Sperm morphology, swimming velocity, and longevity in the house sparrow Passer domesticus

Sperm competition exerts strong selection on males to produce spermatozoa with an optimal morphology that maximizes their fertilization success. Long sperm were first suggested to be favored because they should swim faster. However, studies that investigated the relationship between sperm length and sperm competitive ability or sperm swimming velocity yielded contradictory results. More recently, ratios of the different sections of a spermatozoon (the head, midpiece, and flagellum) were suggested to be more crucial in determining swimming velocity. Additionally, sperm ability to remain and survive in the female storage organs may also influence fertilization success, so that optimal sperm morphology may rather maximize sperm longevity than velocity. In this study, we investigated how sperm morphology is related to sperm velocity and sperm longevity in the house sparrow Passer domesticus. Sperm velocity was found to be correlated with head/flagellum ratio. Sperm with small heads relative to their flagellum showed higher swimming velocity. Additionally, shorter sperm were found to live longer. Finally, we found sperm morphological traits to vary substantially within males and the head/flagellum ratio to be unrelated to total sperm length. We discuss the hypothesis that the substantial within-male variation in sperm morphology reflects a male strategy to produce a diversity of sperm from long, fast-swimming to short, long-living sperm to maximize their fertilization success in a context of sperm competition.

The Trypanosoma brucei cAMP phosphodiesterases TbrPDEB1 and TbrPDEB2: flagellar enzymes that are essential for parasite virulence

Cyclic nucleotide specific phosphodiesterases (PDEs) are pivotal regulators of cellular signaling. They are also important drug targets. Besides catalytic activity and substrate specificity, their subcellular localization and interaction with other cell components are also functionally important. In contrast to the mammalian PDEs, the significance of PDEs in protozoal pathogens remains mostly unknown. The genome of Trypanosoma brucei, the causative agent of human sleeping sickness, codes for five different PDEs. Two of these, TbrPDEB1 and TbrPDEB2, are closely similar, cAMP-specific PDEs containing two GAF-domains in their N-terminal regions. Despite their similarity, these two PDEs exhibit different subcellular localizations. TbrPDEB1 is located in the flagellum, whereas TbrPDEB2 is distributed between flagellum and cytoplasm. RNAi against the two mRNAs revealed that the two enzymes can complement each other but that a simultaneous ablation of both leads to cell death in bloodstream form trypanosomes. RNAi against TbrPDEB1 and TbrPDEB2 also functions in vivo where it completely prevents infection and eliminates ongoing infections. Our data demonstrate that TbrPDEB1 and TbrPDEB2 are essential for virulence, making them valuable potential targets for new PDE-inhibitor based trypanocidal drugs. Furthermore, they are compatible with the notion that the flagellum of T. brucei is an important site of cAMP signaling.--Oberholzer, M., Marti, G., Baresic, M., Kunz, S., Hemphill, A., Seebeck, T. The Trypanosoma brucei cAMP phosphodiesterases TbrPDEB1 and TbrPDEB2: flagellar enzymes that are essential for parasite virulence.

Trypanosomes and mammalian sperm: one of a kind?

Flagellar-mediated motility is an indispensable function for cell types as evolutionarily distant as mammalian sperm and kinetoplastid parasites, a large group of flagellated protozoa that includes several important human pathogens. Despite the obvious importance of flagellar motility, little is known about the signalling processes that direct the frequency and wave shape of the flagellar beat, or those that provide the motile cell with the necessary environmental cues that enable it to aim its movement. Similarly, the energetics of the flagellar beat and the problem of a sufficient ATP supply along the entire length of the beating flagellum remain to be explored. Recent proteome projects studying the flagella of mammalian sperm and kinetoplastid parasites have provided important information and have indicated a surprising degree of similarities between the flagella of these two cell types.

Trypanosomal TAC40 constitutes a novel subclass of mitochondrial -barrel proteins specialized in mitochondrial genome inheritance

Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a β-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum–mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a β-barrel protein of the mitochondrial porin family that mediates a DNA–cytoskeleton linkage that is essential for mitochondrial DNA inheritance.

Morphological evaluation of spermatogenesis in Lake Magadi tilapia (Alcolapia grahami): a fish living on the edge

Spermatogenesis in Lake Magadi tilapia (Alcolapia grahami), a cichlid fish endemic to the highly alkaline and saline Lake Magadi in Kenya, was evaluated using light and transmission electron microscopy. Spermatogenesis, typified by its three major phases (spermatocytogenesis, meiosis and spermiogenesis), was demonstrated by the presence of maturational spermatogenic cells namely spermatogonia, spermatocytes, spermatids and spermatozoa. Primary spermatogonia, the largest of all the germ cells, underwent a series of mitotic divisions producing primary spermatocytes, which then entered two consecutive meiotic divisions to produce secondary spermatocytes and spermatids. Spermatids, in turn, passed through three structurally distinct developmental stages typical of type-I spermiogenesis to yield typical primitive anacrosomal spermatozoa of the externally fertilizing type (aquasperm). The spermatozoon of this fish exhibited a spheroidal head with the nucleus containing highly electron-dense chromatin globules, a midpiece containing ten ovoid mitochondria arranged in two rows and a flagellum formed by the typical 9 + 2 microtubule axoneme. In addition, the midpiece, with no cytoplasmic sheath, appeared to end blindly distally in a lobe-like pattern around the flagellum; a feature that was unique and considered adaptive for the spermatozoon of this species to the harsh external environment. These observations show that the testis of A. grahami often undergoes active spermatogenesis despite the harsh environmental conditions to which it is exposed on a daily basis within the lake. Further, the spermiogenic features and spermatozoal ultrastructure appear to be characteristic of Cichlidae and, therefore, may be of phylogenetic significance.

A conserved mitochondrial outer membrane protein mediates kDNA maintenace in Trypanosoma brucei

Kinetoplastids are defined by the unique organization of their mitochondrial DNA (kDNA). It forms a highly concatenated DNA network that is linked to the basal body of the flagellum by the tripartite attachment complex (TAC). The TAC encompasses intra and extramitochondrial filaments and a highly differentiated region of the two mitochondrial membranes. Here we identify and characterize a mitochondrial outer membrane protein of Trypanosoma brucei that is predominantly localized in the TAC. The protein is essential for growth in both life cycle stages. Immunofluorescence shows that ablation of the protein does not affect kDNA replication but abolishes the segregation of the replicated kDNA network causing rapid loss of kDNA. Besides its role in kDNA maintenance in vivo and in vitro experiments show that the protein is involved in mitochondrial protein import and that it interacts with a recently discovered protein import factor. RNAi experiments in a T. brucei cell line in which the kDNA is dispensable suggest that the essential function is linked to kDNA maintenance. Bioinformatic analysis shows that the studied outer membrane protein has beta-barrel topology and that it belongs to the mitochondrial porin family comprising VDAC, Tom40 and Mdm10. Interestingly, Mdm10 has sofar only been found in yeast. Ist function in protein import and mitochondrial DNA maintenance suggests that the protein in our study is the functional homologue of Mdm10. Thus, the TAC – a defining structure of Kinetoplastids – contains a conserved protein which in yeast and trypanosomes performs the same function. Our study therefore provides an example that trypanosomal biology, rather than being unique, often simply represents a more extreme manifestation of a conserved biological concept.

What a unicellular parasite can tell us about mitochondrial biogenesis?

Most of what we know about mitochondrial biogenesis stems from work in yeast and mammals, which are quite closely related. To understand the conserved features of mitochondria and the evolutionary forces that shaped it, it is important to study a more diverse group of eukaryotes. The parasitic protozoan Trypanosoma brucei and its relatives are excellent systems to do so, since they appear to have diverged from other eukaryotes very early in evolution. This is reflected in a number of unique and extreme features in their mitochondrial biology, including a single continuous mitochondrion that contains a one unit mitochondrial genome that is physically connected across the two membranes with the basal body of the flagellum. Moreover, many mitochondrial transcripts have to be extensively edited in order to become functional mRNAs and organellar translation requires extensive import of cytosolic tRNAs. In my talk I will focus on the discovery and characterization of the elusive mitochondrial protein import system of the mitochondrial outer membrane of trypanosomes. In addition I will present data on a central outer membrane component of the mitochondrial genome inheritance system of T. brucei and compare it to the better characterized system of yeast. - I hope that I can convince you in my talk, that a better understanding of the mitochondrial biology in T. brucei will provide insights into both fundamentally conserved and fundamentally diverged aspects of mitochondrial biogenesis and thus of the evolutionary hstory of mitochondria in general.

A conserved mitochondrial outer membrane protein mediates kDNA maintenace in Trypanosoma brucei

Kinetoplastids are defined by the unique organization of their mitochondrial DNA (kDNA). It forms a highly concatenated DNA network that is linked to the basal body of the flagellum by the tripartite attachment complex (TAC). The TAC encompasses intra and extramitochondrial filaments and a highly differentiated region of the two mitochondrial membranes. Here we identify and characterize a mitochondrial outer membrane protein of Trypanosoma brucei that is predominantly localized in the TAC. The protein is essential for growth in both life cycle stages. Immunofluorescence shows that ablation of the protein does not affect kDNA replication but abolishes the segregation of the replicated kDNA network causing rapid loss of kDNA. Besides its role in kDNA maintenance in vivo and in vitro experiments show that the protein is involved in mitochondrial protein import and that it interacts with a recently discovered protein import factor. RNAi experiments in a T. brucei cell line in which the kDNA is dispensable suggest that the essential function is linked to kDNA maintenance. Bioinformatic analysis shows that the studied outer membrane protein has beta-barrel topology and that it belongs to the mitochondrial porin family comprising VDAC, Tom40 and Mdm10. Interestingly, Mdm10 has so far only been found in yeast. Its function in protein import and mitochondrial DNA maintenance suggests that the protein in our study is the functional homologue of Mdm10. Thus, the TAC – a defining structure of Kinetoplastids – contains a conserved protein which in yeast and trypanosomes performs the same function. Our study therefore provides an example that trypanosomal biology, rather than being unique, often simply represents a more extreme manifestation of a conserved biological concept.

What can a unicellular parasite tell us about mitochondrial biogenesis?

Most of what we know about mitochondrial biogenesis stems from work in yeast and mammals, which are quite closely related. To understand the conserved features of mitochondria and the evolutionary forces that shaped it, it is important to study a more diverse group of eukaryotes. The parasitic protozoan Trypanosoma brucei and its relatives are excellent systems to do so, since they appear to have diverged from other eukaryotes very early in evolution. This is reflected in a number of unique and extreme features in their mitochondrial biology, including a single continuous mitochondrion that contains a one unit mitochondrial genome that is physically connected across the two membranes with the basal body of the flagellum. Moreover, many mitochondrial transcripts have to be extensively edited in order to become functional mRNAs and organellar translation requires extensive import of cytosolic tRNAs. In my talk I will focus on the discovery and characterization of the elusive mitochondrial protein import system of the mitochondrial outer membrane of trypanosomes. In addition I will present data on a central outer membrane component of the mitochondrial genome inheritance system of T. brucei and compare it to the better characterized system of yeast. - I hope that I can convince you in my talk, that a better understanding of the mitochondrial biology in T. brucei will provide insights into both fundamentally conserved and fundamentally diverged aspects of mitochondrial biogenesis and thus of the evolutionary history of mitochondria in general.

A component of the mitochondrial outer membrane proteome of T. brucei probably contains covalent bound fatty acids

A subclass of eukaryotic proteins is subject to modification with fatty acids, the most common of which are palmitic and myristic acid. Protein acylation allows association with cellular membranes in the absence of transmembrane domains. Here we examine POMP39, a protein previously described to be present in the outer mitochondrial membrane proteome (POMP) of the protozoan parasite Trypanosoma brucei. POMP39 lacks canonical transmembrane domains, but is likely both myristoylated and palmitoylated on its N-terminus. Interestingly, the protein is also dually localized on the surface of the mitochondrion as well as in the flagellum of both insect-stage and the bloodstream form of the parasites. Upon abolishing of global protein acylation or mutation of the myristoylation site, POMP39 relocates to the cytosol. RNAi-mediated ablation of the protein neither causes a growth phenotype in insect-stage nor bloodstream form trypanosomes.

What can a unicellular parasite tell us about mitochondrial biogenesis?

In the unicellular parasite Trypanosoma brucei, as in other eukaryotes, more than 95% of all mitochondrial proteins are imported from the cytosol. The recently characterized multisubunit ATOM complex, the functional analogue of the TOM complex of yeast, mediates import of essentially all proteins across the outer mitochondrial membrane in T. brucei. Moreover, an additional protein termed pATOM36, which is loosely associated with the ATOM complex, has been implicated in the import of only a subset of mitochondrial proteins. Here we have investigated more precisely which role pATOM36 plays in mitochondrial protein import. RNAi mediated ablation of pATOM36 specifically depletes a subset of outer mitochondrial membrane proteins including ATOM complex subunits and as a consequence results in the collapse of the ATOM complex as shown by Blue native PAGE. In addition, a SILAC-based global proteomic analysis of uninduced and induced pATOM36 RNAi cells together with in vitro import experiments suggest that pATOM36 might be a novel protein import factor acting on a subset of alpha-helically anchored mitochondrial outer membrane proteins. Identification of pATOM36 interaction partners by co-immunoprecipitation together with immunofluorescence analysis shows that unexpectedly a fraction of the protein is associated with the tripartite attachment complex (TAC). This complex is essential for proper inheritance of the mitochondrial DNA in T. brucei. It forms a physical connection between the single unit mitochondrial DNA and the basal body of the flagellum that is stable throughout the cell cycle. Thus, pATOM36 simultaneously mediates ATOM assembly, and thus protein import, as well as mitochondrial DNA inheritance since it is an essential component of the TAC.

Kinetoplastid-specific pATOM36 mediates membrane insertion of a subset of mitochondrial outer membrane proteins

In trypanosomes, as in other eukaryotes, more than 95% of all mitochondrial proteins are imported into the mitochondrion. The recently characterized multisubunit ATOM complex mediates import of essentially all proteins across the outer mitochondrial membrane in T. brucei. Moreover, an additional protein termed pATOM36, which is loosely associated with the ATOM complex, has been implicated in the import of only a subset of mitochondrial matrix proteins. Here we have investigated more precisely which role pATOM36 plays in mitochondrial protein import. RNAi mediated ablation of pATOM36 specifically depletes a subset of ATOM complex subunits and as a consequence results in the collapse of the ATOM complex as shown by Blue native PAGE. In addition, a SILAC-based global proteomic analysis of uninduced and induced pATOM36 RNAi cells together with in vitro import experiments suggest that pATOM36 might be a novel protein insertase acting on a subset of alpha-helically anchored mitochondrial outer membrane proteins. Identification of pATOM36 interaction partners by co-immunoprecipitation together with immunofluorescence analysis furthermore shows that unexpectedly a fraction of the protein is associated with the tripartite attachment complex (TAC). This complex is essential for proper inheritance of the kDNA as it forms a physical connection between the kDNA and the basal body of the flagellum throughout the cell cycle. Thus, the presence of pATOM36 in the TAC provides an exciting link between mitochondrial protein import and kDNA inheritance.

pATOM36 mediates mitochondrial protein import and mitochondrial DNA inheritance

The multisubunit ATOM complex mediates import of essentially all proteins across the outer mitochondrial membrane in T. brucei. Moreover, an additional protein termed pATOM36, which is loosely associated with the ATOM complex, has been implicated in the import of only a subset of mitochondrial matrix proteins. Here we have investigated more precisely which role pATOM36 plays in mitochondrial protein import. RNAi mediated ablation of pATOM36 specifically depletes a subset of ATOM complex subunits and as a consequence results in the collapse of the ATOM complex as shown by Blue native PAGE. In addition, a SILAC-based global proteomic analysis of uninduced and induced pATOM36 RNAi cells together with in vitro import experiments suggest that pATOM36 might be a novel protein insertase acting on a subset of alpha-helically anchored mitochondrial outer membrane proteins. Identification of pATOM36 interaction partners by co-immunoprecipitation together with immunofluorescence analysis furthermore shows that unexpectedly a fraction of the protein is associated with the tripartite attachment complex (TAC). This complex is essential for proper inheritance of the mtDNA; also called kinetoplast or kDNA; as it forms a physical connection between the kDNA and the basal body of the single flagellum throughout the cell cycle. Thus, the presence of pATOM36 in the TAC provides an exciting link between mitochondrial protein import and kDNA inheritance.

TAC102 Is a Novel Component of the Mitochondrial Genome Segregation Machinery in Trypanosomes

Trypanosomes show an intriguing organization of their mitochondrial DNA into a catenated network, the kinetoplast DNA (kDNA). While more than 30 proteins involved in kDNA replication have been described, only few components of kDNA segregation machinery are currently known. Electron microscopy studies identified a high-order structure, the tripartite attachment complex (TAC), linking the basal body of the flagellum via the mitochondrial membranes to the kDNA. Here we describe TAC102, a novel core component of the TAC, which is essential for proper kDNA segregation during cell division. Loss of TAC102 leads to mitochondrial genome missegregation but has no impact on proper organelle biogenesis and segregation. The protein is present throughout the cell cycle and is assembled into the newly developing TAC only after the pro-basal body has matured indicating a hierarchy in the assembly process. Furthermore, we provide evidence that the TAC is replicated de novo rather than using a semi-conservative mechanism. Lastly, we demonstrate that TAC102 lacks an N-terminal mitochondrial targeting sequence and requires sequences in the C-terminal part of the protein for its proper localization.