Coordination between actin and microtubules is important for numerous cellular processes in diverse eukaryotes. In plants, tip-growing cells require actin for cell expansion and microtubules for orientation of cell expansion, but how the two cytoskeletons are linked is an open question. In tip-growing cells of the moss Physcomitrella patens , we show that an actin cluster near the cell apex dictates the direction of rapid cell expansion. Formation of this structure depends on the convergence of microtubules near the cell tip. We discovered that microtubule convergence requires class VIII myosin function, and actin is necessary for myosin VIII–mediated focusing of microtubules. The loss of myosin VIII function affects both networks, indicating functional connections among the three cytoskeletal components. Our data suggest that microtubules direct localization of formins, actin nucleation factors, that generate actin filaments further focusing microtubules, thereby establishing a positive feedback loop ensuring that actin polymerization and cell expansion occur at a defined site resulting in persistent polarized growth.

Exocytosis, facilitated by the exocyst, is fundamentally important for remodeling cell walls and membranes. Here, we analyzed For1F , a novel gene that encodes a fusion of an exocyst subunit (Sec10) and an actin nucleation factor (formin). We showed that the fusion occurred early in moss evolution and has been retained for more than 170 million years. In Physcomitrella patens , For1F is essential, and the expressed protein is a fusion of Sec10 and formin. Reduction of For1F or actin filaments inhibits exocytosis, and For1F dynamically associates with Sec6, another exocyst subunit, in an actin-dependent manner. Complementation experiments demonstrate that constitutive expression of either half of the gene or the paralogous Sec10b rescues loss of For1F, suggesting that fusion of the two domains is not essential, consistent with findings in yeast, where formin and the exocyst are linked noncovalently. Although not essential, the fusion may have had selective advantages and provides a unique opportunity to probe actin regulation of exocytosis.

Many aspects of cytoskeletal assembly and dynamics can be recapitulated in vitro; yet, how the cytoskeleton integrates signals in vivo across cellular membranes is far less understood. Recent work has demonstrated that the membrane alone, or through membrane-associated proteins, can effect dynamic changes to the cytoskeleton, thereby impacting cell physiology. Having identified mechanistic links between membranes and the actin, microtubule, and septin cytoskeletons, these studies highlight the membrane’s central role in coordinating these cytoskeletal systems to carry out essential processes, such as endocytosis, spindle positioning, and cellular compartmentalization.

Class II formins are key regulators of actin and are essential for polarized plant cell growth. Here, we show that the class II formin N-terminal phosphatase and tensin (PTEN) domain binds phosphoinositide-3,5-bisphosphate (PI(3,5)P 2 ). Replacing the PTEN domain with polypeptides of known lipid-binding specificity, we show that PI(3,5)P 2 binding was required for formin-mediated polarized growth. Via PTEN, formin also localized to the cell apex, phragmoplast, and to the cell cortex as dynamic cortical spots. We show that the cortical localization driven by binding to PI(3,5)P 2 was required for function. Silencing the kinases that produce PI(3,5)P 2 reduced cortical targeting of formin and inhibited polarized growth. We show a subset of cortical formin spots moved in actin-dependent linear trajectories. We observed that the linearly moving subpopulation of cortical formin generated new actin filaments de novo and along preexisting filaments, providing evidence for formin-mediated actin bundling in vivo. Taken together, our data directly link PI(3,5)P 2 to generation and remodeling of the cortical actin array.

Fission yeast myo1 + encodes a myosin-I with all three tail homology domains (TH1, 2, 3) found in typical long-tailed myosin-Is. Myo1p tail also contains a COOH-terminal acidic region similar to the A-domain of WASp/Scar proteins and other fungal myosin-Is. Our analysis shows that Myo1p and Wsp1p, the fission yeast WASp-like protein, share functions and cooperate in controlling actin assembly. First, Myo1p localizes to cortical patches enriched at tips of growing cells and at sites of cell division. Myo1p patches partially colocalize with actin patches and are dependent on an intact actin cytoskeleton. Second, although deletion of myo1 + is not lethal, Δmyo1 cells have actin cytoskeletal defects, including loss of polarized cell growth, delocalized actin patches, and mating defects. Third, additional disruption of wsp1 + is synthetically lethal, suggesting that these genes may share functions. In mapping the domains of Myo1p tail that share function with Wsp1p, we discovered that a Myo1p construct with just the head and TH1 domains is sufficient for cortical localization and to rescue all Δmyo1 defects. However, it fails to rescue the Δmyo1 Δwsp1 lethality. Additional tail domains, TH2 and TH3, are required to complement the double mutant. Fourth, we show that a recombinant Myo1p tail binds to Arp2/3 complex and activates its actin nucleation activity.