Biology subjects

Meissner, M.

Publications and source records attributed to Meissner, M..

12 recordsLinked to original sources

bioRxiv2025

Tracking maternal proteins uncovers a central role for the residual body in organelle recycling during Toxoplasma gondii replication

Toxoplasma gondii replicates through endodyogeny, an unconventional form of internal budding in which two daughter cells are assembled within a single mother cell. During this process, daughter cells must acquire a full complement of organelles, which may be inherited from the mother, formed de novo, or assembled through a combination of both mechanisms. To date, the fate of maternal components during replication remains poorly understood. We previously showed that F-actin driven dynamics generate an intravacuolar network, associated with the residual body (RB) and facilitates the recycling of microneme proteins. However, the inheritance and recycling of other organelles have not been systematically analysed. To address this, we employed a dual HaloTag-based pulse-chase fluorescence labelling strategy to distinguish between de novo-synthesized and maternally inherited proteins in replicating tachyzoites. This approach reveals three distinct modes of organelle inheritance: (1) inheritance of intact maternal organelles (e.g., rhoptries and micronemes), (2) expansion and division of pre-existing maternal organelles with incorporation of newly synthesized components (e.g., Golgi apparatus and apicoplast), and (3) degradation of maternal structures followed by de novo assembly (e.g., inner membrane complex). Furthermore, we identify Myosin F (MyoF) as an important factor in the redistribution of maternal micronemes and rhoptries. In the absence of MyoF, these organelles accumulate in the RB, while restoration of MyoF permits accumulated maternal micronemes to redistribute to daughter parasites. Together, these findings support a role for the RB as a dynamic compartment in the recycling and redistribution of maternal organelles.

cell biology↗

bioRxiv2025

Localisation of Apicomplexa motor Myosin A with axial nanometric precision using graphene energy transfer

Migration in Apicomplexan parasites, a phylum that includes Plasmodium spp. responsible for malaria and the zoonotic Toxoplasma, is explained by the linear migration motor model. This model predicts that Myosin A is confined beneath the plasma membrane and above the inner membrane complex (IMC), a membranous barrier separating the cell membrane and the cytoplasm. Cumulative data, using mutant cell lines and biochemical data support this model. Paradoxically, a proof of the precise localization of Myosin A is still lacking due to limitations in resolving the motor with nanometric precision within the space comprising the membrane and the IMC. Here, we implement graphene energy transfer (GET), a novel axial nanometric ruler with a resolution of [~]1 nm, to determine the relative axial position of Myosin A and IMC1 (a reference protein defining IMC position). Using GET, we estimate the IMC dimensions with precision matching electron microscopy (EM) data and the added advantage of identifying specific bauplan proteins. We complement these measurements with 2D STED microscopy in MIRA confiners, uExMIC, and cryo-immunolabeling. Our data present the first direct localization of Myosin A populations with nanometric resolution in the third dimension, which is compatible with the linear motor model. One sentence summaryUsing the novel GET method, this study presents the first direct localization of Myosin A populations in Toxoplasma gondii with 1-4 nm axial precision, supporting the linear model.

biophysics↗

bioRxiv2024

Hijacked Immune Cells Traverse Microenvironmental Barriers by Positioning and Pushing their Intracellular Parasite Cargo

Cell migration is fundamental to development, homeostasis, and immunity, and requires cells to traverse space-limited microenvironments by deforming intracellular organelles such as the nucleus. Although nuclear mechanics are established as a rate-limiting factor during confined migration, how cells transport bulky foreign cargo remains unclear. Here, using Toxoplasma gondii, a disease-causing parasite that hijacks motile immune cells for dissemination while replicating into large intracellular assemblies, we discover that parasitic cargoes exceed the size and stiffness of host nuclei, and therefore constitute a primary bottleneck for migration. We uncover a mechanoadaptive response to parasite size, characterized by size-dependent intracellular repositioning, myosin-II contractility, and bleb-based protrusions. This reprogramming of forces and subcellular architecture enables the transport of oversized, mechanically rigid cargo through space-limited microenvironments. These findings establish a mechanobiological principle of how cells size-dependently adapt forces and their subcellular architecture to accommodate intracellular objects that challenge the physical limits of cell migration.

cell biology↗

bioRxiv2022

Evolution of lineage-specific trafficking proteins and a novel post-Golgi trafficking pathway in Apicomplexa

The Organelle Paralogy Hypothesis (OPH) posits a mechanism to explain the evolution of non-endosymbiotically derived organelles, predicting that lineage-specific pathways organelles should result when identity-encoding membrane trafficking components duplicate and co-evolve. Here we investigate the presence of such lineage-specific membrane-trafficking machinery paralogs in the globally important lineage of parasites, the Apicomplexa. Using a new phylogenetic workflow, we are able to identify 18 novel paralogs of known membrane-trafficking machinery, the emergence of several of which correlate with the presence of new endomembrane organelles in apicomplexans or their larger lineage. Gene coregulation analysis of a large set of membrane-trafficking proteins in Toxoplasma both corroborate known molecular cell biological interactions between characterized machinery and suggest involvement of many of these new components into established pathways for biogenesis of or trafficking to the microneme and rhoptry invasion organelles. Moreover, focused molecular parasitological analysis of the apicomplexan Arf-like small GTPases, and the ArlX3 protein specifically, revealed a novel post-Golgi trafficking pathways involved in delivery of proteins to micronemes and rhoptries, with knock down demonstrating reduced invasion capacity. The totality of our data has identified an unforeseen post-Golgi trafficking pathway in apicomplexans and is consistent with the OPH mechanism acting to produce novel endomembrane pathways or organelles at various evolutionary stages across the Alveolate lineage. Significance statementThe mechanism of non-endosymbiotic organelle evolution has been relatively poorly explored and yet is relevant to many eukaryotic compartments, including the endomembrane system. The Organelle Paralogy Hypothesis predicts novel lineage-specific paralogs evolutionarily concurrent with emergence of new endomembrane organelles or pathways. By investigating this phenomenon in the apicomplexan parasites and their relatives, we identify and profile over a dozen new trafficking factors, several correlating with emergence of lineage-specific organelles. Cell biological study of one such factor demonstrates the existence of a novel post-Golgi trafficking pathway for components to the invasion organelles in the parasite Toxoplasma gondii. This work reveals how non-endosymbiotic organelle evolution has shaped cellular novelty in this lineage, relevant both to global health and fundamental evolutionary biology.

microbiology↗

bioRxiv2022

A central protein complex essential for Invasionin Toxoplasma gondii

Apicomplexa are obligate intracellular parasites. While most species are restricted to specific hosts and cell types, Toxoplasma gondii can invade every nucleated cell derived from warm-blooded animals. This broad host range suggests that this parasite can recognize multiple host cell ligands or structures, leading to the activation of a central protein complex, which should be conserved in all apicomplexans. During invasion, the unique secretory organelles (micronemes and rhoptries) are sequentially released and several micronemal proteins have been suggested to be required for host cell recognition and invasion. However, to date only few micronemal proteins have been demonstrated to be essential for invasion. Cysteine Repeat Modular Proteins (CRMPs) are a family of apicomplexan specific proteins. In Toxoplasma gondii, two CRMPs are present in the genome. The Kringle domain containing protein (CRMPA) and the GCC2 GCC3 domain containing protein (CRMPB). Here we demonstrate that both proteins form a complex that contains additional micronemal proteins. Disruption of this complex results in a block of rhoptry secretion and parasites being unable to invade the host cell. In conclusion, this complex is a central invasion complex conserved in all apicomplexans.

cell biology↗

bioRxiv2021

Malaria transmission relies on concavin-mediated maintenance of Plasmodium sporozoite cell shape

During transmission of malaria-causing parasites from mosquitoes to mammals, Plasmodium sporozoites migrate rapidly in the skin to search for a blood vessel. The high migratory speed and narrow passages taken by the parasites suggest considerable strain on the sporozoites to maintain their shape. Here we report on a newly identified protein, concavin, that is important for maintenance of the sporozoite shape inside salivary glands of mosquitoes and during migration in the skin. Concavin-GFP localized at the cytoplasmic periphery of sporozoites and concavin(-) sporozoites progressively rounded up upon entry of salivary glands. These rounded concavin(-) sporozoites failed to pass through the narrow salivary ducts and were hence rarely ejected by mosquitoes. However, normally shaped concavin(-) sporozoites could be transmitted and migrated in the skin or skin like environments. Strikingly, motile concavin(-) sporozoites could disintegrate while migrating through narrow strictures in the skin leading to parasite arrest or death and decreased transmission efficiency. We suggest that concavin contributes to cell shape maintenance by riveting the plasma membrane to the subtending inner membrane complex. SIGNIFICANCEMalaria parasites are transmitted by Anopheles mosquitoes and rely on rapid migration for establishing an infection. We identified and characterized a protein, named concavin, essential for maintaining the shape of the sporozoite. Concavin is a membrane associated protein facing the cytoplasm suggesting that it contributes to riveting the plasma membrane to the subtending inner membrane complex. Sporozoites lacking concavin can round up in the salivary glands, are less well transmitted to mice and disintegrate while migrating in the skin. Hence, concavin is essential for parasite transmission and infectivity. Highlights- A membrane associated protein is essential for Plasmodium shape maintenance -Migrating parasites disintegrate in the absence of concavin -First protein essential for cellular integrity of Plasmodium sporozoites -Thickened and deformed Plasmodium sporozoites fail to be transmitted by mosquitoes

microbiology↗

bioRxiv2021

A phenotypic screen using splitCas9 identifies essential genes required for actin regulation during host cell egress and invasion by Toxoplasma gondii

Apicomplexan parasites, such as Toxoplasma gondii, possess unique organelles, cytoskeletal structures, signalling cascades, replicate by internal budding within a specialised compartment and actively invade and exit the host cell, to name a few aspects of the unique biology that characterise this phylum. Due to their huge phylogenetic distance from well established model organisms, such as opisthokonts, comparative genomics has a limited capacity to infer gene functions and conserved proteins can fulfil different roles in apicomplexans. Indeed, approximately 30% of all genes are annotated as hypothetical and many had a crucial role during the asexual life cycle in genome-wide screens. While the current CRISPR/Cas9-based screens allow the identification of fitness conferring genes, only little information about the respective functions can be obtained. To overcome this limitation, and to group genes of interest into functional groups, we established a conditional Cas9-system in T. gondii that allows phenotypic screens. Using an indicator strain for F-actin dynamics and apicoplast segregation, we identified critical genes required for defined steps during the asexual life cycle. The detailed characterisation of two of these candidates revealed them to be critical for host cell egress and invasion and to act at different time points in the disassembly of the intravacuolar F-actin network. While the signalling linking factor (SLF) is an integral part of a signalling complex required for early induction of egress, a novel conoid protein (conoid gliding protein, CGP) acts late during egress and is required for the activation of gliding motility.

microbiology↗

bioRxiv2019

Rab11A regulates the constitutive secretory pathway during Toxoplasma gondii invasion of host cells and parasite replication.

Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of DG and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile parasites suggesting that a Rab11A-dependent apically polarized transport of cargo regulates parasite motility.

microbiology↗

bioRxiv2019

Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion

The obligate intracellular parasites Toxoplasma gondii and Plasmodium spp. invade host cells by injecting a protein complex into the membrane of the targeted cell that bridges the two cells through the assembly of a ring-like junction. This circular junction stretches while the parasites applies a traction force to pass through; a step that typically concurs with transient constriction of the parasite body. Here we show that the junction can oppose resistance to the passage of the parasites nucleus. Super-resolution microscopy and real time imaging highlighted an F-actin pool at the apex of pre-invading parasite, an F-actin ring at the junction area during invasion but also networks of perinuclear and posteriorly localized F-actin. Mutant parasites with dysfunctional acto-myosin showed significant decrease of junctional and perinuclear F-actin and are coincidently affected in nuclear passage through the junction. We propose that the F-actin machinery eases nuclear passage by stabilising the junction and pushing the nucleus through the constriction, providing first evidence for a dual contribution of actin-forces during host cell invasion by apicomplexan parasites.

cell biology↗

bioRxiv2018

Formin-2 drives intracellular polymerisation of actin filaments enabling correct segregation of apicoplasts in Plasmodium falciparum and Toxoplasma gondii.

Pathogenic obligate-intracellular apicomplexan parasites possess an essential chloroplast-like organelle called the apicoplast that undergoes division and segregation during replication. Parasite actin is essential during intracellular development, implicated in vesicular transport, parasite replication and apicoplast inheritance. However, the inability to visualise live actin dynamics in apicomplexan parasites limited functional characterisation of both filamentous-actin (F-actin) and actin regulatory factors. Apicomplexans possess at least two distinct formins, Formin-1 and Formin-2, predicted to serve as actin-nucleating factors, and previously implicated in regulating gliding motility and host cell invasion. Here, we expressed chromobodies and validated them as F-actin-binding sensors in Plasmodium falciparum and characterised the in vivo dynamics of the F-actin network. The F-actin network could be modulated chemically and disrupted by conditionally deleting the actin-1 gene. In a comparative approach, we demonstrate that Formin-2 is closely associated with apicoplasts and with the F-actin network in P. falciparum and Toxoplasma gondii. Consequently, disruption of Formin-2 resulted not only in an apicoplast segregation defect, but also in complete abrogation of F-actin dynamics in intracellular parasites. Together, our results strongly indicate that Formin-2-mediated filament formation is the common primary mechanism for F-actin nucleation during apicomplexan intracellular growth effecting apicoplast segregation.

cell biology↗

bioRxiv2018

Apicomplexan motility depends on the operation of an endocytic-secretory cycle

Apicomplexan parasites invade host cells in an active process, involving their ability to move by gliding motility and invasion. While the acto-myosin-system of the parasite plays a crucial role in the formation and release of attachment sites during this process, there are still open questions, such as how the force powering motility is generated. In many eukaryotes a secretory-endocytic cycle leads to recycling of receptors (integrins), necessary to form attachment sites, regulation of surface area during motility and generation of retrograde membrane flow. Here we demonstrate that endocytosis operates during gliding motility in Toxoplasma gondii and appears to be crucial for the establishment of retrograde membrane flow, since inhibition of endocytosis blocks retrograde flow and motility. We identified lysophosphatidic acid (LPA) as a potent stimulator of endocytosis and demonstrate that extracellular parasites can efficiently incorporate exogenous material, such as nanogold particles. Furthermore, we show that surface proteins of the parasite are recycled during this process. Interestingly, the endocytic and secretory pathways of the parasite converge, and endocytosed material is subsequently secreted, demonstrating the operation of an endocytic-secretory cycle. Together our data consolidate previous findings and we propose a novel model that reconciles parasite motility with observations in other eukaryotes: the fountain-flow-model for apicomplexan parasite motility.

molecular biology↗

bioRxiv2018

Tobacco TGA7 mediates gene expression dependent and independent of salicylic acid

Basic region leucine zipper (bZIP) transcription factors of the TGA family control gene expression in response to diverse stimuli. Arabidopsis clade II and clade III TGA factors mediate salicylic acid (SA)-induced expression of PATHOGENESIS-RELATED GENE1 (PR-1) via interplay with NONEXPRESSOR OF PR GENES1 (NPR1, a.k.a. NIM1). Interaction with TGA factors occurs through the central ankyrin repeat domain of NPR1. In a yeast two-hybrid screen with the NPR1 bait, we identified TGA7, a novel member of the tobacco (Nt) TGA family grouping to clade III. TGA7 is most similar to NtTGA1a, and, like NtTGA1a, TGA7 displays transcription activity in yeast. Unexpectedly, TGA7 preferentially and uniquely interacts with the SA-sensitive C-terminal region of NtNPR1, demonstrating that NtNPR1 harbors multiple distinct TGA factor binding sites. Interaction with NPR1 impairs TGA7 transcription activity in yeast. Furthermore, TGA7 binding to the NtNPR1 C-terminus is outcompeted by SA-induced type 2 NIM1-INTERACTING (NIMIN) proteins. In tobacco plants, a TGA7-Gal4 DNA-binding domain chimeric protein (TGA7GBD) mediates SA-responsive reporter gene expression in young leaf tissue and spontaneous reporter activation in older leaves displaying PR-1 gene expression. Astonishingly, TGA7GBD is also able to activate the reporter independent from PR-1 gene expression in noninduced cotyledons of tobacco seedlings. Together, our findings support a model in which TGA7 mediates both SA-dependent and SA-independent gene activation controlled by the plants developmental stage and by the C-terminal region of constitutively accumulating NtNPR1.

plant biology↗