Biology subjects

Engelberg, K.

Publications and source records attributed to Engelberg, K..

7 recordsLinked to original sources

bioRxiv2025

Toxoplasma gondii AAP7 is essential for functionally connecting the IMC embedded apical annuli to the plasma membrane

The Toxoplasma gondii cytoskeleton contains an intermediate filament network, supporting a quilt of alveolar sheets forming the inner membrane complex (IMC), undergirded by 22 subpellicular microtubules (SPMTs). Embedded within the IMC are the apical annuli: 5-6 ring-shaped pores facilitating dense granule exocytosis. Here we describe a novel apical annuli protein, AAP7. AAP7 depletion causes a severe fitness defect. In stable AAP7-depleted (ATc-resistant) parasites, LMBD3 no longer traffics to the annuli, but accumulates among the secretory organelles. This suggests AAP7 is required to traffic LMDB3 to the plasma membrane through a novel route. Moreover, it indicated that AAP7 connects plasma membrane embedded LMDB3 to the AAP proteins embedded in the IMC sutures. Functionally, AAP7 depletion results in reduced secretion of dense granule proteins. Specifically, parasitophorous vacuole membrane pore forming GRA17 secretion is reduced, causing bubble vacuoles. GRA17 overexpression overcomes AAP7 depletion and reduces bubble vacuoles revealing the critical defect. An additional AAP7 depletion phenotype is the accumulation of polyglutamylated SPMTs at the basal end, indicating slow turnover. Lastly, from a comparative angle, we investigated annuli in Sarcocystis neurona, revealing 6 apical annuli. This is surprising considering S. neuronas 11 alveolar vesicles and expected 11 annuli. Ergo, annuli architecture does not take cues from IMC suture positioning. In summary, our analysis of AAP7 led to equally versatile and novel insights in apical annuli architecture, their assembly (uncovering a potentially novel trafficking process), how they interface with the IMC and impact the SPMTs, and their critical function in facilitating GRA17 secretion required for the pore across the parasitophorous vacuole membrane

microbiology↗

bioRxiv2024

Co-dependent formation of the Toxoplasma gondii subpellicular microtubules and inner membrane skeleton

One of the defining features of apicomplexan parasites is their cytoskeleton composed of alveolar vesicles, known as the inner membrane complex (IMC) undergirded by intermediate-like filament network and an array of subpellicular microtubules (SPMTs). In Toxoplasma gondii, this specialized cytoskeleton is involved in all aspects of the disease-causing lytic cycle, and notably acting as a scaffold for parasite offspring in the internal budding process. Despite advances in our understanding of the architecture and molecular composition, insights pertaining to the coordinated assembly of the scaffold are still largely elusive. Here, T. gondii tachyzoites were dissected by advanced, iterative expansion microscopy (pan-ExM) revealing new insights into the very early sequential formation steps of the tubulin scaffold. A comparative study of the related parasite Sarcocystis neurona revealed that different MT bundling organizations of the nascent SPMTs correlate with the number of central and basal alveolar vesicles. In absence of a so far identified MT nucleation mechanism, we genetically dissected T. gondii {gamma}-tubulin and {gamma}-tubulin complex protein 4 (GCP4). While {gamma}-tubulin depletion abolished the formation of the tubulin scaffold, a set of MTs still formed that suggests SPMTs are nucleated at the outer core of the centrosome. Depletion of GCP4 interfered with the correct assembly of SPMTs into the forming daughter buds, further indicating that the parasite utilizes the {gamma}-tubulin complex in tubulin scaffold formation.

microbiology↗

bioRxiv2022

Toxoplasma gondii-s basal complex: the other apicomplexan business end is multifunctional

The Apicomplexa are famously named for their apical complex, a constellation of organelles at their apical end dedicated to invasion of their host cells. In contrast, at the other end of the cell, the basal complex (BC) has been in the limelight since it is much less prominent and specific functions were not immediately obvious. However, in the past decade a staggering array of functions have been associated with the BC, and strides have been made in understanding its structure. Here, these collective insights are supplemented with new data to provide an overview of the understanding of the BC in Toxoplasma gondii. The emerging picture is that the BC is a dynamic and multifunctional complex, with a series of (putative) functions. The BC has multiple roles in cell division: it is the site where building blocks are added to the cytoskeleton scaffold; it exerts a two-step stretch and constriction mechanism as contractile ring; and it is key in organelle division. Furthermore, the BC has numerous putative roles in import, such as the recycling of mother cell remnants, the acquisition of host-derived vesicles, possibly the uptake of lipids derived from the extracellular medium, and the endocytosis of micronemal proteins. The latter process ties the BC to motility, whereas an additional role in motility is conferred by Myosin C. Furthermore, the BC acts on the assembly and/or function of the intravacuolar network, which may directly or indirectly contribute to the establishment of chronic tissue cysts. Here we provide experimental support for molecules acting in several of these processes, and identify several new BC proteins critical to maintaining the cytoplasmic bridge between divided parasites. However, the dispensable nature of many BC components leaves many questions unanswered regarding its function. In conclusion, the BC in T. gondii is a dynamic and multifunctional structure at the posterior end of the parasite.

cell biology↗

bioRxiv2021

A more complex basal complex: novel components mapped to the Toxoplasma gondii cytokinesis machinery portray an expanded hierarchy of its assembly and function

The basal complex (BC) of Toxoplasma gondii has an essential role in cell division but details on the mechanism are lacking. To promote insights in this process, reciprocal proximity based biotinylation was used to map the basal complex proteome. An assembled protein map was interrogated by spatiotemporal characterization of critical components as well as functionally by disrupting the expression of the components. Spatially, this revealed four proteins sub-complexes with distinct sub-structural BC localization. Temporally, several patterns were differentiated based on their first appearance and/or disappearance from the BC corresponding with different steps in BC development (initiation, expansion, constriction, maturation). We also identified a protein pre-ceding BC formation (BCC0) laid out in a 5-fold symmetry. This symmetry marks the apical annuli and site of alveolar suture formation. From here, it was determined that the apical cap is assembled in the apical direction, whereas the rest of the IMC expands in the basal direction, inspiring a new bi-directional daughter budding process. Furthermore, we discovered BCC4, an essential protein exclusively localizing to the BC during cell division. Although depletion of BCC4 did not prevent BC formation, it led to BC fragmentation at the mid-point of cell division. Based on these data, a model is presented wherein BCC4 and MORN1 stabilize each other and form a rubber band that implies an essential role for the BC in preventing the fraying of the basal end of the assembling daughter cytoskeleton scaffolds. Furthermore, one new component of the Myosin J and Centrin2 cluster was BCC1, a hypothetical protein whose depletion prevents the non-essential last step of BC constriction. Overall, the BC is a highly dynamic, multi-functional structure that is critical to the hierarchical assembly of the daughter parasites.

microbiology↗

bioRxiv2020

Fussing about fission: defining variety among mainstream and exotic apicomplexan cell division modes

Cellular reproduction defines life, yet our textbook-level understanding of cell division is limited to a small number of model organisms centered around humans. The horizon on cell division variants is expanded here by advancing insights on the fascinating cell division modes found in the Apicomplexa, a key group of protozoan parasites. The Apicomplexa display remarkable variation in offspring number, whether karyokinesis follows each S/M-phase or not, and whether daughter cells bud in the cytoplasm or bud from the cortex. We find that the terminology used to describe the various manifestations of asexual apicomplexan cell division emphasizes either the number of offspring or site of budding, which are not directly comparable features and has led to confusion in the literature. Division modes have been primarily studied in two human pathogenic Apicomplexa, malaria-causing Plasmodium spp. and Toxoplasma gondii, a major cause of opportunistic infections. Plasmodium spp. divide asexually by schizogony, producing multiple daughters per division round through a cortical budding process, though at several life-cycle nuclear amplifications are not followed by karyokinesis. T. gondii divides by endodyogeny producing two internally budding daughters per division round. Here we add to this diversity in replication mechanisms by considering the cattle parasite Babesia bigemina and the pig parasite Cystoisospora suis. B. bigemina produces two daughters per division round by a binary fission mechanism whereas C. suis produces daughters through both endodyogeny and multiple internal budding known as endopolygeny. In addition, we provide new data from the causative agent of equine protozoal myeloencephalitis (EPM), Sarcocystis neurona, which also undergoes endopolygeny but differs from C. suis by maintaining a single multiploid nucleus. Overall, we operationally define two principally different division modes: internal budding found in cyst-forming Coccidia (comprising endodyogeny and two forms of endopolygeny) and external budding found in the other parasites studied (comprising the two forms of schizogony, binary fission and multiple fission). Progressive insights into the principles defining the molecular and cellular requirements for internal versus external budding, as well as variations encountered in sexual stages are discussed. The evolutionary pressures and mechanisms underlying apicomplexan cell division diversification carries relevance across Eukaryota. Contribution to the FieldMechanisms of cell division vary dramatically across the Tree of Life, but the mechanistic basis has only been mapped for several model organisms. Here we present cell division strategies across Apicomplexa, a group of obligate intracellular parasites with significant impact on humans and domesticated animals. Asexual apicomplexan cell division is organized around assembly of daughter buds, but division forms differ in the cellular site of budding, number of offspring per division round, whether each S-phase follows karyokinesis and if mitotic rounds progress synchronously. This varies not just between parasites, but also between different life-cycle stages of a given species. We discuss the historical context of terminology describing division modes, which has led to confusion on how different modes relate to each other. Innovations in cell culture and genetics together with light microscopy advances have opened up cell biological studies that can shed light on this puzzle. We present new data for three division modes barely studied before. Together with existing data, we show how division modes are organized along phylogenetic lines and differentiate along external and internal budding mechanisms. We also discuss new insights into how the variations in division mode are regulated at the molecular level, and possess unique cell biological requirements.

microbiology↗

bioRxiv2019

The apical annuli of Toxoplasma gondii are composed of coiled-coil and signaling proteins embedded in the IMC sutures

The apical annuli are among the most intriguing and understudied structures in the cytoskeleton of the apicomplexan parasite Toxoplasma gondii. We mapped the proteome of the annuli in Toxoplasma by reciprocal proximity biotinylation (BioID), and validated five apical annuli proteins (AAP1-5), Centrin2 and a methyltransferase (AAMT). Moreover, Inner Membrane Complex (IMC) suture proteins connecting the alveolar vesicles were also detected and support annuli residence within the sutures. Super-resolution microscopy (SR-SIM) identified a concentric organization comprising four rings with diameters ranging from 200-400 nm. The high prevalence of domain signatures shared with centrosomal proteins in the AAPs together with Centrin2 suggest that the annuli are related and/or derived from the centrosomes. Phylogenetic analysis revealed the AAPs are conserved narrowly in Coccidian, apicomplexan parasites that multiply by an internal budding mechanism. This suggests a role in replication, for example, to provide pores in the mother IMC permitting exchange of building blocks and waste products. However, presence of multiple signaling domains and proteins are suggestive of additional functions. Knockout of AAP4, the most conserved compound forming the largest ring-like structure, modestly decreased parasite fitness in vitro but had no significant impact on acute virulence in vivo. In conclusion, the apical annuli are composed of coiled-coil and signaling proteins assembled in a pore-like structure crossing the IMC barrier maintained during internal budding.

cell biology↗

bioRxiv2018

A member of the ferlin calcium sensor family is essential for Toxoplasma gondii rhoptry secretion

Invasion of host cells by apicomplexan parasites such as Toxoplasma gondii is critical for their infectivity and pathogenesis. In Toxoplasma, secretion of essential egress, motility and invasion-related proteins from microneme organelles is regulated by oscillations of intracellular Ca2+. Later stages of invasion are considered Ca2+-independent, including the secretion of proteins required for host cell entry and remodeling from the parasites rhoptries. We identified a family of three Toxoplasma proteins with homology to the ferlin family of double C2 domain-containing Ca2+ sensors. In humans and model organisms such Ca2+ sensors orchestrate Ca2+-dependent exocytic membrane fusion with the plasma membrane. One ferlin that is conserved across the Apicomplexa, TgFER2, localizes to the parasites cortical membrane skeleton, apical end, and rhoptries. Unexpectedly, conditionally TgFER2-depleted parasites secreted their micronemes normally and were completely motile. However, these parasites were unable to invade host cells and were therefore not viable. Specifically, knockdown of TgFER2 prevented rhoptry secretion and these parasites failed to form the moving junction on the parasite-host interface necessary for host cell invasion. Collectively, these data demonstrate that the putative Ca2+ sensor TgFER2 is required for the secretion of rhoptries. These findings provide the first regulatory and mechanistic insights into this critical yet poorly understood aspect of apicomplexan host cell invasion.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC=\"FIGDIR/small/304048_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (58K):\norg.highwire.dtl.DTLVardef@505202org.highwire.dtl.DTLVardef@138400aorg.highwire.dtl.DTLVardef@1f468beorg.highwire.dtl.DTLVardef@1089d34_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗