In vivo analysis of formin dynamics in the moss P. patens reveals functional class diversification.

Formins are actin regulators critical for diverse processes across eukaryotes. With many formins in plants and animals, it has been challenging to determine formin function in vivo We found that the phylogenetically distinct class I integral membrane formins (denoted For1) from the moss P.patens enrich at sites of membrane turnover, with For1D more tightly associated with the plasma membrane than For1A. To probe formin function, we generated formin-null lines with greatly reduced formin complexity. We found that For1A and For1D help to anchor actin near the cell apex, with For1A contributing to formation of cytosolic actin, while For1D contributes to plasma membrane-associated actin. At the cortex, For1A and For1D localized to motile puncta and differentially impacted actin dynamics. We found that class I cortical formin mobility depended on microtubules and only moderately on actin, whereas class II formin (denoted For2) mobility solely depended on actin. Moreover, cortical For2A tightly correlated with the puncta labeled by the endocytic membrane dye FM4-64, and null mutants in class I formins did not affect uptake of a similar dye, FM1-43, suggesting that class I and II formins are involved in distinct membrane trafficking pathways.

Actin interacting protein1 and actin depolymerizing factor drive rapid actin dynamics in Physcomitrella patens.

The remodeling of actin networks is required for a variety of cellular processes in eukaryotes. In plants, several actin binding proteins have been implicated in remodeling cortical actin filaments (F-actin). However, the extent to which these proteins support F-actin dynamics in planta has not been tested. Using reverse genetics, complementation analyses, and cell biological approaches, we assessed the in vivo function of two actin turnover proteins: actin interacting protein1 (AIP1) and actin depolymerizing factor (ADF). We report that AIP1 is a single-copy gene in the moss Physcomitrella patens. AIP1 knockout plants are viable but have reduced expansion of tip-growing cells. AIP1 is diffusely cytosolic and functions in a common genetic pathway with ADF to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin remodeling, AIP1 knockout lines accumulate F-actin bundles, have fewer dynamic ends, and have reduced severing frequency. Importantly, we demonstrate that AIP1 promotes and ADF is essential for cortical F-actin dynamics.

An ancient Sec10-formin fusion provides insights into actin-mediated regulation of exocytosis.

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.

Rapid screening for temperature-sensitive alleles in plants.

We developed a simple and fast method to identify temperature-sensitive alleles of essential plant genes. We used primary and tertiary structure information to identify residues in the core of the protein of interest. These residues were mutated and tested for temperature sensitivity, taking advantage of the exceptionally rapid 1-week complementation assay in the moss Physcomitrella patens. As test molecules, we selected the actin-binding proteins profilin and actin-depolymerizing factor, because they are essential and their loss-of-function phenotype can be fully rescued. Screening a small number of candidate mutants, we successfully identified temperature-sensitive alleles of both profilin and actin-depolymerizing factor. Plants harboring these alleles grew well at the permissive temperature of 20 degrees C to 25 degrees C but showed a complete loss of function at the restrictive temperature of 32 degrees C. Notably, the profilin mutation identified in the moss gene can be transferred to profilins from other plant species, also rendering them temperature sensitive. The ability to routinely generate temperature-sensitive alleles of essential plant proteins provides a powerful tool for the study of gene function in plants.