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Eukaryotic cells contain branched actin networks that are essential for endocytosis, motility, and other key cellular processes. These networks, which are formed by filamentous actin and the Arp2/3 complex, must subsequently be debranched to allow network remodeling and to recycle the Arp2/3 complex. Debranching appears to be catalyzed by two different members of the actin depolymerizing factor homology protein family: cofilin and glial maturation factor (GMF). However, their mechanisms of debranching are only partially understood. Here, we used single-molecule fluorescence imaging of Arp2/3 complex and actin filaments under physiological ionic conditions to observe debranching by GMF and cofilin. We demonstrate that cofilin, like GMF, is an authentic debrancher independent of its filament-severing activity and that the debranching activities of the two proteins are additive. While GMF binds directly to the Arp2/3 complex, cofilin selectively accumulates on branch–junction daughter filaments in tropomyosin-decorated networks just prior to debranching events. Quantitative comparison of debranching rates with the known kinetics of cofilin–actin binding suggests that cofilin occupancy of a particular single actin site at the branch junction is sufficient to trigger debranching. In rare cases in which the order of departure could be resolved during GMF- or cofilin-induced debranching, the Arp2/3 complex left the branch junction bound to the pointed end of the daughter filament, suggesting that both GMF and cofilin can work by destabilizing the mother filament–Arp2/3 complex interface. Taken together, these observations suggest that GMF and cofilin promote debranching by distinct yet complementary mechanisms.

 

Microtubule-based active matter provides insight into the self-organization of motile interacting constituents. We describe several formulations of microtubule-based 3D active isotropic fluids. Dynamics of these fluids is powered by three types of kinesin motors: a processive motor, a non-processive motor, and a motor which is permanently linked to a microtubule backbone. Another modification uses a specific microtubule crosslinker to induce bundle formation instead of a non-specific polymer depletant. In comparison to the already established system, each formulation exhibits distinct properties. These developments reveal the temporal stability of microtubule-based active fluids while extending their reach and the applicability. 

Cellular actin networks grow by ATP-actin addition at filament barbed ends and have long been presumed to depolymerize at their pointed ends, primarily after filaments undergo “aging” (ATP hydrolysis and Pi release). The cytosol contains high levels of actin monomers, which favors assembly over disassembly, and barbed ends are enriched in ADP-Pi actin. For these reasons, the potential for a barbed end depolymerization mechanism in cells has received little attention. Here, using microfluidics-assisted TIRF microscopy, we show that mouse twinfilin, a member of the ADF-homology family, induces depolymerization of ADP-Pi barbed ends even under assembly-promoting conditions. Indeed, we observe in single reactions containing micromolar concentrations of actin monomers the simultaneous rapid elongation of formin-bound barbed ends and twinfilin-induced depolymerization of free barbed ends. The data show that twinfilin catalyzes dissociation of subunits from ADP-Pi barbed ends and thereby bypasses filament aging prerequisites to disassemble newly polymerized actin filaments.

 

Cellular actin networks can be rapidly disassembled and remodeled in a few seconds, yet in vitro actin filaments depolymerize slowly over minutes. The cellular mechanisms enabling actin to depolymerize this fast have so far remained obscure. Using microfluidics-assisted TIRF, we show that Cyclase-associated protein (CAP) and Cofilin synergize to processively depolymerize actin filament pointed ends at a rate 330-fold faster than spontaneous depolymerization. Single molecule imaging further reveals that hexameric CAP molecules interact with the pointed ends of Cofilin-decorated filaments for several seconds at a time, removing approximately 100 actin subunits per binding event. These findings establish a paradigm, in which a filament end-binding protein and a side-binding protein work in concert to control actin dynamics, and help explain how rapid actin network depolymerization is achieved in cells.

 

RNA polymerases (RNAPs) transcribe genes through a cycle of recruitment to promoter DNA, initiation, elongation, and termination. After termination, RNAP is thought to initiate the next round of transcription by detaching from DNA and rebinding a new promoter. Here we use single-molecule fluorescence microscopy to observe individual RNAP molecules after transcript release at a terminator. Following termination, RNAP almost always remains bound to DNA and sometimes exhibits one-dimensional sliding over thousands of basepairs. Unexpectedly, the DNA-bound RNAP often restarts transcription, usually in reverse direction, thus producing an antisense transcript. Furthermore, we report evidence of this secondary initiation in live cells, using genome-wide RNA sequencing. These findings reveal an alternative transcription cycle that allows RNAP to reinitiate without dissociating from DNA, which is likely to have important implications for gene regulation.

 

Formation and turnover of branched actin networks underlies cell migration and other essential force-driven processes. Type I nucleation-promoting factors (NPFs) such as WASP recruit actin monomers to Arp2/3 complex to stimulate nucleation. In contrast, mechanisms of type II NPFs such as Abp1 (also known as HIP55 and Drebrin-like protein) are less well understood. Here, we use single-molecule analysis to investigate yeast Abp1 effects on Arp2/3 complex, and find that Abp1 strongly enhances Arp2/3-dependent branch nucleation by stabilizing Arp2/3 on sides of mother filaments. Abp1 binds dynamically to filament sides, with sub-second lifetimes, yet associates stably with branch junctions. Further, we uncover a role for Abp1 in protecting filament junctions from GMF-induced debranching by competing with GMF for Arp2/3 binding. These data, combined with EM structures of Abp1 dimers bound to Arp2/3 complex in two different conformations, expand our mechanistic understanding of type II NPFs.