The distal end of the eukaryotic flagellum/cilium is important for axonemal growth and signaling and has distinct biomechanical properties. Specific flagellum tip structures exist, yet their composition, dynamics, and functions are largely unknown. We used biochemical approaches to identify seven constituents of the flagella connector at the tip of an assembling trypanosome flagellum and three constituents of the axonemal capping structure at the tips of both assembling and mature flagella. Both tip structures contain evolutionarily conserved as well as kinetoplastid-specific proteins, and component assembly into the structures occurs very early during flagellum extension. Localization and functional studies reveal that the flagella connector membrane junction is attached to the tips of extending microtubules of the assembling flagellum by a kinesin-15 family member. On the opposite side, a kinetoplastid-specific kinesin facilitates attachment of the junction to the microtubules in the mature flagellum. Functional studies also suggest roles of several other components and the definition of subdomains in the tip structures.

• Klíčová slova
• axonemal capping structure, flagella connector, flagellar distal end, structure immunoprecipitation, trypanosome,
• MeSH
• axonema chemie   metabolismus   MeSH
• flagella chemie   metabolismus   MeSH
• kineziny chemie   metabolismus   MeSH
• protozoální proteiny chemie   metabolismus   MeSH
• Trypanosoma brucei brucei chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kineziny MeSH
• protozoální proteiny MeSH

The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure - the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms.

• Klíčová slova
• Flagellum attachment zone, Morphogenesis, Trypanosomes,
• MeSH
• cilie genetika   metabolismus   MeSH
• cytokineze genetika   MeSH
• cytoskelet genetika   metabolismus   MeSH
• flagella genetika   metabolismus   MeSH
• mikrotubuly genetika   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• stadia vývoje genetika   MeSH
• Trypanosoma brucei brucei genetika   růst a vývoj   MeSH
• tvar buňky genetika   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH

Differentiation of Trypanosoma brucei, a flagellated protozoan parasite, between life cycle stages typically occurs through an asymmetric cell division process, producing two morphologically distinct daughter cells. Conversely, proliferative cell divisions produce two daughter cells, which look similar but are not identical. To examine in detail differences between the daughter cells of a proliferative division of procyclic T. brucei we used the recently identified constituents of the flagella connector. These segregate asymmetrically during cytokinesis allowing the new-flagellum and the old-flagellum daughters to be distinguished. We discovered that there are distinct morphological differences between the two daughters, with the new-flagellum daughter in particular re-modelling rapidly and extensively in early G1. This re-modelling process involves an increase in cell body, flagellum and flagellum attachment zone length and is accompanied by architectural changes to the anterior cell end. The old-flagellum daughter undergoes a different G1 re-modelling, however, despite this there was no difference in G1 duration of their respective cell cycles. This work demonstrates that the two daughters of a proliferative division of T. brucei are non-equivalent and enables more refined morphological analysis of mutant phenotypes. We suggest all proliferative divisions in T. brucei and related organisms will involve non-equivalence.

• MeSH
• buněčné dělení MeSH
• cytokineze MeSH
• flagella genetika   metabolismus   MeSH
• proliferace buněk MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• stadia vývoje MeSH
• Trypanosoma brucei brucei cytologie   genetika   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH

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. 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 morphogenesis of the anterior cell tip. 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 to the membrane organisation at the anterior cell tip, resulting in cells that were connected to each other by a membranous bridge structure between their flagella. Moreover, the FAZ2 null mutant was unable to develop and proliferate in sand flies and had 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.

• MeSH
• buněčná membrána MeSH
• cytokineze MeSH
• cytoskelet metabolismus   MeSH
• flagella fyziologie   ultrastruktura   MeSH
• interakce hostitele a parazita * MeSH
• Leishmania růst a vývoj   ultrastruktura   MeSH
• leishmanióza parazitologie   MeSH
• morfogeneze * MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• Psychodidae parazitologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH

Unc-51-like kinase (ULK) family serine-threonine protein kinase homologues have been linked to the function of motile cilia in diverse species. Mutations in Fused/STK36 and ULK4 in mice resulted in hydrocephalus and other phenotypes consistent with ciliary defects. How either protein contributes to the assembly and function of motile cilia is not well understood. Here we studied the phenotypes of ULK4 and Fused gene knockout (KO) mutants in the flagellated protist Leishmania mexicana. Both KO mutants exhibited a variety of structural defects of the flagellum cytoskeleton. Biochemical approaches indicate spatial proximity of these proteins and indicate a direct interaction between the N-terminus of LmxULK4 and LmxFused. Both proteins display a dispersed localization throughout the cell body and flagellum, with enrichment near the flagellar base and tip. The stable expression of LmxULK4 was dependent on the presence of LmxFused. Fused/STK36 was previously shown to localize to mammalian motile cilia, and we demonstrate here that ULK4 also localizes to the motile cilia in mouse ependymal cells. Taken together these data suggest a model where the pseudokinase ULK4 is a positive regulator of the kinase Fused/ STK36 in a pathway required for stable assembly of motile cilia.

• MeSH
• cilie metabolismus   MeSH
• flagella * metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• myši MeSH
• protein-serin-threoninkinasy * metabolismus   MeSH
• savci metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein-serin-threoninkinasy * MeSH
• Stk36 protein, mouse MeSH Prohlížeč

The factors affecting the inter-individual differences in sperm freezability is a major line of research in spermatology. Poor sperm freezability is mainly characterised by a low sperm velocity, which in turn is associated with low fertility rates in most animal species. Studies concerning the implications of sperm morphometry on freezability are quite limited, and most of them are based on sperm head size regardless of the structural parts of the flagellum, which provides sperm motility. Here, for the first time, we determined the volumes of the flagellum structures in fresh epididymal red deer spermatozoa using a stereological method under phase contrast microscopy. Sperm samples from thirty-three stags were frozen and classified as good freezers (GF) or bad freezers (BF) at two hours post-thawing using three sperm kinetic parameters which are strongly correlated with fertility in this species. Fourteen stags were clearly identified as GF, whereas nineteen were BF. No significant difference in sperm head size between the two groups was found. On the contrary, the GF exhibited a lower principal piece volume than the BF (6.13 µm3 vs 6.61 µm3, respectively, p = 0.006). The volume of the flagellum structures showed a strong negative relationship with post-thawing sperm velocity. For instance, the volume of the sperm principal piece was negatively correlated with sperm velocity at two hours post-thawing (r = -0.60; p<0.001). Our results clearly show that a higher volume of the sperm principal piece results in poor freezability, and highlights the key role of flagellum size in sperm cryopreservation success.

• MeSH
• analýza hlavních komponent MeSH
• bičík spermie fyziologie   MeSH
• epididymis cytologie   MeSH
• hlavička spermie fyziologie   MeSH
• kinetika MeSH
• kryoprezervace metody   MeSH
• mikroskopie fázově kontrastní MeSH
• motilita spermií fyziologie   MeSH
• organely fyziologie   MeSH
• shluková analýza MeSH
• spermie klasifikace   fyziologie   MeSH
• uchování spermatu metody   MeSH
• vysoká zvěř MeSH
• zmrazování * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Motility analysis of spermatozoa relies on the investigation of either head trajectories or flagellum characteristics. Those two sets of parameters are far from being independent, the flagellum playing the role of motor, whereas the head plays a passive role of cargo. Therefore, quantitative descriptions of head trajectories represent a simplification of the complex pattern of whole sperm cell motion, resulting from the waves developed by the flagellum. The flagellum itself responds to a large variety of signals that precisely control its axoneme to allow activation, acceleration, slowing down or reorientation of the whole spermatozoon. Thus, it is obvious that analysis of flagellum characteristics provides information on the original source of movement and orientation of the sperm cell and presents additional parameters that enrich the panoply of quantitative descriptors of sperm motility. In this review, we briefly describe the methodologies used to obtain good-quality images of fish spermatozoa (head and especially flagellum) while they move fast and the methods developed for their analysis. The paper also aims to establish a link between classical analyses by computer-aided sperm analysis (CASA) and the descriptors generated by fish sperm flagellum analysis, and emphasises the information to be gained regarding motility performance from flagellum motion data.

• MeSH
• analýza spermatu MeSH
• axonema fyziologie   MeSH
• bičík spermie fyziologie   MeSH
• motilita spermií fyziologie   MeSH
• počítačové zpracování obrazu MeSH
• ryby fyziologie   MeSH
• software MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

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.

• MeSH
• flagella metabolismus   MeSH
• kineziny metabolismus   MeSH
• Leishmania mexicana patogenita   fyziologie   MeSH
• leishmanióza metabolismus   MeSH
• morfogeneze MeSH
• myši MeSH
• proliferace buněk MeSH
• protozoální proteiny metabolismus   MeSH
• Psychodidae MeSH
• virulence fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kineziny MeSH
• protozoální proteiny MeSH

Leishmania kinetoplastid 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 for Leishmania pathogenicity. The Leishmania FP 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.

• Klíčová slova
• Leishmania, flagellar pocket, morphogenesis, pathogenicity,
• MeSH
• buněčná membrána metabolismus   MeSH
• cilie genetika   fyziologie   ultrastruktura   MeSH
• delece genu MeSH
• endocytóza MeSH
• flagella genetika   fyziologie   ultrastruktura   MeSH
• interakce hostitele a parazita MeSH
• Leishmania genetika   patogenita   fyziologie   ultrastruktura   MeSH
• mezibuněčné spoje MeSH
• myši MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• Psychodidae parazitologie   MeSH
• virulence genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny MeSH

Azospirillum brasilense has the ability of swimming and swarming motility owing to the work of a constitutive polar flagellum and inducible lateral flagella, respectively. The interplay between these flagellar systems is poorly understood. One of the key elements of the flagellar export apparatus is the protein FlhB. Two predicted flhB genes are present in the genome of A. brasilense Sp245 (accession nos. HE577327-HE577333). Experimental evidence obtained here indicates that the chromosomal coding sequence (CDS) AZOBR_150177 (flhB1) of Sp245 is essential for the production of both types of flagella. In an flhB1:: Omegon-Km mutant, Sp245.1063, defects in polar and lateral flagellar assembly and motility were complemented by expressing the wild-type flhB1 gene from plasmid pRK415. It was found that Sp245.1063 lost the capacity for slight but statistically significant decrease in mean cell length in response to transfer from solid to liquid media, and vice versa; in the complemented mutant, this capacity was restored. It was also shown that after the acquisition of the pRK415-harbored downstream CDS AZOBR_150176, cells of Sp245 and Sp245.1063 ceased to elongate on solid media. These initial data suggest that the AZOBR_150176-encoded putative multisensory hybrid sensor histidine kinase-response regulator, in concert with FlhB1, plays a role in morphological response of azospirilla to changes in the hardness of a milieu.

• Klíčová slova
• Azospirillum brasilense, Mixed flagellation, Motility,
• MeSH
• Azospirillum brasilense genetika   metabolismus   MeSH
• bakteriální chromozomy genetika   metabolismus   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• flagella chemie   genetika   metabolismus   MeSH
• plazmidy genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH