Intraflagellar Transport Components Research Articles

IFT cargo and motors associate sequentially with IFT trains to enter cilia of C. elegans

Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport cargo proteins into the cilium, across the crowded transition zone. How trains assemble at the base and how proteins associate with them is far from understood. Here, we use single-molecule imaging in the cilia of C. elegans chemosensory neurons to directly visualize the entry of kinesin-2 motors, kinesin-II and OSM-3, as well as anterograde cargo proteins, IFT dynein and tubulin. Single-particle tracking shows that IFT components associate with trains sequentially, both in time and space. Super-resolution maps of IFT components in wild-type and mutant worms reveal ciliary ultrastructure and show that kinesin-II is essential for axonemal organization. Finally, imaging cilia lacking kinesin-II and/or transition zone function uncovers the interplay of kinesin-II and OSM-3 in driving efficient transport of IFT trains across the transition zone.

Characterization of Chlamydomonas voltage-gated calcium channel and its interaction with photoreceptor support VGCC modulated photobehavioral response in the green alga

Calcium (Ca2+) signaling plays a major role in regulating multiple processes in living cells. The photoreceptor potential in Chlamydomonas triggers the generation of all or no flagellar Ca2+ currents that cause membrane depolarization across the eyespot and flagella. Modulation in membrane potential causes changes in the flagellar waveform, and hence, alters the beating patterns of Chlamydomonas flagella. The rhodopsin-mediated eyespot membrane potential is generated by the photoreceptor Ca2+ current or P-current however, the flagellar Ca2+ currents are mediated by unidentified voltage-gated calcium (VGCC or CaV) and potassium channels (VGKC). The voltage-gated ion channel that associates with ChRs to generate Ca2+ influx across the flagella and its cellular distribution has not yet been identified. Here, we identified putative VGCCs from algae and predicted their novel properties through insilico analysis. We further present experimental evidence on Chlamydomonas reinhardtii VGCCs to predict their novel physiological roles. Our experimental evidences showed that CrVGCC4 localizes to the eyespot and flagella of Chlamydomonas and associates with channelrhodopsins (ChRs). Further in silico interactome analysis of CrVGCCs suggested that they putatively interact with photoreceptor proteins, calcium signaling, and intraflagellar transport components. Expression analysis indicated that these VGCCs and their putative interactors can be perturbed by light stimuli. Collectively, our data suggest that VGCCs in general, and VGCC4 in particular, might be involved in the regulation of the photobehavioral response of Chlamydomonas.

A single-molecule view on intraflagellar transport in C. elegans chemosensory cilia.

In our cells, motor proteins do not work on their own: cargoes are often transported by multiple motors, of the same type, but often also of opposite directionality. To study motor cooperation, we have been focusing on intraflagellar transport (IFT), which is essential for the assembly and maintenance of cilia. In particular, we study the chemosensory cilia of C. elegans. To visualize IFT with live fluorescence microscopy, we generate mutant worms expressing fluorescent versions of the proteins of interest. Our fluorescence and image-analysis approaches allow us to visualize, track and quantify trains of IFT components moving together, as well as individual motors or IFT proteins. In C. elegans, IFT is driven by groups of tens of three different motor proteins: 2 kinesin-2's (the slow kinesin-II and the faster OSM-3), which drive transport of cargo trains from base to tip of the cilium, and IFT dynein, which drives transport back to the base. Cargo trains move in one go from base to tip and from tip to base. Speed and directionality appear to be regulated by motor proteins docking on and off the trains. Here I present new single-molecule data focusing on the entry of IFT components, including motor proteins, into the cilium. We observe that different IFT components attach to IFT trains in a sequential way, both in time and in location. Pooling the localization information of many individual molecules allows mapping of the 3D ultrastructure of the axoneme with a precision of several tens of nanometers, in wild-type as well as mutant strains lacking e.g. motors. These maps provide new insights in ciliary structure at the base, train-docking dynamics and the interplay of kinesin-II and OSM-3. Our results provide important new insights into how motors cooperate to drive intracellular transport.

Deletion of CEP164 in mouse photoreceptors post-ciliogenesis interrupts ciliary intraflagellar transport (IFT).

Centrosomal protein of 164 kDa (CEP164) is located at distal appendages of primary cilia and is necessary for basal body (BB) docking to the apical membrane. To investigate the function of photoreceptor CEP164 before and after BB docking, we deleted CEP164 during retina embryonic development (Six3Cre), in postnatal rod photoreceptors (iCre75) and in mature retina using tamoxifen induction (Prom1-ETCre). BBs dock to the cell cortex during postnatal day 6 (P6) to extend a connecting cilium (CC) and an axoneme. P6 retina-specific knockouts (retCep164-/-) are unable to dock BBs, thereby preventing formation of CC or outer segments (OSs). In rod-specific knockouts (rodCep164-/-), Cre expression starts after P7 and CC/OS form. P16 rodCep164-/- rods have nearly normal OS lengths, and maintain OS attachment through P21 despite loss of CEP164. Intraflagellar transport components (IFT88, IFT57 and IFT140) were reduced at P16 rodCep164-/- BBs and CC tips and nearly absent at P21, indicating impaired intraflagellar transport. Nascent OS discs, labeled with a fluorescent dye on P14 and P18 and harvested on P19, showed continued rodCep164-/- disc morphogenesis but absence of P14 discs mid-distally, indicating OS instability. Tamoxifen induction with PROM1ETCre;Cep164F/F (tamCep164-/-) adult mice affected maintenance of both rod and cone OSs. The results suggest that CEP164 is key towards recruitment and stabilization of IFT-B particles at the BB/CC. IFT impairment may be the main driver of ciliary malfunction observed with hypomorphic CEP164 mutations.

TTC30A and TTC30B Redundancy Protects IFT Complex B Integrity and Its Pivotal Role in Ciliogenesis.

Intraflagellar transport (IFT) is a microtubule-based system that supports the assembly and maintenance of cilia. The dysfunction of IFT leads to ciliopathies of variable severity. Two of the IFT-B components are the paralogue proteins TTC30A and TTC30B. To investigate whether these proteins constitute redundant functions, CRISPR/Cas9 was used to generate single TTC30A or B and double-knockout hTERT-RPE1 cells. Ciliogenesis assays showed the redundancy of both proteins while the polyglutamylation of cilia was affected in single knockouts. The localization of other IFT components was not affected by the depletion of a single paralogue. A loss of both proteins led to a severe ciliogenesis defect, resulting in no cilia formation, which was rescued by TTC30A or B. The redundancy can be explained by the highly similar interaction patterns of the paralogues; both equally interact with the IFT-B machinery. Our study demonstrates that a loss of one TTC30 paralogue can mostly be compensated by the other, thus preventing severe ciliary defects. However, cells assemble shorter cilia, which are potentially limited in their function, especially because of impaired polyglutamylation. A complete loss of both proteins leads to a deficit in IFT complex B integrity followed by disrupted IFT and subsequently no cilia formation.

MEIG1 determines the manchette localization of IFT20 and IFT88, two intraflagellar transport components in male germ cells

Sperm flagella formation is a complex process that requires cargo transport systems to deliver structural proteins for sperm flagella assembly. Two cargo transport systems, the intramanchette transport (IMT) and intraflagellar transport (IFT), have been shown to play critical roles in spermatogenesis and sperm flagella formation. IMT exists only in elongating spermatids, while IFT is responsible for delivering cargo proteins in the developing cilia/flagella. Our laboratory discovered that mouse meiosis expressed gene 1 (MEIG1), a gene essential for sperm flagella formation, is present in the manchette of elongating spermatids. IFT complex components, IFT20 and IFT88, are also present in the manchette of the elongating spermatids. Given that the three proteins have the same localization in elongating spermatids and are essential for normal spermatogenesis and sperm flagella formation, we hypothesize that they are in the same complex, which is supported by co-immunoprecipitation assay using mouse testis extracts. In the Meig1 knockout mice, neither IFT20 nor IFT88 was present in the manchette in the elongating spermatids even though their localizations were normal in spermatocytes and round spermatids. However, MEIG1 was still present in the manchette in elongating spermatids of the conditional Ift20 knockout mice. In the sucrose gradient assay, both IFT20 and IFT88 proteins drifted from higher density fractions to lighter ones in the Meig1 knockout mice. MEIG1 distribution was not changed in the conditional Ift20 knockout mice. Finally, testicular IFT20 and IFT88 protein and mRNA levels were significantly reduced in Meig1 knockout mice. Our data suggests that MEIG1 is a key protein in determining the manchette localization of certain IFT components, including IFT20 and IFT88, in male germ cells.

A single-molecule view on intracellular transport in C. elegans chemosensory cilia

In our cells, motor proteins do not work on their own: cargoes are often transported by multiple motors, of the same type, but often also of opposite directionality. To study motor cooperation, we have focused on a particular transport process, intraflagellar transport (IFT), which takes place in cilia and flagella and is essential for the assembly and maintenance of these organelles. As a model system we use IFT in the chemosensory cilia of the nematode C. elegans. In order to visualize IFT components and cargoes with fluorescence microscopy on living animals, we generate mutant-nematodes expressing fluorescent versions of the proteins of interest.

WDR60-mediated dynein-2 loading into cilia powers retrograde IFT and transition zone crossing.

The dynein-2 motor complex drives retrograde intraflagellar transport (IFT), playing a pivotal role in the assembly and functions of cilia. However, the mechanisms that regulate dynein-2 motility remain poorly understood. Here, we identify the Caenorhabditis elegans WDR60 homologue, WDR-60, and dissect the roles of this intermediate chain using genome editing and live imaging of endogenous dynein-2/IFT components. We find that loss of WDR-60 impairs dynein-2 recruitment to cilia and its incorporation onto anterograde IFT trains, reducing retrograde motor availability at the ciliary tip. Consistent with this, we show that fewer dynein-2 motors power WDR-60-deficient retrograde IFT trains, which move at reduced velocities and fail to exit cilia, accumulating on the distal side of the transition zone. Remarkably, disrupting the transition zone's NPHP module almost fully restores ciliary exit of underpowered retrograde trains in wdr-60 mutants. This work establishes WDR-60 as a major contributor to IFT, and the NPHP module as a roadblock to dynein-2 passage through the transition zone.

In vivo imaging shows continued association of several IFT-A, IFT-B and dynein complexes while IFT trains U-turn at the tip.

Flagellar assembly depends on intraflagellar transport (IFT), a bidirectional motility of protein carriers, the IFT trains. The trains are periodic assemblies of IFT-A and IFT-B subcomplexes and the motors kinesin-2 and IFT dynein. At the tip, anterograde trains are remodeled for retrograde IFT, a process that in Chlamydomonas involves kinesin-2 release and train fragmentation. However, the degree of train disassembly at the tip remains unknown. Here, we performed two-color imaging of fluorescent protein-tagged IFT components, which indicates that IFT-A and IFT-B proteins from a given anterograde train usually return in the same set of retrograde trains. Similarly, concurrent turnaround was typical for IFT-B proteins and the IFT dynein subunit D1bLIC-GFP but severance was observed as well. Our data support a simple model of IFT turnaround, in which IFT-A, IFT-B and IFT dynein typically remain associated at the tip and segments of the anterograde trains convert directly into retrograde trains. Continuous association of IFT-A, IFT-B and IFT dynein during tip remodeling could balance protein entry and exit, preventing the build-up of IFT material in flagella.

Kinesin-2 transports Orco into the olfactory cilium of Drosophila melanogaster at specific developmental stages.

The cilium, the sensing centre for the cell, displays an extensive repertoire of receptors for various cell signalling processes. The dynamic nature of ciliary signalling indicates that the ciliary entry of receptors and associated proteins must be regulated and conditional. To understand this process, we studied the ciliary localisation of the odour-receptor coreceptor (Orco), a seven-pass transmembrane protein essential for insect olfaction. Little is known about when and how Orco gets into the cilia. Here, using Drosophila melanogaster, we show that the bulk of Orco selectively enters the cilia on adult olfactory sensory neurons in two discrete, one-hour intervals after eclosion. A conditional loss of heterotrimeric kinesin-2 during this period reduces the electrophysiological response to odours and affects olfactory behaviour. We further show that Orco binds to the C-terminal tail fragments of the heterotrimeric kinesin-2 motor, which is required to transfer Orco from the ciliary base to the outer segment and maintain within an approximately four-micron stretch at the distal portion of the ciliary outer-segment. The Orco transport was not affected by the loss of critical intraflagellar transport components, IFT172/Oseg2 and IFT88/NompB, respectively, during the adult stage. These results highlight a novel developmental regulation of seven-pass transmembrane receptor transport into the cilia and indicate that ciliary signalling is both developmentally and temporally regulated.

The decrease of intraflagellar transport impairs sensory perception and metabolism in ageing

Sensory perception and metabolic homeostasis are known to deteriorate with ageing, impairing the health of aged animals, while mechanisms underlying their deterioration remain poorly understood. The potential interplay between the declining sensory perception and the impaired metabolism during ageing is also barely explored. Here, we report that the intraflagellar transport (IFT) in the cilia of sensory neurons is impaired in the aged nematode Caenorhabditis elegans due to a daf-19/RFX-modulated decrease of IFT components. We find that the reduced IFT in sensory cilia thus impairs sensory perception with ageing. Moreover, we demonstrate that whereas the IFT-dependent decrease of sensory perception in aged worms has a mild impact on the insulin/IGF-1 signalling, it remarkably suppresses AMP-activated protein kinase (AMPK) signalling across tissues. We show that upregulating daf-19/RFX effectively enhances IFT, sensory perception, AMPK activity and autophagy, promoting metabolic homeostasis and longevity. Our study determines an ageing pathway causing IFT decay and sensory perception deterioration, which in turn disrupts metabolism and healthy ageing.

IFT20 is critical for collagen biosynthesis in craniofacial bone formation

Intraflagellar transport (IFT) is essential for assembling primary cilia required for bone formation. Disruption of IFT frequently leads to bone defects in humans. While it has been well studied about the function of IFT in osteogenic cell proliferation and differentiation, little is known about its role in collagen biosynthesis during bone formation. Here we show that IFT20, the smallest IFT protein in the IFT-B complex, is important for collagen biosynthesis in mice. Deletion of Ift20 in craniofacial osteoblasts displayed bone defects in the face. While collagen protein levels are unaffected by loss of Ift20, collagen cross-linking was significantly altered. In both Ift20:Wnt1-Cre and Ift20:Ocn-Cre mice the bones exhibit increased hydroxylysine-aldehyde deived cross-linking, and decreased lysine-aldehyde derived cross-linking. To obtain insight into the molecular mechanisms, we examined the expression levels of telopeptidyl lysyl hydroxylase 2 (LH2), and associated chaperone complexes. The results demonstrated that, while LH2 levels were unaffected by loss of Ift20, its chaperone, FKBP65, was significantly increased in Ift20:Wnt1-Cre and Ift20:Ocn-Cre mouse calvaria as well as femurs. These results suggest that IFT20 plays a pivotal role in collagen biosynthesis by regulating, in part, telopeptidyl lysine hydroxylation and cross-linking in bone. To the best of our knowledge, this is the first to demonstrate that the IFT components control collagen post-translational modifications. This provides a novel insight into the craniofacial bone defects associated with craniofacial skeletal ciliopathies.

Genetic reduction of cilium length by targeting intraflagellar transport 88 protein impedes kidney and liver cyst formation in mouse models of autosomal polycystic kidney disease

Polycystin-1 (PC1) and -2 (PC2), products of the PKD1 and PKD2 genes, are mutated in autosomal dominant polycystic kidney disease (ADPKD). They localize to the primary cilia; however, their ciliary function is in dispute. Loss of either the primary cilia or PC1 or PC2 causes cyst formation. However, loss of both cilia and PC1 or PC2 inhibits cyst growth via an unknown pathway. To help define a pathway, we studied cilium length in human and mouse kidneys. We found cilia are elongated in kidneys from patients with ADPKD and from both Pkd1 and Pkd2 knockout mice. Cilia elongate following polycystin inactivation. The role of intraflagellar transport proteins in Pkd1-deficient mice is also unknown. We found that inactivation of Ift88 (a gene expressing a core component of intraflagellar transport) in Pkd1 knockout mice, as well as in a new Pkd2 knockout mouse, shortened the elongated cilia, impeded kidney and liver cystogenesis, and reduced cell proliferation. Multi-stage invivo analysis of signaling pathways revealed β-catenin activation as a prominent, early, and sustained event in disease onset and progression in Pkd2 single knockout but not in Pkd2.Ift88 double knockout mouse kidneys. Additionally, AMPK, mTOR and ERK pathways were altered in Pkd2 single knockout mice but only AMPK and mTOR pathway alteration were rescued in Pkd2.Ift88 double knockout mice. Thus, our findings advocate an essential role of polycystins in the structure and function of the primary cilia and implicate β-catenin as a key inducer of cystogenesis downstream of the primary cilia. Our data suggest that modulating cilium length and/or its associated signaling events may offer novel therapeutic approaches for ADPKD.

Ubiquitin links smoothened to intraflagellar transport to regulate Hedgehog signaling.

In the absence of Hedgehog ligand, patched-1 (Ptch1) localizes to cilia and prevents ciliary accumulation and activation of smoothened (Smo). Upon ligand binding, Ptch1 is removed from cilia, and Smo is derepressed and accumulates in cilia where it activates signaling. The mechanisms regulating these dynamic movements are not well understood, but defects in intraflagellar transport components, including Ift27 and the BBSome, cause Smo to accumulate in cilia without pathway activation. We find that in the absence of ligand-induced pathway activation, Smo is ubiquitinated and removed from cilia, and this process is dependent on Ift27 and BBSome components. Activation of Hedgehog signaling decreases Smo ubiquitination and ciliary removal, resulting in its accumulation. Blocking ubiquitination of Smo by an E1 ligase inhibitor or by mutating two lysine residues in intracellular loop three causes Smo to aberrantly accumulate in cilia without pathway activation. These data provide a mechanism to control Smo's ciliary level during Hedgehog signaling by regulating the ubiquitination state of the receptor.

The essential role of intraflagellar transport protein IFT81 in male mice spermiogenesis and fertility.

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism that is indispensable for the formation and maintenance of cilia and flagella; however, the implications and functions of IFT81 remain unknown. In this study, we disrupted IFT81 expression in male germ cells starting from the spermatocyte stage. As a result, homozygous mutant males were completely infertile and displayed abnormal sperm parameters. In addition to oligozoospermia, spermatozoa presented dysmorphic and nonfunctional flagella. Histological examination of testes from homozygous mutant mice revealed abnormal spermiogenesis associated with sloughing of germ cells and the presence of numerous multinucleated giant germ cells (symblasts) in the lumen of seminiferous tubules and epididymis. Moreover, only few elongated spermatids and spermatozoa were seen in analyzed cross sections. Transmission electron microscopy showed a complete disorganization of the axoneme and para-axonemal structures such as the mitochondrial sheath, fibrous sheath, and outer dense fibers. In addition, numerous vesicles that contain unassembled microtubules were observed within developing spermatids. Acrosome structure analysis showed normal appearance, thus excluding a crucial role of IFT81 in acrosome biogenesis. These observations showed that IFT81 is an important member of the IFT process during spermatogenesis and that its absence is associated with abnormal flagellum formation leading to male infertility. The expression levels of several IFT components in testes, including IFT20, IFT25, IFT27, IFT57, IFT74, and IFT88, but not IFT140, were significantly reduced in homozygous mutant mice. Overall, our study demonstrates that IFT81 plays an essential role during spermatogenesis by modulating the assembly and elongation of the sperm flagella.

Murine germ cell-specific disruption of Ift172 causes defects in spermiogenesis and male fertility.

Intraflagellar transport (IFT) is a conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. IFT172 is a component of the IFT complex. Global disruption of mouse Ift172 gene caused typical phenotypes of ciliopathy. Mouse Ift172 gene appears to translate two major proteins; the full-length protein is highly expressed in the tissues enriched in cilia and the smaller 130 kDa one is only abundant in the testis. In male germ cells, IFT172 is highly expressed in the manchette of elongating spermatids. A germ cell-specific Ift172 mutant mice were generated, and the mutant mice did not show gross abnormalities. There was no difference in testis/body weight between the control and mutant mice, but more than half of the adult homozygous mutant males were infertile and associated with abnormally developed germ cells in the spermiogenesis phase. The cauda epididymides in mutant mice contained less developed sperm that showed significantly reduced motility, and these sperm had multiple defects in ultrastructure and bent tails. In the mutant mice, testicular expression levels of some IFT components, including IFT20, IFT27, IFT74, IFT81 and IFT140, and a central apparatus protein SPAG16L were not changed. However, expression levels of ODF2, a component of the outer dense fiber, and AKAP4, a component of fibrous sheath, and two IFT components IFT25 and IFT57 were dramatically reduced. Our findings demonstrate that IFT172 is essential for normal male fertility and spermiogenesis in mice, probably by modulating specific IFT proteins and transporting/assembling unique accessory structural proteins into spermatozoa.

Trypanosomes have divergent kinesin-2 proteins that function differentially in flagellum biosynthesis and cell viability.

Trypanosoma brucei, the causative agent of African sleeping sickness, has a flagellum that is crucial for motility, pathogenicity, and viability. In most eukaryotes, the intraflagellar transport (IFT) machinery drives flagellum biogenesis, and anterograde IFT requires kinesin-2 motor proteins. In this study, we investigated the function of the two T. brucei kinesin-2 proteins, TbKin2a and TbKin2b, in bloodstream form trypanosomes. We found that, compared to kinesin-2 proteins across other phyla, TbKin2a and TbKin2b show greater variation in neck, stalk and tail domain sequences. Both kinesins contributed additively to flagellar lengthening. Silencing TbKin2a inhibited cell proliferation, cytokinesis and motility, whereas silencing TbKin2b did not. TbKin2a was localized on the flagellum and colocalized with IFT components near the basal body, consistent with it performing a role in IFT. TbKin2a was also detected on the flagellar attachment zone, a specialized structure that connects the flagellum to the cell body. Our results indicate that kinesin-2 proteins in trypanosomes play conserved roles in flagellar biosynthesis and exhibit a specialized localization, emphasizing the evolutionary flexibility of motor protein function in an organism with a large complement of kinesins.

Emerging Roles of the Intraflagellar Transport System in the Orchestration of Cellular Degradation Pathways.

Ciliated cells exploit a specific transport system, the intraflagellar transport (IFT) system, to ensure the traffic of molecules from the cell body to the cilium. However, it is now clear that IFT activity is not restricted to cilia-related functions. This is strikingly exemplified by the observation that IFT proteins play important roles in cells lacking a primary cilium, such as lymphocytes. Indeed, in T cells the IFT system regulates the polarized transport of endosome-associated T cell antigen receptors and signaling mediators during assembly of the immune synapse, a specialized interface that forms on encounter with a cognate antigen presenting cell and on which T cell activation and effector function crucially depend. Cellular degradation pathways have recently emerged as new extraciliary functions of the IFT system. IFT proteins have been demonstrated to regulate autophagy in ciliated cells through their ability to recruit the autophagy machinery to the base of the cilium. We have now implicated the IFT component IFT20 in another central degradation process that also controls the latest steps in autophagy, namely lysosome function, by regulating the cation-independent mannose-6-phosphate receptor (CI-MPR)-dependent lysosomal targeting of acid hydrolases. This involves the ability of IFT20 to act as an adaptor coupling the CI-MPR to dynein for retrograde transport to the trans-Golgi network. In this short review we will discuss the emerging roles of IFT proteins in cellular degradation pathways.

Abnormal fertility, acrosome formation, IFT20 expression and localization in conditional Gmap210 knockout mice.

GMAP210 (TRIP11) is a cis-Golgi network-associated protein and a Golgi membrane receptor for IFT20, an intraflagellar transport component essential for male fertility and spermiogenesis in mice. To investigate the role of GMAP210 in male fertility and spermatogenesis, floxed Gmap210 mice were bred with Stra8-iCre mice so that the Gmap210 gene is disrupted in spermatocytes and spermatids in this study. The Gmap210flox/flox: Stra8-iCre mutant mice showed no gross abnormalities and survived to adulthood. In adult males, testis and body weights showed no difference between controls and mutant mice. Low-magnification histological examination of the testes revealed normal seminiferous tubule structure, but sperm counts and fertility were significantly reduced in mutant mice compared with controls. Higher resolution examination of the mutant seminiferous epithelium showed that nearly all sperm had more oblong, abnormally shaped heads, while the sperm tails appeared to have normal morphology. Electron microscopy also revealed abnormally shaped sperm heads but normal axoneme core structure; some sperm showed membrane defects in the midpiece. In mutant mice, expression levels of IFT20 and other selective acrosomal proteins were significantly reduced, and their localization was also affected. Peanut-lectin, an acrosome maker, was almost absent in the spermatids and epididymal sperm. Mitochondrion staining was highly concentrated in the heads of sperm, suggesting that the midpieces were coiling around or aggregating near the heads. Defects in acrosome biogenesis were further confirmed by electron microscopy. Collectively, our findings suggest that GMAP210 is essential for acrosome biogenesis, normal mitochondrial sheath formation, and male fertility, and it determines expression levels and acrosomal localization of IFT20 and other acrosomal proteins.

Cilia drive developmental plasticity and are essential for efficient prey detection in predatory nematodes.

Cilia are complex organelles involved in a broad array of functions in eukaryotic organisms. Nematodes employ cilia for environmental sensing, which shapes developmental decisions and influences morphologically plastic traits and adaptive behaviours. Here, we assess the role of cilia in the nematode Pristionchus pacificus, and determine their importance in regulating the developmentally plastic mouth-form decision in addition to predatory feeding and self-recognition behaviours, all of which are not present in Caenorhabditis elegans. An analysis of a multitude of cilia-related mutants including representatives of the six protein subcomplexes required in intraflagellar transport (IFT) plus the regulatory factor X transcription factor daf-19 revealed that cilia are essential for processing the external cues influencing the mouth-form decision and for the efficient detection of prey. Surprisingly, we observed that loss-of-function mutations in the different IFT components resulted in contrasting mouth-form phenotypes and different degrees of predation deficiencies. This observation supports the idea that perturbing different IFT subcomplexes has different effects on signalling downstream of the cilium. Finally, self-recognition was maintained in the cilia deficient mutants tested, indicating that the mechanisms triggering self-recognition in P. pacificus may not require the presence of fully functional cilia.