Flagella of Tumor-Targeting Bacteria Trigger Local Hemorrhage to Reprogram Tumor-Associated Macrophages for Improved Antitumor Therapy.

Tumor-associated macrophages (TAMs) exhibit an immunosuppressive M2 phenotype and lead to failure of antitumor therapy. Infiltrated erythrocytes during hemorrhage are recognized as a promising strategy for polarizing TAMs. However, novel materials that precisely induce tumor hemorrhage without affecting normal coagulation still face challenges. Here, tumor-targeting bacteria (flhDC VNP) are genetically constructed to realize precise tumor hemorrhage. FlhDC VNP colonizes the tumor and overexpresses flagella during proliferation. The flagella promote the expression of tumor necrosis factor α, which induces local tumor hemorrhage. Infiltrated erythrocytes during the hemorrhage temporarily polarize macrophages to the M1 subtype. In the presence of artesunate, this short-lived polarization is transformed into a sustained polarization because artesunate and heme form a complex that continuously produces reactive oxygen species. Therefore, the flagella of active tumor-targeting bacteria may open up new strategies for reprogramming TAMs and improving antitumor therapy.

Bi-allelic variants in DNHD1 cause flagellar axoneme defects and asthenoteratozoospermia in humans and mice.

Asthenoteratozoospermia, defined as reduced sperm motility and abnormal sperm morphology, is a disorder with considerable genetic heterogeneity. Although previous studies have identified several asthenoteratozoospermia-associated genes, the etiology remains unknown for the majority of affected men. Here, we performed whole-exome sequencing on 497 unrelated men with asthenoteratozoospermia and identified DNHD1 bi-allelic variants from eight families (1.6%). All detected variants were predicted to be deleterious via multiple bioinformatics tools. Hematoxylin and eosin (H&E) staining revealed that individuals with bi-allelic DNHD1 variants presented striking abnormalities of the flagella; transmission electron microscopy (TEM) further showed flagellar axoneme defects, including central pair microtubule (CP) deficiency and mitochondrial sheath (MS) malformations. In sperm from fertile men, DNHD1 was localized to the entire flagella of the normal sperm; however, it was nearly absent in the flagella of men with bi-allelic DNHD1 variants. Moreover, abundance of the CP markers SPAG6 and SPEF2 was significantly reduced in spermatozoa from men harboring bi-allelic DNHD1 variants. In addition, Dnhd1 knockout male mice (Dnhd1‒/‒) exhibited asthenoteratozoospermia and infertility, a finding consistent with the sperm phenotypes present in human subjects with DNHD1 variants. The female partners of four out of seven men who underwent intracytoplasmic sperm injection therapy subsequently became pregnant. In conclusion, our study showed that bi-allelic DNHD1 variants cause asthenoteratozoospermia, a finding that provides crucial insights into the biological underpinnings of this disorder and should assist with counseling of affected individuals.

TTC12 Loss-of-Function Mutations Cause Primary Ciliary Dyskinesia and Unveil Distinct Dynein Assembly Mechanisms in Motile Cilia Versus Flagella.

Cilia and flagella are evolutionarily conserved organelles whose motility relies on the outer and inner dynein arm complexes (ODAs and IDAs). Defects in ODAs and IDAs result in primary ciliary dyskinesia (PCD), a disease characterized by recurrent airway infections and male infertility. PCD mutations in assembly factors have been shown to cause a combined ODA-IDA defect, affecting both cilia and flagella. We identified four loss-of-function mutations in TTC12, which encodes a cytoplasmic protein, in four independent families in which affected individuals displayed a peculiar PCD phenotype characterized by the absence of ODAs and IDAs in sperm flagella, contrasting with the absence of only IDAs in respiratory cilia. Analyses of both primary cells from individuals carrying TTC12 mutations and human differentiated airway cells invalidated for TTC12 by a CRISPR-Cas9 approach revealed an IDA defect restricted to a subset of single-headed IDAs that are different in flagella and cilia, whereas TTC12 depletion in the ciliate Paramecium tetraurelia recapitulated the sperm phenotype. Overall, our study, which identifies TTC12 as a gene involved in PCD, unveils distinct dynein assembly mechanisms in human motile cilia versus flagella.

Nexin-Dynein regulatory complex component DRC7 but not FBXL13 is required for sperm flagellum formation and male fertility in mice.

Flagella and cilia are evolutionarily conserved cellular organelles. Abnormal formation or motility of these organelles in humans causes several syndromic diseases termed ciliopathies. The central component of flagella and cilia is the axoneme that is composed of the '9+2' microtubule arrangement, dynein arms, radial spokes, and the Nexin-Dynein Regulatory Complex (N-DRC). The N-DRC is localized between doublet microtubules and has been extensively studied in the unicellular flagellate Chlamydomonas. Recently, it has been reported that TCTE1 (DRC5), a component of the N-DRC, is essential for proper sperm motility and male fertility in mice. Further, TCTE1 has been shown to interact with FBXL13 (DRC6) and DRC7; however, functional roles of FBXL13 and DRC7 in mammals have not been elucidated. Here we show that Fbxl13 and Drc7 expression are testes-enriched in mice. Although Fbxl13 knockout (KO) mice did not show any obvious phenotypes, Drc7 KO male mice were infertile due to their short immotile spermatozoa. In Drc7 KO spermatids, the axoneme is disorganized and the '9+2' microtubule arrangement was difficult to detect. Further, other N-DRC components fail to incorporate into the flagellum without DRC7. These results indicate that Drc7, but not Fbxl13, is essential for the correct assembly of the N-DRC and flagella.

IFT52 mutations destabilize anterograde complex assembly, disrupt ciliogenesis and result in short rib polydactyly syndrome.

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.

Canonical and noncanonical intraflagellar transport regulates craniofacial skeletal development.

The primary cilium is a cellular organelle that coordinates signaling pathways critical for cell proliferation, differentiation, survival, and homeostasis. Intraflagellar transport (IFT) plays a pivotal role in assembling primary cilia. Disruption and/or dysfunction of IFT components can cause multiple diseases, including skeletal dysplasia. However, the mechanism by which IFT regulates skeletogenesis remains elusive. Here, we show that a neural crest-specific deletion of intraflagellar transport 20 (Ift20) in mice compromises ciliogenesis and intracellular transport of collagen, which leads to osteopenia in the facial region. Whereas platelet-derived growth factor receptor alpha (PDGFRα) was present on the surface of primary cilia in wild-type osteoblasts, disruption of Ift20 down-regulated PDGFRα production, which caused suppression of PDGF-Akt signaling, resulting in decreased osteogenic proliferation and increased cell death. Although osteogenic differentiation in cranial neural crest (CNC)-derived cells occurred normally in Ift20-mutant cells, the process of mineralization was severely attenuated due to delayed secretion of type I collagen. In control osteoblasts, procollagen was easily transported from the endoplasmic reticulum (ER) to the Golgi apparatus. By contrast, despite having similar levels of collagen type 1 alpha 1 (Col1a1) expression, Ift20 mutants did not secrete procollagen because of dysfunctional ER-to-Golgi trafficking. These data suggest that in the multipotent stem cells of CNCs, IFT20 is indispensable for regulating not only ciliogenesis but also collagen intracellular trafficking. Our study introduces a unique perspective on the canonical and noncanonical functions of IFT20 in craniofacial skeletal development.

Ciliopathies with skeletal anomalies and renal insufficiency due to mutations in the IFT-A gene WDR19.

A subset of ciliopathies, including Sensenbrenner, Jeune, and short-rib polydactyly syndromes are characterized by skeletal anomalies accompanied by multiorgan defects such as chronic renal failure and retinitis pigmentosa. Through exome sequencing we identified compound heterozygous mutations in WDR19 in a Norwegian family with Sensenbrenner syndrome. In a Dutch family with the clinically overlapping Jeune syndrome, a homozygous missense mutation in the same gene was found. Both families displayed a nephronophthisis-like nephropathy. Independently, we also identified compound heterozygous WDR19 mutations by exome sequencing in a Moroccan family with isolated nephronophthisis. WDR19 encodes IFT144, a member of the intraflagellar transport (IFT) complex A that drives retrograde ciliary transport. We show that IFT144 is absent from the cilia of fibroblasts from one of the Sensenbrenner patients and that ciliary abundance and morphology is perturbed, demonstrating the ciliary pathogenesis. Our results suggest that isolated nephronophthisis, Jeune, and Sensenbrenner syndromes are clinically overlapping disorders that can result from a similar molecular cause.

Epididymal hypo-osmolality induces abnormal sperm morphology and function in the estrogen receptor alpha knockout mouse.

Estrogen receptor-alpha (ESR1) is highly expressed in the efferent ductules of all species studied as well as in the epididymal epithelium in mice and other select species. Male mice lacking ESR1 (Esr1KO) are infertile, but transplantation studies demonstrated that Esr1KO germ cells are capable of fertilization when placed in a wild-type reproductive tract. These results suggest that extratesticular regions, such as the efferent ductules and epididymis, are the major source of pathological changes in Esr1KO males. Previous studies have shown alterations in ion and fluid transporters in the efferent duct and epididymal epithelia of Esr1KO males, leading to misregulation of luminal fluid pH. To determine the effect of an altered epididymal milieu on Esr1KO sperm, we assayed sperm morphology in the different regions of the epididymis. Sperm recovered from the epididymis exhibited abnormal flagellar coiling and increased incidence of spontaneous acrosome reactions, both of which are consistent with exposure to abnormal epididymal fluid. Analysis of the epididymal fluid revealed that the osmolality of the Esr1KO fluid was reduced relative to wild type, consistent with prior reports of inappropriate fluid absorption from the efferent ductules. This, along with the finding that morphological defects increased with transit through the epididymal duct, suggests that the anomalies in sperm are a consequence of the abnormal luminal environment. Consistent with this, incubating Esr1KO sperm in a more wild-type-like osmotic environment significantly rescued the abnormal flagellar coiling. This work demonstrates that Esr1KO mice exhibit an abnormal fluid environment in the lumen of the efferent ducts and epididymis, precluding normal sperm maturation and instead resulting in progressive deterioration of sperm that contributes to infertility.

Participation of the inositol phospholipid signaling pathway in the increase in cytosolic calcium induced by tributyltin chloride intoxication of chlorophyllous protozoa Euglena gracilis Z and its achlorophyllous mutant SM-ZK.

Exposure of tri-n-butyl tin chloride (TBTCl) as a stressor to Euglena gracilis Z causes rapid alteration of cell morphology followed by deflagellation. The present study was undertaken to reveal the mechanism of the cell response at a molecular level. Chlamydomonas reinhardtii, in this study E. gracilis Z and its achlorophyllous mutant SM-ZK, gave similar results when subject to the same stressor. Indeed, similar results were obtained with both strains. Next, assuming that the morphological alteration caused by TBTCl is mediated by the inositide phosphate-lipid signaling pathway, the effects of signal transduction and Ca2+ release reagents (mastoparan as a G-protein activator, neomycin as a phospholipase C inhibitor, verapamil as a Ca2+ channel blocker, and A23187 as a Ca2+ ionophore) on morphology and intracellular Ca2+ levels were examined with or without TBTCl. The data strongly suggest that the morphological alteration is mediated by an increase in Ca2+ linked to the inositol phosphatide pathway. The cellular response to signal transduction inducing reagents was compared between the E. gracilis chlorophyllous Z strain and its achlorophyllous mutant SM-ZK strain. Significant differences were observed between the Z and SM-ZK strains in terms of the stress response and intracellular Ca2+ level.

Cilia and disease.

Cilia are classified according to their microtubule components as 9+2 (motile) and 9+0 (primary) cilia. Disruption of 9+2 cilia, which move mucus across respiratory epithelia, leads to rhinitis, sinusitis and bronchiectasis. Approximately half of the patients with primary ciliary dyskinesia (PCD) have situs inversus, providing a link between left-right asymmetry and cilia. 9+0 cilia at the embryonic node are also motile and involved in establishing left-right asymmetry. Most 9+0 cilia, however, act as antennae, sensing the external environment. Defective 9+0 cilia of principal cells of the nephron cause cystic diseases of the kidney. In the rods and cones of the retina, photoreceptor discs and visual pigments are synthesized in the inner segment and transported to the distal outer segment through a narrow 9+0 connecting cilium; defects in this process lead to retinitis pigmentosa. Although the function of primary cilia in some organs is being elucidated, in many other organs they have not been studied at all. It is probable that many more cilia-related disorders remain to be discovered.

Further studies on knockout mice lacking a functional dynein heavy chain (MDHC7). 1. Evidence for a structural deficit in the axoneme.

Male mice had previously been generated in which the inner dynein arm heavy chain 7 gene (MDHC7) was inactivated by the substitution of four exons encoding the ATP-binding site (P1-loop) with the neomycin resistance gene, giving a putative non-functional gene product. We have used additional techniques of electron microscopy to determine what effect the truncated, non-functional heavy chain has on the assembly of the inner dynein arm complex. From a comparison of MDHC7-/- with the wild-type morphology, we have found that the expected loss of a C-terminal (globular) domain is associated with inner dynein arm 3, a change from two visible "heads" to one. This deficit was seen in replicas of rapidly-frozen, deeply-etched spermatozoa, and was confirmed in filtered images of 20-nm-thin sections, cut in longitudinal planes. Assembly of the other IDAs appeared unaffected. This study is the first to reveal the location of a specific dynein heavy chain within the 96-nm repeat pattern of the inner dynein arms of the mammalian axoneme.

Further studies on knockout mice lacking a functional dynein heavy chain (MDHC7). 2. A developmental explanation for the asthenozoospermia.

Male mice had been previously generated in which the inner dynein arm heavy chain 7 gene (MDHC7) was disrupted. MDHC7-/- animals show asthenozoospermia and are sterile. Very few of their spermatozoa can achieve forward progression, but for those that can, we add here the information (1) that the three-dimensional aspects of their movement are normal; (2) that their maximum velocity is less than that of wild-type controls; and (3) that they are entirely unable to penetrate media of raised viscosity (25-4,000 cP). However, the large majority of the spermatozoa can achieve only a low amplitude vibration. In these sperm we find, using electron microscopy, that the outer dense fibres retain attachments to the inner surface of the mitochondria. Such attachments are present in normal epididymal mouse spermatozoa but are broken down as soon as the sperm become motile on release from the epididymis. The attachments are presumed to be essential during midpiece development and, afterwards, to require a threshold level of force to loosen them and so permit the sliding displacements necessary for normal bending. We presume that the disruption of the inner dynein arm heavy chain gene, MDHC7, means that there is insufficient force to overcome the attachments, for all but a few spermatozoa.

The diagnosis of trichomonas vaginalis in liquid-based Pap tests: morphological characteristics.

We investigated the morphological features of Trichomonas vaginalis in liquid-based Papanicolaou (Pap) (LBP) tests, in order to assess the impact of this new slide preparation system on recognition of T. vaginalis organisms. We reviewed 88 LBP test slides previously interpreted as showing T. vaginalis and assessed morphological characteristics of the organisms and the presence of secondary features. Eighty-six of 88 slides showed, at least focally, organisms with cytoplasmic granules, nuclei, and flagella allowing for confident recognition of T. vaginalis. Compared with 58 controls, cases of T. vaginalis were more likely to have squamous cells with perinuclear halos, "ghost cells," and coccobacilli. The lack of a "dirty" background or cytolysis allowed a more straightforward identification of the organisms. We concluded that T. vaginalis organisms are morphologically distinct in LBP tests, which may lead to an increased specificity when they are identified in such tests.

Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella.

Intraflagellar transport (IFT) is a rapid movement of multi-subunit protein particles along flagellar microtubules and is required for assembly and maintenance of eukaryotic flagella. We cloned and sequenced a Chlamydomonas cDNA encoding the IFT88 subunit of the IFT particle and identified a Chlamydomonas insertional mutant that is missing this gene. The phenotype of this mutant is normal except for the complete absence of flagella. IFT88 is homologous to mouse and human genes called Tg737. Mice with defects in Tg737 die shortly after birth from polycystic kidney disease. We show that the primary cilia in the kidney of Tg737 mutant mice are shorter than normal. This indicates that IFT is important for primary cilia assembly in mammals. It is likely that primary cilia have an important function in the kidney and that defects in their assembly can lead to polycystic kidney disease.

Cytoplasmic dynein heavy chain 1b is required for flagellar assembly in Chlamydomonas.

A second cytoplasmic dynein heavy chain (cDhc) has recently been identified in several organisms, and its expression pattern is consistent with a possible role in axoneme assembly. We have used a genetic approach to ask whether cDhc1b is involved in flagellar assembly in Chlamydomonas. Using a modified PCR protocol, we recovered two cDhc sequences distinct from the axonemal Dhc sequences identified previously. cDhc1a is closely related to the major cytoplasmic Dhc, whereas cDhc1b is closely related to the minor cDhc isoform identified in sea urchins, Caenorhabditis elegans, and Tetrahymena. The Chlamydomonas cDhc1b transcript is a low-abundance mRNA whose expression is enhanced by deflagellation. To determine its role in flagellar assembly, we screened a collection of stumpy flagellar (stf) mutants generated by insertional mutagenesis and identified two strains in which portions of the cDhc1b gene have been deleted. The two mutants assemble short flagellar stumps (<1-2 micrometer) filled with aberrant microtubules, raft-like particles, and other amorphous material. The results indicate that cDhc1b is involved in the transport of components required for flagellar assembly in Chlamydomonas.

Ultrastructural pathology of the sperm flagellum: association between flagellar pathology and fertility prognosis in severely asthenozoospermic men.

An ultrastructural study of spermatozoa in a series of 247 severely asthenozoospermic patients disclosed two kinds of anomalies. The first was dysplasia of the fibrous sheath, a primary defect of spermatozoa with hypertrophy and hyperplasia of the fibrous sheath, associated axonemal anomalies, familial incidence and chronic respiratory disease. The patients could be divided into two subgroups: the complete form (all spermatozoa affected) and the incomplete form (alterations in 70-80% spermatozoa). There were no spontaneous or in-vitro fertilization (IVF) pregnancies. Intracytoplasmic sperm injection (ICSI) in six patients resulted in successful fertilizations, but only two pregnancies were obtained. These features configure a phenotype that suggests a genetic origin. The second anomaly was non-specific flagellar anomaly (NSFA), random secondary flagellar alterations affecting variable numbers of spermatozoa, without respiratory disease or familial incidence. 54 men with NSFA were followed for 2-6 years. Of these, 18 achieved conception, either spontaneous or by means of assisted fertilization, followed by 14 pregnancies and 12 live births. Their sperm motility significantly increased during the follow-up period. In the remaining 36 men motility did not change during the follow-up period and there were no fertilizations or pregnancies. We conclude that in severe asthenozoospermia, ultrastructural examination of spermatozoa has an effective prognostic value, identifying two syndromes with very different flagellar alterations and fertility potentials.

Structural heterogeneity of the axonemes of respiratory cilia and sperm flagella in normal men.

The ultrastructure of normal human cilia and flagella was examined and quantitatively assessed to determine the normal variations in the structure of the axoneme. Ciliated respiratory epithelial cells and spermatozoa from 10 normal, nonsmoking male volunteers who had normal semen parameters were fixed for electron microscopy. Tannic acid and MgSO4 were included during fixation to enhance, in particular, axonemal components. In 75 axonemal cross sections per sample, the number of outer doublet and central singlet microtubules, outer and inner dynein arms, and radial spokes were recorded. Statistical analysis of the results showed a marked reduction, from the expected value of nine, in the numbers of inner dynein arms (mean +/- SE, cilia, 5.31 +/- 0.13; sperm, 5.38 +/- 0.16) and radial spokes (cilia, 4.95 +/- 0.22; sperm, 5.80 +/- 0.19). The ideal axoneme with all its structural components was seen in only 0.13% of cilia and 0.80% of sperm tails. Significantly more doublet microtubules (P less than 0.05) and less central microtubules (P less than 0.01) and radial spokes (P less than 0.01) were seen in cilia than in sperm tail axonemes. Between subjects there was little variation in the mean number of a structure seen per axoneme. However, within each sample, the variation was considerably higher, particularly for the inner and outer dynein arms and radial spokes. The doublet microtubules had significantly greater standard deviations in the sperm tails compared with the cilia (P less than 0.01), and furthermore, a significantly greater number of sperm tails compared with cilia showed the incorrect number of doublet microtubules (P less than 0.02). In one semen sample, with normal semen analysis, 20% of the sperm tails showed incorrect numbers of doublet microtubules, ranging from 12 + 2 to 5 + 2 compared with only 1.3% in cilia from this subject. This study has demonstrated that the ideal axoneme is rarely seen even in normal samples, probably because of the technical difficulties in resolution and visualization, and stresses the need for thorough documentation of axonemal ultrastructure. This work provides a normal data base for comparison with patients who have chronic respiratory disease and suspected infertility.