Biology subjects

Shortt, E.

Publications and source records attributed to Shortt, E..

4 recordsLinked to original sources

bioRxiv2023

Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma

Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.

microbiology↗

bioRxiv2022

CDPK2A and CDPK1 form a signaling module upstream of Toxoplasma motility

The transition between parasite replication and dissemination is regulated in apicomplexan parasites by fluctuations in cytosolic calcium concentrations, effectuated by calcium-dependent protein kinases (CDPKs). We examined the role of CDPK2A in the lytic cycle of Toxoplasma, analyzing its role in the regulation of cellular processes associated with parasite motility. We used chemical-genetic approaches and conditional depletion to determine that CDPK2A contributes to the initiation of parasite motility through microneme discharge. We demonstrate that the N-terminal extension of CDPK2A is necessary for the proteins function. Conditional depletion revealed an epistatic interaction between CDPK2A and CDPK1, suggesting that the two kinases work together to mediate motility in response to certain stimuli. This signaling module appears distinct from that of CDPK3 and PKG, which also controls egress. CDPK2A is revealed as an important regulator of the Toxoplasma kinetic phase, linked to other kinases that govern this critical transition. Our work uncovers extensive interconnectedness between the signaling pathways that govern parasite motility. IMPORTANCEThis work uncovers interactions between various signaling pathways that govern Toxoplasma gondii egress. Specifically, we compare the function of three canonical calcium dependent protein kinases (CDPKs) using chemical-genetic and conditional-depletion approaches. We describe the function of a previously uncharacterized CDPK, CDPK2A, in the Toxoplasma lytic cycle, demonstrating it contributes to parasite fitness through regulation of microneme discharge, gliding motility, and egress from infected host cells. Comparison of analog-sensitive (AS) kinase alleles and conditionally-depleted alleles uncovered epistasis between CDPK2A and CDPK1 implying a partial functional redundancy. Understanding the topology of signaling pathways underlying key events in the parasite life cycle can aid in efforts targeting parasite kinases for anti-parasitic therapies.

microbiology↗

bioRxiv2021

High-throughput functionalization of the Toxoplasma kinome uncovers a novel regulator of invasion and egress

Protein kinases regulate fundamental aspects of cell biology in all eukaryotes, making them attractive chemotherapeutic targets in Apicomplexan parasites such as the causative agents of malaria (Plasmodium spp.) and toxoplasmosis (Toxoplasma gondii). However, the precise roles of individual parasite kinases cannot be inferred simply from sequence identity, due to rewiring of signaling pathways and the shifting repertoire of kinases across species. To systematically examine the parasite kinome, we developed a high-throughput (HiT) CRISPR-mediated tagging strategy to endogenously label all predicted cytosolic protein kinases with a synthetic sequence encoding the minimal auxin-inducible degron (mAID) linked to a fluorophore and epitope tag. The system enables the assembly of thousands of tagging vectors from synthetic sequences in a single reaction and the pooled generation of mutants to examine kinase localization and function. We examined the phenotypes associated with kinase knock-down in 1,160 arrayed clones by replica-plating in the presence or absence of auxin and found broad defects across the lytic cycle for 109 clonal isolates, assigning localizations to 39 proteins, and associating 15 kinases within 6 distinct morphological phenotypes. The relative fitness of tagged alleles was also examined by tracking the relative abundance of individual guide RNAs as parasite populations progressed through the lytic cycle, in the presence or absence of auxin. Pooled screening had a high predictive value and differentiated between delayed and acute death. Demonstrating the value of this resource, we identified a novel kinase associated with delayed death as a novel regulator of invasion and egress. We call the previously unstudied kinase Store Potentiating/Activating Regulatory Kinase (SPARK), based on its impact on intracellular Ca2+ stores at key moments during the lytic cycle. Despite having a similar kinase domain to the mammalian PDK1, SPARK lacks the canonical lipid-binding domain and we find no indication SPARK positively regulates other AGC kinases, suggesting a rewiring of signaling pathways to accommodate parasite adaptations. The HiT vector screening system extends the applications of genome-wide screens into complex cellular phenotypes, providing a scalable and versatile platform for the dissection of apicomplexan cell biology.

microbiology↗

bioRxiv2020

A PEROXO-Tag enables rapid isolation of peroxisomes from human cells

Peroxisomes are metabolic organelles that perform a diverse array of critical functions in human physiology. Traditional isolation methods for peroxisomes can take more than one hour to complete and can be laborious to implement. To address this, we have now extended our prior work on rapid organellar isolation to peroxisomes via the development of a peroxisomally-localized 3XHA epitope tag ("PEROXO-Tag") and associated immunoprecipitation ("PEROXO-IP") workflow. Our PEROXO-IP workflow has excellent reproducibility, is easy to implement, and achieves highly rapid (~10 minutes post-homogenization) and specific isolation of human peroxisomes, which we characterize here via proteomic profiling. By offering speed, specificity, reproducibility, and ease of use, the PEROXO-IP workflow should facilitate studies on the biology of peroxisomes.

cell biology↗