The kinesin of the flagellum attachment zone in Leishmania is required for cell morphogenesis, cell division and virulence in the mammalian host

Leishmania parasites possess a unique and complex cytoskeletal structure termed flagellum attachment zone (FAZ) connecting the base of the flagellum to one side of the flagellar pocket (FP), an invagination of the cell body membrane and the sole site for endocytosis and exocytosis. This structure is involved in FP architecture and cell morphogenesis, but its precise role and molecular composition remain enigmatic. Here, we characterized Leishmania FAZ7, the only known FAZ protein containing a kinesin motor domain, and part of a clade of trypanosomatid-specific kinesins with unknown functions. The two paralogs of FAZ7, FAZ7A and FAZ7B, display different localizations and functions. FAZ7A localizes at the basal body, while FAZ7B localizes at the distal part of the FP, where the FAZ structure is present in Leishmania. While null mutants of FAZ7A displayed normal growth rates, the deletion of FAZ7B impaired cell growth in both promastigotes and amastigotes of Leishmania. The kinesin activity is crucial for its function. Deletion of FAZ7B resulted in altered cell division, cell morphogenesis (including flagellum length), and FP structure and function. Furthermore, knocking out FAZ7B induced a mis-localization of two of the FAZ proteins, and disrupted the molecular organization of the FP collar, affecting the localization of its components. Loss of the kinesin FAZ7B has important consequences in the insect vector and mammalian host by reducing proliferation in the sand fly and pathogenicity in mice. Our findings reveal the pivotal role of the only FAZ kinesin as part of the factors important for a successful life cycle of Leishmania.

Development of a Modal-Based Turbomachine Blade-Disk Attachment Inspection Technique

Turbine blade failures are among the leading causes of steam turbine failure. Failure types typically include cracking, rubbing, blade fouling, and foreign object damage. There is currently a range of non-destructive testing methods used to detect damage at the blade-disk attachment zone, all of which involve disassembling of the blade from the disk for periodic inspection. Evidence indicate that a method to detect damage at the blade-disk attachment zone using a non-contact, non-destructive in-situ off-line modal-based structural health monitoring technique could be useful under some circumstances. Such a technique would have the advantage of eliminating the necessity to disassemble blades during inspection. This would result in significant cost savings. Also, defects associated with the disassembly and reassembly of blades would be avoided. Thus, the aim of this study was to develop a modal-based turbomachinery blade disk attachment inspection technique. Modal parameters were acquired from a robust experimental modal analysis of freely supported low-pressure steam turbine blade-disk segment assemblies. Artificial single-location cracks were intentionally introduced into the turbine blades by cutting a 1 mm thickness notch at three probable damage locations, namely, at the upper pinhole on the leading-edge pressure side, above the root at the base of the aerofoil on the leading-edge and on the trailing-edge. In this work, a finite element analysis of the bladed disk segment assemblies was carried out with and without damage. To validate the reliability of the numerical models, the numerical results were correlated with the measured values, the results of which showed a strong correlation. Finally, a parametric study was conducted in which various healthy and damaged blade-disk cases were systematically investigated. This was done to examine the sensitivity of the blade natural frequency to damage. The artificial damage above the root was found to cause the largest changes in natural frequency. These changes were even more pronounced for assemblies with two blades. Receiver operating characteristic curves were used to assess the discriminatory ability of the results. Each damage case was found to be unique and therefore identifiable from its corresponding healthy case.

Maintenance of hook complex integrity and centrin arm assembly facilitates flagellum inheritance in Trypanosoma brucei

Inheritance of the newly assembled flagellum in the human parasite Trypanosoma brucei depends on the faithful duplication and segregation of multiple flagellum-associated cytoskeletal structures, including the hook complex and its associated centrin arm. The biological functions of this unique hook complex–centrin arm assembly remain poorly understood. Here, we report a hook complex–associated protein named BOH2 that plays an essential role in promoting flagellum inheritance. BOH2 localizes to the hooked part of the hook complex by bridging the hook complex, the centrin arm, and the flagellum attachment zone filament. Depletion of BOH2 caused the loss of the shank part of the hook complex and its associated protein TbSmee1, disrupted the assembly of the centrin arm and the recruitment of centrin arm–associated protein CAAP1, inhibited the assembly of the flagellum attachment zone, and caused flagellum mispositioning and detachment. These results demonstrate crucial roles of BOH2 in maintaining hook complex integrity and promoting centrin arm formation and suggest that proper assembly of the hook complex–centrin arm structure facilitates flagellum inheritance.

Role for the flagellum attachment zone in the resolution of cell membrane morphogenesis during Leishmania cell division

The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum and these are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenetic resolution of the anterior cell tip during cell division. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption in membrane organisation at the anterior cell tip, resulting in cells that late in division were connected to each other by a membranous bridge structure between their flagella. These changes had a great impact in vivo with the FAZ2 null mutant unable to develop and proliferate in sand flies and causing a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division.Author summaryLeishmania are parasites that cause leishmaniasis in humans with symptoms ranging from mild cutaneous lesions to severe visceral disease. The life cycle of these parasites alternates between the human host and the sand fly vector, with distinct forms in both. These different forms have different cell shapes that are adapted for survival in these different environments. Leishmania parasites have an elongated cell shape with a flagellum extending from one end and this shape is due to a protein skeleton beneath the cell membrane. This skeleton is made up of different units one of which is called the flagellum attachment zone (FAZ), that connects the flagellum to the cell body. We have found that one of the proteins in the FAZ called FAZ2 is important for generating the shape of the cell at the point where the flagellum exits the cell. When we deleted FAZ2 we found that the cell membrane at the tip of the was distorted resulting in unusual connections between the flagella of different cells. We found that the disruption to the cell shape reduces the ability of the parasite to infect mice and develop in the sand fly, which shows the importance of the parasite shape.

In situ structural analysis of the flagellum attachment zone in Trypanosoma brucei using cryo-scanning transmission electron tomography

SummaryThe flagellum of Trypanosoma brucei is a 20 µm-long organelle responsible for locomotion and cell morphogenesis. The flagellum attachment zone (FAZ) is a multi-protein complex whose function is to attach the flagellum to the cell body but also to guide cytokinesis. Cryo-transmission electron microscopy is a tool of choice to access the structure of the FAZ in a close-to-native state. However, because of the large dimension of the cell body, the whole FAZ cannot be structurally studied in situ at high resolution in 3D using classical transmission electron microscopy approaches. In the present work, cryo-scanning transmission electron tomography, a new method capable of investigating cryo-fixed thick biological samples, has been used to study whole T. brucei cells at the bloodstream stage. The method has been used to visualise and characterise the structure and organisation of the FAZ filament. It is composed of an array of cytoplasmic stick-like structures. These sticks are heterogeneously distributed between the posterior part and the anterior tip of the cell. This cryo-STET investigation provides new insights in the structure the FAZ filament. In combination with protein structure predictions, this work proposes a new model for the elongation of the FAZ.HighlightsFlagellar and cellular membranes are in close contact next to the FAZ filamentSticks are heterogeneously distributed along the FAZ filament lengthThin appendages are present between the FAZ filament sticks to neighbouring microtubulesFAZ elongation could originate from the force exerted by dynein motors on subpellicular microtubules

Leishmaniaflagellum attachment zone is critical for flagellar pocket shape, development in the sand fly, and pathogenicity in the host

Leishmaniakinetoplastid parasites infect millions of people worldwide and have a distinct cellular architecture depending on location in the host or vector and specific pathogenicity functions. An invagination of the cell body membrane at the base of the flagellum, the flagellar pocket (FP), is an iconic kinetoplastid feature, and is central to processes that are critical forLeishmaniapathogenicity. TheLeishmaniaFP has a bulbous region posterior to the FP collar and a distal neck region where the FP membrane surrounds the flagellum more closely. The flagellum is attached to one side of the FP neck by the short flagellum attachment zone (FAZ). We addressed whether targeting the FAZ affects FP shape and its function as a platform for host–parasite interactions. Deletion of the FAZ protein, FAZ5, clearly altered FP architecture and had a modest effect in endocytosis but did not compromise cell proliferation in culture. However, FAZ5 deletion had a dramatic impact in vivo: Mutants were unable to develop late-stage infections in sand flies, and parasite burdens in mice were reduced by >97%. Our work demonstrates the importance of the FAZ for FP function and architecture. Moreover, we show that deletion of a single FAZ protein can have a large impact on parasite development and pathogenicity.

Breeding biology of the Helmeted Manakin Antilophia galeata in an ecotone between the Atlantic Forest and the Cerrado

Manakins (Pipridae) are well-known by their promiscuous mating system. Nonetheless, scarce evidence suggests that the Helmeted Manakin Antilophia galeata, the only dichromatic manakin widely distributed in the South American Cerrado, is monogamic. We studied the breeding biology of the Helmeted Manakin in the state of Minas Gerais, southeastern Brazil. We found 17 nests, which are built in the forest undergrowth (usually below 3 m height). Nests are a cup attached by its top lip usually in the angle of a forked branch. The nest attachment zone is made mostly of spider silk. The structural zone is constructed with dry broad leaves, leaf petioles and horsehair fungus, which were bind together by a considerable amount of spider silk. The outer (decorative) zone is made with some hanging dry broad leaves, frequently forming a tail. Clutch size is always two (n = 12), and eggs are long oval, with a ground color in different shades of beige, marked with irregular spot, blotches and, sometimes, streaks ranging from light to dark brown. Mean eggs measurements were 23.9 x 16.3 mm (n = 14), weighting 3.3 g (n = 10). Eggs are laid once each day and hatching is synchronous. Females are the sole responsible for nest building (which usually took 10 days), incubation (18.5 days), and nestling care (18.3 days). The simple percentage of successful nests (n = 11) was 27% and predation was the main cause of nest failure. The breeding season extends from the second half of August to the first half of January. Renesting after loss of a first clutch is a common strategy and we recorded up to three nesting attempts for a single female. We present evidence that the Helmeted Manakin is promiscuous, as usual for a dichromatic manakin.