Molecular underpinnings of cytoskeletal cross-talk.

Cross-talk between the microtubule and actin networks has come under intense scrutiny following the realization that it is crucial for numerous essential processes, ranging from cytokinesis to cell migration. It is becoming increasingly clear that proteins long-considered highly specific for one or the other cytoskeletal system do, in fact, make use of both filament types. How this functional duality of "shared proteins" has evolved and how their coadaptation enables cross-talk at the molecular level remain largely unknown. We previously discovered that the mammalian adaptor protein melanophilin of the actin-associated myosin motor is one such "shared protein," which also interacts with microtubules in vitro. In a hypothesis-driven in vitro and in silico approach, we turn to early and lower vertebrates and ask two fundamental questions. First, is the capability of interacting with microtubules and actin filaments unique to mammalian melanophilin or did it evolve over time? Second, what is the functional consequence of being able to interact with both filament types at the cellular level? We describe the emergence of a protein domain that confers the capability of interacting with both filament types onto melanophilin. Strikingly, our computational modeling demonstrates that the regulatory power of this domain on the microscopic scale alone is sufficient to recapitulate previously observed behavior of pigment organelles in amphibian melanophores. Collectively, our dissection provides a molecular framework for explaining the underpinnings of functional cross-talk and its potential to orchestrate the cell-wide redistribution of organelles on the cytoskeleton.

Global assessment of marine and freshwater recreational fish reveals mismatch in climate change vulnerability and conservation effort.

Recreational fisheries contribute substantially to the sociocultural and economic well-being of coastal and riparian regions worldwide, but climate change threatens their sustainability. Fishery managers require information on how climate change will impact key recreational species; however, the absence of a global assessment hinders both directed and widespread conservation efforts. In this study, we present the first global climate change vulnerability assessment of recreationally targeted fish species from marine and freshwater environments (including diadromous fishes). We use climate change projections and data on species' physiological and ecological traits to quantify and map global climate vulnerability and analyze these patterns alongside the indices of socioeconomic value and conservation effort to determine where efforts are sufficient and where they might fall short. We found that over 20% of recreationally targeted fishes are vulnerable to climate change under a high emission scenario. Overall, marine fishes had the highest number of vulnerable species, concentrated in regions with sensitive habitat types (e.g., coral reefs). However, freshwater fishes had higher proportions of species at risk from climate change, with concentrations in northern Europe, Australia, and southern Africa. Mismatches in conservation effort and vulnerability were found within all regions and life-history groups. A key pattern was that current conservation effort focused primarily on marine fishes of high socioeconomic value rather than on the freshwater and diadromous fishes that were predicted to be proportionately more vulnerable. While several marine regions were notably lacking in protection (e.g., Caribbean Sea, Banda Sea), only 19% of vulnerable marine species were without conservation effort. By contrast, 72% of freshwater fishes and 33% of diadromous fishes had no measures in place, despite their high vulnerability and cultural value. The spatial and taxonomic analyses presented here provide guidance for the future conservation and management of recreational fisheries as climate change progresses.

Feeling the force: formin's role in mechanotransduction.

Fundamental cellular processes such as division, polarization, and motility require the tightly regulated spatial and temporal assembly and disassembly of the underlying actin cytoskeleton. The actin cytoskeleton has been long viewed as a central player facilitating diverse mechanotransduction pathways due to the notion that it is capable of receiving, processing, transmitting, and generating mechanical stresses. Recent work has begun to uncover the roles of mechanical stresses in modulating the activity of key regulatory actin-binding proteins and their interactions with actin filaments, thereby controlling the assembly (formin and Arp2/3 complex) and disassembly (ADF/Cofilin) of actin filament networks. In this review, we will focus on discussing the current molecular understanding of how members of the formin protein family sense and respond to forces and the potential implications for formin-mediated mechanotransduction in cells.

Mechanoregulated inhibition of formin facilitates contractile actomyosin ring assembly.

Cytokinesis physically separates dividing cells by forming a contractile actomyosin ring. The fission yeast contractile ring has been proposed to assemble by Search-Capture-Pull-Release from cytokinesis precursor nodes that include the molecular motor type-II myosin Myo2 and the actin assembly factor formin Cdc12. By successfully reconstituting Search-Capture-Pull in vitro, we discovered that formin Cdc12 is a mechanosensor, whereby myosin pulling on formin-bound actin filaments inhibits Cdc12-mediated actin assembly. We mapped Cdc12 mechanoregulation to its formin homology 1 domain, which facilitates delivery of new actin subunits to the elongating actin filament. Quantitative modeling suggests that the pulling force of the myosin propagates through the actin filament, which behaves as an entropic spring, and thereby may stretch the disordered formin homology 1 domain and impede formin-mediated actin filament elongation. Finally, live cell imaging of mechano-insensitive formin mutant cells established that mechanoregulation of formin Cdc12 is required for efficient contractile ring assembly in vivo.The fission yeast cytokinetic ring assembles by Search-Capture-Pull-Release from precursor nodes that include formin Cdc12 and myosin Myo2. The authors reconstitute Search-Capture-Pull in vitro and find that Myo2 pulling on Cdc12-associated actin filaments mechano-inhibits Cdc12-mediated assembly, which enables proper ring assembly in vivo.

In Vitro Biochemical Characterization of Cytokinesis Actin-Binding Proteins.

Characterizing the biochemical and biophysical properties of purified proteins is critical to understand the underlying molecular mechanisms that facilitate complicated cellular processes such as cytokinesis. Here we outline in vitro assays to investigate the effects of cytokinesis actin-binding proteins on actin filament dynamics and organization. We describe (1) multicolor single-molecule TIRF microscopy actin assembly assays, (2) "bulk" pyrene actin assembly/disassembly assays, and (3) "bulk" sedimentation actin filament binding and bundling assays.

Actin age orchestrates myosin-5 and myosin-6 run lengths.

Unlike a static and immobile skeleton, the actin cytoskeleton is a highly dynamic network of filamentous actin (F-actin) polymers that continuously turn over. In addition to generating mechanical forces and sensing mechanical deformation, dynamic F-actin networks serve as cellular tracks for myosin motor traffic. However, much of our mechanistic understanding of processive myosins comes from in vitro studies in which motility was studied on pre-assembled and artificially stabilized, static F-actin tracks. In this work, we examine the role of actin dynamics in single-molecule myosin motility using assembling F-actin and two highly processive motors, myosin-5 and myosin-6. These two myosins have distinct functions in the cell and travel in opposite directions along actin filaments [1-3]. Myosin-5 walks toward the barbed ends of F-actin, traveling to sites of actin polymerization at the cell periphery [4]. Myosin-6 walks toward the pointed end of F-actin [5], traveling toward the cell center along older segments of the actin filament. We find that myosin-5 takes 1.3- to 1.5-fold longer runs on ADP•Pi (young) F-actin, whereas myosin-6 takes 1.7- to 3.6-fold longer runs along ADP (old) F-actin. These results suggest that conformational differences between ADP•Pi and ADP F-actin tailor these myosins to walk farther toward their preferred actin filament end. Taken together, these experiments define a new mechanism by which myosin traffic may sort to different F-actin networks depending on filament age.

Disassembly activity of actin-depolymerizing factor (ADF) is associated with distinct cellular processes in apicomplexan parasites.

Proteins of the actin-depolymerizing factor (ADF)/cofilin family have been shown to be crucial for the motility and survival of apicomplexan parasites. However, the mechanisms by which ADF proteins fulfill their function remain poorly understood. In this study, we investigate the comparative activities of ADF proteins from Toxoplasma gondii and Plasmodium falciparum, the human malaria parasite, using a conditional T. gondii ADF-knockout line complemented with ADF variants from either species. We show that P. falciparum ADF1 can fully restore native TgADF activity, demonstrating functional conservation between parasites. Strikingly, mutation of a key basic residue (Lys-72), previously implicated in disassembly in PfADF1, had no detectable phenotypic effect on parasite growth, motility, or development. In contrast, organelle segregation was severely impaired when complementing with a TgADF mutant lacking the corresponding residue (Lys-68). Biochemical analyses of each ADF protein confirmed the reduced ability of lysine mutants to mediate actin depolymerization via filament disassembly although not severing, in contrast to previous reports. These data suggest that actin filament disassembly is essential for apicomplexan parasite development but not for motility, as well as pointing to genus-specific coevolution between ADF proteins and their native actin.

In vitro reconstitution of an mRNA-transport complex reveals mechanisms of assembly and motor activation.

The assembly and composition of ribonucleic acid (RNA)-transporting particles for asymmetric messenger RNA (mRNA) localization is not well understood. During mitosis of budding yeast, the Swi5p-dependent HO expression (SHE) complex transports a set of mRNAs into the daughter cell. We recombinantly reconstituted the core SHE complex and assessed its properties. The cytoplasmic precomplex contains only one motor and is unable to support continuous transport. However, a defined interaction with a second, RNA-bound precomplex after its nuclear export dimerizes the motor and activates processive RNA transport. The run length observed in vitro is compatible with long-distance transport in vivo. Surprisingly, SHE complexes that either contain or lack RNA cargo show similar motility properties, demonstrating that the RNA-binding protein and not its cargo activates motility. We further show that SHE complexes have a defined size but multimerize into variable particles upon binding of RNAs with multiple localization elements. Based on these findings, we provide an estimate of number, size, and composition of such multimeric SHE particles in the cell.

Diffusion of myosin V on microtubules: a fine-tuned interaction for which E-hooks are dispensable.

Organelle transport in eukaryotes employs both microtubule and actin tracks to deliver cargo effectively to their destinations, but the question of how the two systems cooperate is still largely unanswered. Recently, in vitro studies revealed that the actin-based processive motor myosin V also binds to, and diffuses along microtubules. This biophysical trick enables cells to exploit both tracks for the same transport process without switching motors. The detailed mechanisms underlying this behavior remain to be solved. By means of single molecule Total Internal Reflection Microscopy (TIRFM), we show here that electrostatic tethering between the positively charged loop 2 and the negatively charged C-terminal E-hooks of microtubules is dispensable. Furthermore, our data indicate that in addition to charge-charge interactions, other interaction forces such as non-ionic attraction might account for myosin V diffusion. These findings provide evidence for a novel way of myosin tethering to microtubules that does not interfere with other E-hook-dependent processes.