scholarly journals Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin

2013 ◽  
Vol 24 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Faisal Chaudhry ◽  
Dennis Breitsprecher ◽  
Kristin Little ◽  
Grigory Sharov ◽  
Olga Sokolova ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Genetic Analysis ◽  
Fluorescence Microscopy ◽  
Actin Filament ◽  
Total Internal Reflection ◽  
Cellular Function ◽  
Internal Reflection ◽  
Actin Networks ◽  
C Terminus

Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert.

scholarly journals Tropomyosin isoforms differentially tune actin filament length and disassembly

2019 ◽  
Vol 30 (5) ◽  
pp. 671-679 ◽  
Author(s):  
Silvia Jansen ◽  
Bruce L. Goode
Keyword(s):  
Fluorescence Microscopy ◽  
Actin Filament ◽  
Actin Filaments ◽  
Total Internal Reflection ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Filament Length ◽  
Actin Networks ◽  
Tropomyosin Isoforms ◽  
Specific Effects

Cellular actin networks exhibit diverse filamentous architectures and turnover dynamics, but how these differences are specified remains poorly understood. Here, we used multicolor total internal reflection fluorescence microscopy to ask how decoration of actin filaments by five biologically prominent Tropomyosin (TPM) isoforms influences disassembly induced by Cofilin alone, or by the collaborative effects of Cofilin, Coronin, and AIP1 (CCA). TPM decoration restricted Cofilin binding to pointed ends, while not interfering with Coronin binding to filament sides. Different isoforms of TPM provided variable levels of protection against disassembly, with the strongest protection by Tpm3.1 and the weakest by Tpm1.6. In biomimetic assays in which filaments were simultaneously assembled by formins and disassembled by CCA, these TPM isoform–specific effects persisted, giving rise to filaments with different lengths and treadmilling behavior. Together, our data reveal that TPM isoforms have quantitatively distinct abilities to tune actin filament length and turnover.

scholarly journals Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation

Science ◽  
2018 ◽  
Vol 361 (6404) ◽  
pp. eaau1504 ◽  
Author(s):  
Annapurna Vemu ◽  
Ewa Szczesna ◽  
Elena A. Zehr ◽  
Jeffrey O. Spector ◽  
Nikolaus Grigorieff ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Total Internal Reflection ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
High Density ◽  
Guanosine Triphosphate ◽  
Microtubule Severing

Spastin and katanin sever and destabilize microtubules. Paradoxically, despite their destructive activity they increase microtubule mass in vivo. We combined single-molecule total internal reflection fluorescence microscopy and electron microscopy to show that the elemental step in microtubule severing is the generation of nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers. These damage sites are repaired spontaneously by guanosine triphosphate (GTP)–tubulin incorporation, which rejuvenates and stabilizes the microtubule shaft. Consequently, spastin and katanin increase microtubule rescue rates. Furthermore, newly severed ends emerge with a high density of GTP-tubulin that protects them against depolymerization. The stabilization of the newly severed plus ends and the higher rescue frequency synergize to amplify microtubule number and mass. Thus, severing enzymes regulate microtubule architecture and dynamics by promoting GTP-tubulin incorporation within the microtubule shaft.

scholarly journals Myosin 1b Flattens and Prunes Branched Actin Filaments

2020 ◽  
Author(s):  
Julien Pernier ◽  
Antoine Morchain ◽  
Valentina Caorsi ◽  
Aurélie Bertin ◽  
Hugo Bousquet ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Molecular Motor ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Actin Network ◽  
Tirf Microscopy ◽  
Actin Networks ◽  
Cellular Processes

AbstractMotile and morphological cellular processes require a spatially and temporally coordinated branched actin network that is controlled by the activity of various regulatory proteins including the Arp2/3 complex, profilin, cofilin and tropomyosin. We have previously reported that myosin 1b regulates the density of the actin network in the growth cone. Using in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we show in this report that this molecular motor flattens the Arp2/3-dependent actin branches up to breaking them and reduces the probability to form new branches. This experiment reveals that myosin 1b can produce force sufficient enough to break up the Arp2/3-mediated actin junction. Together with the former in vivo studies, this work emphasizes the essential role played by myosins in the architecture and in the dynamics of actin networks in different cellular regions.Short summaryUsing in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we show that myosin flattens the Arp2/3-dependent actin branches up to breaking them and reduces the probability to form new branches

scholarly journals Synergy between Wsp1 and Dip1 may initiate assembly of endocytic actin networks

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Connor J Balzer ◽  
Michael L James ◽  
Heidy Y Narvaez-Ortiz ◽  
Luke A Helgeson ◽  
Vladimir Sirotkin ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Schizosaccharomyces Pombe ◽  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Total Internal Reflection ◽  
Actin Assembly ◽  
Actin Network ◽  
Actin Networks ◽  
Co Activation

The actin filament nucleator Arp2/3 complex is activated at cortical sites in Schizosaccharomyces pombe to assemble branched actin networks that drive endocytosis. Arp2/3 complex activators Wsp1 and Dip1 are required for proper actin assembly at endocytic sites, but how they coordinately control Arp2/3-mediated actin assembly is unknown. Alone, Dip1 activates Arp2/3 complex without preexisting actin filaments to nucleate ‘seed’ filaments that activate Wsp1-bound Arp2/3 complex, thereby initiating branched actin network assembly. In contrast, because Wsp1 requires preexisting filaments to activate, it has been assumed to function exclusively in propagating actin networks by stimulating branching from preexisting filaments. Here we show that Wsp1 is important not only for propagation but also for initiation of endocytic actin networks. Using single molecule total internal reflection fluorescence microscopy we show that Wsp1 synergizes with Dip1 to co-activate Arp2/3 complex. Synergistic co-activation does not require preexisting actin filaments, explaining how Wsp1 contributes to actin network initiation in cells.

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