Base Of Cilium Research Articles

TAOK2 Drives Opposing Cilia Length Deficits in 16p11.2 Deletion and Duplication Carriers.

Copy number variation (CNV) in the 16p11.2 (BP4-BP5) genomic locus is strongly associated with autism. Carriers of 16p11.2 deletion and duplication exhibit several common behavioral and social impairments, yet, show opposing brain structural changes and body mass index. To determine cellular mechanisms that might contribute to these opposing phenotypes, we performed quantitative tandem mass tag (TMT) proteomics on human dorsal forebrain neural progenitor cells (NPCs) differentiated from induced pluripotent stem cells (iPSC) derived from 16p11.2 CNV carriers. Differentially phosphorylated proteins between unaffected individuals and 16p11.2 CNV carriers were significantly enriched for centrosomal and cilia proteins. Deletion patient-derived NPCs show increased primary cilium length compared to unaffected individuals, while stunted cilium growth was observed in 16p11.2 duplication NPCs. Through cellular shRNA and overexpression screens in human iPSC derived NPCs, we determined the contribution of genes within the 16p11.2 locus to cilium length. TAOK2, a serine threonine protein kinase, and PPP4C, a protein phosphatase, were found to regulate primary cilia length in a gene dosage-dependent manner. We found TAOK2 was localized at centrosomes and the base of the primary cilium, and NPCs differentiated from TAOK2 knockout iPSCs had longer cilia. In absence of TAOK2, there was increased pericentrin at the basal body, and aberrant accumulation of IFT88 at the ciliary distal tip. Further, pharmacological inhibition of TAO kinase activity led to increased ciliary length, indicating that TAOK2 negatively controls primary cilium length through its catalytic activity. These results implicate aberrant cilia length in the pathophysiology of 16p11.2 CNV, and establish the role of TAOK2 kinase as a regulator of primary cilium length.

Regulation of adipose progenitors and fibro-adipogenic progenitors through primary cilia

The primary cilium, an antenna-like structure of cell membrane, detects various signals and regulates cellular functions such as proliferation and differentiation. The impairment of primary cilium is associated with the etiologies of diseases including cancer, obesity, and congenital anomalies. In this review, novel functions of trichoplein, a suppressor of ciliogenesis, on the regulation of adipose progenitors and fibro-adipogenic progenitors are focused. Trichoplein-knockout mice show resistance to high-fat diet-induced obesity and accelerated regeneration after skeletal muscle injury. The primary cilia of adipose progenitors from trichoplein-knockout mice are elongated, leading to the inhibitions of the accumulation of lipid raft to the base of primary cilia and the phosphorylation of AKT. The primary cilia of fibro-adipogenic progenitors from trichoplein-knockout mice are also elongated, causing the increased expression of IL-13 through IL-33 receptor signaling. These mechanisms are involved in the resistance to diet-induced obesity and improved regeneration. These findings suggest that targeting the primary cilia of specific cells may be a novel therapeutic approach through modulating cellular functions.

A cilia-bound unconventional secretory pathway for Drosophila odorant receptors

BackgroundPost-translational transport is a vital process which ensures that each protein reaches its site of function. Though most do so via an ordered ER-to-Golgi route, an increasing number of proteins are now shown to bypass this conventional secretory pathway.ResultsIn the Drosophila olfactory sensory neurons (OSNs), odorant receptors (ORs) are trafficked from the ER towards the cilia. Here, we show that Or22a, a receptor of various esters and alcoholic compounds, reaches the cilia partially through unconventional means. Or22a frequently present as puncta at the somatic cell body exit and within the dendrite prior to the cilia base. These rarely coincide with markers of either the intermediary ER-Golgi-intermediate-compartment (ERGIC) or Golgi structures. ERGIC and Golgi also displayed axonal localization biases, a further indication that at least some measure of OR transport may occur independently of their involvement. Additionally, neither the loss of several COPII genes involved in anterograde trafficking nor ERGIC itself affected puncta formation or Or22a transport to the cilium. Instead, we observed the consistent colocalization of Or22a puncta with Grasp65, the sole Drosophila homolog of mammalian GRASP55/Grh1, a marker of the unconventional pathway. The numbers of both Or22a and Grasp65-positive puncta were furthermore increased upon nutritional starvation, a condition known to enhance Golgi-bypassing secretory activity.ConclusionsOur results demonstrate an alternative route of Or22a transport, thus expanding the repertoire of unconventional secretion mechanisms in neurons.

INPP5E Regulates the Distribution of Phospholipids on Cilia in RPE1 Cells.

Primary cilia are static microtubule-based structures protruding from the cell surface and present on most vertebrate cells. The appropriate localization of phospholipids is essential for cilia formation and stability. INPP5E is a cilia-localized inositol 5-phosphatase; its deletion alters the phosphoinositide composition in the ciliary membrane, disrupting ciliary function. The EGFP-2xP4MSidM, PHPLCδ1-EGFP, and SMO-tRFP plasmids were constructed by the Gateway system to establish a stable RPE1 cell line. The INPP5E KO RPE1 cell line was constructed with the CRISPR/Cas9 system. The localization of INPP5E and the distribution of PI(4,5)P2 and PI4P were examined by immunofluorescence microscopy. The fluorescence intensity co-localized with cilia was quantified by ImageJ. In RPE1 cells, PI4P is localized at the ciliary membrane, whereas PI(4,5)P2 is localized at the base of cilia. Knocking down or knocking out INPP5E alters this distribution, resulting in the distribution of PI(4,5)P2 along the ciliary membrane and the disappearance of PI4P from the cilia. Meanwhile, PI(4,5)P2 is located in the ciliary membrane labeled by SMO-tRFP. INPP5E regulates the distribution of phosphoinositide on cilia. PI(4,5)P2 localizes at the ciliary membrane labeled with SMO-tRFP, indicating that ciliary pocket membrane contains PI(4,5)P2, and phosphoinositide composition in early membrane structures may differ from that in mature ciliary membrane.

The CPLANE protein Fuzzy regulates ciliogenesis by suppressing actin polymerization at the base of the primary cilium via p190A RhoGAP.

The primary cilium decorates most eukaryotic cells and regulates tissue morphogenesis and maintenance. Structural or functional defects of primary cilium result in ciliopathies, congenital human disorders affecting multiple organs. Pathogenic variants in the ciliogenesis and planar cell polarity effectors (CPLANE) genes FUZZY, INTU and WDPCP disturb ciliogenesis, causing severe ciliopathies in humans and mice. Here, we show that the loss of Fuzzy in mice results in defects of primary cilia, accompanied by increased RhoA activity and excessive actin polymerization at the basal body. We discovered that, mechanistically, Fuzzy interacts with and recruits the negative actin regulator ARHGAP35 (also known as p190A RhoGAP) to the basal body. We identified genetic interactions between the two genes and found that a mutant ArhGAP35 allele increases the severity of phenotypic defects observed in Fuzzy-/- mice. Based on our findings, we propose that Fuzzy regulates ciliogenesis by recruiting ARHGAP35 to the basal body, where the latter likely restricts actin polymerization and modifies the actin network. Our study identifies a mechanism whereby CPLANE proteins control both actin polymerization and primary cilium formation.

Early insights into the role of Exoc6B associated with spondyloepimetaphyseal dysplasia with joint laxity type 3 in primary ciliogenesis and chondrogenic differentiation in vitro.

Spondyloepimetaphyseal dysplasia with joint laxity type 3 (SEMDJL3) is a rare skeletal dysplasia associated with EXOC6B, a component of the exocyst complex, involved in vesicle tethering and exocytosis at the plasma membrane. So far, EXOC6B and the pathomechanisms underlying SEMDJL3 remain obscure. Exoc6b was detected largely at the perinuclear regions and the primary cilia base in ATDC5 prechondrocytes. Its shRNA lentiviral knockdown impeded primary ciliogenesis. In Exoc6b silenced prechondrocytes, Hedgehog signaling was attenuated, including when stimulated with Smoothened agonist. Exoc6b knockdown deregulated the mRNA and protein levels of Col2a1, a marker of chondrocyte proliferation at 7- and 14-days following differentiation. It led to the upregulation of Ihh another marker of proliferative chondrocytes. The levels of Col10a1, a marker of chondrocyte hypertrophy was enhanced at 14 days of differentiation. Congruently, Axin2, a canonical Wnt pathway modulator that inhibits chondrocyte hypertrophy was repressed. The expression of Mmp13 and Adamts4 that are terminal chondrocyte hypertrophy markers involved in extracellular matrix (ECM) remodelling were downregulated at 7 and 14 days of chondrogenesis. Bglap that encodes for the most abundant non-collagenous bone matrix constituent and promotesECM calcification was suppressed at 14 days of chondrocyte differentiation. ECM mineralization was assessed by Alizarin Red staining. Gene expression and ciliogenesis were investigated by reverse transcription quantitative real-time PCR, immunoblotting, and immunocytochemistry. These findings provide initial insights into the potential role of Exoc6b in primary ciliogenesis and chondrogenic differentiation, contributing towards a preliminary understanding of the molecular pathomechanisms underlying SEMDJL3.

TMEM216 promotes primary ciliogenesis and Hedgehog signaling through the SUFU-GLI2/GLI3 axis.

Primary cilia are enriched in signaling receptors, and defects in their formation or function can induce conditions such as polycystic kidney disease, postaxial hexadactyly, and microphthalmia. Mammalian Hedgehog (Hh) signaling is important in the development of primary cilia, and TMEM216, a transmembrane protein that localizes to the base of cilia, is also implicated in ciliogenesis in zebrafish. Here, we found that Tmem216-deficient mice had impaired Hh signaling and displayed typical ciliopathic phenotypes. These phenomena were also observed in cells deficient in TMEM216. Furthermore, TMEM216 interacted with core Hh signaling proteins, including SUFU, a negative regulator of Hh, and GLI2/GLI3, transcription factors downstream of Hh. The competition between TMEM216 and SUFU for binding to GLI2/GLI3 inhibited the cleavage of GLI2/GLI3 into their repressor forms, which resulted in the nuclear accumulation of full-length GLI2 and the decreased nuclear localization of cleaved GLI3, ultimately leading to the activation of Hh signaling. Together, these data suggest that the TMEM216-SUFU-GLI2/GLI3 axis plays a role in TMEM216 deficiency-induced ciliopathies and Hh signaling abnormalities.

Primary cilia and actin regulatory pathways in renal ciliopathies.

Ciliopathies are a group of rare genetic disorders caused by defects to the structure or function of the primary cilium. They often affect multiple organs, leading to brain malformations, congenital heart defects, and anomalies of the retina or skeletal system. Kidney abnormalities are among the most frequent ciliopathic phenotypes manifesting as smaller, dysplastic, and cystic kidneys that are often accompanied by renal fibrosis. Many renal ciliopathies cause chronic kidney disease and often progress to end-stage renal disease, necessitating replacing therapies. There are more than 35 known ciliopathies; each is a rare hereditary condition, yet collectively they account for a significant proportion of chronic kidney disease worldwide. The primary cilium is a tiny microtubule-based organelle at the apex of almost all vertebrate cells. It serves as a "cellular antenna" surveying environment outside the cell and transducing this information inside the cell to trigger multiple signaling responses crucial for tissue morphogenesis and homeostasis. Hundreds of proteins and unique cellular mechanisms are involved in cilia formation. Recent evidence suggests that actin remodeling and regulation at the base of the primary cilium strongly impacts ciliogenesis. In this review, we provide an overview of the structure and function of the primary cilium, focusing on the role of actin cytoskeleton and its regulators in ciliogenesis. We then describe the key clinical, genetic, and molecular aspects of renal ciliopathies. We highlight what is known about actin regulation in the pathogenesis of these diseases with the aim to consider these recent molecular findings as potential therapeutic targets for renal ciliopathies.

SHCBP1 Overexpression Aggravates Pancreatitis by Triggering the Loss of Primary Cilia.

Primary cilia are microtubule-based organelles that mediate various biological processes. Pancreatic cells are typically ciliated; however, the role of primary cilia in acute pancreatitis (AP) is largely unknown. Here, we report that the loss of primary cilia, mediated by SHCBP1 (SHC1 binding protein), exerted a provocative effect on AP. Primary cilia are extensively lost in inflamed pancreatic cells in vitro and in mouse tissues with AP in vivo. Abrogation of primary cilia aggravated lipopolysaccharide (LPS)-induced inflammation in pancreatic cells. Mechanistically, AP induced the overexpression of SHCBP1 mitotic factor, which is localized to the base of primary cilia. SHCBP1 deficiency relieved LPS- and cerulein-induced pancreatitis by preventing the loss of primary cilia in vitro and in vivo. Collectively, we reveal that inflammation-induced loss of primary cilia aggravates AP. Furthermore, abrogating SHCBP1 to prevent primary cilia loss is an efficient strategy to combat AP.

The evolution of centriole degradation in mouse sperm

Centrioles are subcellular organelles found at the cilia base with an evolutionarily conserved structure and a shock absorber-like function. In sperm, centrioles are found at the flagellum base and are essential for embryo development in basal animals. Yet, sperm centrioles have evolved diverse forms, sometimes acting like a transmission system, as in cattle, and sometimes becoming dispensable, as in house mice. How the essential sperm centriole evolved to become dispensable in some organisms is unclear. Here, we test the hypothesis that this transition occurred through a cascade of evolutionary changes to the proteins, structure, and function of sperm centrioles and was possibly driven by sperm competition. We found that the final steps in this cascade are associated with a change in the primary structure of the centriolar inner scaffold protein FAM161A in rodents. This information provides the first insight into the molecular mechanisms and adaptive evolution underlying a major evolutionary transition within the internal structure of the mammalian sperm neck.

TTLL12 is required for primary ciliary axoneme formation in polarized epithelial cells

The primary cilium is a critical sensory organelle that is built of axonemal microtubules ensheathed by a ciliary membrane. In polarized epithelial cells, primary cilia reside on the apical surface and must extend these microtubules directly into the extracellular space and remain a stable structure. However, the factors regulating cross-talk between ciliation and cell polarization, as well as axonemal microtubule growth and stabilization in polarized epithelia, are not fully understood. In this study, we find TTLL12, a previously uncharacterized member of the Tubulin Tyrosine Ligase-Like (TTLL) family, localizes to the base of primary cilia and is required for cilia formation in polarized renal epithelial cells. We also show that TTLL12 directly binds to the α/β-tubulin heterodimer in vitro and regulates microtubule dynamics, stability, and post-translational modifications (PTMs). While all other TTLLs catalyze the addition of glutamate or glycine to microtubule C-terminal tails, TTLL12 uniquely affects tubulin PTMs by promoting both microtubule lysine acetylation and arginine methylation. Together, this work identifies a novel microtubule regulator and provides insight into the requirements for apical extracellular axoneme formation.

GPRC5C regulates the composition of cilia in the olfactory system

BackgroundOlfactory sensory neurons detect odourants via multiple long cilia that protrude from their dendritic endings. The G protein-coupled receptor GPRC5C was identified as part of the olfactory ciliary membrane proteome, but its function and localization is unknown.ResultsHigh-resolution confocal and electron microscopy revealed that GPRC5C is located at the base of sensory cilia in olfactory neurons, but not in primary cilia of immature neurons or stem cells. Additionally, GPRC5C localization in sensory cilia parallels cilia formation and follows the formation of the basal body. In closer examination, GPRC5C was found in the ciliary transition zone. GPRC5C deficiency altered the structure of sensory cilia and increased ciliary layer thickness. However, primary cilia were unaffected. Olfactory sensory neurons from Gprc5c-deficient mice exhibited altered localization of olfactory signalling cascade proteins, and of ciliary phosphatidylinositol-4,5-bisphosphat. Sensory neurons also exhibited increased neuronal activity as well as altered mitochondrial morphology, and knockout mice had an improved ability to detect food pellets based on smell.ConclusionsOur study shows that GPRC5C regulates olfactory cilia composition and length, thereby controlling odour perception.

Circadian oscillation in primary cilium length by clock genes regulates fibroblast cell migration.

Various mammalian cells have autonomous cellular clocks that are produced by the transcriptional cycle of clock genes. Cellular clocks provide circadian rhythms for cellular functions via transcriptional and cytoskeletal regulation. The vast majority of mammalian cells possess a primary cilium, an organelle protruding from the cell surface. Here, we investigated the little-known relationship between circadian rhythm and primary cilia. The length and number of primary cilia showed circadian dynamics both in vitro and in vivo. The circadian rhythm of primary cilium length was abolished by SR9011 and Bmal1 knockout. A centrosomal protein, pericentrin, transiently accumulates in centriolar satellites, the base of primary cilia at the shortest cilia phase, and induces elongation of primary ciliaat the longest cilia phase in the circadian rhythm of primary cilia. In addition, rhythmic cell migration during wound healing depends on the length of primary cilia and affects the rate of wound healing. Our findings demonstrate that the circadian dynamics of primary cilium length by clock genes control fibroblast migration and could provide new insights into chronobiology.

Enteric Nervous System: Identification of a Novel Neuronal Sensory Network in the Duodenal Epithelium.

The communication between the intestinal epithelium and the enteric nervous system has been considered indirect. Mechanical or chemical stimuli activate enteroendocrine cells inducing hormone secretion, which act on sub-epithelial nerve ends, activating the enteric nervous system. However, we identified an epithelial cell that expresses NKAIN4, a neuronal protein associated with the β-subunit of Na+/K+-ATPase. This cell overexpresses Na+/K+-ATPase and ouabain-insensitive Na+-ATPase, enzymes involved in active sodium transport. NKAIN4-positive cells also express neuronal markers as NeuN, acetylcholine-esterase, acetylcholine-transferase, α3- and α7-subunits of ACh receptors, glutamic-decarboxylase, and serotonin-receptor-7, suggesting they are neurons. NKAIN4-positive cells show a polarized shape with an oval body, an apical process finished in a knob-like terminal in contact with the lumen, a basal cilia body at the base of the apical extension, and basal axon-like soma projections connecting sub-epithelial nerve terminals, lymphoid nodules, glial cells, and enterochromaffin cells, forming a network that reaches the epithelial surface. We also showed, using retrograde labeling and immunofluorescence, that these cells receive afferent signals from the enteric nervous system. Finally, we demonstrated that acetylcholine activates NKAIN4-positive cells inducing Ca2+ mobilization and probably serotonin secretion in enterochromaffin cells. NKAIN4-positive cells are neurons that would form a part of a duodenal sensory network for physiological or noxious luminal stimuli.

Damaged insect ciliated neurons regrow axons as effectively as other neurons

Most animal neurons pick up information through branching structures, called dendrites, before sending sensory signals to the neuron's axon, which then passes the signal to the central nervous system for processing. But not all neurons are constructed this way. The neurons of many invertebrates pick up sensory signals through slender structures called cilia, which can communicate the senses of vision, smell and body position. While the damaged axons of conventional neurons are capable of regeneration, no one knew whether injured ciliated neuron axons could recover. As Drosophila larvae are packed full of sensory ciliated neurons and the insects come complete with a box of molecular tools – which allows researchers to answer specific physiological questions – Melissa Rolls and her colleagues, Michelle Stone and Abigail Mauger, all from Pennsylvania State University, USA, decided to find out whether the severed axons of Drosophila larva ciliated neurons could recover and, if so, how.But first the trio had to select the ideal type of neuron for their experiment. Realising that they would require one that was close to the surface of the larva's body, so they could see it with ease, they selected the lateral monoscolopidial chordotonal organ (lch1), which projects cilia into the side of the larva and detects stretching as it moves. Then, the team severed several lch1 axons with a high-precision UV laser, to be sure that the neuron could survive the injury. Fortunately, 24 h after the procedure, the neurons were still alive. Cutting them had not been fatal, but could they regrow a new axon?Cutting the lch1 axons of 33 larvae near the tip – 70 μm along the length of each – the team was impressed to see that more than half of the severed axon stumps regrew, with some re-joining the same bundle of nerves, while others joined a different nerve nearby, just like the regrowing axons of neurons with dendrites. But what would happen if a ciliated neuron's axon was removed entirely, after being severed close to the cell body at the heart of the neuron? Could the ciliated neuron recover from this more severe damage?Impressively, yes. Almost all of the severed ciliated neurons began sprouting new structures called neurites – which can eventually grow into axons – 96 h later and they did this from different locations on the neuron, including the cell body, the remaining axon stump and even the base of the sensory cilium. The team admits that this was surprising, as conventional neurons regrow axons from the base of dendrites. In addition, newly grown axons from neurons with dendrites follow the path of other axons and are unbranched, whereas the ciliated neuron's regrown neurites were branched. And when the team checked whether the ciliated neurons were using the same cellular mechanism as conventional neurons to trigger neurite growth, they found they were, even though the resulting neurite growth looked so different from conventional axon regrowth.But would the new neurites of ciliated neurons eventually develop into axons or some other cellular component, such as a dendrite? Checking for signs that the neurites were destined to become axons, the team found strong evidence that they were, although some of the branching structures also had the characteristics of dendrites.Having suspected that insect ciliated neurons wouldn't be capable of regrowing damaged axons as effectively as conventional neurons, Rolls and her colleagues were impressed by the cell's versatility. Not only can they regenerate completely severed axons, but they do so using the same mechanism as damaged conventional neurons to successfully rebuild injured insect sensory systems.

Nephron-specific deletion of ALMS1 (Alstrom syndrome 1) increases surface NKCC2 expression and NaCl reabsorption in the Thick Ascending Limb

Mutations in the ALMS1 gene in humans cause Alström syndrome, characterized by progressive metabolic alterations that include obesity, hypertension, and chronic kidney disease (CKD). SNPs in the ALMS1 gene in the general population are associated with lower GFR, hypertension and metabolic syndrome. ALMS1 is widely expressed where it localizes to endosomes, centrosomes and the base of cilia. Our lab has previously shown that ALMS1 is expressed in renal tubules of the Thick Ascending Limb (TAL) where NKCC2 is the primary apical NaCl transporter. Whole animal gene deletion of ALMS1 increases blood pressure, and inhibits NKCC2 endocytosis, increasing its surface expression and renal NaCl reabsorption. However, whole animal ALMS1 KO rats or mice develop obesity, affecting the interpretation of organ-specific data. We hypothesized that ALMS1 is a part of protein complex that binds apical NKCC2 in TALs and promotes its endocytosis. To study the role of ALMS1 in the nephron without influence of obesity we generated a loxP flanked exon 7 ALMS1 transgenic mouse line, that we crossed with nephron specific inducible Pax8-rtTA Cre mice (Pax8-ALMS1 KO). 4 weeks after inducing Cre expression with doxycycline (2mg/ml in 2% sucrose), ALMS1 expression in TAL suspensions decreased by 75% (p<0.01). To study the role of ALMS1 in TAL function, we measured surface to total NKCC2 ratio, which was increased by 53±15% in TALs from Pax8-ALMS1 KO compared to control floxed ALMS1 (p<0.01). Recombinant ALMS1 (GST-Carboxyl terminus-ALMS1), or immunoprecipitation of ALMS1, pulled down NKCC2 from WT TAL lysates (n=3), suggesting that the mechanism by which ALMS1 regulates surface NKCC2 involves protein-protein interactions. 4 weeks after inducing Cre expression with doxycycline we measured bumetanide-induced natriuresis (4h) as an index of NKCC2-mediated NaCl absorption. We found that bumetanide induced UNa excretion was higher in Pax8-ALMS1 KO, compared to control (control: 115±19 vs Pax8-ALMS1 KO: 146±30 μmols Na/4h, p<0.05). Since whole animal ALMS1 deletion induced hypertension, we measured systolic blood pressure (SBP) by tail cuff one month after inducible deletion of ALMS1 or in floxed ALMS1 mice treated with doxycycline as controls. Baseline SBP in normal Na diet (0.4%) was similar between strains. However, SBP increased by 12±3 mmHg in Pax8-ALMS1 KO, but not in control floxed ALMS1 littermates after 9 days of high Na diet (WT: 100±10, Pax8-ALMS1 KO: 115±11 mmHg, p<0.05 from baseline, n=4). We conclude that ALMS1, binds NKCC2 to regulate its surface expression. Depletion of ALMS1 in the nephron enhances surface NKCC2, TAL NaCl reabsorption, and induces salt-sensitivity of blood pressure. Our data indicate that regulation of the ALMS1-NKCC2 interaction in the TAL is important for renal NaCl reabsorption and regulation of blood pressure. Funding: NIH R56DK131114, Henry Ford Fund This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Bacterial TLR2/6 Ligands Block Ciliogenesis, Derepress Hedgehog Signaling, and Expand the Neocortex.

Microbial components have a range of direct effects on the fetal brain. However, little is known about the cellular targets and molecular mechanisms that mediate these effects. Neural progenitor cells (NPCs) control the size and architecture of the brain and understanding the mechanisms regulating NPCs is crucial to understanding brain developmental disorders. We identify ventricular radial glia (vRG), the primary NPC, as the target of bacterial cell wall (BCW) generated during the antibiotic treatment of maternal pneumonia. BCW enhanced proliferative potential of vRGs by shortening the cell cycle and increasing self-renewal. Expanded vRGs propagated to increase neuronal output in all cortical layers. Remarkably, Toll-like receptor 2 (TLR2), which recognizes BCW, localized at the base of primary cilia in vRGs and the BCW-TLR2 interaction suppressed ciliogenesis leading to derepression of Hedgehog (HH) signaling and expansion of vRGs. We also show that TLR6 is an essential partner of TLR2 in this process. Surprisingly, TLR6 alone was required to set the number of cortical neurons under healthy conditions. These findings suggest that an endogenous signal from TLRs suppresses cortical expansion during normal development of the neocortex and that BCW antagonizes that signal through the TLR2/cilia/HH signaling axis changing brain structure and function. IMPORTANCE Fetal brain development in early gestation can be impacted by transplacental infection, altered metabolites from the maternal microbiome, or maternal immune activation. It is less well understood how maternal microbial subcomponents that cross the placenta, such as bacterial cell wall (BCW), directly interact with fetal neural progenitors and neurons and affect development. This scenario plays out in the clinic when BCW debris released during antibiotic therapy of maternal infection traffics to the fetal brain. This study identifies the direct interaction of BCW with TLR2/6 present on the primary cilium, the signaling hub on fetal neural progenitor cells (NPCs). NPCs control the size and architecture of the brain and understanding the mechanisms regulating NPCs is crucial to understanding brain developmental disorders. Within a window of vulnerability before the appearance of fetal immune cells, the BCW-TLR2/6 interaction results in the inhibition of ciliogenesis, derepression of Sonic Hedgehog signaling, excess proliferation of neural progenitors, and abnormal cortical architecture. In the first example of TLR signaling linked to Sonic Hedgehog, BCW/TLR2/6 appears to act during fetal brain morphogenesis to play a role in setting the total cell number in the neocortex.

Septin-mediated RhoA activation engages the exocyst complex to recruit the cilium transition zone.

Septins are filamentous GTPases that play important but poorly characterized roles in ciliogenesis. Here, we show that SEPTIN9 regulates RhoA signaling at the base of cilia by binding and activating the RhoA guanine nucleotide exchange factor, ARHGEF18. GTP-RhoA is known to activate the membrane targeting exocyst complex, and suppression of SEPTIN9 causes disruption of ciliogenesis and mislocalization of an exocyst subunit, SEC8. Using basal body-targeted proteins, we show that upregulating RhoA signaling at the cilium can rescue ciliary defects and mislocalization of SEC8 caused by global SEPTIN9 depletion. Moreover, we demonstrate that the transition zone components, RPGRIP1L and TCTN2, fail to accumulate at the transition zone in cells lacking SEPTIN9 or depleted of the exocyst complex. Thus, SEPTIN9 regulates the recruitment of transition zone proteins on Golgi-derived vesicles by activating the exocyst via RhoA to allow the formation of primary cilia.

Primary cilia sense glutamine availability and respond via asparagine synthetase

Depriving cells of nutrients triggers an energetic crisis, which is resolved by metabolic rewiring and organelle reorganization. Primary cilia are microtubule-based organelles at the cell surface, capable of integrating multiple metabolic and signalling cues, but their precise sensory function is not fully understood. Here we show that primary cilia respond to nutrient availability and adjust their length via glutamine-mediated anaplerosis facilitated by asparagine synthetase (ASNS). Nutrient deprivation causes cilia elongation, mediated by reduced mitochondrial function, ATP availability and AMPK activation independently of mTORC1. Of note, glutamine removal and replenishment is necessary and sufficient to induce ciliary elongation or retraction, respectively, under nutrient stress conditions both in vivo and in vitro by restoring mitochondrial anaplerosis via ASNS-dependent glutamate generation. Ift88-mutant cells lacking cilia show reduced glutamine-dependent mitochondrial anaplerosis during metabolic stress, due to reduced expression and activity of ASNS at the base of cilia. Our data indicate a role for cilia in responding to, and possibly sensing, cellular glutamine levels via ASNS during metabolic stress.

A role for Shugoshin in human cilia?

We have recently described a novel role for the conserved centromeric/kinetochore protein and cohesin protector, Shugoshin, in cilia of C. elegans. Worms are unusual in that the sole Shugoshin protein ( SGO-1 ) is dispensable for chromosome segregation but required for cilia function in fully differentiated sensory neurons. Depletion of sgo-1 leads to an array of sensory defects observed in other cilia mutants with a compromised diffusion barrier. Accordingly, SGO-1 loads to the base of cilia in sensory neurons and can be observed occupying the transition zone, the critical ciliary domain that regulates trafficking in and out of ciliary compartments. Here we start to address a potential conserved role in cilia for vertebrate Shugoshin by asking whether human Shugoshin can: (1) localize to cilia and (2) rescue defects due to Shugoshin depletion in C. elegans . Our preliminary results suggest that human Shugoshin is detectable in the cilia base but show limited functional conservation when expressed in C. elegans sensory neurons.