Kinesins are an ancient superfamily of microtubule dependent motors. They participate in an extensive and diverse list of essential cellular functions, including mitosis, cytokinesis, cell polarization, cell elongation, flagellar development, and intracellular transport. Based on phylogenetic relationships, the kinesin superfamily has been subdivided into 14 families, which are represented in most eukaryotic phyla. The functions of these families are sometimes conserved between species, but important variations in function across species have been observed. Plants possess most kinesin families including a few plant specific families. With the availability of an ever increasing number of genome sequences from plants, it is important to document the complete complement of kinesins present in a given organism. This will help develop a molecular framework to explore the function of each family using genetics, biochemistry, and cell biology. The moss Physcomitrella patens has emerged as a powerful model organism to study gene function in plants, which makes it a key candidate to explore complex gene families, such as the kinesin superfamily. Here we report a detailed phylogenetic characterization of the 71 kinesins of the kinesin superfamily in Physcomitrella. We found a remarkable conservation of families and subfamily classes with Arabidopsis, which is important for future comparative analysis of function. Some of the families, such as kinesins 14s are composed of fewer members in moss, while other families, such as the kinesin 12s are greatly expanded. To improve the comparison between species, and to simplify communication between research groups, we propose a classification of subfamilies based on our phylogenetic analysis.

SUMMARYTip growth is essential for land colonization by bryophytes, plant sexual reproduction and water and nutrient uptake. Because this specialized form of polarized cell growth requires both a dynamic actin cytoskeleton and active secretion, it has been proposed that the F-actin-associated motor myosin XI is essential for this process. Nevertheless, a spatial and temporal relationship between myosin XI and F-actin during tip growth is not known in any plant cell. Here, we use the highly polarized cells of the moss Physcomitrella patens to show that myosin XI and F-actin localize, in vivo, at the same apical domain and that both signals fluctuate. Surprisingly, phase analysis shows that increase in myosin XI anticipates that of F-actin; in contrast, myosin XI levels at the tip fluctuate in identical phase with a vesicle marker. Pharmacological analysis using a low concentration of the actin polymerization inhibitor latrunculin B showed that the F-actin at the tip can be significantly diminished while myosin XI remains elevated in this region, suggesting that a mechanism exists to cluster myosin XI-associated structures at the cell's apex. In addition, this approach uncovered a mechanism for actin polymerization-dependent motility in the moss cytoplasm, where myosin XI-associated structures seem to anticipate and organize the actin polymerization machinery. From our results, we inferred a model where the interaction between myosin XI-associated vesicular structures and F-actin polymerization-driven motility function at the cell's apex to maintain polarized cell growth. We hypothesize this is a general mechanism for the participation of myosin XI and F-actin in tip growing cells.

Members of the cofilin/ADF family of proteins sever actin filaments, increasing the number of filament ends available for polymerization or depolymerization. Cofilin binds actin filaments with positive cooperativity, forming clusters of contiguously bound cofilin along the filament lattice. Filament severing occurs preferentially at boundaries between bare and cofilin-decorated (cofilactin) segments and is biased at 1 side of a cluster. A molecular understanding of cooperative binding and filament severing has been impeded by a lack of structural data describing boundaries. Here, we apply methods for analyzing filament cryo-electron microscopy (cryo-EM) data at the single subunit level to directly investigate the structure of boundaries within partially decorated cofilactin filaments. Subnanometer resolution maps of isolated, bound cofilin molecules and an actin-cofilactin boundary indicate that cofilin-induced actin conformational changes are local and limited to subunits directly contacting bound cofilin. An isolated, bound cofilin compromises longitudinal filament contacts of 1 protofilament, consistent with a single cofilin having filament-severing activity. An individual, bound phosphomimetic (S3D) cofilin with weak severing activity adopts a unique binding mode that does not perturb actin structure. Cofilin clusters disrupt both protofilaments, consistent with a higher severing activity at boundaries compared to single cofilin. Comparison of these structures indicates that this disruption is substantially greater at pointed end sides of cofilactin clusters than at the barbed end. These structures, with the distribution of bound cofilin clusters, suggest that maximum binding cooperativity is achieved when 2 cofilins occupy adjacent sites. These results reveal the structural origins of cooperative cofilin binding and actin filament severing.

Networks of branched actin filaments formed by Arp2/3 complex generate and experience mechanical forces during essential cellular functions, including cell motility and endocytosis. External forces regulate the assembly and architecture of branched actin networks both in vitro and in cells. Considerably less is known about how mechanical forces influence the disassembly of actin filament networks, specifically, the dissociation of branches. We used microfluidics to apply force to branches formed from purified muscle actin and fission yeast Arp2/3 complex and observed debranching events in real time with total internal reflection fluorescence microscopy. Low forces in the range of 0 pN to 2 pN on branches accelerated their dissociation from mother filaments more than two orders of magnitude, from hours to <1 min. Neither force on the mother filament nor thermal fluctuations in mother filament shape influenced debranching. Arp2/3 complex at branch junctions adopts two distinct mechanical states with different sensitivities to force, which we name “young/strong” and “old/weak.” The “young/strong” state 1 has adenosine 5′-diphosphate (ADP)−Pibound to Arp2/3 complex. Phosphate release converts Arp2/3 complex into the “old/weak” state 2 with bound ADP, which is 20 times more sensitive to force than state 1. Branches with ADP−Arp2/3 complex are more sensitive to debranching by fission yeast GMF (glia maturation factor) than branches with ADP−Pi−Arp2/3 complex. These findings suggest that aging of branch junctions by phosphate release from Arp2/3 complex and mechanical forces contribute to disassembling “old” actin filament branches in cells.

(L.V.).F-actin has been shown to be essential for tip growth in an array of plant models, including Physcomitrella patens. One hypothesis is that diffusion can transport secretory vesicles, while actin plays a regulatory role during secretion. Alternatively, it is possible that actin-based transport is necessary to overcome vesicle transport limitations to sustain secretion. Therefore, a quantitative analysis of diffusion, secretion kinetics, and cell geometry is necessary to clarify the role of actin in polarized growth. Using fluorescence recovery after photobleaching analysis, we first show that secretory vesicles move toward and accumulate at the tip in an actin-dependent manner. We then depolymerized F-actin to decouple vesicle diffusion from actin-mediated transport and measured the diffusion coefficient and concentration of vesicles. Using these values, we constructed a theoretical diffusion-based model for growth, demonstrating that with fast-enough vesicle fusion kinetics, diffusion could support normal cell growth rates. We further refined our model to explore how experimentally extrapolated vesicle fusion kinetics and the size of the secretion zone limit diffusion-based growth. This model predicts that diffusion-mediated growth is dependent on the size of the region of exocytosis at the tip and that diffusion-based growth would be significantly slower than normal cell growth. To further explore the size of the secretion zone, we used a cell wall degradation enzyme cocktail and determined that the secretion zone is smaller than 6 mm in diameter at the tip. Taken together, our results highlight the requirement for active transport in polarized growth and provide important insight into vesicle secretion during tip growth.

In plants, light determines chloroplast position; these organelles show avoidance and accumulation responses in high and low fluence-rate light, respectively. Chloroplast motility in response to light is driven by cytoskeletal elements. The actin cytoskeleton mediates chloroplast photorelocation responses in Arabidopsis thaliana. In contrast, in the moss Physcomitrella patens, both, actin filaments and microtubules can transport chloroplasts. Because of the surprising evidence that two kinesin-like proteins (called KACs) are important for actin-dependent chloroplast photorelocation in vascular plants, we wanted to determine the cytoskeletal system responsible for the function of these proteins in moss. We performed genespecific silencing using RNA interference in P. patens. We confirmed existing reports using gene knockouts, that PpKAC1 and PpKAC2 are required for chloroplast dispersion under uniform white light conditions, and that the two proteins are functionally equivalent. To address the specific cytoskeletal elements responsible for motility, this loss-offunction approach was combined with cytoskeleton-targeted drug studies. We found that, in P. patens, these KACs mediate the chloroplast light-avoidance response in an actin filamentdependent, rather than a microtubule-dependent manner. Using correlation-decay analysis of cytoskeletal dynamics, we found that PpKAC stabilizes cortical actin filaments, but has no effect on microtubule dynamics.

Fluorescence Recovery After Photobleaching (FRAP) is an important tool used by cell biologists to study the diffusion and binding kinetics of vesicles, proteins, and other molecules in the cytoplasm, nucleus or cell membrane. While many FRAP models have been developed over the past decades, the influence of the complex boundaries of three-dimensional cellular geometries on the recovery curves, in conjunction with ROI and optical effects (imaging, photobleaching, photoswitching, and scanning), has not been well studied. Here, we developed a three-dimensional computational model of the FRAP process that incorporates particle diffusion, cell boundary effects, and the optical properties of the scanning confocal microscope, and validated this model using the tip-growing cells of Physcomitrella patens. We then show how these cell boundary and optical effects confound the interpretation of FRAP recovery curves, including the number of dynamic states of a given fluorescent protein, in a wide range of cellular geometries-both in two and three dimensions-namely nuclei, filopodia, and lamellipodia of mammalian cells, and in cell types such as the budding yeast, S. pombe, and tip-growing plant cells. We explored the performance of existing analytical and algorithmic FRAP models in these various cellular geometries, and determined that the VCell VirtualFRAP tool provides the best accuracy to measure diffusion coefficients. Our computational model is not limited only to these cells types, but can easily be extended to other cellular geometries via the graphical Java-based application we also provide. This particle-based simulation-called the Digital Confocal Microscopy Suite, DCMS-can also perform fluorescence dynamics assays, such as Number and Brightness (N&B), Fluorescence Correlation Spectroscopy (FCS), Raster Image Correlation Spectroscopy (RICS), and could help shape the way these techniques are interpreted.

This protocol describes a quantitative analysis of the morphology of small plants from the moss Physcomitrella patens. The protocol can be used for the analysis of growth phenotypes produced by transient RNA interference or for the analysis of stable mutant plants. Information is presented to guide the investigator in the choice of vectors and basic conditions to perform transient RNA interference in moss. Detailed directions and examples for fluorescence image acquisition of small regenerating moss plants are provided. Instructions for the use of an ImageJ-based macro for quantitative morphological analysis of these plants are also provided.