Meriem Garfa-Traore

Mutations in TRAF3IP1/IFT54 reveal a new role for IFT proteins in microtubule stabilization

Ciliopathies are a large group of clinically and genetically heterogeneous disorders caused by defects in primary cilia. Here we identified mutations in TRAF3IP1 (TNF Receptor-Associated Factor Interacting Protein 1) in eight patients from five families with nephronophthisis (NPH) and retinal degeneration, two of the most common manifestations of ciliopathies. TRAF3IP1 encodes IFT54, a subunit of the IFT-B complex required for ciliogenesis. The identified mutations result in mild ciliary defects in patients but also reveal an unexpected role of IFT54 as a negative regulator of microtubule stability via MAP4 (microtubule-associated protein 4). Microtubule defects are associated with altered epithelialization/polarity in renal cells and with pronephric cysts and microphthalmia in zebrafish embryos. Our findings highlight the regulation of cytoplasmic microtubule dynamics as a role of the IFT54 protein beyond the cilium, contributing to the development of NPH-related ciliopathies.

Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging from a Hydrolethalus Phenotype to Short-Rib Polydactyly Syndrome

KIAA0586, the human ortholog of chicken TALPID3, is a centrosomal protein that is essential for primary ciliogenesis. Its disruption in animal models causes defects attributed to abnormal hedgehog signaling; these defects include polydactyly and abnormal dorsoventral patterning of the neural tube. Here, we report homozygous mutations of KIAA0586 in four families affected by lethal ciliopathies ranging from a hydrolethalus phenotype to short-rib polydactyly. We show defective ciliogenesis, as well as abnormal response to SHH-signaling activation in cells derived from affected individuals, consistent with a role of KIAA0586 in primary cilia biogenesis. Whereas centriolar maturation seemed unaffected in mutant cells, we observed an abnormal extended pattern of CEP290, a centriolar satellite protein previously associated with ciliopathies. Our data show the crucial role of KIAA0586 in human primary ciliogenesis and subsequent abnormal hedgehog signaling through abnormal GLI3 processing. Our results thus establish that KIAA0586 mutations cause lethal ciliopathies.

G-CSF mobilizes CD34+ regulatory monocytes that inhibit graft-versus-host disease

G-SCF–mobilized CD34+ monocytes inhibit graft-versus-host disease by the production of nitric oxide and the induction of regulatory T cells. Monocytes suppress GVHD Hematopoietic stem cell transplantation is a highly successful therapy used in patients with bone marrow dysfunction. However, when the stem cell donor cannot be matched to the recipient, the transplanted cells may attack the host in a process called graft-versus-host disease (GVHD). D’Aveni et al. now show that granulocyte colony-stimulating factor (G-CSF)–mobilized stem cells contain a previously uncharacterized population of immunosuppressive CD34+ cells transcriptionally similar to mature monocytes. These cells induce allogeneic T cell death in response to interferon-γ, resulting in regulatory T cell expansion and immunosuppression. The fraction of these CD34+ monocytes in peripheral blood inversely correlates with GVHD in patients, suggesting that expanding these cells before transplant may decrease the risk of GVHD. Granulocyte colony-stimulating factor (G-CSF) is routinely used to collect peripheral blood stem cells (PBSCs) from healthy donors for allogeneic hematopoietic stem cell transplantation (allo-HSCT). We show that, in both humans and mice, G-CSF mobilizes a subset of CD34+ cells with mature monocyte features. These cells, which are phenotypically and functionally conserved in mice and humans, are transcriptionally distinct from myeloid and monocytic precursors but similar to mature monocytes and endowed with immunosuppressive properties. In response to interferon-γ released by activated T cells, these cells produce nitric oxide, which induces allogeneic T cell death both in vitro and in vivo. These apoptotic T cells are engulfed by macrophages that release transforming growth factor–β and promote regulatory T cell expansion. Indeed, the fraction of CD34+ monocytes in peripheral blood CD34+ cells inversely correlates with the incidence of acute graft-versus-host disease (GVHD) in humans. Therefore, G-CSF–mobilized cells are an attractive candidate population to be expanded ex vivo for cellular therapy against GVHD.

Massive Diversification in Aging Colonies of Escherichia coli

The evolutionary success of bacteria depends greatly on their capacity to continually generate phenotypic diversity. Structured environments are particularly favorable for diversification because of attenuated clonal interference, which renders selective sweeps nearly impossible and enhances opportunities for adaptive radiation. We examined at the microscale level the emergence and the spatial and temporal dynamics of phenotypic diversity and their underlying causes in Escherichia coli colonies. An important dynamic heterogeneity in the growth, metabolic activity, morphology, gene expression patterns, stress response induction, and death patterns among cells within colonies was observed. Genetic analysis indicated that the phenotypic variation resulted mostly from mutations and that indole production, oxidative stress, and the RpoS-regulated general stress response played an important role in the generation of diversity. We observed the emergence and persistence of phenotypic variants within single colonies that exhibited variable fitness compared to the parental strain. Some variants showed improved capacity to produce biofilms, whereas others were able to use different nutrients or to tolerate antibiotics or oxidative stress. Taken together, our data show that bacterial colonies provide an ecological opportunity for the generation and maintenance of vast phenotypic diversity, which may increase the probability of population survival in unpredictable environments.

IFT81 , encoding an IFT-B core protein, as a very rare cause of a ciliopathy phenotype

Background Bidirectional intraflagellar transport (IFT) consists of two major protein complexes, IFT-A and IFT-B. In contrast to the IFT-B complex, all components of IFT-A have recently been linked to human ciliopathies when defective. We therefore hypothesised that mutations in additional IFT-B encoding genes can be found in patients with multisystemic ciliopathies. Methods We screened 1628 individuals with reno-ocular ciliopathies by targeted next-generation sequencing of ciliary candidate genes, including all IFT-B encoding genes. Results Consequently, we identified a homozygous mutation in IFT81 affecting an obligatory donor splice site in an individual with nephronophthisis and polydactyly. Further, we detected a loss-of-stop mutation with extension of the deduced protein by 10 amino acids in an individual with neuronal ceroid lipofuscinosis-1. This proband presented with retinal dystrophy and brain lesions including cerebellar atrophy, a phenotype to which the IFT81 variant might contribute. Cultured fibroblasts of this latter affected individual showed a significant decrease in ciliated cell abundance compared with controls and increased expression of the transcription factor GLI2 suggesting deranged sonic hedgehog signalling. Conclusions This work describes identification of mutations of IFT81 in individuals with symptoms consistent with the clinical spectrum of ciliopathies. It might represent the rare case of a core IFT-B complex protein found associated with human disease. Our data further suggest that defects in the IFT-B core are an exceedingly rare finding, probably due to its indispensable role for ciliary assembly in development.

Human Fucci Pancreatic Beta Cell Lines: New Tools to Study Beta Cell Cycle and Terminal Differentiation

Regulation of cell cycle in beta cells is poorly understood, especially in humans. We exploited here the recently described human pancreatic beta cell line EndoC-βH2 to set up experimental systems for cell cycle studies. We derived 2 populations from EndoC-βH2 cells that stably harbor the 2 genes encoding the Fucci fluorescent indicators of cell cycle, either from two vectors, or from a unique bicistronic vector. In proliferating non-synchronized cells, the 2 Fucci indicators revealed cells in the expected phases of cell cycle, with orange and green cells being in G1 and S/G2/M cells, respectively, and allowed the sorting of cells in different substeps of G1. The Fucci indicators also faithfully red out alterations in human beta cell proliferative activity since a mitogen-rich medium decreased the proportion of orange cells and inflated the green population, while reciprocal changes were observed when cells were induced to cease proliferation and increased expression of some beta cell genes. In the last situation, acquisition of a more differentiated beta cell phenotype correlates with an increased intensity in orange fluorescence. Hence Fucci beta cell lines provide new tools to address important questions regarding human beta cell cycle and differentiation.

IRAP+ endosomes restrict TLR9 activation and signaling

The retention of intracellular Toll-like receptors (TLRs) in the endoplasmic reticulum prevents their activation under basal conditions. TLR9 is activated by sensing ligands in specific endosomal-lysosomal compartments. Here we identified IRAP+ endosomes as major cellular compartments for the early steps of TLR9 activation in dendritic cells (DCs). Both TLR9 and its ligand, the dinucleotide CpG, were present as cargo in IRAP+ endosomes. In the absence of the aminopeptidase IRAP, the trafficking of CpG and TLR9 to lysosomes and signaling via TLR9 were enhanced in DCs and in mice following bacterial infection. IRAP stabilized CpG-containing endosomes by interacting with the actin-nucleation factor FHOD4, which slowed the trafficking of TLR9 toward lysosomes. Thus, endosomal retention of TLR9 via the interaction of IRAP with the actin cytoskeleton is a mechanism that prevents hyper-activation of TLR9 in DCs.

UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in dendritic cells

Dendritic cells (DC) have the unique ability to present exogenous antigens via the major histocompatibility complex class I pathway to stimulate naive CD8+ T cells. In DCs with a non-functional mutation in Unc93b1 (3d mutation), endosomal acidification, phagosomal maturation, antigen degradation, antigen export to the cytosol and the function of the store-operated-Ca2+-entry regulator STIM1 are impaired. These defects result in compromised antigen cross-presentation and anti-tumor responses in 3d-mutated mice. Here, we show that UNC93B1 interacts with the calcium sensor STIM1 in the endoplasmic reticulum, a critical step for STIM1 oligomerization and activation. Expression of a constitutively active STIM1 mutant, which no longer binds UNC93B1, restores antigen degradation and cross-presentation in 3d-mutated DCs. Furthermore, ablation of STIM1 in mouse and human cells leads to a decrease in cross-presentation. Our data indicate that the UNC93B1 and STIM1 cooperation is important for calcium flux and antigen cross-presentation in DCs.STIM proteins sense Ca2+ depletion in the ER and activate store-operated Ca2+ entry in response, a process associated with dendritic cell (DC) functions. Here, the authors show that optimal antigen cross-presentation in DCs requires the association of the chaperone molecule UNC93B1 with STIM1.

Ependymal cilia beating induces an actin network to protect centrioles against shear stress

Multiciliated ependymal cells line all brain cavities. The beating of their motile cilia contributes to the flow of cerebrospinal fluid, which is required for brain homoeostasis and functions. Motile cilia, nucleated from centrioles, persist once formed and withstand the forces produced by the external fluid flow and by their own cilia beating. Here, we show that a dense actin network around the centrioles is induced by cilia beating, as shown by the disorganisation of the actin network upon impairment of cilia motility. Moreover, disruption of the actin network, or specifically of the apical actin network, causes motile cilia and their centrioles to detach from the apical surface of ependymal cell. In conclusion, cilia beating controls the apical actin network around centrioles; the mechanical resistance of this actin network contributes, in turn, to centriole stability.Ependymal ciliary beating contributes to the flow of cerebrospinal fluid in the brain ventricles and these cilia resist the flow forces. Here the authors show that the assembly of a dense actin network around the centrioles is induced by cilia beating to protect centrioles against the shear stress generated by ciliary motility.

WDR81 mutations cause extreme microcephaly and impair mitotic progression in human fibroblasts and Drosophila neural stem cells

Microlissencephaly is a rare brain malformation characterized by congenital microcephaly and lissencephaly. Microlissencephaly is suspected to result from abnormalities in the proliferation or survival of neural progenitors. Despite the recent identification of six genes involved in microlissencephaly, the pathophysiological basis of this condition remains poorly understood. We performed trio-based whole exome sequencing in seven subjects from five non-consanguineous families who presented with either microcephaly or microlissencephaly. This led to the identification of compound heterozygous mutations in WDR81, a gene previously associated with cerebellar ataxia, intellectual disability and quadrupedal locomotion. Patient phenotypes ranged from severe microcephaly with extremely reduced gyration with pontocerebellar hypoplasia to moderate microcephaly with cerebellar atrophy. In patient fibroblast cells, WDR81 mutations were associated with increased mitotic index and delayed prometaphase/metaphase transition. Similarly, in vivo, we showed that knockdown of the WDR81 orthologue in Drosophila led to increased mitotic index of neural stem cells with delayed mitotic progression. In summary, we highlight the broad phenotypic spectrum of WDR81-related brain malformations, which include microcephaly with moderate to extremely reduced gyration and cerebellar anomalies. Our results suggest that WDR81 might have a role in mitosis that is conserved between Drosophila and humans.