The presynaptic terminal contains a complex network of filaments whose precise organization and functions are not yet understood. The cryoelectron tomography experiments reported in this study ...indicate that these structures play a prominent role in synaptic vesicle release. Docked synaptic vesicles did not make membrane to membrane contact with the active zone but were instead linked to it by tethers of different length. Our observations are consistent with an exocytosis model in which vesicles are first anchored by long (>5 nm) tethers that give way to multiple short tethers once vesicles enter the readily releasable pool. The formation of short tethers was inhibited by tetanus toxin, indicating that it depends on soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor complex assembly. Vesicles were extensively interlinked via a set of connectors that underwent profound rearrangements upon synaptic stimulation and okadaic acid treatment, suggesting a role of these connectors in synaptic vesicle mobilization and neurotransmitter release.
Crista junctions (CJs) are important for mitochondrial organization and function, but the molecular basis of their formation and architecture is obscure. We have identified and characterized a ...mitochondrial membrane protein in yeast, Fcj1 (formation of CJ protein 1), which is specifically enriched in CJs. Cells lacking Fcj1 lack CJs, exhibit concentric stacks of inner membrane in the mitochondrial matrix, and show increased levels of F₁FO-ATP synthase (F₁FO) supercomplexes. Overexpression of Fcj1 leads to increased CJ formation, branching of cristae, enlargement of CJ diameter, and reduced levels of F₁FO supercomplexes. Impairment of F₁FO oligomer formation by deletion of its subunits e/g (Su e/g) causes CJ diameter enlargement and reduction of cristae tip numbers and promotes cristae branching. Fcj1 and Su e/g genetically interact. We propose a model in which the antagonism between Fcj1 and Su e/g locally modulates the F₁FO oligomeric state, thereby controlling membrane curvature of cristae to generate CJs and cristae tips.
Abstract
Neurofilament light chain (NfL), released during central nervous injury, has evolved as a powerful serum marker of disease severity in many neurological disorders, including infectious ...diseases. So far NfL has not been assessed in cerebral malaria in human or its rodent model experimental cerebral malaria (ECM), a disease that can lead to fatal brain edema or reversible brain edema. In this study we assessed if NfL serum levels can also grade disease severity in an ECM mouse model with reversible (n = 11) and irreversible edema (n = 10). Blood–brain-barrier disruption and brain volume were determined by magnetic resonance imaging. Neurofilament density volume as well as structural integrity were examined by electron microscopy in regions of most severe brain damage (olfactory bulb (OB), cortex and brainstem). NfL plasma levels in mice with irreversible edema (317.0 ± 45.01 pg/ml) or reversible edema (528.3 ± 125.4 pg/ml) were significantly increased compared to controls (103.4 ± 25.78 pg/ml) by three to five fold, but did not differ significantly in mice with reversible or irreversible edema. In both reversible and irreversible edema, the brain region most affected was the OB with highest level of blood–brain-barrier disruption and most pronounced decrease in neurofilament density volume, which correlated with NfL plasma levels (r = − 0.68, p = 0.045). In cortical and brainstem regions neurofilament density was only decreased in mice with irreversible edema and strongest in the brainstem. In reversible edema NfL plasma levels, MRI findings and neurofilament volume density normalized at 3 months’ follow-up. In conclusion, NfL plasma levels are elevated during ECM confirming brain damage. However, NfL plasma levels fail short on reliably indicating on the final outcomes in the acute disease stage that could be either fatal or reversible. Increased levels of plasma NfL during the acute disease stage are thus likely driven by the anatomical location of brain damage, the olfactory bulb, a region that serves as cerebral draining pathway into the nasal lymphatics.
Plasmodium falciparum infections can cause severe malaria, but not every infected person develops life-threatening complications. In particular, carriers of the structural haemoglobinopathies S and C ...and infants are protected from severe disease. Protection is associated with impaired parasite-induced host actin reorganization, required for vesicular trafficking of parasite-encoded adhesins, and reduced cytoadherence of parasitized erythrocytes in the microvasculature. Here we show that aberrant host actin remodelling and the ensuing reduced cytoadherence result from a redox imbalance inherent to haemoglobinopathic and fetal erythrocytes. We further show that a transient oxidative insult to wild-type erythrocytes before infection with P. falciparum induces the phenotypic features associated with the protective trait of haemoglobinopathic and fetal erythrocytes. Moreover, pretreatment of mice with the pro-oxidative nutritional supplement menadione mitigate the development of experimental cerebral malaria. Our results identify redox imbalance as a causative principle of protection from severe malaria, which might inspire host-directed intervention strategies.
Mitochondrial protein import (MPI) is essential for the biogenesis of mitochondria in all eukaryotes. Current models of MPI are predominantly based on experiments with one group of eukaryotes, the ...opisthokonts. Although fascinating genome database-driven hypotheses on the evolution of the MPI machineries have been published, previous experimental research on non-opisthokonts usually focused on the analysis of single pathways or components in, for example, plants and parasites. In this study, we have established the kinetoplastid parasite Leishmania tarentolae as a model organism for the comprehensive analysis of non-opisthokont MPI into all four mitochondrial compartments. We found that opisthokont marker proteins are efficiently imported into isolated L. tarentolae mitochondria. Vice versa, L. tarentolae marker proteins of all compartments are also imported into mitochondria from yeast. The results are remarkable because only a few of the more than 25 classical components of the opisthokont MPI machineries are found in parasite genome databases. Our results demonstrate that different MPI pathways are functionally conserved among eukaryotes despite significant compositional differences of the MPI machineries. Moreover, our model system could lead to the identification of significantly altered or even novel MPI components in non-opisthokonts. Such differences might serve as starting points for drug development against parasitic protists.
PfEMP1 (erythrocyte membrane protein 1) adhesins play a pivotal role in the pathophysiology of falciparum malaria, by mediating sequestration of
-infected erythrocytes in the microvasculature. PfEMP1 ...variants are expressed by
genes and are presented on membrane elevations, termed knobs. However, the organization of PfEMP1 on knobs is largely unclear. Here, we use super-resolution microscopy and genetically altered parasites expressing a modified
a gene in which the coding sequence of the photoactivatable mEOS2 was inserted to determine the number and distribution of PfEMP1 on single knobs. The data were verified by quantitative fluorescence-activated cell sorting analysis and immuno-electron microscopy together with stereology methods. We show that knobs contain 3.3 ± 1.7 and 4.3 ± 2.5 PfEMP1 molecules, predominantly placed on the knob tip, in parasitized erythrocytes containing wild type and sickle haemoglobin, respectively. The ramifications of our findings for cytoadhesion and immune evasion are discussed.
Following invasion of human red blood cells (RBCs) by the malaria parasite, Plasmodium falciparum, a remarkable process of remodeling occurs in the host cell mediated by trafficking of several ...hundred effector proteins to the RBC compartment. The exported virulence protein, P falciparum erythrocyte membrane protein 1 (PfEMP1), is responsible for cytoadherence of infected cells to host endothelial receptors. Maurer clefts are organelles essential for protein trafficking, sorting, and assembly of protein complexes. Here we demonstrate that disruption of PfEMP1 trafficking protein 1 (PfPTP1) function leads to severe alterations in the architecture of Maurer’s clefts. Furthermore, 2 major surface antigen families, PfEMP1 and STEVOR, are no longer displayed on the host cell surface leading to ablation of cytoadherence to host receptors. PfPTP1 functions in a large complex of proteins and is required for linking of Maurer’s clefts to the host actin cytoskeleton.
•Maurer’s clefts are P falciparum–derived membranous structures within the host erythrocyte that are essential for parasite survival.•PfPTP1 functions in a large complex of proteins and is required for linking of Maurer’s clefts to the host actin cytoskeleton.
The pathology associated with malaria infection is largely due to the ability of infected human RBCs to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon ...is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin. ATPase assays on recombinant protein verify a functional interaction between PFA66 and residual host cell HSP70. Taken together, our data reveal a role for PFA66 in host cell modification, strongly implicate human HSP70s as being essential in this process and uncover a new KAHRP-independent molecular factor required for correct knob biogenesis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Bacteria can be propelled in liquids by flagellar filaments that are attached to and moved by flagellar motors. These motors are rotary nanomachines that use the electrochemical potential from ion ...gradients. The motor can spin in both directions with specific proteins regulating the direction in response to chemotactic stimuli. Here we investigated the structure of flagellar motors of
Borrelia spirochetes, the causative agents of Lyme disease in humans. We revealed the structure of the motor complex at 4.6-nm resolution by sub-volume averaging of cryo-electron tomograms and subsequently imposing rotational symmetry. This allowed direct visualisation of individual motor components, the connection between the stator and the peptidoglycan as well as filamentous linkers between the stator and the rod. Two different motor assemblies seem to co-exist at a single bacterial pole. While most motors were completely assembled, a smaller fraction appeared to lack part of the C-ring, which plays a role in protein export and switching the directionality of rotation. Our data suggest a novel mechanism that bacteria may use to control the direction of movement.
Spirochetes of the Borrelia burgdorferi sensu lato group, the causative agents of Lyme borreliosis, exhibit a complex biology evolved in its zoonotic cycle. Cryo-electron tomography was used to ...investigate structural features of three species, B. burgdorferi, B. garinii and B. afzelii, known to cause different clinical manifestations in humans. All three organisms revealed an overall similar architecture and showed different numbers of periplasmic flagellar filaments, polar periplasmic void regions, vesicles budding from the outer membrane sheath, which was covered by an amorphous slime layer. The latter was shown to be distinct in its density when comparing the three human-pathogenic Lyme disease spirochetes and Borrelia hermsii, a species causing relapsing fever. Tomograms of dividing bacteria revealed vesicles near the site of division and new basal bodies that were attached at each end of newly establishing cytoplasmic cylinder poles, while periplasmic flagellar filaments still passed the impending site of division. Two different kinds of cytoplasmic filaments showed similarities to MreB or FtsZ filaments of other bacteria. The similar and distinct structural features of Borrelia and the previously investigated pathogenic and non-pathogenic Treponema species emphasize the importance of further studying phylogenetically distant spirochetes.
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