scholarly journals Dissection of two parallel pathways for formin-mediated actin filament elongation

2018 ◽  
Vol 293 (46) ◽  
pp. 17917-17928 ◽  
Author(s):  
Laura A. Sherer ◽  
Mark E. Zweifel ◽  
Naomi Courtemanche
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Total Internal Reflection ◽  
Actin Polymerization ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
The Other ◽  
Tirf Microscopy ◽  
Parallel Pathways

Formins direct the elongation of unbranched actin filaments that are incorporated into a diverse set of cytoskeletal structures. Elongation of formin-bound filaments occurs along two parallel pathways. The formin homology 2 (FH2) pathway allows actin monomers to bind directly to barbed ends bound by dimeric FH2 domains. The formin homology 1 (FH1) pathway involves transfer of profilin-bound actin to the barbed end from polyproline tracts located in the disordered FH1 domains. Here, we used a total internal reflection fluorescence (TIRF) microscopy-based fluorescence approach to determine the fraction of actin subunits incorporated via the FH1 and FH2 pathways during filament elongation mediated by two formins. We found that the fraction of filament elongation that occurs via each pathway directly depends on the efficiency of the other pathway, indicating that these two pathways compete with each other for subunit addition by formins. We conclude that this competition allows formins to compensate for changes in the efficiency of one pathway by adjusting the frequency of subunit addition via the other, thus increasing the overall robustness of formin-mediated actin polymerization.

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 Total Internal Reflection Fluorescence (TIRF) Microscopy of Chlamydomonas Flagella

2009 ◽  
pp. 157-177 ◽  
Author(s):  
Benjamin D. Engel ◽  
Karl-Ferdinand Lechtreck ◽  
Tsuyoshi Sakai ◽  
Mitsuo Ikebe ◽  
George B. Witman ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Tirf Microscopy

scholarly journals Identification of novel insulin mimetic drugs by quantitative total internal reflection fluorescence ( TIRF ) microscopy

2014 ◽  
Vol 171 (23) ◽  
pp. 5237-5251 ◽  
Author(s):  
Peter Lanzerstorfer ◽  
Verena Stadlbauer ◽  
Lilia A Chtcheglova ◽  
Renate Haselgrübler ◽  
Daniela Borgmann ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Tirf Microscopy ◽  
Insulin Mimetic

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

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