2017

Current Biology

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SummaryA living cell’s ability to assemble actin filaments in intracellular motile processes is directly dependent on the availability of polymerizable actin monomers, which feed polarized filament growth [1, 2]. Continued generation of the monomer pool by filament disassembly is therefore crucial. Disassemblers like actin depolymerizing factor (ADF)/cofilin and filament cappers like capping protein (CP) are essential agonists of motility [3, 4, 5, 6, 7, 8], but the exact molecular mechanisms by which they accelerate actin polymerization at the leading edge and filament turnover has been debated for over two decades [9, 10, 11, 12]. Whereas filament fragmentation by ADF/cofilin has long been demonstrated by total internal reflection fluorescence (TIRF) [13, 14], filament depolymerization was only inferred from bulk solution assays [15]. Using microfluidics-assisted TIRF microscopy, we provide the first direct visual evidence of ADF’s simultaneous severing and rapid depolymerization of individual filaments. Using a conceptually novel assay to directly visualize ADF’s effect on a population of pre-assembled filaments, we demonstrate how ADF’s enhanced pointed-end depolymerization causes an increase in polymerizable actin monomers, thus promoting faster barbed-end growth. We further reveal that ADF-enhanced depolymerization synergizes with CP’s long-predicted “monomer funneling” [16] and leads to skyrocketing of filament growth rates, close to estimated lamellipodial rates. The “funneling model” hypothesized, on thermodynamic grounds, that at high enough extent of capping, the few non-capped filaments transiently grow much faster [15], an effect proposed to be very important for motility. We provide the first direct microscopic evidence of monomer funneling at the scale of individual filaments. These results significantly enhance our understanding of the turnover of cellular actin networks.

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Regulators of actin filament barbed ends at a glance

Cited by 82 publications

References 71 publications

Actin Turnover in Lamellipodial Fragments

Actin Turnover in Lamellipodial Fragments

CARMIL family proteins as multidomain regulators of actin-based motility

Enhanced Depolymerization of Actin Filaments by ADF/Cofilin and Monomer Funneling by Capping Protein Cooperate to Accelerate Barbed-End Growth

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