Direct interactions of intraflagellar transport complex B proteins IFT88, IFT52, and IFT46.
ABSTRACT: Intraflagellar transport (IFT) particles of Chlamydomonas reinhardtii contain two distinct protein complexes, A and B, composed of at least 6 and 15 protein subunits, respectively. As isolated from C. reinhardtii flagella, IFT complex B can be further reduced to a approximately 500-kDa core that contains IFT88, 2x IFT81, 2x IFT74/72, IFT52, IFT46, IFT27, IFT25, and IFT22. In this study, yeast-based two-hybrid analysis was combined with bacterial coexpression to show that three of the core B subunits, IFT88, IFT52, and IFT46, interact directly with each other and, together, are capable of forming a ternary complex. Chemical cross-linking results support the IFT52-IFT88 interaction and provide additional evidence of an association between IFT27 and IFT81. With previous studies showing that IFT81 and IFT74/72 interact to form a (IFT81)(2)(IFT74/72)(2) heterotetramer and that IFT27 and IFT25 form a heterodimer, the architecture of complex B is revealing itself. Last, electroporation of recombinant IFT46 was used to rescue flagellar assembly of a newly identified ift46 mutant and to monitor in vivo localization and movement of the IFT particles.
Project description:Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27: Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survived to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymides contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the "9+2? axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the conditional Ift25 knockout mice, appeared to be normal in the conditional Ift27 knockout mice. Our findings suggest that like IFT25, IFT27, even though not required for ciliogenesis in somatic cells, is essential for sperm flagella formation, sperm function, and male fertility in mice. IFT25 and IFT27 control sperm formation/function through many common mechanisms, but IFT25 has additional roles beyond IFT27.
Project description:BACKGROUND: Intraflagellar transport (IFT) is the bidirectional movement of IFT particles between the cell body and the distal tip of a flagellum. Organized into complexes A and B, IFT particles are composed of at least 18 proteins. The function of IFT proteins in flagellar assembly has been extensively investigated. However, much less is known about the molecular mechanism of how IFT is regulated. METHODOLOGY/PRINCIPAL FINDINGS: We herein report the identification of a novel IFT particle protein, IFT25, in Chlamydomonas. Dephosphorylation assay revealed that IFT25 is a phosphoprotein. Biochemical analysis of temperature sensitive IFT mutants indicated that IFT25 is an IFT complex B subunit. In vitro binding assay confirmed that IFT25 binds to IFT27, a Rab-like small GTPase component of the IFT complex B. Immunofluorescence staining showed that IFT25 has a punctuate flagellar distribution as expected for an IFT protein, but displays a unique distribution pattern at the flagellar base. IFT25 co-localizes with IFT27 at the distal-most portion of basal bodies, probably the transition zones, and concentrates in the basal body region by partially overlapping with other IFT complex B subunits, such as IFT46. Sucrose density gradient centrifugation analysis demonstrated that, in flagella, the majority of IFT27 and IFT25 including both phosphorylated and non-phosphorylated forms are cosedimented with other complex B subunits in the 16S fractions. In contrast, in cell body, only a fraction of IFT25 and IFT27 is integrated into the preassembled complex B, and IFT25 detected in complex B is preferentially phosphorylated. CONCLUSION/SIGNIFICANCE: IFT25 is a phosphoprotein component of IFT particle complex B. IFT25 directly interacts with IFT27, and these two proteins likely form a subcomplex in vivo. We postulate that the association and disassociation between the subcomplex of IFT25 and IFT27 and complex B might be involved in the regulation of IFT.
Project description:Intraflagellar transport protein 74 (IFT74) is a component of the core intraflagellar transport complex, a bidirectional movement of large particles along the axoneme microtubules for cilia formation. In this study, we investigated its role in sperm flagella formation and discovered that mice deficiency in Ift74 gene in male germ cells were infertile with low sperm count and immotile sperm. The few developed spermatozoa displayed misshaped heads and short tails. Transmission electron microscopy revealed abnormal flagellar axonemes in the seminiferous tubules where sperm are made. Clusters of unassembled microtubules were present in the spermatids. Testicular expression levels of IFT27, IFT57, IFT81, IFT88, and IFT140 proteins were significantly reduced in the conditional Ift74 mutant mice, with the exception of IFT20 and IFT25. The levels of outer dense fiber 2 and sperm-associated antigen 16L proteins were also not changed. However, the processed A-Kinase anchor protein, a major component of the fibrous sheath, a unique structure of sperm tail, was significantly reduced. Our study demonstrates that IFT74 is essential for mouse sperm formation, probably through assembly of the core axoneme and fibrous sheath, and suggests that IFT74 may be a potential genetic factor affecting male reproduction in man.
Project description:Cilia and flagella are complex structures emanating from the surface of most eukaroytic cells and serve important functions including motility, signaling, and sensory reception. A process called intraflagellar transport (IFT) is of central importance to ciliary assembly and maintenance. The IFT complex is required for this transport and consists of two distinct multisubunit subcomplexes, IFT-A and IFT-B. Despite the importance of the IFT complex, little is known about its overall architecture. This paper presents a biochemical dissection of the molecular interactions within the IFT-B core complex. Two stable subcomplexes consisting of IFT88/70/52/46 and IFT81/74/27/25 were recombinantly co-expressed and purified. We identify a novel interaction between IFT70/52 and map the interaction domains between IFT52 and the other subunits within the IFT88/70/52/46 complex. Additionally, we show that IFT52 binds directly to the IFT81/74/27/25 complex, indicating that it could mediate the interaction between the two subcomplexes. Our data lead to an improved architectural map for the IFT-B core complex with new interactions as well as domain resolution mapping for several subunits.
Project description:The assembly and maintenance of most cilia and flagella rely on intraflagellar transport (IFT). Recent in vitro studies have suggested that, together, the calponin-homology domain within the IFT81 N-terminus and the highly basic N-terminus of IFT74 form a module for IFT of tubulin. By using Chlamydomonas mutants for IFT81 and IFT74, we tested this hypothesis in vivo. Modification of the predicted tubulin-binding residues in IFT81 did not significantly affect basic anterograde IFT and length of steady-state flagella but slowed down flagellar regeneration, a phenotype similar to that seen in a strain that lacks the IFT74 N-terminus. In both mutants, the frequency of tubulin transport by IFT was greatly reduced. A double mutant that combined the modifications to IFT81 and IFT74 was able to form only very short flagella. These results indicate that, together, the IFT81 and IFT74 N-termini are crucial for flagellar assembly, and are likely to function as the main module for IFT of tubulin.
Project description:The short-rib polydactyly syndromes (SRPS) encompass a radiographically and genetically heterogeneous group of skeletal ciliopathies that are characterized by a long narrow chest, short extremities, and variable occurrence of polydactyly. Radiographic abnormalities include undermineralization of the calvarium, shortened and bowed appendicular bones, trident shaped acetabula and polydactyly. In a case of SRPS we identified compound heterozygosity for mutations in IFT52, which encodes a component of the anterograde intraflagellar transport complex. The IFT52 mutant cells synthesized a significantly reduced amount of IFT52 protein, leading to reduced synthesis of IFT74, IFT81, IFT88 and ARL13B, other key anterograde complex members. Ciliogenesis was also disrupted in the mutant cells, with a 60% reduction in the presence of cilia on mutant cells and loss of cilia length regulation for the cells with cilia. These data demonstrate that IFT52 is essential for anterograde complex integrity and for the biosynthesis and maintenance of cilia. The data identify a new locus for SRPS and show that IFT52 mutations result in a ciliopathy with primary effects on the skeleton.
Project description:Intraflagellar transport (IFT) is a conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. However, IFT25, a component of the IFT complex, is not required for the formation of cilia in somatic tissues. In mice, the gene is highly expressed in the testis, and its expression is upregulated during the final phase when sperm flagella are formed. To investigate the role of IFT25 in sperm flagella formation, the gene was specifically disrupted in male germ cells. All homozygous knockout mice survived to adulthood and did not show any gross abnormalities. However, all homozygous knockout males were completely infertile. Sperm numbers were reduced and these sperm were completely immotile. Multiple morphological abnormalities were observed in sperm, including round heads, short and bent tails, with some tails showing branched flagella and others with frequent abnormal thicknesses, as well as swollen tips of the tail. Transmission electron microscopy revealed that flagellar accessory structures, including the fibrous sheath and outer dense fibers, were disorganized, and most sperm had also lost the "9+2" microtubule structure. In the testis, IFT25 forms a complex with other IFT proteins. In Ift25 knockout testes, IFT27, an IFT25 binding partner, was missing, and IFT20 and IFT81 levels were also reduced. Our findings suggest that IFT25, although not necessary for the formation of cilia in somatic cells, is indispensable for sperm flagellum formation and male fertility in mice.
Project description:Intraflagellar transport (IFT) particles are composed of polyprotein complexes IFT-A and IFT-B as well as cargo adaptors such as the BBSome. Two IFT-B subunits, IFT25 and IFT27 were found to form a heterodimer, which is essential in exporting the BBSome out of the cilium but not involved in flagellar assembly and cytokinesis in vertebrates. Controversial results were, however, recorded to show that defects in IFT, flagellar assembly and even cytokinesis were caused by IFT27 knockdown in Chlamydomonas reinhardtii Using C. reinhardtii as a model organism, we report that depletion of IFT25 has no effect on flagellar assembly and does not affect the entry of the BBSome into the flagellum, but IFT25 depletion did impair BBSome movement out of the flagellum, clarifying the evolutionally conserved role of IFT25 in regulating the exit of the BBSome from the flagellum cross species. Interestingly, depletion of IFT25 causes dramatic reduction of IFT27 as expected, which does not cause defects in flagellar assembly and cytokinesis in C. reinhardtii Our data thus support that Chlamydomonas IFT27, like its vertebrate homologues, is not involved in flagellar assembly and cytokinesis.
Project description:Cilia are microtubule-based organelles that assemble via intraflagellar transport (IFT) and function as signaling hubs on eukaryotic cells. IFT relies on molecular motors and IFT complexes that mediate the contacts with ciliary cargo. To elucidate the architecture of the IFT-B complex, we reconstituted and purified the nonameric IFT-B core from Chlamydomonas reinhardtii and determined the crystal structures of C. reinhardtii IFT70/52 and Tetrahymena IFT52/46 subcomplexes. The 2.5-Å resolution IFT70/52 structure shows that IFT52330-370 is buried deeply within the IFT70 tetratricopeptide repeat superhelix. Furthermore, the polycystic kidney disease protein IFT88 binds IFT52281-329 in a complex that interacts directly with IFT70/IFT52330-381 in trans. The structure of IFT52C/IFT46C was solved at 2.3 Å resolution, and we show that it is essential for IFT-B core integrity by mediating interaction between IFT88/70/52/46 and IFT81/74/27/25/22 subcomplexes. Consistent with this, overexpression of mammalian IFT52C in MDCK cells is dominant-negative and causes IFT protein mislocalization and disrupted ciliogenesis. These data further rationalize several ciliogenesis phenotypes of IFT mutant strains.
Project description:Intraflagellar transport (IFT) of ciliary precursors such as tubulin from the cytoplasm to the ciliary tip is involved in the construction of the cilium, a hairlike organelle found on most eukaryotic cells. However, the molecular mechanisms of IFT are poorly understood. Here, we found that the two core IFT proteins IFT74 and IFT81 form a tubulin-binding module and mapped the interaction to a calponin homology domain of IFT81 and a highly basic domain in IFT74. Knockdown of IFT81 and rescue experiments with point mutants showed that tubulin binding by IFT81 was required for ciliogenesis in human cells.