CPAP promotes timely cilium disassembly to maintain neural progenitor pool

A mutation in the centrosomal‐P4.1‐associated protein (CPAP) causes Seckel syndrome with microcephaly, which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However, mechanisms of NPCs maintenance remain unclear. Here, we report an unexpected role for the cilium in NPCs maintenance and identify CPAP as a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly, CPAP provides a scaffold for the cilium disassembly complex (CDC), which includes Nde1, Aurora A, and OFD1, recruited to the ciliary base for timely cilium disassembly. In contrast, mutated CPAP fails to localize at the ciliary base associated with inefficient CDC recruitment, long cilia, retarded cilium disassembly, and delayed cell cycle re‐entry leading to premature differentiation of patient iPS‐derived NPCs. Aberrant CDC function also promotes premature differentiation of NPCs in Seckel iPS‐derived organoids. Thus, our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control.     

Primary cilium alterations and expression changes of Patched1 proteins in niemann‐pick type C disease

Niemann‐Pick type C disease (NPC) is a disorder characterized by abnormal intracellular accumulation of unesterified cholesterol and glycolipids. Two distinct disease‐causing genes have been isolated, NPC1 and NPC2. The NPC1 protein is involved in the sorting and recycling of cholesterol and glycosphingolipids in the late endosomal/lysosomal system. It has extensive homology with the Patched1 (Ptc1) receptor, a transmembrane protein localized in the primary cilium, and involved in the Hedgehog signaling (Shh) pathway. We assessed the presence of NPC1 and Ptc1 proteins and evaluated the relative distribution and morphology of primary cilia in fibroblasts from five NPC1 patients and controls, and in normal fibroblasts treated with 3‐ß‐[2‐(diethylamino)ethoxy]androst‐5‐en‐17‐one (U18666A), a cholesterol transport‐inhibiting drug that is widely used to mimic NPC. Immunofluorescence and western blot analyses showed a significant decrease in expression of NPC1 and Ptc1 in NPC1 fibroblasts, while they were normally expressed in U18666A‐treated fibroblasts. Moreover, fibroblasts from NPC1 patients and U18666A‐treated cells showed a lower percentage distribution of primary cilia and a significant reduction in median cilia length with respect to controls. These are the first results demonstrating altered cytoplasmic expression of Ptc1 and reduced number and length of primary cilia, where Ptc1 is located, in fibroblasts from NPC1 patients. We suggest that the alterations in Ptc1 expression in cells from NPC1 patients are closely related to NPC1 expression deficit, while the primary cilia alterations observed in NPC1 and U18666A‐treated fibroblasts may represent a secondary event derived from a defective metabolic pathway.     

The primary cilium of telocytes in the vasculature: electron microscope imaging

Blood vessels are highly organized and complex structure, which are far more than simple tubes conducting the blood to almost any tissue of the body. The fine structure of the wall of blood vessels has been studied previously using the electron microscope, but the presence the telocytes associated with vasculature, a specific new cellular entity, has not been studied in depth. Interestingly, telocytes have been recently found in the epicardium, myocardium, endocardium, human term placenta, duodenal lamina propria and pleura. We show the presence of telocytes located on the extracellular matrix of blood vessels (arterioles, venules and capillaries) by immunohistochemistry and transmission electron microscopy. Also, we demonstrated the first evidence of a primary cilium in telocytes. Several functions have been proposed for these cells. Here, the telocyte-blood vessels cell proximity, the relationship between telocytes, exosomes and nervous trunks may have a special significance.     

Molecular cloning and characterization of LKv1, a novel voltage‐gated potassium channel in leech

We have cloned a novel voltage‐gated K channel, LKv1, in two species of leech. The properties of LKv1 expressed in transiently transfected HEK293 cells is that of a delayed rectifier current. LKv1 may be a major modulator of excitability in leech neurons, since antibody localization studies show that LKv1 is expressed in the soma and axons of all neurons in both the central and peripheral nervous systems. Comparison of the biophysical and pharmacological properties of LKv1 with native voltage‐gated conductances in leech neurons suggests that LKv1 may correspond to the previously characterized delayed rectifier current, I_K. Phylogenetic analysis of LKv1 shows that it is related to the Shaker subfamily of voltage‐gated K channels although it occupies a separate branch from that of the monophyletic Shaker clade composed of the flatworm, Aplysia, Drosophila, and mammalian Shaker homologs as well as from that of two recently identified Shaker‐related K channels in jellyfish. Thus, this analysis indicates that this group of voltage‐gated K channels contains several evolutionarily divergent lineages. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 287–299, 1999     

Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes

K_V10.1 is a voltage-gated potassium channel expressed selectively in the mammalian brain but also aberrantly in cancer cells. In this study we identified short splice variants of K_V10.1 resulting from exon-skipping events (E65 and E70) in human brain and cancer cell lines. The presence of the variants was confirmed by Northern blot and RNase protection assays. Both variants completely lacked the transmembrane domains of the channel and produced cytoplasmic proteins without channel function. In a reconstituted system, both variants co-precipitated with the full-length channel and induced a robust down-regulation of K_V10.1 current when co-expressed with the full-length form, but their effect was mechanistically different. E65 required a tetramerization domain and induced a reduction in the overall expression of full-length K_V10.1, whereas E70 mainly affected its glycosylation pattern. E65 triggered the activation of cyclin-dependent kinases in Xenopus laevis oocytes, suggesting a role in cell cycle control. Our observations highlight the relevance of noncanonical functions for the oncogenicity of K_V10.1, which need to be considered when ion channels are targeted for cancer therapy.     

Cortactin Controls Surface Expression of the Voltage-gated Potassium Channel KV10.1

K_V10.1 is a voltage-gated potassium channel aberrantly expressed in many cases of cancer, and participates in cancer initiation and tumor progression. Its action as an oncoprotein can be inhibited by a functional monoclonal antibody, indicating a role for channels located at the plasma membrane, accessible to the antibody. Cortactin is an actin-interacting protein implicated in cytoskeletal architecture and often amplified in several types of cancer. In this study, we describe a physical and functional interaction between cortactin and K_V10.1. Binding of these two proteins occurs between the C terminus of K_V10.1 and the proline-rich domain of cortactin, regions targeted by many post-translational modifications. This interaction is specific for K_V10.1 and does not occur with K_V10.2. Cortactin controls the abundance of K_V10.1 at the plasma membrane and is required for functional expression of K_V10.1 channels.     

Auto‐phosphorylation of a voltage‐gated K+ channel controls non‐associative learning

Here, we characterize a new K⁺ channel–kinase complex that operates in the metazoan Caenorhabditis elegans to control learning behaviour. This channel is composed of a pore‐forming subunit, dubbed KHT‐1 (73% homology to human Kv3.1), and the accessory subunit MPS‐1, which shows kinase activity. Genetic, biochemical and electrophysiological evidence show that KHT‐1 and MPS‐1 form a complex in vitro and in native mechanosensory PLM neurons, and that KHT‐1 is a substrate for the kinase activity of MPS‐1. Behavioural analysis further shows that the kinase activity of MPS‐1 is specifically required for habituation to repetitive mechanical stimulation. Thus, worms bearing an inactive MPS‐1 variant (D178N) respond normally to touch on the body but do not habituate to repetitive mechanical stimulation such as tapping on the side of the Petri dish. Hence, the phosphorylation status of KHT‐1–MPS‐1 seems to be linked to distinct behavioural responses. In the non‐phosphorylated state the channel is necessary for the normal function of the touch neurons. In the auto‐phosphorylated state the channel acts to induce neuronal adaptation to mechanical stimulation. Taken together, these data establish a new mechanism of dynamic regulation of electrical signalling in the nervous system.     

Role of voltage‐gated potassium channels in the fate determination of embryonic stem cells

Embryonic stem cells (ESCs) possess two unique characteristics: self‐renewal and pluripotency. In this study, roles of voltage‐gated potassium channels (K_v) in maintaining mouse (m) ESC characteristics were investigated. Tetraethylammonium (TEA⁺), a K_v blocker, attenuated cell proliferation in a concentration‐dependent manner. Possible reasons for this attenuation, including cytotoxicity, cell cycle arrest and differentiation, were examined. Blocking K_v did not change the viability of mESCs. Interestingly, K_v inhibition increased the proportion of cells in G₀/G₁ phase and decreased that in S phase. This change in cell cycle distribution can be attributed to cell cycle arrest or differentiation. Loss of pluripotency as determined at both molecular and functional levels was detected in mESCs with K_v blockade, indicating that K_v inhibition in undifferentiated mESCs directs cells to differentiate instead of to self‐renew and progress through the cell cycle. Membrane potential measurement revealed that K_v blockade led to depolarization, consistent with the role of K_v as the key determinant of membrane potential. The present results suggest that membrane potential changes may act as a “switch” for ESCs to decide whether to proliferate or to differentiate: hyperpolarization at G₁ phase would favor ESCs to enter S phase while depolarization would favor ESCs to differentiate. Consistent with this notion, S‐phase‐synchronized mESCs were found to be more hyperpolarized than G₀/G₁‐phase‐synchronized mESCs. Moreover, when mESCs differentiated, the differentiation derivatives depolarized at the initial stage of differentiation. This investigation is the first study to provide evidence that K_v and membrane potential affect the fate determination of ESCs. J. Cell. Physiol. 224:165–177, 2010 © 2010 Wiley‐Liss, Inc.     

A novel Cre‐inducible knock‐in ARL13B‐tRFP fusion cilium reporter

Cilia play a major role in the regulation of numerous signaling pathways and are essential for embryonic development. Mutations in genes affecting ciliary function can cause a variety of diseases in humans summarized as ciliopathies. To facilitate the detection and visualization of cilia in a temporal and spatial manner in mouse tissues, we generated a Cre‐inducible cilium‐specific reporter mouse line expressing an ARL13B‐tRFP fusion protein driven by a CMV enhancer/chicken β actin promotor (pCAG) from the Hprt locus. We detected bright and specific ciliary signals by immunostainings of various mono‐ and multiciliated tissues and by time‐lapse live‐cell analysis of cultured embryos and organ explant cultures. Additionally, we monitored cilium assembly and disassembly in embryonic fibroblast cells using live‐cell imaging. Thus, the ARL13B‐tRFP reporter mouse strain is a valuable tool for the investigation of ciliary structure and function in a tissue‐specific manner to understand processes, such as ciliary protein trafficking or cilium‐dependent signaling in vitro and in vivo.     

Tetrodotoxin‐resistant voltage‐gated sodium channel Nav1.8 constitutively interacts with ankyrin G

The tetrodotoxin‐resistant (TTX‐R) voltage‐gated sodium channel Na_v1.8 is predominantly expressed in peripheral afferent neurons, but in case of neuronal injury an ectopic and detrimental expression of Na_v1.8 occurs in neurons of the CNS. In CNS neurons, Na_v1.2 and Na_v1.6 channels accumulate at the axon initial segment, the site of the generation of the action potential, through a direct interaction with the scaffolding protein ankyrin G (ankG). This interaction is regulated by protein kinase CK2 phosphorylation. In this study, we quantitatively analyzed the interaction between Na_v1.8 and ankG. GST pull‐down assay and surface plasmon resonance technology revealed that Na_v1.8 strongly and constitutively interacts with ankG, in comparison to what observed for Na_v1.2. An ion channel bearing the ankyrin‐binding motif of Na_v1.8 displaced the endogenous Na_v1 accumulation at the axon initial segment of hippocampal neurons. Finally, Na_v1.8 and ankG co‐localized in skin nerves fibers. Altogether, these results indicate that Na_v1.8 carries all the information required for its localization at ankG micro‐domains. The constitutive binding of Na_v1.8 with ankG could contribute to the pathological aspects of illnesses where Na_v1.8 is ectopically expressed in CNS neurons.

     

Detergent screening of the human voltage‐gated proton channel using fluorescence‐detection size‐exclusion chromatography

The human voltage‐gated proton channel (Hv1) is a membrane protein consisting of four transmembrane domains and intracellular amino‐ and carboxy‐termini. The protein is activated by membrane depolarization, similar to other voltage‐sensitive proteins. However, the Hv1 proton channel lacks a traditional ion pore. The human Hv1 proton channel has been implicated in mediating sperm capacitance, stroke, and most recently as a biomarker/mediator of cancer metastasis. Recently, the three‐dimensional structures for homologues of this voltage‐gated proton channel were reported. However, it is not clear what artificial environment is needed to facilitate the isolation and purification of the human Hv1 proton channel for structural study. In the present study, we generated a chimeric protein that placed an enhanced green fluorescent protein (EGFP) to the amino‐terminus of the human Hv1 proton channel (termed EGFP‐Hv1). The chimeric protein was expressed in a baculovirus expression system using Sf9 cells and subjected to detergent screening using fluorescence‐detection size‐exclusion chromatography. The EGFP‐Hv1 proton channel can be solubilized in the zwitterionic detergent Anzergent 3–12 and the nonionic n‐dodecyl‐β‐d ‐maltoside (DDM) with little protein aggregation and a prominent monomeric protein peak at 48 h postinfection. Furthermore, we demonstrate that the chimeric protein exhibits a monomeric protein peak, which is distinguishable from protein aggregates, at the final size‐exclusion chromatography purification step. Taken together, we can conclude that solubilization in DDM will provide a useable final product for further structural characterization of the full‐length human Hv1 proton channel.     

Characterization of the voltage‐gated sodium channel of the Asian citrus psyllid,Diaphorina citri

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important insect pest of citrus. It is the vector of ‘Candidatus’ Liberibacter asiaticus, a phloem‐limited bacterium that infects citrus, resulting in the disease Huanglongbing (HLB). Disease management relies heavily on suppression of D. citri populations with insecticides, including pyrethroids. In recent annual surveys to monitor insecticide resistance, reduced susceptibility to fenpropathrin was identified in several field populations of D. citri. The primary target of pyrethroids is the voltage‐gated sodium channel (VGSC). The VGSC is prone to target‐site insensitivity because of mutations that either reduce pyrethroid binding and/or alter gating kinetics. These mutations, known as knockdown resistance or kdr, have been reported in a wide diversity of arthropod species. Alternative splicing, in combination with kdr mutations, has been also associated with reduced pyrethroid efficacy. Here we report the molecular characterization of the VGSC in D. citri along with a survey of alternative splicing across developmental stages of this species. Previous studies demonstrated that D. citri has an exquisite enzymatic arsenal to detoxify insecticides resulting in reduced efficacy. The results from the current investigation demonstrate that target‐site insensitivity is also a potential basis for insecticide resistance to pyrethroids in D. citri. The VGSC sequence and its molecular characterization should facilitate early elucidation of the underlying cause of an established case of resistance to pyrethroids. This is the first characterization of a VGSC from a hemipteran to this level of detail, with the majority of the previous studies on dipterans and lepidopterans.