Two Essential Light Chains Regulate the MyoA Lever Arm To Promote
            <i>Toxoplasma</i>
            Gliding Motility

Williams, Melanie J.; Alonso, Hernán; Enciso, Marta; Egarter, Saskia; Sheiner, Lilach; Meissner, Markus; Striepen, Boris; Smith, Brian J.; Tonkin, Christopher J.

doi:10.1128/mbio.00845-15

Cited by 45 publications

(83 citation statements)

References 55 publications

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“…PfMyoA can bind an essential type light chain in addition to MTIP. We speculated that PfMyoA may bind a second light chain, based on the observation that the class XIV MyoA from Toxoplasma gondii binds an essential type light chain (ELC) in addition to MLC1, the homolog of MTIP (7,14,15). For proof of principle, PfMyoA heavy chains, MTIP, and TgELC were coexpressed.…”

Section: Expression Of Pfmyoa In Sf9 Cells Requires Co-expression Witmentioning

confidence: 99%

Binding of a Newly Identified Essential Light Chain to ExpressedPlasmodium falciparumClass XIV Myosin Enhances Actin Motility

Bookwalter

Tay

McCrorie

et al. 2017

Preprint

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Motility of the apicomplexan parasite Plasmodium falciparum, the causative agent of malaria, is enabled by the glideosome, a multi-protein complex containing the class XIV myosin motor, PfMyoA. Parasite motility is necessary for invasion into host cells and for virulence. Here we show that milligram quantities of functional PfMyoA can be expressed using the baculovirus/Sf9 cell expression system, provided that a UCS (UNC-45/CRO1/She4p) family myosin co-chaperone from Plasmodium spp. is co-expressed with the heavy chain. The homologous chaperone from the apicomplexan Toxoplasma gondii does not functionally substitute. We expressed a functional full-length PfMyoA with bound myosin tail interacting protein (MTIP), the only known light chain of PfMyoA. We then identified an additional "essential" light chain (PfELC) that co-purified with PfMyoA isolated from parasite lysates. PfMyoA expressed with both light chains moved actin at ~3.8 µm/sec, more than twice that of PfMyoA-MTIP (~1.7 µm/sec), consistent with the light chain binding domain acting as a lever arm to amplify nucleotide-dependent motions in the motor domain. Surprisingly, PfMyoA moved skeletal actin or expressed P. falciparum actin at the same speed. Duty ratio estimates suggest that PfMyoA may be able to move actin at maximal speed with as few as 6 motors. Under unloaded conditions, neither phosphorylation of Ser19 of the heavy chain, phosphorylation of several Ser residues in the N-terminal extension of MTIP, or calcium affected the speed of actin motion. These studies provide the essential framework for targeting the glideosome as a potential drug target to inhibit invasion by the malaria parasite.

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Section: Expression Of Pfmyoa In Sf9 Cells Requires Co-expression Witmentioning

confidence: 99%

Binding of a Newly Identified Essential Light Chain to ExpressedPlasmodium falciparumClass XIV Myosin Enhances Actin Motility

Bookwalter

Tay

McCrorie

et al. 2017

Preprint

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“…Apicomplexan motility functions through a conserved, actomyosin motor that forms a complex termed the glideosome, the main molecular machinery involved in parasite motility, egress, and invasion (9). Within the glideosome, Regulatory Light Chains (MLC1), Essential Light Chains 1 and 2 (ELC1 and ELC2), Ca 2+ binding proteins, interact either directly with Ca 2+ or via Ca 2+ dependent phosphorylation events to stimulate motility (41). The binding affinity of ELC is 37 µM This scenario supports the importance of Ca 2+ influx for the stimulation of motility and egress, necessary to activate the glideosome while the overall cytosolic Ca 2+ concentration would be maintained low.…”

Section: Discussionmentioning

confidence: 99%

The Role of Potassium and Host Calcium Signaling inToxoplasma gondiiegress

Vella

Moore

et al. 2020

Preprint

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Toxoplasma gondii, an obligate intracellular parasite, is capable of invading virtually any nucleated cell. Active invasion by the parasite is a precise process and intracellular parasites reside and replicate inside a parasitophorous vacuole (PV). The membrane of the PV functions as a molecular sieve allowing for its ionic milieu to be in equilibrium with the host cytosol. The parasite would thus be exposed to the ionic fluctuations of the host cytoplasm, such as increases in host cytosolic Ca 2+ during Ca 2+ signaling events. Ca 2+ is a universal signaling molecule and both the host cell and T. gondii will utilize Ca 2+ signaling to stimulate diverse cellular functions. Using T. gondii tachyzoites and host cells expressing genetically encoded Ca 2+ indicators, we demonstrate that host Ca 2+ signaling impacts intracellular Ca 2+ levels of the parasite. We propose that Ca 2+ influx derived from host Ca 2+ signaling into the parasite is utilized to maintain the intracellular Ca 2+ stores replenished as the parasite replicates within the low Ca 2+ environment of the host cell.Intracellular Ca 2+ store(s) release is needed to reach the initiation spike in Ca 2+ that meets the threshold of Ca 2+ needed to initiate the tightly coordinated process of egress. Post activation of the signal and subsequent microneme secretion, would cause breakdown of the host cell and allow for extracellular Ca 2+ influx, causing a second, larger spike in Ca 2+ and activation of the glideosome, the main motility machinery. To directly deliver precise concentrations of ions needed for egress, we used patch-clamped infected host cells and monitored parasite egress. In doing so, we discovered an alternative role for K + in timing parasite egress. This is the first study using wholecell patch clamp to study the role of ions such as K + and Ca 2+ in T. gondii egress. Author SummaryToxoplasma gondii is an intracellular parasite that causes disease by engaging in multiple rounds of a lytic cycle which consists of invasion, replication inside a parasitophorous vacuole (PV) and egress, resulting in lysis of the host cell. In intracellular parasites, the release of Ca 2+ from intracellular stores forms part of a series of signaling events that culminate in stimulation of motility and egress. This means that intracellular parasites must be able to uptake Ca 2+ while replicating to keep their intracellular stores filled to be used during egress. This is not an insignificant task considering that they are inside a PV surrounded by the host cytosol which contains Ca 2+ below 100 nM. We present data showing that the host Ca 2+ increases are able to 3 reach the parasite cytosol through a Ca 2+ influx mechanism sensitive to nifedipine. This influx causes oscillations that are the result of Ca 2+ increase and re-uptake by intracellular stores. A Ca 2+ threshold was needed for egress, extracellular Ca 2+ influx was relevant for egress and potassium had a modulatory effect on egress. We used infected patched host cells, a novel approach that leaves the host ...

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“…It is unlikely that these CaM-like proteins will bind Ca 2+ given that most of their EF-hands are degenerate. Two myosin regulatory light chain proteins, essential light chains 1 and 2 (ELC1, ELC2), which form part of the glideosome, have been studied and ELC1 has been shown to bind Ca 2+ as estimated by modeling studies and thermal shift assays [71, 72]. Their affinity for Ca 2+ of this protein is low (37 ± 9 μM) suggesting that for this interaction to be physiologically relevant it would have to be in a high Ca 2+ concentration microdomain.…”

Section: Calcium Binding Proteins In T Gondiimentioning

confidence: 99%

“…MLC1 and/or ELC1 or ELC2 interact with Ca 2+ directly or via Ca 2+ dependent phosphorylation events to stimulate motility. The glideosome was adapted from [71]. …”

Section: Figurementioning

confidence: 99%

Calcium signaling and the lytic cycle of the Apicomplexan parasite Toxoplasma gondii

Triana

Márquez-Nogueras

Vella

et al. 2018

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Toxoplasma gondii has a complex life cycle involving different hosts and is dependent on fast responses, as the parasite reacts to changing environmental conditions. T. gondii causes disease by lysing the host cells that it infects and it does this by reiterating its lytic cycle, which consists of host cell invasion, replication inside the host cell, and egress causing host cell lysis. Calcium ion (Ca2+) signaling triggers activation of molecules involved in the stimulation and enhancement of each step of the parasite lytic cycle. Ca2+ signaling is essential for the cellular and developmental changes that support T. gondii parasitism. The characterization of the molecular players and pathways directly activated by Ca2+ signaling in Toxoplasma is sketchy and incomplete. The evolutionary distance between Toxoplasma and other eukaryotic model systems makes the comparison sometimes not informative. The advent of new genomic information and new genetic tools applicable for studying Toxoplasma biology is rapidly changing this scenario. The Toxoplasma genome reveals the presence of many genes potentially involved in Ca2+ signaling, even though the role of most of them is not known. The use of Genetically Encoded Calcium Indicators (GECIs) has allowed studies on the role of novel calcium-related proteins on egress, an essential step for the virulence and dissemination of Toxoplasma. In addition, the discovery of new Ca2+ players is generating novel targets for drugs, vaccines, and diagnostic tools and a better understanding of the biology of these parasites.

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Two Essential Light Chains Regulate the MyoA Lever Arm To Promote Toxoplasma Gliding Motility

Cited by 45 publications

References 55 publications

Binding of a Newly Identified Essential Light Chain to ExpressedPlasmodium falciparumClass XIV Myosin Enhances Actin Motility

Binding of a Newly Identified Essential Light Chain to ExpressedPlasmodium falciparumClass XIV Myosin Enhances Actin Motility

The Role of Potassium and Host Calcium Signaling inToxoplasma gondiiegress

Calcium signaling and the lytic cycle of the Apicomplexan parasite Toxoplasma gondii

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