-
David T Riglar, Kelly L Rogers,
Eric Hanssen,
Lynne Turnbull,
Hayley E Bullen,
Sarah C Charnaud,
Jude Przyborski,
Paul R Gilson,
Cynthia B Whitchurch,
Brendan S Crabb,
Jake Baum,
Alan F Cowman
[show abstract]
[hide abstract]
ABSTRACT: Export of proteins into the infected erythrocyte is critical for malaria parasite survival. The majority of effector proteins are thought to export via a proteinaceous translocon, resident in the parasitophorous vacuole membrane surrounding the parasite. Identification of the Plasmodium translocon of exported proteins and its biochemical association with exported proteins suggests it performs this role. Direct evidence for this, however, is lacking. Here using viable purified Plasmodium falciparum merozoites and three-dimensional structured illumination microscopy, we investigate remodelling events immediately following parasite invasion. We show that multiple complexes of the Plasmodium translocon of exported proteins localize together in foci that dynamically change in clustering behaviour. Furthermore, we provide conclusive evidence of spatial association between exported proteins and exported protein 2, a core component of the Plasmodium translocon of exported proteins, during native conditions and upon generation of translocation intermediates. These data provide the most direct cellular evidence to date that protein export occurs at regions of the parasitophorous vacuole membrane housing the Plasmodium translocon of exported proteins complex.
Nature Communications 01/2013; 4:1415. · 7.40 Impact Factor
-
Marlyse A Debrincat,
Emma C Josefsson,
Chloé James,
Katya J Henley,
Sarah Ellis,
Marion Lebois,
Kelly L Betterman,
Rachael M Lane, Kelly L Rogers,
Michael J White,
Andrew W Roberts,
Natasha L Harvey,
Donald Metcalf,
Benjamin T Kile
[show abstract]
[hide abstract]
ABSTRACT: Mature megakaryocytes depend on the function of Bcl-x(L), a member of the Bcl-2 family of prosurvival proteins, to proceed safely through the process of platelet shedding. Despite this, loss of Bcl-x(L) does not prevent the growth and maturation of megakaryocytes, suggesting redundancy with other prosurvival proteins. We therefore generated mice with a megakaryocyte-specific deletion of Mcl-1, which is known to be expressed in megakaryocytes. Megakaryopoiesis, platelet production, and platelet lifespan were unperturbed in Mcl-1(Pf4Δ/Pf4Δ) animals. However, treatment with ABT-737, a BH3 mimetic compound that inhibits the prosurvival proteins Bcl-2, Bcl-x(L), and Bcl-w resulted in the complete ablation of megakaryocytes and platelets. Genetic deletion of both Mcl-1 and Bcl-x(L) in megakaryocytes resulted in preweaning lethality. Megakaryopoiesis in Bcl-x(Pf4Δ/Pf4Δ) Mcl-1(Pf4Δ/Pf4Δ) embryos was severely compromised, and these animals exhibited ectopic bleeding. Our studies indicate that the combination of Bcl-x(L) and Mcl-1 is essential for the viability of the megakaryocyte lineage.
Blood 02/2012; 119(24):5850-8. · 9.90 Impact Factor
-
Elizabeth S Zuccala,
Alexander M Gout,
Chaitali Dekiwadia,
Danushka S Marapana,
Fiona Angrisano,
Lynne Turnbull,
David T Riglar, Kelly L Rogers,
Cynthia B Whitchurch,
Stuart A Ralph,
Terence P Speed,
Jake Baum
[show abstract]
[hide abstract]
ABSTRACT: Host cell infection by apicomplexan parasites plays an essential role in lifecycle progression for these obligate intracellular pathogens. For most species, including the etiological agents of malaria and toxoplasmosis, infection requires active host-cell invasion dependent on formation of a tight junction - the organising interface between parasite and host cell during entry. Formation of this structure is not, however, shared across all Apicomplexa or indeed all parasite lifecycle stages. Here, using an in silico integrative genomic search and endogenous gene-tagging strategy, we sought to characterise proteins that function specifically during junction-dependent invasion, a class of proteins we term invasins to distinguish them from adhesins that function in species specific host-cell recognition. High-definition imaging of tagged Plasmodium falciparum invasins localised proteins to multiple cellular compartments of the blood stage merozoite. This includes several that localise to distinct subcompartments within the rhoptries. While originating from the same organelle, however, each has very different dynamics during invasion. Apical Sushi Protein and Rhoptry Neck protein 2 release early, following the junction, whilst a novel rhoptry protein PFF0645c releases only after invasion is complete. This supports the idea that organisation of proteins within a secretory organelle determines the order and destination of protein secretion and provides a localisation-based classification strategy for predicting invasin function during apicomplexan parasite invasion.
PLoS ONE 01/2012; 7(9):e46160. · 4.09 Impact Factor
-
Emma C Josefsson,
Chloé James,
Katya J Henley,
Marlyse A Debrincat, Kelly L Rogers,
Mark R Dowling,
Michael J White,
Elizabeth A Kruse,
Rachael M Lane,
Sarah Ellis,
Paquita Nurden,
Kylie D Mason,
Lorraine A O'Reilly,
Andrew W Roberts,
Donald Metcalf,
David C S Huang,
Benjamin T Kile
[show abstract]
[hide abstract]
ABSTRACT: It is believed that megakaryocytes undergo a specialized form of apoptosis to shed platelets. Conversely, a range of pathophysiological insults, including chemotherapy, are thought to cause thrombocytopenia by inducing the apoptotic death of megakaryocytes and their progenitors. To resolve this paradox, we generated mice with hematopoietic- or megakaryocyte-specific deletions of the essential mediators of apoptosis, Bak and Bax. We found that platelet production was unperturbed. In stark contrast, deletion of the prosurvival protein Bcl-x(L) resulted in megakaryocyte apoptosis and a failure of platelet shedding. This could be rescued by deletion of Bak and Bax. We examined the effect on megakaryocytes of three agents that activate the intrinsic apoptosis pathway in other cell types: etoposide, staurosporine, and the BH3 mimetic ABT-737. All three triggered mitochondrial damage, caspase activation, and cell death. Deletion of Bak and Bax rendered megakaryocytes resistant to etoposide and ABT-737. In vivo, mice with a Bak(-/-) Bax(-/-) hematopoietic system were protected against thrombocytopenia induced by the chemotherapeutic agent carboplatin. Thus, megakaryocytes do not activate the intrinsic pathway to generate platelets; rather, the opposite is true: they must restrain it to survive and progress safely through proplatelet formation and platelet shedding.
Journal of Experimental Medicine 09/2011; 208(10):2017-31. · 13.85 Impact Factor
-
Ben A Croker,
Joanne A O'Donnell,
Cameron J Nowell,
Donald Metcalf,
Grant Dewson,
Kirsteen J Campbell, Kelly L Rogers,
Yifang Hu,
Gordon K Smyth,
Jian-Guo Zhang,
Michael White,
Kurt Lackovic,
Louise H Cengia,
Lorraine A O'Reilly,
Philippe Bouillet,
Suzanne Cory,
Andreas Strasser,
Andrew W Roberts
[show abstract]
[hide abstract]
ABSTRACT: During immune responses, neutrophils must integrate survival and death signals from multiple sources to regulate their lifespan. Signals that activate either the Bcl-2- or death receptor-regulated apoptosis pathways can provide powerful stimuli for neutrophils to undergo cell death, but whether they act cooperatively in parallel or directly cross-talk in neutrophils is not known. Previous studies suggested that Bcl-2 family proteins are not required for Fas-induced cell death in neutrophils, but did not examine whether they could modulate its rapid onset. By monitoring the rate of change in neutrophil viability associated with activation of the Fas-triggered death receptor pathway using real-time cell imaging, we show that the Bcl-2-related proteins Bid, Bax, and Bak accelerate neutrophil apoptosis but are not essential for cell death. Increased Bcl-2 or Mcl-1 expression prevents efficient induction of apoptosis by Fas stimulation indicating that the Bcl-2-regulated apoptosis pathway can directly interfere with Fas-triggered apoptosis. Fas has been shown to initiate NFκB activation and gene transcription in cell lines, however gene transcription is not altered in Fas-activated Bid(-/-) neutrophils, indicating that apoptosis occurs independently of gene transcription in neutrophils. The specification of kinetics of neutrophil apoptosis by Bid impacts on the magnitude of neutrophil IL-1β production, implicating a functional role for the Bcl-2-regulated pathway in controlling neutrophil responses to FasL. These data demonstrate that the intrinsic apoptosis pathway directly controls the kinetics of Fas-triggered apoptosis in neutrophils.
Proceedings of the National Academy of Sciences 08/2011; 108(32):13135-40. · 9.68 Impact Factor
-
David T Riglar,
Dave Richard,
Danny W Wilson,
Michelle J Boyle,
Chaitali Dekiwadia,
Lynne Turnbull,
Fiona Angrisano,
Danushka S Marapana, Kelly L Rogers,
Cynthia B Whitchurch,
James G Beeson,
Alan F Cowman,
Stuart A Ralph,
Jake Baum
[show abstract]
[hide abstract]
ABSTRACT: Erythrocyte invasion by the merozoite is an obligatory stage in Plasmodium parasite infection and essential to malaria disease progression. Attempts to study this process have been hindered by the poor invasion synchrony of merozoites from the only in vitro culture-adapted human malaria parasite, Plasmodium falciparum. Using fluorescence, three-dimensional structured illumination, and immunoelectron microscopy of filtered merozoites, we analyze cellular and molecular events underlying each discrete step of invasion. Monitoring the dynamics of these events revealed that commitment to the process is mediated through merozoite attachment to the erythrocyte, triggering all subsequent invasion events, which then proceed without obvious checkpoints. Instead, coordination of the invasion process involves formation of the merozoite-erythrocyte tight junction, which acts as a nexus for rhoptry secretion, surface-protein shedding, and actomyosin motor activation. The ability to break down each molecular step allows us to propose a comprehensive model for the molecular basis of parasite invasion.
Cell host & microbe 01/2011; 9(1):9-20. · 13.02 Impact Factor
