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Eukaryotic cells across the tree of life organize their subcellular components via intracellular transport mechanisms. In canonical transport, myosin, kinesin, and dynein motor proteins interact with cargos via adaptor proteins and move along filamentous actin or microtubule tracks. In contrast to this canonical mode, hitchhiking is a newly discovered mode of intracellular transport in which a cargo attaches itself to an already-motile cargo rather than directly associating with a motor protein itself. Many cargos including messenger RNAs, protein complexes, and organelles hitchhike on membrane-bound cargos. Hitchhiking-like behaviors have been shown to impact cellular processes including local protein translation, long-distance signaling, and organelle network reorganization. Here, we review instances of cargo hitchhiking in fungal, animal, and plant cells and discuss the potential cellular and evolutionary importance of hitchhiking in these different contexts.

In the filamentous fungus Aspergillus nidulans, peroxisomes move by hitchhiking on early endosomes. Here, it is shown that the Woronin body, a peroxisome-derived organelle that plugs incomplete septa upon injury to hyphae, also hitchhikes on early endosomes. While hitchhiking is required for Woronin body distribution and motility, it is dispensable for septal localization and plugging.

The proper functioning of organelles depends on their intracellular localization, mediated by motor protein-dependent transport on cytoskeletal tracks. Rather than directly associating with a motor protein, peroxisomes move by hitchhiking on motile early endosomes in the filamentous fungusAspergillus nidulans. However, the cellular function of peroxisome hitchhiking is unclear. Peroxisome hitchhiking requires the protein PxdA, which is conserved within the fungal subphylum Pezizomycotina, but absent from other fungal clades. Woronin bodies are specialized peroxisomes that are also unique to the Pezizomycotina. In these fungi, multinucleate hyphal segments are separated by incomplete cell walls called septa that possess a central pore enabling cytoplasmic exchange. Upon damage to a hyphal segment, Woronin bodies plug septal pores to prevent wide-spread leakage. Here, we tested if peroxisome hitchhiking is important for Woronin body motility, distribution, and function inA. nidulans. We show that Woronin body proteins are present within all motile peroxisomes and hitchhike on PxdA-labeled early endosomes during bidirectional, long-distance movements. Loss of peroxisome hitchhiking by knocking outpxdAsignificantly affected Woronin body distribution and motility in the cytoplasm, but Woronin body hitchhiking is ultimately dispensable for septal localization and plugging.

Cellular functions such as autophagy, cell signaling and vesicular trafficking involve the retrograde transport of motor-driven cargo along microtubules. Typically, newly formed cargo engages in slow diffusive movement from its point of origin before attaching to a microtubule. In some cell types, cargo destined for delivery to the perinuclear region relies on capture at dynein-enriched loading zones located near microtubule plus-ends. Such systems include extended cell regions of neurites and fungal hyphae, where the efficiency of the initial diffusive loading process depends on the axial distribution of microtubule plus-ends relative to the initial cargo position. We use analytic mean first passage time calculations and numerical simulations to model diffusive capture processes in tubular cells, exploring how the spatial arrangement of microtubule plus-ends affects the efficiency of retrograde cargo transport. Our model delineates the key features of optimal microtubule ar-rangements that minimize mean cargo capture times. Namely, we show that configurations with a single long microtubule and broad distribution of additional microtubule plus-ends allow for efficient capture in a variety of different scenarios for retrograde transport. Live-cell imaging of microtubule plus-ends in Aspergillus nidulans hyphae indicates that their distributions exhibit these optimal qualitative features. Our results highlight important coupling effects between microtubule length distribution and retrograde cargo transport, providing guiding principles for the spatial arrangement of microtubules within tubular cell regions.

In contrast to the canonical picture of transport by direct attachment to motor proteins, recent evidence shows that a number of intracellular "cargos" navigate the cytoplasm by hitchhiking on motor-driven "carrier" organelles. We describe a quantitative model of intracellular cargo transport via hitchhiking, examining the efficiency of hitchhiking initiation as a function of geometric and mechanical parameters. We focus specifically on the parameter regime relevant to the hitchhiking motion of peroxisome organelles in fungal hyphae. Our work predicts the dependence of transport initiation rates on the distribution of cytoskeletal tracks and carrier organelles, as well as the number, length, and flexibility of the linker proteins that mediate contact between the carrier and the hitchhiking cargo. Furthermore, we demonstrate that attaching organelles to microtubules can result in a substantial enhancement of the hitchhiking initiation rate in tubular geometries such as those found in fungal hyphae. This enhancement is expected to increase the overall transport rate of hitchhiking organelles and lead to greater efficiency in organelle dispersion. Our results leverage a quantitative physical model to highlight the importance of organelle encounter dynamics in noncanonical intracellular transport.

In eukaryotic cells, intracellular components are organized by the microtubule motors cytoplasmic dynein-1 (dynein) and kinesins, which are linked to cargos via adaptor proteins. While ~40 kinesins transport cargo toward the plus end of microtubules, a single dynein moves cargo in the opposite direction. How dynein transports a wide variety of cargos remains an open question. The FTS–Hook–FHIP (‘FHF’) cargo adaptor complex links dynein to cargo in humans and fungi. As human cells have three Hooks and four FHIP proteins, we hypothesized that the combinatorial assembly of different Hook and FHIP proteins could underlie dynein cargo diversity. Using proteomic approaches, we determine the protein ‘interactome’ of each FHIP protein. Live-cell imaging and biochemical approaches show that different FHF complexes associate with distinct motile cargos. These complexes also move with dynein and its cofactor dynactin in single-molecule in vitro reconstitution assays. Complexes composed of FTS, FHIP1B, and Hook1/Hook3 colocalize with Rab5-tagged early endosomes via a direct interaction between FHIP1B and GTP-bound Rab5. In contrast, complexes composed of FTS, FHIP2A, and Hook2 colocalize with Rab1A-tagged ER-to-Golgi cargos and FHIP2A is involved in the motility of Rab1A tubules. Our findings suggest that combinatorial assembly of different FTS–Hook–FHIP complexes is one mechanism dynein uses to achieve cargo specificity.

In eukaryotic cells, intracellular components are organized by the microtubule motors cytoplasmic dynein-1 (dynein) and kinesins, which are linked to cargos via adaptor proteins. While ∼40 kinesins transport cargo toward the plus end of microtubules, a single dynein moves cargo in the opposite direction. How dynein transports a wide variety of cargos remains an open question. The FTS-Hook-FHIP (“FHF”) cargo adaptor complex links dynein to cargo in mammals and fungi. As human cells have three Hooks and four FHIP proteins, we hypothesized that the combinatorial assembly of different Hook and FHIP proteins could underlie dynein cargo diversity. Using proteomic approaches, we determine the protein ‘interactome’ of each FHIP protein. Live-cell imaging and biochemical approaches show that different FHF complexes associate with distinct motile cargos. These complexes also move with dynein and its cofactor dynactin in single-molecule in vitro reconstitution assays. Complexes composed of FTS, FHIP1B, and Hook1/Hook3 co-localize with Rab5-tagged early endosomes via a direct interaction between FHIP1B and GTP-bound Rab5. In contrast, complexes composed of FTS, FHIP2A and Hook2 colocalize with Rab1A-tagged ER-to-Golgi cargos and FHIP2A is involved in the motility of Rab1A tubules. Our findings suggest that combinatorial assembly of different FTS-Hook-FHIP complexes is one mechanism dynein uses to achieve cargo specificity.

The regulated assembly of multiple filamentous actin (F-actin) networks from an actin monomer pool is important for a variety of cellular processes. Chlamydomonas reinhardtii is a unicellular green alga expressing a conventional and divergent actin that is an emerging system for investigating the complex regulation of actin polymerization. One actin network that contains exclusively conventional F-actin in Chlamydomonas is the fertilization tubule, a mating structure at the apical cell surface in gametes. In addition to two actin genes, Chlamydomonas expresses a profilin (PRF1) and four formin genes (FOR1-4), one of which (FOR1) we have characterized for the first time. We found that unlike typical profilins, PRF1 prevents unwanted actin assembly by strongly inhibiting both F-actin nucleation and barbed-end elongation at equimolar concentrations to actin. However, FOR1 stimulates the assembly of rapidly elongating actin filaments from PRF1-bound actin. Furthermore, for1 and prf1-1 mutants, as well as the small molecule formin inhibitor SMIFH2, prevent fertilization tubule formation in gametes, suggesting that polymerization of F-actin for fertilization tubule formation is a primary function of FOR1. Together, these findings indicate that FOR1 and PRF1 cooperate to selectively and rapidly assemble F-actin at the right time and place.

Mitochondrial anchors have functions that extend beyond simply positioning mitochondria. In budding yeast, mitochondria drive the assembly of the mitochondrial anchor protein Num1 into clusters, which serve to anchor mitochondria as well as dynein to the cell cortex. Here, we explore a conserved role for mitochondria in dynein anchoring by examining the tethering functions of the evolutionarily distant Schizosaccharomyces pombe Num1 homologue. In addition to its function in dynein anchoring, we find that S. pombe Num1, also known as Mcp5, interacts with and tethers mitochondria to the plasma membrane in S. pombe and Saccharomyces cerevisiae. Thus, the mitochondria and plasma membrane-binding domains of the Num1 homologues, as well as the membrane features these domains recognize, are conserved. In S. pombe, we find that mitochondria impact the assembly and cellular distribution of Num1 clusters and that Num1 clusters actively engaged in mitochondrial tethering serve as cortical attachment sites for dynein. Thus, mitochondria play a critical and conserved role in the formation and distribution of dynein-anchoring sites at the cell cortex and, as a consequence, impact dynein function. These findings shed light on an ancient mechanism of mitochondria-dependent dynein anchoring that is conserved over more than 450 million years of evolution, raising the intriguing possibility that the role mitochondria play in dynein anchoring and function extends beyond yeast to higher eukaryotes.

In canonical microtubule-based transport, adaptor proteins link cargos to the molecular motors dynein and kinesin. Recently, an alternative mode of transport known as ‘hitchhiking’ was discovered, in which a cargo achieves motility by hitching a ride on an already-motile cargo, rather than attaching to a motor protein. Hitchhiking has been best-studied in two filamentous fungi, Aspergillus nidulans and Ustilago maydis. In U. maydis, ribonucleoprotein complexes, peroxisomes, lipid droplets, and endoplasmic reticulum all hitchhike on early endosomes. In A. nidulans, peroxisomes hitchhike using a putative molecular linker, PxdA, that associates with early endosomes. However, whether other organelles use PxdA to hitchhike on early endosomes is unclear, as are the molecular mechanisms that regulate hitchhiking in A. nidulans. Here we find that the proper distribution of lipid droplets, mitochondria and autophagosomes do not require PxdA, suggesting that PxdA is a molecular linker specific to peroxisomes. We also identify two new pxdA alleles, including a point mutation (R2044P) that disrupts PxdA’s ability to associate with early endosomes and reduces peroxisome movement. Finally, we identify a novel regulator of peroxisome hitchhiking, the phosphatase DipA. DipA co-localizes with early endosomes and its early endosome-association relies on PxdA.

In contrast to the canonical picture of transport by direct attachment to motor proteins, recent evidence shows that a number of intracellular ‘cargos’ navigate the cytoplasm by hitchhiking on motor-driven ‘carrier’ organelles. We describe a quantitative model of intracellular cargo transport via hitchhiking, examining the efficiency of hitchhiking initiation as a function of geometric and mechanical parameters. We focus specifically on the parameter regime relevant to the hitchhiking motion of peroxisome organelles in fungal hyphae. Our work predicts the dependence of transport initiation rates on the distribution of cytoskeletal tracks and carrier organelles, as well as the number, length and flexibility of the linker proteins that mediate contact between the carrier and the hitchhiking cargo. Furthermore, we demonstrate that attaching organelles to microtubules can result in a substantial enhancement of the hitchhiking initiation rate in tubular geometries such as those found in fungal hyphae. This enhancement is expected to increase the overall transport rate of hitchhiking organelles, and lead to greater efficiency in organelle dispersion. Our results leverage a quantitative physical model to highlight the importance of organelle encounter dynamics in non-canonical intracellular transport.SIGNIFICANCEA variety of cellular components are transported via hitchhiking by attaching to other motile organelles. Defects in the molecular machinery responsible for organelle hitchhiking may be linked with neurodegenerative disorders. To date, no comprehensive physical models of this non-canonical mode of transport have been developed. In particular, the connection between molecular- and organelle-scale properties of hitchhiking components and their effect on cellular-scale transport has remained unclear. Here, we investigate the mechanics of hitchhiking initiation and explore organelle interactions that can modulate the efficiency of this process.

The highly dynamic bundling activity of fission yeast α-actinin SpAin1 was biochemically characterized, and its importance for contractile ring formation in vivo was tested. Investigation of a mutant with higher bundling activity, SpAin1(216E), revealed that dynamic SpAin1-mediated bundling is crucial for proper ring assembly and constriction., The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction.

A budding yeast profilin ScPFY mutant library was engineered and used to select for mutants that complement the fission yeast profilin temperature-sensitive mutant cdc3-124. ScPFY(9-Mut) rescues the ability of profilin to facilitate actin assembly by the cytokinesis formin Cdc12 in vitro and allow contractile ring assembly in vivo., The evolutionarily conserved small actin-monomer binding protein profilin is believed to be a housekeeping factor that maintains a general pool of unassembled actin. However, despite similar primary sequences, structural folds, and affinities for G-actin and poly-l-proline, budding yeast profilin ScPFY fails to complement fission yeast profilin SpPRF temperature-sensitive mutant cdc3-124 cells. To identify profilin's essential properties, we built a combinatorial library of ScPFY variants containing either WT or SpPRF residues at multiple positions and carried out a genetic selection to isolate variants that support life in fission yeast. We subsequently engineered ScPFY(9-Mut), a variant containing nine substitutions in the actin-binding region, which complements cdc3-124 cells. ScPFY(9-Mut), but not WT ScPFY, suppresses severe cytokinesis defects in cdc3-124 cells. Furthermore, the major activity rescued by ScPFY(9-Mut) is the ability to enhance cytokinesis formin Cdc12-mediated actin assembly in vitro, which allows cells to assemble functional contractile rings. Therefore an essential role of profilin is to specifically facilitate formin-mediated actin assembly for cytokinesis in fission yeast.

The fission yeast actin cytoskeleton is an ideal, simplified system to investigate fundamental mechanisms behind cellular self-organization. By focusing on the stabilizing protein tropomyosin Cdc8, bundling protein fimbrin Fim1, and severing protein coffin Adf1, we examined how their pairwise and collective interactions with actin filaments regulate their activity and segregation to functionally diverse F-actin networks. Utilizing multi-color TIRF microscopy of in vitro reconstituted F-actin networks, we observed and characterized two distinct Cdc8 cables loading and spreading cooperatively on individual actin filaments. Furthermore, Cdc8, Fim1, and Adf1 all compete for association with F-actin by different mechanisms, and their cooperative association with actin filaments affects their ability to compete. Finally, competition between Fim1 and Adf1 for F-actin synergizes their activities, promoting rapid displacement of Cdc8 from a dense F-actin network. Our findings reveal that competitive and cooperative interactions between actin binding proteins help define their associations with different F-actin networks. DOI: http://dx.doi.org/10.7554/eLife.23152.001, eLife digest Cells use a protein called actin to provide shape, to generate the forces needed for cells to divide, and for many other essential processes. Inside a cell, individual actin proteins join up to form long filaments. These actin filaments are organized in different ways to make networks that have distinct properties, each tailored for a specific process. For instance, bundles of straight actin filaments help a cell to divide, whereas a network of branched actin filaments allows cells to move. The different proteins that bind to actin filaments influence how quickly actin filaments are assembled and organized into networks. Therefore, many of the properties of an actin filament network are due to the actin binding proteins that are associated with it. Two actin binding proteins called fimbrin and cofilin associate with a type of actin filament network known as the actin patch. A third actin binding protein called tropomyosin associates with a different network that forms a ring. It is not known how particular actin binding proteins choose to associate with one actin network instead of another. Christensen et al. used a fluorescence microscopy technique to study how fimbrin, cofilin and tropomyosin associate with different actin networks in a single-celled organism called fission yeast. This technique involved incubating actin and actin binding proteins together in a microscope chamber. The experiments show that some actin binding proteins, like tropomyosin, cooperate to bind to actin. Individual tropomyosin molecules find it difficult to bind actin filaments on their own, but once one tropomyosin molecule is attached to the filament, others rapidly join to coat the filament. On the other hand, some actin-binding proteins compete for binding to filaments. For example, the binding of fimbrin to actin filaments causes tropomyosin to be removed from the actin network. Further experiments revealed that fimbrin and cofilin work with each other to rapidly generate a dense actin network and displace tropomyosin. Together, the findings of Christensen et al. suggest that competitions between actin binding proteins determine which actin binding proteins are associated with an actin network. The next challenge is to understand how the most competitive actin-binding proteins are kept off actin networks where they do not belong. Further studies will shed light on how these interactions cause large changes in how the cell is organized. DOI: http://dx.doi.org/10.7554/eLife.23152.002

Cells assemble and maintain functionally distinct actin cytoskeleton networks with various actin filament organizations and dynamics through the coordinated action of different sets of actin-binding proteins. The biochemical and functional properties of diverse actin-binding proteins, both alone and in combination, have been increasingly well studied. Conversely, how different sets of actin-binding proteins properly sort to distinct actin filament networks in the first place is not nearly as well understood. Actin-binding protein sorting is critical for the self-organization of diverse dynamic actin cytoskeleton networks within a common cytoplasm. Using in vitro reconstitution techniques including biomimetic assays and single-molecule multi-color total internal reflection fluorescence microscopy, we discovered that sorting of the prominent actin-bundling proteins fascin and α-actinin to distinct networks is an intrinsic behavior, free of complicated cellular signaling cascades. When mixed, fascin and α-actinin mutually exclude each other by promoting their own recruitment and inhibiting recruitment of the other, resulting in the formation of distinct fascin- or α-actinin-bundled domains. Subdiffraction-resolution light microscopy and negative-staining electron microscopy revealed that fascin domains are densely packed, whereas α-actinin domains consist of widely spaced parallel actin filaments. Importantly, other actin-binding proteins such as fimbrin and espin show high specificity between these two bundle types within the same reaction. Here we directly observe that fascin and α-actinin intrinsically segregate to discrete bundled domains that are specifically recognized by other actin-binding proteins.

Street, Chicago,IL 60637, USASummaryControlling the quantity and size of organelles throughcompetition for a limited supply of components is quicklyemerging as an important cellular regulatory mechanism[1]. Cells assemble diverse actin filament (F-actin) net-works for fundamental processes including division,motility, and polarization [2–4]. F-actin polymerization istightly regulated by activation of assembly factors suchas the Arp2/3 complex and formins at specific times andplaces. We directly tested an additional hypothesis thatdiverse F-actin networks are in homeostasis, wherebycompetition for actin monomers (G-actin) is critical forregulating F-actin network size. Here we show that inhibi-tion of Arp2/3 complex in the fission yeast Schizosacchar-omyces pombe not only depletes Arp2/3-complex-mediatedendocytic actin patches, but also induces a dramaticexcess of formin-assembled F-actin. Conversely, disrup-tion of formin increases the density of Arp2/3-complex-mediated patches. Furthermore, modification of actin levelssignificantly perturbs the fission yeast actin cytoskeleton.Increasing actin favors Arp2/3-complex-mediated actin as-sembly, whereas decreasing actin favors formin-mediatedcontractile rings. Therefore, the specific actin concentra-tion in a cell is critical, and competition for G-actin helpsregulate the proper amount of F-actin assembly for diverseprocesses.Results and DiscussionTo control F-actin network density, actin polymerization istightly regulated through the activation of assembly (nucle-ation)factorsbyGTPasesignaling cascades, therateat whichF-actin barbed ends are capped, the rate at which assemblyfactors are turned off, and F-actin disassembly factors[2, 3, 5]. The supply of unassembled G-actin is not generallyconsidered to be limiting [6, 7]. Alternatively, it is possiblethat the actin cytoskeleton is homeostatic with a limitedconcentration of G-actin, which is competed for by assemblyfactors to help regulate its incorporation into diverse F-actinnetworks [3, 8–10]. However, this intriguing additional hypoth-esis has not been systematically tested.Fission yeast forms three F-actin network structures bythree different assembly factors [9]. The Arp2/3 complexassemblesshort-branched F-actinin endocytic actin patches,whereas the formins For3 and Cdc12 assemble long-straightF-actin in polarizing actin cables and the cytokinetic contrac-tile ring, respectively. The amount of actin and other compo-nents incorporated into actin patches and contractile rings isremarkably consistent, varying less than 50% for each struc-ture [11–13]. Although measuring the composition of actincables has been technically challenging, they may be similarlyconsistent. Of the w1 million actin molecules per cell, w35%to 50% are evenly distributed between 30 to 50 actin patches,w10% are incorporated into contractile rings, and perhaps asmuch as 15% are estimated to be consumed by actin cables[11–15].To directly test the hypothesis that assembly factors com-peteforG-actin,weinvestigatedtheconsequencesofsystem-atically disrupting individual assembly factors in fission yeastcells. Initially, we treated cells expressing the general F-actinmarker Lifeact-GFP with a range of concentrations of theArp2/3 complex inhibitor CK-666 [16], causing a dose-depen-dent decrease in the number of actin patches (Figures 1Aand 1B and Figure S1A available online), reduction in patchmotility, and increase in patch lifetime (Table S1). Strikingly,actin patch depletion coincides with the dramatic formationof new ectopic cable-like F-actin (Figures 1A and S1A), satu-rating at w100 mM CK-666 (Figure 1B). CK-666 treatmentfacilitates ectopic F-actin assembly in both minimal and richgrowth media, is visible with different general F-actin markersincluding rhodamine-phalloidin (Figures S1B–S1F), and isinhibited by the G-actin sequestering drug LatA (Figure S1G).Observation of cells in a microfluidic chamber revealedthat depletion of actin patches and the concomitant assemblyof ectopic F-actin occurs in w10–20 min after addition ofsaturating concentrations of CK-666 (Figure 1C and 1D andMovie S1). Ectopic F-actin rapidly disassembles upon washout of CK-666 with a corresponding reassembly of actinpatches in w10–40 min (Figures 1C and 1D). Actin patchproteins ArpC5-mCherry (Arp2/3 complex component) andAcp2-GFP (actin capping protein) are released into the cyto-plasm by CK-666 treatment, but do not incorporate into theectopic F-actin (Figures S1H–S1J).Genetic disruption of Arp2/3 complex also leads to ectopicF-actin assembly, albeit less prominently than with CK-666since actin patches are not depleted completely under theseconditions (Figures 1E–1H). Compared to wild-type (WT) cells,at the restrictive temperature of 19 C Arp2/3 complex cold-sensitive mutant arp3-C1 cells [17] have approximately halfthenumberofpatchesandacorrespondingstatisticallysignif-icant3-foldincreasein ectopicF-actin(p

Tropomyosins modulate the activity of actin binding proteins---including myosin, cofilin, the Arp2/3 complex, and formins---by themselves binding and coating actin filaments. A single tropomyosin protein is a coiled-coil capable of end-to-end association, and one segment of an actin filament is capable of simultaneously accommodating two tropomyosin cables. The coating of actin filaments by tropomyosin is known to be highly cooperative, however the details of this process are not well understood. Using labeled actin and labeled fission yeast tropomyosin, Cdc8, we can observe by TIRF microscopy the formation of small tropomyosin Cdc8 ‘seeds’ that appear to initially associate with actin filaments and then subsequently extend from both ends. Additionally, we are capable of observing two distinct tropomyosin cables associated with a single actin filament. Our setup allows us to study in detail the interactions of tropomyosin Cdc8 with the actin filament and with other tropomyosin Cdc8 molecules. To interpret the experimental observations, we have used statistical approaches, kinetic master equations, and a new kinetic Ising model. Theory and modeling lends insight into the mechanism underlying Cdc8's highly cooperative binding kinetics. Our results are consistent with a small amount of “face cooperativity,” that is, a slight preference for tropomyosin Cdc8 molecules to associate with the actin filament opposite to an already bound tropomyosin cable. These findings suggest that some tropomyosin binding cooperativity is mediated through the actin filament, and not only via end-to-end binding of Cdc8 molecules.

SummaryFission yeast cells use Arp2/3 complex and formin to assemble diverse filamentous actin (F-actin) networks within a common cytoplasm for endocytosis, division, and polarization. Although these homeostatic F-actin networks are usually investigated separately, competition for a limited pool of actin monomers (G-actin) helps to regulate their size and density. However, the mechanism by which G-actin is correctly distributed between rival F-actin networks is not clear. Using a combination of cell biological approaches and in vitro reconstitution of competition between actin assembly factors, we found that the small G-actin binding protein profilin directly inhibits Arp2/3 complex-mediated actin assembly. Profilin is therefore required for formin to compete effectively with excess Arp2/3 complex for limited G-actin and to assemble F-actin for contractile ring formation in dividing cells.