Insights into the origin of metazoan filopodia and microvilli

Abstract

Filopodia are fine actin-based cellular projections used for both environmental sensing and cell motility, and they are essential organelles for metazoan cells. In this study, we reconstruct the origin of metazoan filopodia and microvilli. We first report on the evolutionary assembly of the filopodial molecular toolkit and show that homologs of many metazoan filopodial components, including fascin and myosin X, were already present in the unicellular or colonial progenitors of metazoans. Furthermore, we find that the actin crosslinking protein fascin localizes to filopodia-like structures and microvilli in the choanoflagellate Salpingoeca rosetta. In addition, homologs of filopodial genes in the holozoan Capsaspora owczarzaki are upregulated in filopodia-bearing cells relative to those that lack them. Therefore, our findings suggest that proteins essential for metazoan filopodia and microvilli are functionally conserved in unicellular and colonial holozoans and that the last common ancestor of metazoans bore a complex and specific filopodial machinery.

Keywords: Capsaspora; Cdc42; choanoflagellate; fascin; filopodia; formin evolution; gelsolin evolution; pseudopodia.

Fig. 1.

Phylogenetic distribution of diverse proteins associated with filopodia and related actin-based cellular protrusions. A black dot indicates the presence of clear homologs, whereas absence of a dot indicates that a homolog was not detected in that taxon. ¹Based on Boureux et al. (2007). ²Only present in the excavates Naegleria gruberi and Trichomonas vaginalis. ³Plants have a villin-like protein that, despite having the same domain architecture as metazoans, is not phylogenetically related with holozoan villin (supplementary fig. S5, Supplementary Material online). ⁴Rac1 RhoGTPases are key filopodia-inducers in amoebozoans, but not in metazoans (Dumontier et al. 2000).

Fig. 2.

Filopodia-like structures in close relatives of metazoans. (A) Capsaspora owczarzaki filopodiated cells bear multiple long bundles of actin microfilaments, as revealed by staining with phalloidin (green). The cell periphery is revealed by staining with antibodies against beta-tubulin (red). SEM shows the presence of multiple long filopodia-like structures in C. owczarzaki filopodiated cells (B), but not in nonfilopodiated, cystic cells (C). (D) In Salpingoeca rosetta, attached cells bear actin microfilaments in the apical collar of microvilli and in basally positioned long cellular protrusions that resemble filopodia. Salpingoeca rosetta cells were stained with phalloidin (green) and antibodies against beta-tubulin (red). (E) TEM of thin sections through a choanoflagellate shows the presence of basally positioned cellular processes (indicated with black rectangle), shown in higher magnification in (F). Scale bars (A–E: 1 µM, F: 200 nm).

Fig. 3.

Subcellular localization of Fascin in Salpingoeca rosetta. (A) Phase contrast microscopy shows the morphology of a fixed S. rosetta cell. (B, C) Immunolocalization studies reveal that Fascin localizes to a basal filopodia-like structure (fp) and to the apical actin filled collar (c). (D) Western blot analysis shows that S. rosetta cell lysate probed with Fascin antibodies detect a single band of approximately 55 kDa (+). No signal was detected when primary Fascin antibody was omitted (−). f, flagellum; c, microvilli collar; fp, filopodia. Scale bar: 1 µM.

Fig. 4.

Expression of filopodial and related genes in unicellular holozoans. (A) Log₂-fold expression (see Materials and Methods) of Capsaspora owczarzaki filopodial genes between filopodiated and cystic stages. (B) Log₂-fold expression of Salpingoeca rosetta filopodial genes between attached and colonial stages. Red lines highlight 2-fold expression differences. For clarity, negative values indicate overexpression in one stage compared with the other, and vice versa.

Fig. 5.

Evolution of the metazoan filopodial toolkit. Schematic representation of the metazoan filopodial toolkit (left), with colors indicating the inferred evolutionary origin of each gene (white, eukaryotes; blue, Amorphea; yellow, opisthokonts + apusozoans; orange, holozoans; green, choanoflagellates + metazoans; red, metazoans). The cladogram (right) represents gains and losses and the presence of filopodia and microvilli in different groups. Dashed filopodia in other eukaryotes represent the presence of filopodia only in some groups (see main text).