- Email: [email protected]
Actin Nanobodies Uncover the Mystery of Actin Filament Dynamics in Toxoplasma gondii
TREPAR 1659 No. of Pages 3
Spotlight
Actin Nanobodies Uncover the Mystery of Actin Filament Dynamics in Toxoplasma gondii Isabelle Tardieux1,* While the intracellular parasite Toxoplasma relies on a divergent actomyosin motor to support unique speeds in directional movement, the dynamics and architecture of parasite actin filaments remain a much-discussed issue. Using actin chromobodies, Periz et al. started to unveil how networks of dynamic F-actin connect Toxoplasma progeny and expand in the replicative vacuole. Actin isoforms, owing to their abundance and to their intrinsic ability to transition between a monomeric and a polymeric form, [17_TD$IF]are involved in a remarkable array of functions in eukaryotic cells. Understanding how actin contributes to highly coordinated and diverse processes – such as vesicle trafficking and secretion, cell division and differentiation, gene expression, cell-to-cell communication, and tissue morphogenesis – remains a major focus across multiple disciplines in life science research. Over the years, hundreds of actin partners were identified. These directly or indirectly couple the actin cytoskeleton to plasma and intracellular membranes, to microtubules, and to intermediate filaments, in order to provide scaffolds and to fulfill specific functions in a timely and localized fashion. Aside from the biochemical and structural knowledge of actin, which relies on purified native and recombinant actins, an enormous effort has been made over
recent decades to probe the behavior of actin in live cells. To that end, the ectopic expression of fluorescently tagged actin, or the micro-injection of fluorescent exogenous actin, in conjunction with high-speed and highly sensitive fluorescence microscopy, has allowed computer-assisted quantification of actin turnover and actin flow at the leading edge of a crawling cell [1]. Total internal reflection fluorescence (TIRF) microscopy has also been instrumental in monitoring actin cycles at focal adhesion sites during cell [18_TD$IF]crawling and spreading on substrates. Despite their informative value, such fluorescent actin fusion products may not behave like native actin in their assembly kinetics in living cells, and they may also often lack the functionality of native actin. Furthermore, because they coexist with endogenous actin, these fluorescent actin versions provide limited resolution of actin pools and of actin distribution in cells. To improve actin detection in live cells, new fluorogenic probes – derived from the F-actin binding domain of proteins, or from F-actin binding compound – were developed. They include, for example, the cell-permeant SiR, a silicon–rhodamine molecule fused to the F-actin binding compound jasplakinolide, and the genetically encoded Lifeact derived from the 17 first amino acids of the yeast ABP140 [2,3]. These actin probes have the advantage of preserving actin dynamics, and they provide a high signal-to-noise ratio that allows analysis of the dynamic features of actin-related processes, and possibly their dysfunctions under pathological settings, or through hijacking, by invasive microbes, in the course of specific invasive microbe– cell interactions. Unfortunately SiR and Lifeact [19_TD$IF]do not label the actin cytoskeleton – in particular, F-actin of the protozoan parasites Toxoplasma gondii and Plasmodium spp. T. gondii and Plasmodium spp. are pre-eminent single-celled eukaryotic parasites, clustered within the phylum Apicomplexa, that display notorious actin-dependent motile and invasive skills [4]. While it is acknowledged
that actin dynamics is tightly regulated to promote transient F-actin assembly, we have only incomplete mechanistic details of how Toxoplasma [120_TD$IF]actin product TgACT1 and Plasmodium isoform PfACT1 (which is expressed in invasive stages) support motile and invasive functions. This is, in part, due to a significant divergence between parasite and mammalian actin sequences likely accounting for functional singularities. TgACT1 has only 83% sequence identity with mammalian actin and 93.1% with PfACT1 [5]. These differences may have contributed to the difficulty to (i) produce functional recombinant actin in vitro based on protocols for mammalian actins, (ii) collect a population of actin filaments by ultracentrifugation, and (iii) visualize actin filaments in situ. However, the two first limitations have been overcome in Toxoplasma and have led Skillman and collaborators to propose unusual noncooperative isodesmic polymerization kinetics for Toxoplasma actin in vitro [6] – in contrast to the conventional cooperative nucleation– elongation mechanism of eukaryotic actin polymerization[12_TD$IF]. On the other hand, the assembly kinetics of actin filaments in the context of live cells in Toxoplasma remained quite elusive until Periz and colleagues [7] decided to take advantage of the recently developed actin chromobody (Cb) strategy, which was reported to have low cytotoxicity and to preserve the full dynamic properties of actin [8]. Periz and colleagues engineered stable T. gondii lines that express an actin Cb in fusion with either Emerald GFP or Halotag; both lines contain similar amounts of actin as the parental one and maintain intact gliding skills and growth potential in vitro. With high-resolution imaging, Periz and colleagues provided unprecedented resolution of F-actin inside live, intracellular T. gondii tachyzoites, pointing to previously unforeseen roles of actin over their developmental program within a parasitophorous vacuole (PV). The authors discovered a peculiar filamentous and thick structure that
Trends in Parasitology, Month Year, Vol. xx, No. yy
1
TREPAR 1659 No. of Pages 3
connected individual cells and that spectacularly extended as a long tube or cable-like network within the PV housing the replicating zoites. Because Toxoplasma actin filaments were thought to be transient and short, the authors brought convincing evidence that the actin Cb did label intact and functional F-actin populations using actin-regulatory drugs and conditional mutants for TgACT1, and the actin-depolymerising factor (TgADF) that concomitantly disrupted or stabilized F-actin and the Cbpositive network. In addition to static imaging, dynamic imaging of the intravacuolar network, in the course of parasite replication, revealed that the Cb-positive F-actin network emerged at, or in close vicinity to, a mother residual body, spread while the progeny expanded inside the enlarging PV, and eventually collapsed when the tachyzoite progeny left the PV. Interestingly, fluorescence recovery after photobleaching (FRAP) data support the notion that the network is made of dynamic actin filaments. Moreover, imaging vesicles moving along the network suggest a yet unknown mode of material exchange between two connected parasites or between the parasites and the PV space or PV membrane. These results raise a myriad of new questions, such as: What is so unique about actin nucleation and elongation in T. gondii that leads to filament assembly into a cablelike, bundled structure? What drives the organization of bundled filaments in the absence of a well defined actin-bundling factor? Do actin bundles interact with the lipid– protein tubulo-vesicular network that occupies the PV space – and, if so, how? Among the 11 myosins identified in Toxoplasma – some being ideally restricted to the rear end of the parasite, or at the residual body – which ones can sustain vesicular trafficking along the filaments? How do they transport cargo to their destination? [12_TD$IF] It is worth mentioning that at the time of writing this review, an interesting study
2
Trends in Parasitology, Month Year, Vol. xx, No. yy
from Frénal et al. [9] came out, which identified TgMyoI and TgMyoJ at the basal end of tachyzoites, where they appeared to promote formation or to maintain the connection between daughter cells within the PV, thereby opening the way to new investigations. To answer a few of these questions, although technically challenging, the extraction of the Cb-Halo-actin fraction from isolated PVs should provide an exquisite informative material for proteomic and lipidomic analysis, as well as a novel way to assess actin dynamic properties in vitro. Of note, considering the extensive F-actin network, G-actin may be much less abundant in intracellular T. gondii tachyzoites than in free ones.
to G-actin. This F-actin population may represent individual, or a few, crosslinked filaments working differently in space and time during replication or during the free life of tachyzoites. Indeed, according to the phenotypic analysis of the inducible knockout actin [123_TD$IF][10], these filaments should contribute to apicoplast inheritance and vesicular trafficking but also to extracellular gliding and cell invasion. Monitoring how and where these filaments emerge, elongate, and recycle will certainly require future imaging adjustments to increase signal sensitivity, speed, and resolution. Thanks to the great achievement in generating a tool that all the community was waiting for, and in conjunction with photoactivation and inactivation-based approaches, and super-resolution live-cell imaging, the mystery of the actin contribution to the lytic cycle of tachyzoites might be finally elucidated in the near future.
The Toxoplasma Cb-actin line also revealed a second pool of F-actin inside intracellular and extracellular tachyzoites. It was detected as discrete entities that nevertheless remained discernible from [ eriz et al. [7] now describe unique Fthe diffuse cytoplasmic signal possibly 124_TD$IF]P caused by the weak binding of Cb-actin actin networks that (1) connect one 1- Tachyzoite asexual reproducon by endodyogeny within an intracellular parasitophorous vacuole (PV) PV
Key: F-acn Micronemes
Rhoptries
Microtubules
Nucleus
Apicoplast
Dense granules
Conoid
2- Acve egress of the tachyzoite progeny from the PV before host cell exit
Figure 1. Actin Networks Form, Expand, and Collapse during Intracellular Development of Toxoplasma gondii Tachyzoites.[15_TD$IF] Following invasion of the host cell via a membrane-bound vacuole termed the parasitophorous vacuole (PV), the polarized tachyzoite cell starts to replicate by endodyogeny within the PV. Endodyogeny proceeds with the biogenesis of two daughter parasites within the mother, and their final budding that coincides with the collapse of the maternal material into a residual body (RB) at the posterior end of the daughter parasites. The tachyzoite progeny progressively organizes into a polarized rosette around the RB. Once the progeny fills most of the host cell, tachyzoites actively exit from the PV and the host cell to repeat the lytic cycle.
TREPAR 1659 No. of Pages 3
daughter to the other, and presumably contribute to the rosette pattern while allowing interparasite communication, and (2) collapse while F-actin retreats to the RB before the networks reform again and expand throughout the PV. Breakdown of the network also precedes tachyzoite egress from the PV and the host cell while residual F actin can still be seen within the RB [125_TD$IF](Figure 1). 1
Institute of Advanced BioSciences, Institut National de la Santé et de la Recherche Médicale U1209, Centre National de la Recherche Scientifique UMR 5309, Université Grenoble Alpes, 38000, Grenoble, France
*Correspondence: [email protected] (I. Tardieux). http://dx.doi.org/10.1016/j.pt.2017.06.007 References 1. Danuser, G. and Waterman-Storer, C.M. (2006) Quantitative fluorescent speckle microscopy of cytoskeleton dynamics. Annu. Rev. Biophys. Biomol. Struct. 35, 361– 387 2. Lukinavi9 cius, G. et al. (2014) Fluorogenic probes for livecell imaging of the cytoskeleton. Nat. Methods 11, 731– 733 3. Riedl, J. et al. (2008) Lifeact: a versatile marker to visualize F-actin. Nat. Methods 5, 605–607 4. Tardieux, I. and Baum, J. (2016) Reassessing the mechanics of parasite motility and host-cell invasion. J. Cell Biol. 214, 507–515 5. Dobrowolski, J.M. et al. (1997) Actin in the parasite Toxoplasma gondii is encoded by a single copy gene, ACT1,
and exists primarily in a globular form. Cell Motil. Cytoskelet. 37, 253–262 6. Skillman, K.M. et al. (2013) The unusual dynamics of parasite actin result from isodesmic polymerization. Nat. Commun. 4, 2285 7. Periz, J. et al. (2017) Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation. Elife 6, pii e24119 8. Panza, P. et al. (2015) Live imaging of endogenous protein dynamics in zebrafish using chromobodies. Development 142, 1879–1884 9. Frénal, K. et al. (2017) Myosin-dependent cell-cell communication controls synchronicity of division in acute and chronic stages of Toxoplasma gondii. Nat. Commun. 8, 15710 10. Whitelaw, J.A. et al. (2017) Surface attachment, promoted by the actomyosin system of Toxoplasma gondii is important for efficient gliding motility and invasion. BMC Biol. 15, 1
Trends in Parasitology, Month Year, Vol. xx, No. yy
3
Spotlight
Actin Nanobodies Uncover the Mystery of Actin Filament Dynamics in Toxoplasma gondii Isabelle Tardieux1,* While the intracellular parasite Toxoplasma relies on a divergent actomyosin motor to support unique speeds in directional movement, the dynamics and architecture of parasite actin filaments remain a much-discussed issue. Using actin chromobodies, Periz et al. started to unveil how networks of dynamic F-actin connect Toxoplasma progeny and expand in the replicative vacuole. Actin isoforms, owing to their abundance and to their intrinsic ability to transition between a monomeric and a polymeric form, [17_TD$IF]are involved in a remarkable array of functions in eukaryotic cells. Understanding how actin contributes to highly coordinated and diverse processes – such as vesicle trafficking and secretion, cell division and differentiation, gene expression, cell-to-cell communication, and tissue morphogenesis – remains a major focus across multiple disciplines in life science research. Over the years, hundreds of actin partners were identified. These directly or indirectly couple the actin cytoskeleton to plasma and intracellular membranes, to microtubules, and to intermediate filaments, in order to provide scaffolds and to fulfill specific functions in a timely and localized fashion. Aside from the biochemical and structural knowledge of actin, which relies on purified native and recombinant actins, an enormous effort has been made over
recent decades to probe the behavior of actin in live cells. To that end, the ectopic expression of fluorescently tagged actin, or the micro-injection of fluorescent exogenous actin, in conjunction with high-speed and highly sensitive fluorescence microscopy, has allowed computer-assisted quantification of actin turnover and actin flow at the leading edge of a crawling cell [1]. Total internal reflection fluorescence (TIRF) microscopy has also been instrumental in monitoring actin cycles at focal adhesion sites during cell [18_TD$IF]crawling and spreading on substrates. Despite their informative value, such fluorescent actin fusion products may not behave like native actin in their assembly kinetics in living cells, and they may also often lack the functionality of native actin. Furthermore, because they coexist with endogenous actin, these fluorescent actin versions provide limited resolution of actin pools and of actin distribution in cells. To improve actin detection in live cells, new fluorogenic probes – derived from the F-actin binding domain of proteins, or from F-actin binding compound – were developed. They include, for example, the cell-permeant SiR, a silicon–rhodamine molecule fused to the F-actin binding compound jasplakinolide, and the genetically encoded Lifeact derived from the 17 first amino acids of the yeast ABP140 [2,3]. These actin probes have the advantage of preserving actin dynamics, and they provide a high signal-to-noise ratio that allows analysis of the dynamic features of actin-related processes, and possibly their dysfunctions under pathological settings, or through hijacking, by invasive microbes, in the course of specific invasive microbe– cell interactions. Unfortunately SiR and Lifeact [19_TD$IF]do not label the actin cytoskeleton – in particular, F-actin of the protozoan parasites Toxoplasma gondii and Plasmodium spp. T. gondii and Plasmodium spp. are pre-eminent single-celled eukaryotic parasites, clustered within the phylum Apicomplexa, that display notorious actin-dependent motile and invasive skills [4]. While it is acknowledged
that actin dynamics is tightly regulated to promote transient F-actin assembly, we have only incomplete mechanistic details of how Toxoplasma [120_TD$IF]actin product TgACT1 and Plasmodium isoform PfACT1 (which is expressed in invasive stages) support motile and invasive functions. This is, in part, due to a significant divergence between parasite and mammalian actin sequences likely accounting for functional singularities. TgACT1 has only 83% sequence identity with mammalian actin and 93.1% with PfACT1 [5]. These differences may have contributed to the difficulty to (i) produce functional recombinant actin in vitro based on protocols for mammalian actins, (ii) collect a population of actin filaments by ultracentrifugation, and (iii) visualize actin filaments in situ. However, the two first limitations have been overcome in Toxoplasma and have led Skillman and collaborators to propose unusual noncooperative isodesmic polymerization kinetics for Toxoplasma actin in vitro [6] – in contrast to the conventional cooperative nucleation– elongation mechanism of eukaryotic actin polymerization[12_TD$IF]. On the other hand, the assembly kinetics of actin filaments in the context of live cells in Toxoplasma remained quite elusive until Periz and colleagues [7] decided to take advantage of the recently developed actin chromobody (Cb) strategy, which was reported to have low cytotoxicity and to preserve the full dynamic properties of actin [8]. Periz and colleagues engineered stable T. gondii lines that express an actin Cb in fusion with either Emerald GFP or Halotag; both lines contain similar amounts of actin as the parental one and maintain intact gliding skills and growth potential in vitro. With high-resolution imaging, Periz and colleagues provided unprecedented resolution of F-actin inside live, intracellular T. gondii tachyzoites, pointing to previously unforeseen roles of actin over their developmental program within a parasitophorous vacuole (PV). The authors discovered a peculiar filamentous and thick structure that
Trends in Parasitology, Month Year, Vol. xx, No. yy
1
TREPAR 1659 No. of Pages 3
connected individual cells and that spectacularly extended as a long tube or cable-like network within the PV housing the replicating zoites. Because Toxoplasma actin filaments were thought to be transient and short, the authors brought convincing evidence that the actin Cb did label intact and functional F-actin populations using actin-regulatory drugs and conditional mutants for TgACT1, and the actin-depolymerising factor (TgADF) that concomitantly disrupted or stabilized F-actin and the Cbpositive network. In addition to static imaging, dynamic imaging of the intravacuolar network, in the course of parasite replication, revealed that the Cb-positive F-actin network emerged at, or in close vicinity to, a mother residual body, spread while the progeny expanded inside the enlarging PV, and eventually collapsed when the tachyzoite progeny left the PV. Interestingly, fluorescence recovery after photobleaching (FRAP) data support the notion that the network is made of dynamic actin filaments. Moreover, imaging vesicles moving along the network suggest a yet unknown mode of material exchange between two connected parasites or between the parasites and the PV space or PV membrane. These results raise a myriad of new questions, such as: What is so unique about actin nucleation and elongation in T. gondii that leads to filament assembly into a cablelike, bundled structure? What drives the organization of bundled filaments in the absence of a well defined actin-bundling factor? Do actin bundles interact with the lipid– protein tubulo-vesicular network that occupies the PV space – and, if so, how? Among the 11 myosins identified in Toxoplasma – some being ideally restricted to the rear end of the parasite, or at the residual body – which ones can sustain vesicular trafficking along the filaments? How do they transport cargo to their destination? [12_TD$IF] It is worth mentioning that at the time of writing this review, an interesting study
2
Trends in Parasitology, Month Year, Vol. xx, No. yy
from Frénal et al. [9] came out, which identified TgMyoI and TgMyoJ at the basal end of tachyzoites, where they appeared to promote formation or to maintain the connection between daughter cells within the PV, thereby opening the way to new investigations. To answer a few of these questions, although technically challenging, the extraction of the Cb-Halo-actin fraction from isolated PVs should provide an exquisite informative material for proteomic and lipidomic analysis, as well as a novel way to assess actin dynamic properties in vitro. Of note, considering the extensive F-actin network, G-actin may be much less abundant in intracellular T. gondii tachyzoites than in free ones.
to G-actin. This F-actin population may represent individual, or a few, crosslinked filaments working differently in space and time during replication or during the free life of tachyzoites. Indeed, according to the phenotypic analysis of the inducible knockout actin [123_TD$IF][10], these filaments should contribute to apicoplast inheritance and vesicular trafficking but also to extracellular gliding and cell invasion. Monitoring how and where these filaments emerge, elongate, and recycle will certainly require future imaging adjustments to increase signal sensitivity, speed, and resolution. Thanks to the great achievement in generating a tool that all the community was waiting for, and in conjunction with photoactivation and inactivation-based approaches, and super-resolution live-cell imaging, the mystery of the actin contribution to the lytic cycle of tachyzoites might be finally elucidated in the near future.
The Toxoplasma Cb-actin line also revealed a second pool of F-actin inside intracellular and extracellular tachyzoites. It was detected as discrete entities that nevertheless remained discernible from [ eriz et al. [7] now describe unique Fthe diffuse cytoplasmic signal possibly 124_TD$IF]P caused by the weak binding of Cb-actin actin networks that (1) connect one 1- Tachyzoite asexual reproducon by endodyogeny within an intracellular parasitophorous vacuole (PV) PV
Key: F-acn Micronemes
Rhoptries
Microtubules
Nucleus
Apicoplast
Dense granules
Conoid
2- Acve egress of the tachyzoite progeny from the PV before host cell exit
Figure 1. Actin Networks Form, Expand, and Collapse during Intracellular Development of Toxoplasma gondii Tachyzoites.[15_TD$IF] Following invasion of the host cell via a membrane-bound vacuole termed the parasitophorous vacuole (PV), the polarized tachyzoite cell starts to replicate by endodyogeny within the PV. Endodyogeny proceeds with the biogenesis of two daughter parasites within the mother, and their final budding that coincides with the collapse of the maternal material into a residual body (RB) at the posterior end of the daughter parasites. The tachyzoite progeny progressively organizes into a polarized rosette around the RB. Once the progeny fills most of the host cell, tachyzoites actively exit from the PV and the host cell to repeat the lytic cycle.
TREPAR 1659 No. of Pages 3
daughter to the other, and presumably contribute to the rosette pattern while allowing interparasite communication, and (2) collapse while F-actin retreats to the RB before the networks reform again and expand throughout the PV. Breakdown of the network also precedes tachyzoite egress from the PV and the host cell while residual F actin can still be seen within the RB [125_TD$IF](Figure 1). 1
Institute of Advanced BioSciences, Institut National de la Santé et de la Recherche Médicale U1209, Centre National de la Recherche Scientifique UMR 5309, Université Grenoble Alpes, 38000, Grenoble, France
*Correspondence: [email protected] (I. Tardieux). http://dx.doi.org/10.1016/j.pt.2017.06.007 References 1. Danuser, G. and Waterman-Storer, C.M. (2006) Quantitative fluorescent speckle microscopy of cytoskeleton dynamics. Annu. Rev. Biophys. Biomol. Struct. 35, 361– 387 2. Lukinavi9 cius, G. et al. (2014) Fluorogenic probes for livecell imaging of the cytoskeleton. Nat. Methods 11, 731– 733 3. Riedl, J. et al. (2008) Lifeact: a versatile marker to visualize F-actin. Nat. Methods 5, 605–607 4. Tardieux, I. and Baum, J. (2016) Reassessing the mechanics of parasite motility and host-cell invasion. J. Cell Biol. 214, 507–515 5. Dobrowolski, J.M. et al. (1997) Actin in the parasite Toxoplasma gondii is encoded by a single copy gene, ACT1,
and exists primarily in a globular form. Cell Motil. Cytoskelet. 37, 253–262 6. Skillman, K.M. et al. (2013) The unusual dynamics of parasite actin result from isodesmic polymerization. Nat. Commun. 4, 2285 7. Periz, J. et al. (2017) Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation. Elife 6, pii e24119 8. Panza, P. et al. (2015) Live imaging of endogenous protein dynamics in zebrafish using chromobodies. Development 142, 1879–1884 9. Frénal, K. et al. (2017) Myosin-dependent cell-cell communication controls synchronicity of division in acute and chronic stages of Toxoplasma gondii. Nat. Commun. 8, 15710 10. Whitelaw, J.A. et al. (2017) Surface attachment, promoted by the actomyosin system of Toxoplasma gondii is important for efficient gliding motility and invasion. BMC Biol. 15, 1
Trends in Parasitology, Month Year, Vol. xx, No. yy
3