The actin networks of chytrid fungi reveal evolutionary loss of cytoskeletal complexity in the fungal kingdom

Abstract

Cells from across the eukaryotic tree use actin polymers and a number of conserved regulators for a wide variety of functions including endocytosis, cytokinesis, and cell migration. Despite this conservation, the actin cytoskeleton has undergone significant evolution and diversification, highlighted by the differences in the actin cytoskeletal networks of mammalian cells and yeast. Chytrid fungi diverged before the emergence of the Dikarya (multicellular fungi and yeast), and therefore provide a unique opportunity to study the evolution of the actin cytoskeleton. Chytrids have two life stages: zoospore cells that can swim with a flagellum, and sessile sporangial cells that, like multicellular fungi, are encased in a chitinous cell wall. Here we show that zoospores of the amphibian-killing chytrid Batrachochytrium dendrobatidis (Bd) build dynamic actin structures that resemble those of animal cells, including pseudopods, an actin cortex, and filopodia-like actin spikes. In contrast, Bd sporangia assemble actin patches similar to those of yeast, as well as perinuclear actin shells. Our identification of actin cytoskeletal elements in the genomes of five species of chytrid fungi indicate that these actin structures are controlled by both fungalspecific components as well as actin regulators and myosin motors found in animals but not other fungal lineages. The use of specific small molecule inhibitors indicate that nearly all of Bd's actin structures are dynamic and use distinct nucleators: while pseudopods and actin patches are Arp2/3-dependent, the actin cortex appears formin-dependent, and actin spikes require both nucleators. The presence of animal-and yeast-like actin cytoskeletal components in the genome combined with the intermediate actin phenotypes in Bd suggests that the simplicity of the yeast cytoskeleton may be due to evolutionary loss. evolutionary Rosetta Stone with which we can map the simplified actin features of yeast to those of animals.Bd, like other chytrids, has two developmental stages: motile "zoospore" cells that lack a cellwall and swim with a flagellum, and non-motile sporangia that grow and produce new zoospores ( Fig. 1b; (Berger et al. 2005;Longcore et al. 1999). We recently showed that zoospores can also crawl across surfaces using actin-filled, Arp2/3-dependent pseudopods (Fritz-Laylin, Lord, et al. 2017). Other than these pseudopods, actin's role in any developmental stage of Bd has not been studied. To fill this knowledge gap, we identified homologs of key actin regulatory proteins and myosin motors in multiple species of chytrids and related fungi. We also identified new actin structures in zoospores and sporangia, and tested the requirements of Arp2/3 and formin family proteins for their assembly. We find that both the regulatory networks and actin structures of Bd are intermediate in complexity between animals and Dikarya, suggesting that the streamlined actin networks of common model fungi are a result of secondary evolutionary loss of actin network components. RESULTS Bd has dev...

A light-sensing system in the common ancestor of the fungi

“…10 In contrast to what we observed for the Blastocladiomycota zoospores, actin is the main cytoskeletal protein in the cell body of Chytridiomycota studied here and organizes itself into actin patches, which were clearly visible in almost all zoospores observed from both species (Figures 2O and 2S; supplemental information in FigShare). These actin patches have previously been observed in chytrids, 22,23 and they may correspond to what some authors refer to as fibrous areas identified from TEM analysis. 10 In the chytrid Report B.dendrobatidis, these actin patches were observed in a minority of the cells, and it has been argued that these are involved in endocytosis in sporangia.…”

“…Thus, these structures can be interpreted as a signal that zoospores may have initiated their transition to the sporangial growth stage. 23 Some actin signal was also detected in the flagellum of a subset of the R. globosum, B. emersonii, and A. macrogynus zoospores (Figure 2; supplemental information in FigShare), a phenomenon previously observed in other eukaryotes. [27][28][29] Presence of other photoreceptor proteins across the fungi A range of photoreceptive proteins have been identified across the fungi with a diversity of different mechanisms of action and a variety of structures and light sensitivities.…”

“…20,9,21 To stain the lipid components of zoospores, we used Nile red, and to stain the main components of the cytoskeleton, we used a-tubulin DM1A + Alexa Fluor 647 to stain tubulin and Alexa Fluor 488 Phalloidin to stain actin (Figure 2), both of which have previously been used to effectively study zoospore cytoskeletal systems in B. dendrobatidis and R. globosum. 22,23 Our micrographs of Be (Figures 2A-2H; supplemental information in FigShare) show a large SBC located at the base of the flagellum and between the microtubular cytoskeleton of the zoospore and its cellular membrane. The SBC is composed of multiple lipid particles in close association (Figures 2A-2D).…”

A structure-derived mechanism reveals how capping protein promotes nucleation in branched actin networks

“…Heterodimeric capping protein (CP/CapZ) is an evolutionarily ancient actin regulator found in nearly all eukaryotic organisms and cell types (Cooper and Sept, 2008;Edwards et al, 2014;Prostak et al, 2020;Rivero and Cvrcková, 2007) that controls the fate and interactions of actin filament ends in a variety of cellular contexts. Comprised of two closely related α and β subunits, it forms a constitutive heterodimer that tightly binds to the barbed end of actin filaments to terminate their growth.…”

A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks

“…eterodimeric capping protein (CP/CapZ) is an evolutionarily ancient actin regulator found in nearly all eukaryotic organisms and cell types [1][2][3][4] that controls the fate and interactions of actin filament ends in a variety of cellular contexts. Comprised of two closely related α and β subunits, it forms a constitutive heterodimer that tightly binds to the barbed end of actin filaments to terminate their growth.…”