Membrane Water PermeabilityPlasma membranes provide an effective barrier to the extracellular environment. Water was long believed to move through lipid bilayers by simple diffusion; however, membranes from different tissues vary in their permeability to water. The variability is particularly evident in mammalian kidney where proximal tubules and descending thin limbs of Henle's loop have constitutively high water permeability and are responsible for reabsorption of more than 150 liters per day in adult humans. In contrast, ascending thin limbs have very low water permeability. Renal distal tubules empty into collecting ducts where stimulation with vasopressin causes an increase in water permeability (see Ref. 1 for review). These observations led to the suggestion that specialized water transport molecules must exist in membranes with intrinsically high water permeability. Nevertheless, despite extensive studies, the molecular identity of water transport proteins remained elusive until recently.The well defined features of membrane water permeability permitted serendipitous identification of the first known water channel. While purifying the 32-kDa subunit of the red cell Rh blood group antigen, a new 28-kDa polypeptide was discovered (2). Detailed biochemical studies of this newly identified tetrameric membrane protein were made easy by its low solubility in N-lauroylsarcosine, which permitted simple purification (3). The abundance of the protein in rat renal proximal tubules and descending thin limbs (2) sparked the idea that the 28-kDa polypeptide may be the long sought water channel, and its unique N-terminal amino acid sequence permitted cloning from an erythroid cDNA library (4). Functional Analyses of AQP1The Xenopus oocyte expression system has been extremely useful for study of water transport. Oocytes injected with cRNA for the 28-kDa polypeptide exhibit remarkably high osmotic water permeability (P f ϳ200 ϫ 10 Ϫ4 cm s Ϫ1) and rapidly explode in hypotonic buffer (Fig. 1), whereas control oocytes exhibit minimal permeability (5). First referred to as "CHIP28," this protein is now designated aquaporin-1 (AQP1) 1 by the Human Genome Nomenclature Committee (see http://www.gene.ucl.ac.uk/nomenclature). Despite its large water permeability, AQP1 failed to confer a measurable increase in membrane currents (5). Consistent with these results, highly purified AQP1 protein reconstituted into proteoliposomes exhibits high unit water permeability (P f ϳ3 ϫ 10 9 water molecules subunit Ϫ1 s Ϫ1 ), whereas permeation by other small solutes or even protons was undetectable (6). The prevailing view is that AQP1 is a constitutively active, water-selective pore that permits osmotically driven movement of water. A report that forskolin induces a cation current in AQP1-expressing oocytes (7) was not reproduced by multiple other scientific groups. Permeation by CO 2 has recently been proposed, because rates of pH change are about 40% higher in oocytes expressing AQP1 (8). Permeation by O 2 , nitric oxide, and other gases is...

Trypanosoma brucei is a protozoan parasite that is used as a model organism to study such biological phenomena as gene expression, protein trafficking, and cytoskeletal biogenesis. In T. brucei, endocytosis and exocytosis occur exclusively through a sequestered organelle called the flagellar pocket (FP), an invagination of the pellicular membrane. The pocket is the sole site for specific receptors thus maintaining them inaccessible to components of the innate immune system of the mammalian host. The FP is also responsible for the sorting of protective parasite glycoproteins targeted to, or recycling from, the pellicular membrane, and for the removal of host antibodies from the cell surface. Here, we describe the first characterisation of a flagellar pocket cytoskeletal protein, BILBO1. BILBO1 functions to form a cytoskeleton framework upon which the FP is made and which is also required and essential for FP biogenesis and cell survival. Remarkably, RNA interference (RNAi)-mediated ablation of BILBO1 in insect procyclic-form parasites prevents FP biogenesis and induces vesicle accumulation, Golgi swelling, the aberrant repositioning of the new flagellum, and cell death. Cultured bloodstream-form parasites are also nonviable when subjected to BILBO1 RNAi. These results provide the first molecular evidence for cytoskeletally mediated FP biogenesis.

Spermatogenesis defects concern millions of men worldwide, yet the vast majority remains undiagnosed. Here we report men with primary infertility due to multiple morphological abnormalities of the sperm flagella with severe disorganization of the sperm axoneme, a microtubule-based structure highly conserved throughout evolution. Whole-exome sequencing was performed on 78 patients allowing the identification of 22 men with bi-allelic mutations in DNAH1 (n = 6), CFAP43 (n = 10), and CFAP44 (n = 6). CRISPR/Cas9 created homozygous CFAP43/44 male mice that were infertile and presented severe flagellar defects confirming the human genetic results. Immunoelectron and stimulated-emission-depletion microscopy performed on CFAP43 and CFAP44 orthologs in Trypanosoma brucei evidenced that both proteins are located between the doublet microtubules 5 and 6 and the paraflagellar rod. Overall, we demonstrate that CFAP43 and CFAP44 have a similar structure with a unique axonemal localization and are necessary to produce functional flagella in species ranging from Trypanosoma to human.

The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the annulus/horseshoe shaped flagellar pocket collar (FPC). To date the only known component of the FPC is the protein BILBO1, a cytoskeleton protein that has a N-terminus that contains an ubiquitin-like fold, two EF-hand domains, plus a large C-terminal coiled-coil domain. BILBO1 has been shown to bind calcium, but in this work we demonstrate that mutating either or both calcium-binding domains prevents calcium binding. The expression of deletion or mutated forms of BILBO1 in trypanosomes and mammalian cells demonstrate that the coiled-coil domain is necessary and sufficient for the formation of BILBO1 polymers. This is supported by Yeast two-hybrid analysis. Expression of full-length BILBO1 in mammalian cells induces the formation of linear polymers with comma and globular shaped termini, whereas mutation of the canonical calcium-binding domain resulted in the formation of helical polymers and mutation in both EF-hand domains prevented the formation of linear polymers. We also demonstrate that in T. brucei the coiled-coil domain is able to target BILBO1 to the FPC and to form polymers whilst the EF-hand domains influence polymers shape. This data indicates that BILBO1 has intrinsic polymer forming properties and that binding calcium can modulate the form of these polymers. We discuss whether these properties can influence the formation of the FPC.

Male infertility is a major health concern. Among its different causes, multiple morphological abnormalities of the flagella (MMAF) induces asthenozoospermia and is one of the most severe forms of qualitative sperm defects. Sperm of affected men display short, coiled, absent, and/or irregular flagella. To date, six genes (DNAH1, CFAP43, CFAP44, CFAP69, FSIP2, and WDR66) have been found to be recurrently associated with MMAF, but more than half of the cases analyzed remain unresolved, suggesting that many yet-uncharacterized gene defects account for this phenotype. Here, whole-exome sequencing (WES) was performed on 168 infertile men who had a typical MMAF phenotype. Five unrelated affected individuals carried a homozygous deleterious mutation in ARMC2, a gene not previously linked to the MMAF phenotype. Using the CRISPR-Cas9 technique, we generated homozygous Armc2 mutant mice, which also presented an MMAF phenotype, thus confirming the involvement of ARMC2 in human MMAF. Immunostaining experiments in AMRC2-mutated individuals and mutant mice evidenced the absence of the axonemal central pair complex (CPC) proteins SPAG6 and SPEF2, whereas the other tested axonemal and peri-axonemal components were present, suggesting that ARMC2 is involved in CPC assembly and/or stability. Overall, we showed that bi-allelic mutations in ARMC2 cause male infertility in humans and mice by inducing a typical MMAF phenotype, indicating that this gene is necessary for sperm flagellum structure and assembly.

Trypanosoma brucei belongs to a group of unicellular, flagellated parasites that are responsible for human African trypanosomiasis. An essential aspect of parasite pathogenicity is cytoskeleton remodelling, which occurs during the life cycle of the parasite and is accompanied by major changes in morphology and organelle positioning. The flagellum originates from the basal bodies and exits the cell body through the flagellar pocket (FP) but remains attached to the cell body via the flagellum attachment zone (FAZ). The FP is an invagination of the pellicular membrane and is the sole site for endo- and exocytosis. The FAZ is a large complex of cytoskeletal proteins, plus an intracellular set of four specialised microtubules (MtQ) that elongate from the basal bodies to the anterior end of the cell. At the distal end of the FP, an essential, intracellular, cytoskeletal structure called the flagellar pocket collar (FPC) circumvents the flagellum. Overlapping the FPC is the hook complex (HC) (a sub-structure of the previously named bilobe) that is also essential and is thought to be involved in protein FP entry. BILBO1 is the only functionally characterised FPC protein and is necessary for FPC and FP biogenesis. Here, we used a combination of in vitro and in vivo approaches to identify and characterize a new BILBO1 partner protein—FPC4. We demonstrate that FPC4 localises to the FPC, the HC, and possibly to a proximal portion of the MtQ. We found that the C-terminal domain of FPC4 interacts with the BILBO1 N-terminal domain, and we identified the key amino acids required for this interaction. Interestingly, the FPC4 N-terminal domain was found to bind microtubules. Over-expression studies highlight the role of FPC4 in its association with the FPC, HC and FPC segregation. Our data suggest a tripartite association between the FPC, the HC and the MtQ.

The multiple morphological abnormalities of the flagella (MMAF) phenotype is among the most severe forms of sperm defects responsible for male infertility. The phenotype is characterized by the presence in the ejaculate of immotile spermatozoa with severe flagellar abnormalities including flagella being short, coiled, absent, and of irregular caliber. Recent studies have demonstrated that MMAF is genetically heterogeneous, and genes thus far associated with MMAF account for only one-third of cases. Here we report the identification of homozygous truncating mutations (one stop-gain and one splicing variant) in CFAP69 of two unrelated individuals by whole-exome sequencing of a cohort of 78 infertile men with MMAF. CFAP69 encodes an evolutionarily conserved protein found at high levels in the testis. Immunostaining experiments in sperm from fertile control individuals showed that CFAP69 localized to the midpiece of the flagellum, and the absence of CFAP69 was confirmed in both individuals carrying CFPA69 mutations. Additionally, we found that sperm from a Cfap69 knockout mouse model recapitulated the MMAF phenotype. Ultrastructural analysis of testicular sperm from the knockout mice showed severe disruption of flagellum structure, but histological analysis of testes from these mice revealed the presence of all stages of the seminiferous epithelium, indicating that the overall progression of spermatogenesis is preserved and that the sperm defects likely arise during spermiogenesis. Together, our data indicate that CFAP69 is necessary for flagellum assembly/stability and that in both humans and mice, biallelic truncating mutations in CFAP69 cause autosomal-recessive MMAF and primary male infertility.

To perform their multiple functions, cilia and flagella are precisely positioned at the cell surface by mechanisms that remain poorly understood. The protist Trypanosoma brucei possesses a single flagellum that adheres to the cell body where a specific cytoskeletal structure is localised, the flagellum attachment zone (FAZ). Trypanosomes build a new flagellum whose distal tip is connected to the side of the old flagellum by a discrete structure, the flagella connector. During this process, the basal body of the new flagellum migrates towards the posterior end of the cell. We show that separate inhibition of flagellum assembly, base-to-tip motility or flagella connection leads to reduced basal body migration, demonstrating that the flagellum contributes to its own positioning. We propose a model where pressure applied by movements of the growing new flagellum on the flagella connector leads to a reacting force that in turn contributes to migration of the basal body at the proximal end of the flagellum.