Close

Blocking palmitoylation of Toxoplasma gondii myosin light chain 1 disrupts glideosome composition but has little impact on parasite motility

Pramod K. Rompikuntal, Ian T. Foe, Bin Deng, Matthew Bogyo, Gary E. Ward

Preprint posted on 14 August 2020 https://www.biorxiv.org/content/10.1101/2020.08.13.250399v1

Article now published in mSphere at http://dx.doi.org/10.1128/msphere.00823-20

Missing pieces and unexpected findings: Understanding T. gondii gliding motility.

Selected by Mariana De Niz

Categories: cell biology

Background

     Toxoplasma gondii is an apicomplexan parasite that causes toxoplasmosis- a widespread infection that is common among humans. Unfortunately, Toxoplasma gondii causes severe disease in immunocompromised individuals, and in the developing foetus if it infects pregnant women. T. gondii uses gliding motility to invade host cells, and disseminate within the host body throughout infection. The linear model of gliding motility (reviewed in 1) establishes that T. gondii MyoA (a class XIVa myosin), and its associated light chains TgMLC1 and either TgELC1 or TgELC2, are anchored to the parasite’s inner membrane complex (IMC) via TgGAP45, which in turn binds TgGAP40 and TgGAP50 – both transmembrane proteins. TgGAP50 is thought to serve as a fixed anchor against which the motor complex can generate force. This entire complex, is known as the glideosome.

     TgMLC1 is thought to play two key roles within the glideosome: a) amplifying small motions at the myosin active site into larger movements capable of displacing actin filaments due to binding to the C-terminal tail of TgMyoA to reinforce the motor’s lever arm; and b) the interaction of the N-terminal portion of TgMLC1 at the C-terminal portion of TgGAP45 is thought to be the critical link that tethers the motor to the IMC. Ultimately, TgMLC1 has been shown to be essential for 3D motility, invasion, and host cell egress.

     Despite the linear model having dominated the field for a decade, several phenotypic observations are hard to reconcile with the linear model, suggesting alternative motility mechanisms not explained by the model, might exist. In their current work, Rompikuntal et al (2) investigated the phenotypic consequences of mutations that block TgMLC1 palmitoylation.

Figure 1. Schematic illustrations of the glideosome and the TgMLC1 domain structure (from Ref. 2).

 

Key findings and developments

    Palmitoylation is a widespread post-translational modification thought to play an important role in the biology of T. gondii. S-palmitoylation of proteins mediates membrane association and can regulate phenomena such as subcellular localization, trafficking, structure, stability and various aspects of protein function. Various recent proteomic studies have identified several hundreds of putatively palmitoylated proteins, including all components of the glideosome.

C8 and C11 are likely palmitoylation sites on TgMLC1. Moreover, mutations blocking TgMLC1 palmitoylation do not alter its subcellular localization.

    Two of the five cysteine residues of TgMLC1 have been predicted as potential palmitoylation sites, namely C8 and C11. Both sites are located in the N-terminal extension of TgMLC1- the region that binds to TgGAP45. The authors began by experimentally confirming whether C8 and/or C11 are sites of palmitoylation, by replacing the endogenous TgMLC1 gene with mutant alleles that produced either single or double cysteine-to-serine mutations, rendering these sites non-palmitoylatable. FLAG-tagged lines of the single and double mutants, as well as a control line, were grown in the palmitic acid analogue 17-octadecynoic acid (17-ODYA). Using a combination of assays including TgMLC1 immunoprecipitation, rhodamine-azide tagging of 17-ODYA, and fluorescence scanning, the authors measured the amount of rhodamine fluorescence associated with TgMLC1. They found fluorescence was significantly reduced in the C8S and C11S mutants compared to WT, while in the double mutant no fluorescence was detected, demonstrating that the mutations partially or fully block 17-ODYA labelling. This altogether suggests that C8 and C11 are indeed, very likely sites of palmitoylation on TgLMC1.

Exploring TgMLC1 localization.

   The authors then went on to determine the subcellular localization of non-palmitoylatable TgMLC1. While in WT parasites TgMLC1 localizes uniformly to the parasite periphery, the authors found that in the single and double C8 and/or C11 cysteine-to-serine mutants, TgMLC1 localization remains unaltered. Therefore, blocking palmitoylation does not alter TgLMC1 sub-cellular localization.

    The authors then tested whether blocking palmitoylation of TgMLC1 alters its phase partitioning in Triton X-114. Triton X-114 allows separation into aqueous and detergent phases, respectively enriched in hydrophilic and integral membrane proteins. The WT and single C8 and C11 mutants of TgMLC1 partitioned roughly equally into both phases, but the double C8/C11 mutant was found almost entirely in the aqueous phase. This suggests a lack of direct membrane association in the absence of palmitoylation.

Effects of TgMLC1 palmitoylation on the composition of the glideosome

    It has been suggested that most, if not all components of the glideosome are palmitoylated. The authors analysed FLAG pulldowns from the 17-ODYA experiments (described above), from the C8/C11 single and double mutants, and performed Western blots to determine the presence of glideosome components TgGAP45, TgELC1 and TgMyoA. They concluded that blocking TgMLC1 palmitoylation seems to block its ability to interact with TgGAP45, while simultaneously increasing its interaction with TgMyoA and TgELC1.

Blocking TgMLC1 palmitoylation has no effect on parasite motility

   Given the previous results demonstrating that blocking palmitoylation affects interactions between glideosome components, motility defects in the mutants were expected. However, the authors found that the double mutants parasites’ motility was indistinguishable from the WT TgMLC1 line, in terms of motility initiation, mean displacement, mean speed, and maximum speed, while showing little effects on track length. This was unexpected, since the linear motor model would predict that disruption of the interaction between TgMLC1 and TgGAP45 should result in incapacity to generate force required for movement.

    The authors investigated whether the mutants could undergo changes in the composition of the glideosome that could functionally compensate for the lack of TgMLC1-TgGAP45 interactions. Investigation of whether TgGAP45 associates with any new proteins in the absence of its normal interaction with TgMLC1, showed that TgGAP45 does not appear to interact with any myosins or myosin light chains when its interaction with TgMLC1 is disrupted. Investigation of whether TgMyoA associates with other proteins in the mutants, which might serve to anchor TgMyoA to the IMC in the absence of TgMLC1-TgGAP45 interactions, resulted in a similar conclusion. Finally, the authors tested whether TgMLC1 itself might interact with other IMC-anchored proteins in the absence of its interaction with TgGAP45. The authors ultimately concluded that the near normal motility seen in the mutant parasites is not explained by the binding of either TgGAP45 or components of the motor to alternative proteins that could compensate for the lack of interactions between TgMLC1 and TgGAP45.

What I like about this preprint

I find the topic of parasite motility very interesting and  I think the authors address an important gap in knowledge, clearly identifying that we do not yet fully understand gliding motility in Apicomplexan parasites, and doing so is important to understand their pathogenicity within human hosts. I enjoyed reading the manuscript as it is very succinct and clear from beginning to end.

References

  1. Frenal K, et al, 2017,Gliding motility powers invasion and egress in Apicomplexa, Nature Rev Microbiol, 15: 645-660.
  2. Rompikuntal PK, et al, 2020, Blocking palmitoylation of Toxoplasma gondii myosin light chain 1 disrupts glideosome composition but has little impact on parasite motility, bioRxiv.

 

Posted on: 8 December 2020

doi: https://doi.org/10.1242/prelights.26195

Read preprint (No Ratings Yet)

Author's response

Gary Ward shared

Open questions

1.I enjoyed a lot reading your work. Starting with a broad question – you mention in your introduction the relevance of post-translational modifications for parasite biology, and focus on palmitoylation. Are there other post-translational modifications that are equally relevant or important for Toxoplasma gliding motility, invasion and egress?

The post-translational modification that has been studied most extensively in apicomplexans is phosphorylation, which is widespread and has been implicated in motility, invasion and egress. Focusing on motility, each component of the T. gondii glideosome is known to be phosphorylated on multiple sites. While we don’t know the functional significance of most of these phosphorylations, my lab showed several years ago that phosphorylation of recombinant TgMyoA on three serines near its N-terminus (Ser19, 21 ,29) increases its motor activity. This result was consistent with an elegant study on TgMyoA phosphorylation done in parasites a few years earlier in the Arrizabalaga lab. More recent work demonstrated that phosphorylation of Ser19 also alters the motor function of Plasmodium falciparum MyoA. So a role for MyoA phosphorylation in controlling motor function and parasite motility is at this point well established.  As for post-translational modifications other than phosphorylation, Aoife Heaslip and Ke Hu did some intriguing work suggesting a role for lysine methylation in activating Toxoplasma motility. Palmitoylation and acetylation are also widespread in apicomplexans, including on proteins involved in motility and invasion. Altogether, phosphoproteome, palmitome and acetylome studies have identified more than 25,000 sites of post-translational modification on T. gondii proteins; however, in very few cases do we know anything about their functional significance.

2.Another broad question: how do you expect these post-translational modifications to occur and vary within different tissues within the mammalian host? Namely, how would the parasite location (choice of cell and organ) influence this, and therefore the different phenotypic effects possibly observed in different organs or even hosts?

This is a great question about which we know virtually nothing. In the Plasmodium falciparum MyoA phosphorylation story I mentioned above, Robert-Paganin and colleagues showed that unphosphorylated MyoA could generate more force under load than phosphorylated MyoA, but this ability to generate force came at the expense of the speed with which the motor could displace actin filaments. They suggested that the two phosphorylation states of the motor were “tuned” to function either in invasion (which likely requires the ability to generate more force) or dissemination (where speed may be more important). An interesting model, although purely speculative.

3.More to the specifics – you mention in your manuscript that for a decade, the linear model has dominated the field, but that there are observations that are difficult to reconcile with this model. Are there alternative models already in existence that do reconcile those observations, including the ones you provide here in this manuscript? If not, is there one you would suggest given your findings?

Other models have been proposed, although none (including the linear motor model) can fully explain all observations. Most recently, the Meissner group proposed in intriguing “fountain flow” model, in which an endocytic-secretory cycle drives retrograde membrane flow on the parasite surface, contributing to parasite forward movement. I think the most direct way to discriminate between and directly test the various models would be to visualize the forces the parasite produces as it moves. The Frischknecht and Tardieux labs have done some really nice work in this area, providing insight into parasite force generation on 2D polyacrylamide surfaces. Their data suggest the importance of highly focal zones of attachment to the 2D substrate. We are hoping to eventually move traction force mapping into three dimensional extracellular matrices, where the different models of motility predict distinctly different patterns of force generation. I am most interested in exploring the possibility that more than one motility mechanism exists, and that the parasite uses different mechanisms in different settings (e.g., penetrating a tight junction vs moving along a 2D surface vs moving in 3D through the interstitial extracellular matrix). Many mammalian cells have this capability. For example, cells  may move along planar surfaces via ARP2/3-mediated actin polymerization and extension of their leading edge, but when placed in confined spaces the forward movement of these very same cells is instead driven by polarized water permeation.

4.You investigated possible changes in interactions between glideosome components that could result in compensation to the disrupted interaction between TgGAP45 and TgMLC1 upon blocking palmitoylation, resulting in effective motility. Three questions arising from this work, for me, are: what are your alternative hypothesis on how motility remains intact? Did you observe any differences in other phenomena linked to palmitoylation such as invasion and egress? Do you observe altered infectivity in vivo, which could be missed in in vitro assays?

(1) Motility may remain intact because: (a) the linear motor model or its proposed dependence on TgMLC1-TgGAP45 coupling is wrong; (b) there are redundancies in the system that allow the linear motor to continue generating force even in the absence of TgMLC1-TgGAP45 interaction; or (c) there is more than one way for the parasite to move, and when you block one mechanism another takes over. As we acknowledged in the paper, it could also be that the non-palmitoylatable TgMLC1 still interacts with TgGAP45 with sufficient affinity to couple the motor to the IMC, but insufficient affinity to withstand extraction in non-ionic detergent. It would be very difficult experimentally to completely rule out this possibility. I am going to wait to see what parasite-generated forces look like in 3D (see above) before casting my vote for a particular model or deciding that a completely different one is called for. (2, 3) We did not look at the effect of the mutations on other motility-based phenomena such as invasion or egress, or on disease progression in vivo. These experiments are certainly worth doing, and if a clear phenotype is observed this phenotype would be worth following up. For this particular study we thought we would go straight to the heart of the matter and assay motility itself. We were very surprised to see that the ability of the parasites to move was unaffected by the mutation. Even if it turns out that some other phenotype associated with the mutation is revealed by further work we would still be left with the question of how the mutant parasites can move so well in the 3D motility assay when  TgMLC1 appears to be uncoupled from TgGAP45.

5.From what I understood in the description of the linear model, a question that arose in the context of your work is whether interactions between ELC1 or ELC2 and TgGAP45 would be enough for effective motility, even in the absence of the interaction between MLC1 and TgGAP45?

Based on Figures 5 and S3 in our paper, it seems unlikely that ELC1 binds directly to TgGAP45  when TgMLC1-TgGAP45 interaction is disrupted: in the immunoprecipitates of the mutant parasites, ELC1 continues to associate with TgMyoA/TgMLC1 rather than with TgGAP45. We did not probe the western blots for ELC2, but the functions of ELC1 and 2 are reportedly redundant, and as far as I’m aware there is no direct evidence that either interacts directly with TgGAP45.

Have your say

Your email address will not be published.

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the cell biology category:

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage
Close