Structural basis of rapid actin dynamics in the evolutionarily divergent Leishmania parasite

Abstract

Actin polymerization generates forces for cellular processes throughout the eukaryotic kingdom, but our understanding of the ‘ancient’ actin turnover machineries is limited. We show that, despite > 1 billion years of evolution, pathogenic Leishmania major parasite and mammalian actins share the same overall fold and co-polymerize with each other. Interestingly, Leishmania harbors a simple actin-regulatory machinery that lacks cofilin ‘cofactors’, which accelerate filament disassembly in higher eukaryotes. By applying single-filament biochemistry we discovered that, compared to mammalian proteins, Leishmania actin filaments depolymerize more rapidly from both ends, and are severed > 100-fold more efficiently by cofilin. Our high-resolution cryo-EM structures of Leishmania ADP-, ADP-Pi- and cofilin-actin filaments identify specific features at actin subunit interfaces and cofilin-actin interactions that explain the unusually rapid dynamics of parasite actin filaments. Our findings reveal how divergent parasites achieve rapid actin dynamics using a remarkably simple set of actin-binding proteins, and elucidate evolution of the actin cytoskeleton.

Unusual actin-binding mechanism and the role of profilin in actin dynamics of trypanosomatid parasites

“…Because an earlier study on Leishmania actin dynamics demonstrated that rabbit muscle actin can co-polymerize with Leishmania actin [18], we performed pyrene-actin polymerization experiments by using a mix of 95 % LmActin and 5 % pyrene-labeled rabbit muscle actin. As demonstrated before [18], purified LmActin polymerized readily in the absence of any actin filament nucleators, and this is most likely due to rapid spontaneous nucleation of this actin (Fig. 5C).…”

“…L. major profilin (LmProfilin; TriTrypDB ID: LmjF.32.0520) wild-type and mutants were expressed as fusion proteins with an N-terminal double tag (His6x-GST). The recombinant proteins were purified using an approach reported before [18]. Briefly, E.coli BL21(DE3) (Merck Millipore) cells were grown at 22°C in LB autoinduction media (AIMLB0210, Formedium) supplemented with kanamycin (20 µg/mL) for ~24 h. After lysis, recombinant proteins were first purified using a Ni-NTA column (GE HealthCare) and the His-GST tag was removed by cleavage with 3C-PreScision protease and subsequent incubation with Ni 2+ beads to remove the uncleaved protein and the tag from the solution.…”

“…Moreover, depletion of profilin was reported to affect cellular growth and endocytic trafficking [30], and contribute to mitotic spindle orientation and cell cycle progression in L. donovani parasites [31]. However, no structural information of trypanosomatid profilins is available, and all biochemical work so far has been performed by using a heterologous combination of mammalian and Leishmania actin-binding proteins, which were recently demonstrated to be unfavorable substrates for each other [18]. Thus, the mechanisms by which trypanosomatid profilins interact with actin monomers, and regulate actin dynamics together with other proteins, such as formins, have remained elusive.…”

“…However, due to differences in the subunit-subunit interfaces, the parasite actin filaments display more rapid turnover compared to animal actin filaments. The same study also revealed that L. major cofilin fragments LmActin filaments more frequently compared to mammalian cofilin, demonstrating that both actin filaments and their interplay with cofilin display pronounced differences between animals and trypanosomatids [18].…”

Structure and function ofPlasmodiumactin II in the parasite mosquito stages

“…In contrast, structural data on F-form actin has been more challenging to obtain. During the last years, advances in single-particle electron cryo-microscopy (cryo-EM) have enabled determining high-resolution structures of F-actin from different organisms, such as skeletal muscle a-actin from Mus musculus [11], Oryctolagus cuniculus [12], Gallus gallus [13][14][15], Leishmania major [16] and actin I from P. falciparum [17] to resolutions between 2.15-3 Å. The short length and instability of P. falciparum actin I has allowed structure determination only of filaments stabilized by jasplakinolide (JAS).…”