Length control emerges from cytoskeletal network geometry.

Many cytoskeletal networks consist of individual filaments that are organized into elaborate higher-order structures. While it is appreciated that the size and architecture of these networks are critical for their biological functions, much of the work investigating control over their assembly has focused on mechanisms that regulate the turnover of individual filaments through size-dependent feedback. Here, we propose a very different, feedback-independent mechanism to explain how yeast cells control the length of their actin cables.

The roles of yeast formins and their regulators Bud6 and Bil2 in the pheromone response.

In response to pheromone extend a mating projection. This process depends on the formation of polarized actin cables which direct secretion to the mating tip and translocate the nucleus for karyogamy. Here, we demonstrate that proper mating projection formation requires the formin Bni1, as well as the actin nucleation promoting activities of Bud6, but not the formin Bnr1.

Length control emerges from cytoskeletal network geometry.

Many cytoskeletal networks consist of individual filaments that are organized into elaborate higher order structures. While it is appreciated that the size and architecture of these networks are critical for their biological functions, much of the work investigating control over their assembly has focused on mechanisms that regulate the turnover of individual filaments through size-dependent feedback. Here, we propose a very different, feedback-independent mechanism to explain how yeast cells control the length of their actin cables.

Direct observation of cortactin protecting Arp2/3-actin filament branch junctions from GMF-mediated destabilization.

How cells tightly control the formation and turnover of branched actin filament arrays to drive cell motility, endocytosis, and other cellular processes is still not well understood. Here, we investigated the mechanistic relationship between two binding partners of the Arp2/3 complex, glia maturation factor (GMF) and cortactin. Individually, GMF and cortactin have opposite effects on the stability of actin filament branches, but it is unknown how they work in concert with each other to govern branch turnover.

Mechanisms of actin disassembly and turnover.

Cellular actin networks exhibit a wide range of sizes, shapes, and architectures tailored to their biological roles. Once assembled, these filamentous networks are either maintained in a state of polarized turnover or induced to undergo net disassembly. Further, the rates at which the networks are turned over and/or dismantled can vary greatly, from seconds to minutes to hours or even days.

Cyclase-associated protein interacts with actin filament barbed ends to promote depolymerization and formin displacement.

Cyclase-associated protein (CAP) has emerged as a central player in cellular actin turnover, but its molecular mechanisms of action are not yet fully understood. Recent studies revealed that the N terminus of CAP interacts with the pointed ends of actin filaments to accelerate depolymerization in conjunction with cofilin. Here, we use in vitro microfluidics-assisted TIRF microscopy to show that the C terminus of CAP promotes depolymerization at the opposite (barbed) ends of actin filaments.

Cytosolic concentrations of actin binding proteins and the implications for in vivo F-actin turnover.

Understanding how numerous actin-binding proteins (ABPs) work in concert to control the assembly, organization, and turnover of the actin cytoskeleton requires quantitative information about the levels of each component. Here, we measured the cellular concentrations of actin and the majority of the conserved ABPs in Saccharomyces cerevisiae, as well as the free (cytosolic) fractions of each ABP. The cellular concentration of actin is estimated to be 13.

Dynamic remodeling of actin networks by cyclase-associated protein and CAP-Abp1 complexes.

How actin filaments are spatially organized and remodeled into diverse higher-order networks in vivo is still not well understood. Here, we report an unexpected F-actin "coalescence" activity driven by cyclase-associated protein (CAP) and enhanced by its interactions with actin-binding protein 1 (Abp1). We directly observe S.

Bitesize bundles F-actin and influences actin remodeling in syncytial embryo development.

Actin networks undergo rearrangements that influence cell and tissue shape. Actin network assembly and organization is regulated in space and time by a host of actin binding proteins. The Synaptotagmin-like protein, Bitesize (Btsz), is known to organize actin at epithelial cell apical junctions in a manner that depends on its interaction with the actin-binding protein, Moesin.

Tyrosyl-tRNA synthetase has a noncanonical function in actin bundling.

Dominant mutations in tyrosyl-tRNA synthetase (YARS1) and six other tRNA ligases cause Charcot-Marie-Tooth peripheral neuropathy (CMT). Loss of aminoacylation is not required for their pathogenicity, suggesting a gain-of-function disease mechanism. By an unbiased genetic screen in Drosophila, we link YARS1 dysfunction to actin cytoskeleton organization.

Evolutionary tuning of barbed end competition allows simultaneous construction of architecturally distinct actin structures.

How cells simultaneously assemble actin structures of distinct sizes, shapes, and filamentous architectures is still not well understood. Here, we used budding yeast as a model to investigate how competition for the barbed ends of actin filaments might influence this process. We found that while vertebrate capping protein (CapZ) and formins can simultaneously associate with barbed ends and catalyze each other's displacement, yeast capping protein (Cap1/2) poorly displaces both yeast and vertebrate formins.

Single-molecule analysis of actin filament debranching by cofilin and GMF.

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).

Quantitative Analysis of Actin Cable Length in Yeast.

Polarized actin cables in are linear bundles of crosslinked actin filaments that are assembled by two formins, Bnr1 (localized to the bud neck), and Bni1 (localized to the bud tip). Actin is polymerized at these two sites, which results in cables extending along the cell cortex toward the back of the mother cell. These cables serve as polarized tracks for myosin-based transport of secretory vesicles and other cargo, from the mother cell to the growing daughter cell.

Extensile to contractile transition in active microtubule-actin composites generates layered asters with programmable lifetimes.

We study a reconstituted composite system consisting of an active microtubule network interdigitated with a passive network of entangled F-actin filaments. Increasing the concentration of filamentous actin controls the emergent dynamics, inducing a transition from turbulent-like flows to bulk contractions. At intermediate concentrations, where the active stresses change their symmetry from anisotropic extensile to isotropic contracting, the composite separates into layered asters that coexist with the background turbulent fluid.

Single-molecule imaging of IQGAP1 regulating actin filament dynamics.

IQGAP is a conserved family of actin-binding proteins with essential roles in cell motility, cytokinesis, and cell adhesion, yet there remains a limited understanding of how IQGAP proteins directly influence actin filament dynamics. To close this gap, we used single-molecule and single-filament total internal reflection fluorescence microscopy to observe IQGAP regulating actin dynamics in real time. To our knowledge, this is the first study to do so.

Scaling of subcellular actin structures with cell length through decelerated growth.

How cells tune the size of their subcellular parts to scale with cell size is a fundamental question in cell biology. Until now, most studies on the size control of organelles and other subcellular structures have focused on scaling relationships with cell volume, which can be explained by limiting pool mechanisms. Here, we uncover a distinct scaling relationship with cell length rather than volume, revealed by mathematical modeling and quantitative imaging of yeast actin cables.

Bil2 Is a Novel Inhibitor of the Yeast Formin Bnr1 Required for Proper Actin Cable Organization and Polarized Secretion.

Cell growth in budding yeast depends on rapid and on-going assembly and turnover of polarized actin cables, which direct intracellular transport of post-Golgi vesicles to the bud tip. actin cables are polymerized by two formins, Bni1 and Bnr1. Bni1 assembles cables in the bud, while Bnr1 is anchored to the bud neck and assembles cables that specifically extend filling the mother cell.

DAAM2 Variants Cause Nephrotic Syndrome via Actin Dysregulation.

The discovery of >60 monogenic causes of nephrotic syndrome (NS) has revealed a central role for the actin regulators RhoA/Rac1/Cdc42 and their effectors, including the formin INF2. By whole-exome sequencing (WES), we here discovered bi-allelic variants in the formin DAAM2 in four unrelated families with steroid-resistant NS. We show that DAAM2 localizes to the cytoplasm in podocytes and in kidney sections.

Twinfilin bypasses assembly conditions and actin filament aging to drive barbed end depolymerization.

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.

EB1 Directly Regulates APC-Mediated Actin Nucleation.

EB1 was discovered 25 years ago as a binding partner of the tumor suppressor adenomatous polyposis coli (APC) [1]; however, the significance of EB1-APC interactions has remained poorly understood. EB1 functions at the center of a network of microtubule end-tracking proteins (+TIPs) [2-5], and APC binding to EB1 promotes EB1 association with microtubule ends and microtubule stabilization [6, 7]. Whether EB1 interactions govern functions of APC beyond microtubule regulation has not been explored.

Cell-substrate adhesion drives Scar/WAVE activation and phosphorylation by a Ste20-family kinase, which controls pseudopod lifetime.

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated.

Cofilin Loss in Drosophila Muscles Contributes to Muscle Weakness through Defective Sarcomerogenesis during Muscle Growth.

Sarcomeres, the fundamental contractile units of muscles, are conserved structures composed of actin thin filaments and myosin thick filaments. How sarcomeres are formed and maintained is not well understood. Here, we show that knockdown of Drosophila cofilin (DmCFL), an actin depolymerizing factor, disrupts both sarcomere structure and muscle function.

A septin-Hof1 scaffold at the yeast bud neck binds and organizes actin cables.

Cellular actin arrays are often highly organized, with characteristic patterns critical to their in vivo functions, yet the mechanisms for establishing these higher order geometries remain poorly understood. In formin-polymerized actin cables are spatially organized and aligned along the mother-bud axis to facilitate polarized vesicle traffic. Here, we show that the bud neck-associated F-BAR protein Hof1, independent of its functions in regulating the formin Bnr1, binds to actin filaments and organizes actin cables in vivo.