Two ancient membrane pores mediate mitochondrial-nucleus membrane contact sites.

Coordination between nucleus and mitochondria is essential for cell survival, and thus numerous communication routes have been established between these two organelles over eukaryotic cell evolution. One route for organelle communication is via membrane contact sites, functional appositions formed by molecular tethers. We describe a novel nuclear-mitochondrial membrane contact site in the protozoan Toxoplasma gondii. We have identified specific contacts occurring at the nuclear pore and demonstrated an interaction between components of the nuclear pore and the mitochondrial protein translocon, highlighting them as molecular tethers. Genetic disruption of the nuclear pore or the TOM translocon components, TgNup503 or TgTom40, respectively, result in contact site reduction, supporting their potential involvement in this tether. TgNup503 depletion further leads to specific mitochondrial morphology and functional defects, supporting a role for nuclear-mitochondrial contacts in mediating their communication. The discovery of a contact formed through interaction between two ancient mitochondrial and nuclear complexes sets the ground for better understanding of mitochondrial-nuclear crosstalk in eukaryotes.

Evolutionary analysis identifies a Golgi pathway and correlates lineage-specific factors with endomembrane organelle emergence in apicomplexans.

The organelle paralogy hypothesis (OPH) aims to explain the evolution of non-endosymbiotically derived organelles. It predicts that lineage-specific pathways or 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 Apicomplexa, a globally important parasitic lineage. We are able to identify 18 paralogs of known membrane-trafficking machinery, in several cases co-incident with the presence of new endomembrane organelles in apicomplexans or their parent lineage, the Alveolata. Moreover, focused analysis of the apicomplexan Arf-like small GTPases (i.e., ArlX3) revealed a specific post-Golgi trafficking pathway. This pathway appears involved in delivery of proteins to micronemes and rhoptries, with knockdown demonstrating reduced invasion capacity. Overall, our data have identified an unforeseen post-Golgi trafficking pathway in apicomplexans and are consistent with the OPH mechanism acting to produce endomembrane pathways or organelles at various evolutionary stages across the alveolate lineage.

Inhibition of ATR opposes glioblastoma invasion through disruption of cytoskeletal networks and integrin internalization via macropinocytosis.

BACKGROUND: Glioblastomas have highly infiltrative growth patterns that contribute to recurrence and poor survival. Despite infiltration being a critical therapeutic target, no clinically useful therapies exist that counter glioblastoma invasion. Here, we report that inhibition of ataxia telangiectasia and Rad 3 related kinase (ATR) reduces invasion of glioblastoma cells through dysregulation of cytoskeletal networks and subsequent integrin trafficking. METHODS: Glioblastoma motility and invasion were assessed in vitro and in vivo in response to ATR inhibition (ATRi) and ATR overexpression using time-lapse microscopy, two orthotopic glioblastoma models, and intravital imaging. Disruption to cytoskeleton networks and endocytic processing were investigated via high-throughput, super-resolution and intravital imaging. RESULTS: High ATR expression was associated with significantly poorer survival in clinical datasets while histological, protein expression, and spatial transcriptomics using glioblastoma tumor specimens revealed higher ATR expression at infiltrative margins. Pharmacological inhibition with two different compounds and RNAi targeting of ATR opposed the invasion of glioblastoma, whereas overexpression of ATR drove migration. Subsequent investigation revealed that cytoskeletal dysregulation reduced macropinocytotic internalization of integrins at growth-cone-like structures, resulting in a tumor microtube retraction defect. The biological relevance and translational potential of these findings were confirmed using two orthotopic in vivo models of glioblastoma and intravital imaging. CONCLUSIONS: We demonstrate a novel role for ATR in determining invasion in glioblastoma cells and propose that pharmacological targeting of ATR could have far-reaching clinical benefits beyond radiosensitization.

RAD51-mediated R-loop formation acts to repair transcription-associated DNA breaks driving antigenic variation in Trypanosoma brucei.

RNA-DNA hybrids are epigenetic features of all genomes that intersect with many processes, including transcription, telomere homeostasis, and centromere function. Increasing evidence suggests that RNA-DNA hybrids can provide two conflicting roles in the maintenance and transmission of genomes: They can be the triggers of DNA damage, leading to genome change, or can aid the DNA repair processes needed to respond to DNA lesions. Evasion of host immunity by African trypanosomes, such as Trypanosoma brucei, relies on targeted recombination of silent Variant Surface Glycoprotein (VSG) genes into a specialized telomeric locus that directs transcription of just one VSG from thousands. How such VSG recombination is targeted and initiated is unclear. Here, we show that a key enzyme of T. brucei homologous recombination, RAD51, interacts with RNA-DNA hybrids. In addition, we show that RNA-DNA hybrids display a genome-wide colocalization with DNA breaks and that this relationship is impaired by mutation of RAD51. Finally, we show that RAD51 acts to repair highly abundant, localised DNA breaks at the single transcribed VSG and that mutation of RAD51 alters RNA-DNA hybrid abundance at 70 bp repeats both around the transcribed VSG and across the silent VSG archive. This work reveals a widespread, generalised role for RNA-DNA hybrids in directing RAD51 activity during recombination and uncovers a specialised application of this interplay during targeted DNA break repair needed for the critical T. brucei immune evasion reaction of antigenic variation.

Immunoprecipitation of RNA-DNA hybrid interacting proteins in Trypanosoma brucei reveals conserved and novel activities, including in the control of surface antigen expression needed for immune evasion by antigenic variation.

RNA-DNA hybrids are epigenetic features of genomes that provide a diverse and growing range of activities. Understanding of these functions has been informed by characterising the proteins that interact with the hybrids, but all such analyses have so far focused on mammals, meaning it is unclear if a similar spectrum of RNA-DNA hybrid interactors is found in other eukaryotes. The African trypanosome is a single-cell eukaryotic parasite of the Discoba grouping and displays substantial divergence in several aspects of core biology from its mammalian host. Here, we show that DNA-RNA hybrid immunoprecipitation coupled with mass spectrometry recovers 602 putative interactors in T. brucei mammal- and insect-infective cells, some providing activities also found in mammals and some lineage-specific. We demonstrate that loss of three factors, two putative helicases and a RAD51 paralogue, alters T. brucei nuclear RNA-DNA hybrid and DNA damage levels. Moreover, loss of each factor affects the operation of the parasite immune survival mechanism of antigenic variation. Thus, our work reveals the broad range of activities contributed by RNA-DNA hybrids to T. brucei biology, including new functions in host immune evasion as well as activities likely fundamental to eukaryotic genome function.

AAK1-like: A putative pseudokinase with potential roles in cargo uptake in bloodstream form Trypanosoma brucei parasites.

Selection and internalization of cargo via clathrin-mediated endocytosis requires adaptor protein complexes. One complex, AP-2, acts during cargo selection at the plasma membrane. African trypanosomes lack all components of the AP-2 complex, except for a recently identified orthologue of the AP-2-associated protein kinase 1, AAK1. In characterized eukaryotes, AAK1 phosphorylates the µ2 subunit of the AP-2 complex to enhance cargo recognition and uptake into clathrin-coated vesicles. Here, we show that kinetoplastids encode not one, but two AAK1 orthologues: one (AAK1L2) is absent from salivarian trypanosomes, while the other (AAK1L1) lacks important kinase-specific residues in a range of trypanosomes. These AAK1L1 and AAK1L2 novelties reinforce suggestions of functional divergence in endocytic uptake within salivarian trypanosomes. Despite this, we show that AAK1L1 null mutant Trypanosoma brucei, while viable, display slowed proliferation, morphological abnormalities including swelling of the flagellar pocket, and altered cargo uptake. In summary, our data suggest an unconventional role for a putative pseudokinase during endocytosis and/or vesicular trafficking in T. brucei, independent of AP-2.

Fibronectin matrix assembly and TGFß1 presentation for chondrogenesis of patient derived pericytes for microtia repair.

Tissue engineered cartilage for external ear reconstruction of congenital deformities, such as microtia or resulting from trauma, remains a significant challenge for plastic and reconstructive surgeons. Current strategies involve harvesting autologous costal cartilage or expanding autologous chondrocytes ex vivo. However, these procedures often lead to donor site morbidity and a cell source with limited expansion capacity. Stromal stem cells such as perivascular stem cells (pericytes) offer an attractive alternative cell source, as they can be isolated from many human tissues, readily expanded in vitro and possess chondrogenic differentiation potential. Here, we successfully isolate CD146+ pericytes from the microtia remnant from patients undergoing reconstructive surgery (Microtia pericytes; MPs). Then we investigate their chondrogenic potential using the polymer poly(ethyl acrylate) (PEA) to unfold the extracellular matrix protein fibronectin (FN). FN unfolding exposes key growth factor (GF) and integrin binding sites on the molecule, allowing tethering of the chondrogenic GF transforming growth factor beta 1 (TGFß1). This system leads to solid-phase, matrix-bound, GF presentation in a more physiological-like manner than that of typical chondrogenic induction media (CM) formulations that tend to lead to off-target effects. This simple and controlled material-based approach demonstrates similar chondrogenic potential to CM, while minimising proclivity toward hypertrophy, without the need for complex induction media formulations.

Material-driven fibronectin and vitronectin assembly enhances BMP-2 presentation and osteogenesis.

Mesenchymal stem cell (MSC)-based tissue engineering strategies are of interest in the field of bone tissue regenerative medicine. MSCs are commonly investigated in combination with growth factors (GFs) and biomaterials to provide a regenerative environment for the cells. However, optimizing how biomaterials interact with MSCs and efficiently deliver GFs, remains a challenge. Here, via plasma polymerization, tissue culture plates are coated with a layer of poly (ethyl acrylate) (PEA), which is able to spontaneously permit fibronectin (FN) to form fibrillar nanonetworks. However, vitronectin (VN), another important extracellular matrix (ECM) protein forms multimeric globules on the polymer, thus not displaying functional groups to cells. Interestingly, when FN and VN are co-absorbed onto PEA surfaces, VN can be entrapped within the FN fibrillar nanonetwork in the monomeric form providing a heterogeneous, open ECM network. The combination of FN and VN promote MSC adhesion and leads to enhanced GF binding; here we demonstrate this with bone morphogenetic protein-2 (BMP2). Moreover, MSC differentiation into osteoblasts is enhanced, with elevated expression of osteopontin (OPN) and osteocalcin (OCN) quantified by immunostaining, and increased mineralization observed by von Kossa staining. Osteogenic intracellular signalling is also induced, with increased activity in the SMAD pathway. The study emphasizes the need of recapitulating the complexity of native ECM to achieve optimal cell-material interactions.

Myelinated axons are the primary target of hemin-mediated oxidative damage in a model of the central nervous system.

Iron released from oligodendrocytes during demyelination or derived from haemoglobin breakdown products is believed to amplify oxidative tissue injury in multiple sclerosis (MS). However, the pathophysiological significance of iron-containing haemoglobin breakdown products themselves is rarely considered in the context of MS and their cellular specificity and mode of action remain unclear. Using myelinating cell cultures, we now report the cytotoxic potential of hemin (ferriprotoporphyrin IX chloride), a major degradation product of haemoglobin, is 25-fold greater than equimolar concentrations of free iron in myelinating cultures; a model that reproduces the complex multicellular environment of the CNS. At low micro molar concentrations (3.3 - 10 µM) we observed hemin preferentially binds to myelin and axons to initiate a complex detrimental response that results in targeted demyelination and axonal loss but spares neuronal cell bodies, astrocytes and the majority of oligodendroglia. Demyelination and axonal loss in this context are executed by a combination of mechanisms that include iron-dependent peroxidation by reactive oxygen species (ROS) and ferroptosis. These effects are microglial-independent, do not require any initiating inflammatory insult and represent a direct effect that compromises the structural integrity of myelinated axons in the CNS. Our data identify hemin-mediated demyelination and axonal loss as a novel mechanism by which intracerebral degradation of haemoglobin may contribute to lesion development in MS.

Río-Hortega's drawings revisited with fluorescent protein defines a cytoplasm-filled channel system of CNS myelin.

A century ago this year, Pío del Río-Hortega (1921) coined the term 'oligodendroglia' for the 'interfascicular glia' with very few processes, launching an extensive discovery effort on his new cell type. One hundred years later, we review his original contributions to our understanding of the system of cytoplasmic channels within myelin in the context of what we observe today using light and electron microscopy of genetically encoded fluorescent reporters and immunostaining. We use the term myelinic channel system to describe the cytoplasm-delimited spaces associated with myelin; being the paranodal loops, inner and outer tongues, cytoplasm-filled spaces through compact myelin and further complex motifs associated to the sheath. Using a central nervous system myelinating cell culture model that contains all major neural cell types and produces compact myelin, we find that td-tomato fluorescent protein delineates the myelinic channel system in a manner reminiscent of the drawings of adult white matter by Río-Hortega, despite that he questioned whether some cytoplasmic figures he observed represented artefact. Together, these data lead us to propose a slightly revised model of the 'unrolled' sheath. Further, we show that the myelinic channel system, while relatively stable, can undergo subtle dynamic shape changes over days. Importantly, we capture an under-appreciated complexity of the myelinic channel system in mature myelin sheaths.

Depletion of a Toxoplasma porin leads to defects in mitochondrial morphology and contacts with the endoplasmic reticulum.

The voltage-dependent anion channel (VDAC) is a ubiquitous channel in the outer membrane of the mitochondrion with multiple roles in protein, metabolite and small molecule transport. In mammalian cells, VDAC protein, as part of a larger complex including the inositol triphosphate receptor, has been shown to have a role in mediating contacts between the mitochondria and endoplasmic reticulum (ER). We identify VDAC of the pathogenic apicomplexan Toxoplasma gondii and demonstrate its importance for parasite growth. We show that VDAC is involved in protein import and metabolite transfer to mitochondria. Further, depletion of VDAC resulted in significant morphological changes in the mitochondrion and ER, suggesting a role in mediating contacts between these organelles in T. gondii. This article has an associated First Person interview with the first author of the paper.

Direct, Late-Stage Mono-N-arylation of Pentamidine: Method Development, Mechanistic Insight, and Expedient Access to Novel Antiparastitics against Diamidine-Resistant Parasites.

A selective mono-N-arylation strategy of amidines under Chan-Lam conditions is described. During the reaction optimization phase, the isolation of a mononuclear Cu(II) complex provided unique mechanistic insight into the operation of Chan-Lam mono-N-arylation. The scope of the process is demonstrated, and then applied to access the first mono-N-arylated analogues of pentamidine. Sub-micromolar activity against kinetoplastid parasites was observed for several analogues with no cross-resistance in pentamidine and diminazene-resistant trypanosome strains and against Leishmania mexicana. A fluorescent mono-N-arylated pentamidine analogue revealed rapid cellular uptake, accumulating in parasite nuclei and the kinetoplasts. The DNA binding capability of the mono-N-arylated pentamidine series was confirmed by UV-melt measurements using AT-rich DNA. This work highlights the potential to use Chan-Lam mono-N-arylation to develop therapeutic leads against diamidine-resistant trypanosomiasis and leishmaniasis.

Synthesis, biological, and photophysical studies of molecular rotor-based fluorescent inhibitors of the trypanosome alternative oxidase.

We have recently reported on the development and trypanocidal activity of a class of inhibitors of Trypanosome Alternative Oxidase (TAO) that are targeted to the mitochondrial matrix by coupling to lipophilic cations via C14 linkers to enable optimal interaction with the enzyme's active site. This strategy resulted in a much-enhanced anti-parasite effect, which we ascribed to the greater accumulation of the compound at the location of the target protein, i.e. the mitochondrion, but to date this localization has not been formally established. We therefore synthesized a series of fluorescent analogues to visualize accumulation and distribution within the cell. The fluorophore chosen, julolidine, has the remarkable extra feature of being able to function as a viscosity sensor and might thus additionally act as a probe of the cellular glycerol that is expected to be produced when TAO is inhibited. Two series of fluorescent inhibitor conjugates incorporating a cationic julolidine-based viscosity sensor were synthesized and their photophysical and biological properties were studied. These probes display a red emission, with a high signal-to-noise ratio (SNR), using both single- and two-photon excitation. Upon incubation with T. brucei and mammalian cells, the fluorescent inhibitors 1a and 2a were taken up selectively in the mitochondria as shown by live-cell imaging. Efficient partition of 1a in functional isolated (rat liver) mitochondria was estimated to 66 ± 20% of the total. The compounds inhibited recombinant TAO enzyme in the submicromolar (1a, 2c, 2d) to low nanomolar range (2a) and were effective against WT and multidrug-resistant trypanosome strains (B48, AQP1-3 KO) in the submicromolar range. Good selectivity (SI > 29) over mammalian HEK cells was observed. However, no viscosity-related shift could be detected, presumably because the glycerol was produced cytosolically, and released through aquaglyceroporins, whereas the probe was located, virtually exclusively, in the trypanosome's mitochondrion.

Diminazene resistance in Trypanosoma congolense is not caused by reduced transport capacity but associated with reduced mitochondrial membrane potential.

Trypanosoma congolense is a principal agent causing livestock trypanosomiasis in Africa, costing developing economies billions of dollars and undermining food security. Only the diamidine diminazene and the phenanthridine isometamidium are regularly used, and resistance is widespread but poorly understood. We induced stable diminazene resistance in T. congolense strain IL3000 in vitro. There was no cross-resistance with the phenanthridine drugs, melaminophenyl arsenicals, oxaborole trypanocides, or with diamidine trypanocides, except the close analogs DB829 and DB75. Fluorescence microscopy showed that accumulation of DB75 was inhibited by folate. Uptake of [3 H]-diminazene was slow with low affinity and partly but reciprocally inhibited by folate and by competing diamidines. Expression of T. congolense folate transporters in diminazene-resistant Trypanosoma brucei brucei significantly sensitized the cells to diminazene and DB829, but not to oxaborole AN7973. However, [3 H]-diminazene transport studies, whole-genome sequencing, and RNA-seq found no major changes in diminazene uptake, folate transporter sequence, or expression. Instead, all resistant clones displayed a moderate reduction in the mitochondrial membrane potential Ψm. We conclude that diminazene uptake in T. congolense proceed via multiple low affinity mechanisms including folate transporters; while resistance is associated with a reduction in Ψm it is unclear whether this is the primary cause of the resistance.

Genome maintenance functions of a putative Trypanosoma brucei translesion DNA polymerase include telomere association and a role in antigenic variation.

Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation.

Venetoclax causes metabolic reprogramming independent of BCL-2 inhibition.

BH3-mimetics are a new class of anti-cancer drugs that inhibit anti-apoptotic Bcl-2 proteins. In doing so, BH3-mimetics sensitise to cell death. Venetoclax is a potent, BCL-2 selective BH3-mimetic that is clinically approved for use in chronic lymphocytic leukaemia. Venetoclax has also been shown to inhibit mitochondrial metabolism, this is consistent with a proposed role for BCL-2 in metabolic regulation. We used venetoclax to understand BCL-2 metabolic function. Similar to others, we found that venetoclax inhibited mitochondrial respiration. In addition, we also found that venetoclax impairs TCA cycle activity leading to activation of reductive carboxylation. Importantly, the metabolic effects of venetoclax were independent of cell death because they were also observed in apoptosis-resistant BAX/BAK-deficient cells. However, unlike venetoclax treatment, inhibiting BCL-2 expression had no effect on mitochondrial respiration. Unexpectedly, we found that venetoclax also inhibited mitochondrial respiration and the TCA cycle in BCL-2 deficient cells and in cells lacking all anti-apoptotic BCL-2 family members. Investigating the basis of this off-target effect, we found that venetoclax-induced metabolic reprogramming was dependent upon the integrated stress response and ATF4 transcription factor. These data demonstrate that venetoclax affects cellular metabolism independent of BCL-2 inhibition. This off-target metabolic effect has potential to modulate venetoclax cytotoxicity.

Zygote morphogenesis but not the establishment of cell polarity in Plasmodium berghei is controlled by the small GTPase, RAB11A.

Plasmodium species are apicomplexan parasites whose zoites are polarized cells with a marked apical organisation where the organelles associated with host cell invasion and colonization reside. Plasmodium gametes mate in the mosquito midgut to form the spherical and presumed apolar zygote that morphs during the following 24 hours into a polarized, elongated and motile zoite form, the ookinete. Endocytosis-mediated protein transport is generally necessary for the establishment and maintenance of polarity in epithelial cells and neurons, and the small GTPase RAB11A is an important regulator of protein transport via recycling endosomes. PbRAB11A is essential in blood stage asexual of Plasmodium. Therefore, a promoter swap strategy was employed to down-regulate PbRAB11A expression in gametocytes and zygotes of the rodent malaria parasite, Plasmodium berghei which demonstrated the essential role of RAB11A in ookinete development. The approach revealed that lack of PbRAB11A had no effect on gamete production and fertility rates however, the zygote to ookinete transition was almost totally inhibited and transmission through the mosquito was prevented. Lack of PbRAB11A did not prevent meiosis and mitosis, nor the establishment of polarity as indicated by the correct formation and positioning of the Inner Membrane Complex (IMC) and apical complex. However, morphological maturation was prevented and parasites remained spherical and immotile and furthermore, they were impaired in the secretion and distribution of microneme cargo. The data are consistent with the previously proposed model of RAB11A endosome mediated delivery of plasma membrane in Toxoplasma gondii if not its role in IMC formation and implicate it in microneme function.

Trypanosoma brucei ATR Links DNA Damage Signaling during Antigenic Variation with Regulation of RNA Polymerase I-Transcribed Surface Antigens.

Trypanosoma brucei evades mammalian immunity by using recombination to switch its surface-expressed variant surface glycoprotein (VSG), while ensuring that only one of many subtelomeric multigene VSG expression sites are transcribed at a time. DNA repair activities have been implicated in the catalysis of VSG switching by recombination, not transcriptional control. How VSG switching is signaled to guide the appropriate reaction or to integrate switching into parasite growth is unknown. Here, we show that the loss of ATR, a DNA damage-signaling protein kinase, is lethal, causing nuclear genome instability and increased VSG switching through VSG-localized damage. Furthermore, ATR loss leads to the increased transcription of silent VSG expression sites and expression of mixed VSGs on the cell surface, effects that are associated with the altered localization of RNA polymerase I and VEX1. This work shows that ATR acts in antigenic variation both through DNA damage signaling and surface antigen expression control.

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 apply a traction force to pass through, a step that typically concurs with transient constriction of the parasite body. Here we analyse F-actin dynamics during host cell invasion. 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 localised 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. Our analysis suggests that the junction opposes resistance to the passage of the parasite's nucleus and provides the first evidence for a dual contribution of actin-forces during host cell invasion by apicomplexan parasites.

Malaria transmission through the mosquito requires the function of the OMD protein.

Ookinetes, one of the motile and invasive forms of the malaria parasite, rely on gliding motility in order to establish an infection in the mosquito host. Here we characterize the protein PBANKA_0407300 which is conserved in the Plasmodium genus but lacks significant similarity to proteins of other eukaryotes. It is expressed in gametocytes and throughout the invasive mosquito stages of P. berghei, but is absent from asexual blood stages. Mutants lacking the protein developed morphologically normal ookinetes that were devoid of productive motility although some stretching movement could be detected. We therefore named the protein Ookinete Motility Deficient (OMD). Several key factors known to be involved in motility however were normally expressed and localized in the mutant. Importantly, the mutant failed to establish an infection in the mosquito which resulted in a total malaria transmission blockade.