C2cd6-encoded CatSperτ Targets Sperm Calcium Channel to Ca2+ Signaling Domains in the Flagellar Membrane

In mammalian sperm cells, regulation of spatiotemporal Ca2+ signaling relies on the quadrilinear Ca2+ signaling nanodomains in the flagellar membrane. The sperm-specific, multi-subunit CatSper Ca2+ channel, which is crucial for sperm hyperactivated motility and male fertility, organizes the nanodomains. Here, we report CatSperτ, the C2cd6-encoded membrane-associating C2 domain protein, can independently migrate to the flagella and serve as a major targeting component of the CatSper channel complex. CatSperτ loss-of-function in mice demonstrates that it is essential for sperm hyperactivated motility and male fertility. CatSperτ targets the CatSper channel into the quadrilinear nanodomains in the flagella of developing spermatids, whereas it is dispensable for functional channel assembly. CatSperτ interacts with ciliary trafficking machinery in a C2-dependent manner. These findings provide insights into the CatSper channel trafficking to the Ca2+ signaling nanodomains and the shared molecular mechanisms of ciliary and flagellar membrane targeting.

The Trypanosoma brucei Cytoskeletal Protein KHARON Associates with Partner Proteins to Mediate Both Cytokinesis and Trafficking of Flagellar Membrane Proteins

ABSTRACTIn the African trypanosome Trypanosoma brucei, the cytoskeletal protein TbKHARON is required for trafficking of a putative Ca2+ channel to the flagellar membrane, and it is essential for parasite viability in both the mammalian stage bloodstream forms and the tsetse fly procyclic forms. This protein is located at the base of the flagellum, in the pellicular cytoskeleton, and in the mitotic spindle in both life cycle forms, and it likely serves multiple functions for these parasites. To begin to deconvolve the functions of KHARON, we have investigated partners associated with this protein and their roles in parasite biology. One KHARON associated protein, TbKHAP1, is a close interaction partner that can be crosslinked to KHARON by formaldehyde and pulled down in a molecular complex, and it colocalizes with TbKHARON in the basal body at the base of the flagellum. Knockdown of TbKHAP1 mRNA has similar phenotypes to knockdown of its partner TbKHARON, impairing trafficking of the Ca2+ channel to the flagellar membrane and blocking cytokinesis, implying that the TbKHARON/TbKHAP1 complex mediates trafficking of flagellar membrane proteins. Two other KHAPs, TbKHAP2 and TbKHAP3, are in close proximity to TbKHARON, but knockdown of their mRNAs does not affect trafficking of the Ca2+ channel. Two different flagellar membrane proteins, which are extruded from the flagellar membrane into extracellular vesicles, are also dependent upon TbKHARON for flagellar trafficking. These studies confirm that TbKHARON acts in complexes with other proteins to carry out various biological functions, and that some partners are involved in the core activity of targeting membrane proteins to the flagellum.

A cytoskeletal protein complex is essential for division of intracellular amastigotes of Leishmania mexicana

Previous studies in Leishmania mexicana have identified the cytoskeletal protein KHARON as being important for both flagellar trafficking of the glucose transporter GT1 and for successful cytokinesis and survival of infectious amastigote forms inside mammalian macrophages. KHARON is located in three distinct regions of the cytoskeleton: the base of the flagellum, the subpellicular microtubules, and the mitotic spindle. To deconvolve the different functions for KHARON, we have identified two partner proteins, KHAP1 and KHAP2, which associate with KHARON. KHAP1 is located only in the subpellicular microtubules, whereas KHAP2 is located at the subpellicular microtubules and the base of the flagellum. Both KHAP1 and KHAP2 null mutants are unable to execute cytokinesis but are able to traffic GT1 to the flagellum. These results confirm that KHARON assembles into distinct functional complexes and that the subpellicular complex is essential for cytokinesis and viability of disease-causing amastigotes but not for flagellar membrane trafficking.

A Cytoskeletal Protein Complex is Essential for Division of Intracellular Amastigotes of Leishmania mexicana

Previous studies in Leishmania mexicana have identified the cytoskeletal protein KHARON as being important for both flagellar trafficking of the glucose transporter GT1 and for successful cytokinesis and survival of infectious amastigote forms inside mammalian macrophages. KHARON is located in three distinct regions of the cytoskeleton: the base of the flagellum, the subpellicular microtubules, and the mitotic spindle. To deconvolve the different functions for KHARON, we have identified two partner proteins, KHAP1 and KHAP2, that associate with KHARON. KHAP1 is located only in the subpellicular microtubules, while KHAP2 is located at the subpellicular microtubules and the base of the flagellum. Both the KHAP1 and KHAP2 null mutants are unable to execute cytokinesis but are able to traffic GT1 to the flagellum. These results confirm that KHARON assembles into distinct functional complexes and that the subpellicular complex is essential for cytokinesis and viability of disease-causing amastigotes but not for flagellar membrane trafficking.

Touching the Surface: Diverse Roles for the Flagellar Membrane in Kinetoplastid Parasites

SUMMARY While flagella have been studied extensively as motility organelles, with a focus on internal structures such as the axoneme, more recent research has illuminated the roles of the flagellar surface in a variety of biological processes. Parasitic protists of the order Kinetoplastida, which include trypanosomes and Leishmania species, provide a paradigm for probing the role of flagella in host-microbe interactions and illustrate that this interface between the flagellar surface and the host is of paramount importance. An increasing body of knowledge indicates that the flagellar membrane serves a multitude of functions at this interface: attachment of parasites to tissues within insect vectors, close interactions with intracellular organelles of vertebrate cells, transactions between flagella from different parasites, junctions between the flagella and the parasite cell body, emergence of nanotubes and exosomes from the parasite directed to either host or microbial targets, immune evasion, and sensing of the extracellular milieu. Recent whole-organelle or genome-wide studies have begun to identify protein components of the flagellar surface that must mediate these diverse host-parasite interactions. The increasing corpus of knowledge on kinetoplastid flagella will likely prove illuminating for other flagellated or ciliated pathogens as well.

APEX2 proximity proteomics resolves flagellum subdomains and identifies flagellum tip-specific proteins in Trypanosoma brucei

ABSTRACTTrypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. T. brucei has a single flagellum that plays critical roles in parasite biology, transmission and pathogenesis. An emerging concept in flagellum biology is that the organelle is organized into subdomains, each having specialized composition and function. Overall flagellum proteome has been well-studied, but a critical gap in knowledge is the protein composition of individual flagellum subdomains. We have therefore used APEX-based proximity proteomics to examine protein composition of T. brucei flagellum subdomains. To assess effectiveness of APEX-based proximity labeling, we fused APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, an axonemal complex distributed along the flagellum. We found that DRC1-APEX2 directs flagellum-specific biotinylation and purification of biotinylated proteins yields a DRC1 “proximity proteome” showing good overlap with proteomes obtained from purified axonemes. We next employed APEX2 fused to a flagellar membrane protein that is restricted to the flagellum tip, adenylate cyclase 1 (AC1), or a flagellar membrane protein that is excluded from the flagellum tip, FS179. Principal component analysis demonstrated the pools of biotinylated proteins in AC1-APEX2 and FS179-APEX2 samples are distinguished from each other. Comparing proteins in these two pools allowed us to identify an AC1 proximity proteome that is enriched for flagellum tip proteins and includes several proteins involved in signal transduction. Our combined results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can be used to resolve proteome composition of flagellum subdomains that cannot themselves be readily purified.IMPORTANCESleeping sickness is a neglected tropical disease, caused by the protozoan parasite Trypanosoma brucei. The disease disrupts the sleep-wake cycle, leading to coma and death if left untreated. T. brucei motility, transmission, and virulence depend on its flagellum (aka cilium), which consists of several different specialized subdomains. Given the essential and multifunctional role of the T. brucei flagellum, there is need of approaches that enable proteomic analysis of individual subdomains. Our work establishes that APEX2 proximity labeling can, indeed, be implemented in the biochemical environment of T. brucei, and has allowed identification of proximity proteomes for different subdomains. This capacity opens the possibility to study the composition and function of other compartments. We further expect that this approach may be extended to other eukaryotic pathogens, and will enhance the utility of T. brucei as a model organism to study ciliopathies, heritable human diseases in which cilia function is impaired.