INPP5E regulates CD3ζ enrichment at the immune synapse by phosphoinositide distribution control.

The immune synapse, a highly organized structure formed at the interface between T lymphocytes and antigen-presenting cells (APCs), is essential for T cell activation and the adaptive immune response. It has been shown that this interface shares similarities with the primary cilium, a sensory organelle in eukaryotic cells, although the roles of ciliary proteins on the immune synapse remain elusive. Here, we find that inositol polyphosphate-5-phosphatase E (INPP5E), a cilium-enriched protein responsible for regulating phosphoinositide localization, is enriched at the immune synapse in Jurkat T-cells during superantigen-mediated conjugation or antibody-mediated crosslinking of TCR complexes, and forms a complex with CD3ζ, ZAP-70, and Lck. Silencing INPP5E in Jurkat T-cells impairs the polarized distribution of CD3ζ at the immune synapse and correlates with a failure of PI(4,5)P2 clearance at the center of the synapse. Moreover, INPP5E silencing decreases proximal TCR signaling, including phosphorylation of CD3ζ and ZAP-70, and ultimately attenuates IL-2 secretion. Our results suggest that INPP5E is a new player in phosphoinositide manipulation at the synapse, controlling the TCR signaling cascade.

Rab34 is necessary for early stages of intracellular ciliogenesis.

Primary cilia are sensory organelles present on most vertebrate cells and are critical for development and health. Ciliary dysfunction is associated with a large class of human pathologies collectively known as ciliopathies. These include cystic kidneys, blindness, obesity, skeletal malformations, and other organ anomalies. Using a proximity biotinylation with Ift27 as bait, we identified the small guanosine triphosphatase (GTPase) Rab34 as a ciliary protein. Rab34 localizes to the centrosomes near the mother centriole, the axoneme of developed cilia, and highly dynamic tubule structures in the centrosomal region. Rab34 is required for cilia formation in fibroblasts, where we find that Rab34 loss blocks ciliogenesis at an early step of ciliary vesicle formation. In inner medullary collecting duct (IMCD3) epithelial cells, the requirement is more complex, with Rab34 needed in cells grown at low density but becoming less important as cell density increases. Ciliogenesis can proceed by an internal pathway where cilia form in the cytoplasm before being displayed on the ciliary surface or cilia can assemble by an external pathway where the centriole docks on the plasma membrane before ciliary assembly. Fibroblasts are thought to use the internal pathway, although IMCD3 cells are thought to use the external pathway. However, we find that IMCD3 cells can use the internal assembly pathway and significant numbers of internally assembling cilia are observed in low-density cells. Together, our work indicates that Rab34 is required for internal assembly of cilia, but not for cilia built on the cell surface.

Epigenetic modulation of immune synaptic-cytoskeletal networks potentiates γδ T cell-mediated cytotoxicity in lung cancer.

γδ T cells are a distinct subgroup of T cells that bridge the innate and adaptive immune system and can attack cancer cells in an MHC-unrestricted manner. Trials of adoptive γδ T cell transfer in solid tumors have had limited success. Here, we show that DNA methyltransferase inhibitors (DNMTis) upregulate surface molecules on cancer cells related to γδ T cell activation using quantitative surface proteomics. DNMTi treatment of human lung cancer potentiates tumor lysis by ex vivo-expanded Vδ1-enriched γδ T cells. Mechanistically, DNMTi enhances immune synapse formation and mediates cytoskeletal reorganization via coordinated alterations of DNA methylation and chromatin accessibility. Genetic depletion of adhesion molecules or pharmacological inhibition of actin polymerization abolishes the potentiating effect of DNMTi. Clinically, the DNMTi-associated cytoskeleton signature stratifies lung cancer patients prognostically. These results support a combinatorial strategy of DNMTis and γδ T cell-based immunotherapy in lung cancer management.

Super-resolution microscopy reveals coupling between mammalian centriole subdistal appendages and distal appendages.

Subdistal appendages (sDAPs) are centriolar elements that are observed proximal to the distal appendages (DAPs) in vertebrates. Despite the obvious presence of sDAPs, structural and functional understanding of them remains elusive. Here, by combining super-resolved localization analysis and CRISPR-Cas9 genetic perturbation, we find that although DAPs and sDAPs are primarily responsible for distinct functions in ciliogenesis and microtubule anchoring, respectively, the presence of one element actually affects the positioning of the other. Specifically, we find dual layers of both ODF2 and CEP89, where their localizations are differentially regulated by DAP and sDAP integrity. DAP depletion relaxes longitudinal occupancy of sDAP protein ninein to cover the DAP region, implying a role of DAPs in sDAP positioning. Removing sDAPs alter the distal border of centrosomal γ-tubulins, illustrating a new role of sDAPs. Together, our results provide an architectural framework for sDAPs that sheds light on functional understanding, surprisingly revealing coupling between DAPs and sDAPs.

Differential requirements for the EF-hand domains of human centrin 2 in primary ciliogenesis and nucleotide excision repair.

Centrin 2 is a small conserved calcium-binding protein that localizes to the centriolar distal lumen in human cells. It is required for efficient primary ciliogenesis and nucleotide excision repair (NER). Centrin 2 forms part of the xeroderma pigmentosum group C protein complex. To explore how centrin 2 contributes to these distinct processes, we mutated the four calcium-binding EF-hand domains of human centrin 2. Centrin 2 in which all four EF-hands had been mutated to ablate calcium binding (4DA mutant) was capable of supporting in vitro NER and was as effective as the wild-type protein in rescuing the UV sensitivity of centrin 2-null cells. However, we found that mutation of any of the EF-hand domains impaired primary ciliogenesis in human TERT-RPE1 cells to the same extent as deletion of centrin 2. Phenotypic analysis of the 4DA mutant revealed defects in centrosome localization, centriole satellite assembly, ciliary assembly and function and in interactions with POC5 and SFI1. These observations indicate that centrin 2 requires calcium-binding capacity for its primary ciliogenesis functions, but not for NER, and suggest that these functions require centrin 2 to be capable of forming complexes with partner proteins.This article has an associated First Person interview with the first author of the paper.

Phosphorylation of CEP83 by TTBK2 is necessary for cilia initiation.

Primary cilia are microtubule-based organelles that play important roles in development and tissue homeostasis. Tau-tubulin kinase-2 (TTBK2) is genetically linked to spinocerebellar ataxia type 11, and its kinase activity is crucial for ciliogenesis. Although it has been shown that TTBK2 is recruited to the centriole by distal appendage protein CEP164, little is known about TTBK2 substrates associated with its role in ciliogenesis. Here, we perform superresolution microscopy and discover that serum starvation results in TTBK2 redistribution from the periphery toward the root of distal appendages. Our biochemical analyses uncover CEP83 as a bona fide TTBK2 substrate with four phosphorylation sites characterized. We also demonstrate that CEP164-dependent TTBK2 recruitment to distal appendages is required for subsequent CEP83 phosphorylation. Specifically, TTBK2-dependent CEP83 phosphorylation is important for early ciliogenesis steps, including ciliary vesicle docking and CP110 removal. In summary, our results reveal a molecular mechanism of kinase regulation in ciliogenesis and identify CEP83 as a key substrate of TTBK2 during cilia initiation.

Differential Proteomics Reveals Discrete Functions of Proteins Interacting with Hypo- versus Hyper-phosphorylated NS5A of the Hepatitis C Virus.

Protein phosphorylation is a reversible post-translational modification that regulates many biological processes in almost all living forms. In the case of the hepatitis C virus (HCV), the nonstructural protein 5A (NS5A) is believed to transit between hypo- and hyper-phosphorylated forms that interact with host proteins to execute different functions; however, little was known about the proteins that bind either form of NS5A. Here, we generated two high-quality antibodies specific to serine 235 nonphosphorylated hypo- vs serine 235 phosphorylated (pS235) hyper-phosphorylated form of NS5A and for the first time segregated these two forms of NS5A plus their interacting proteins for dimethyl-labeling based proteomics. We identified 629 proteins, of which 238 were quantified in three replicates. Bioinformatics showed 46 proteins that preferentially bind hypo-phosphorylated NS5A are involved in antiviral response and another 46 proteins that bind pS235 hyper-phosphorylated NS5A are involved in liver cancer progression. We further identified a DNA-dependent kinase (DNA-PK) that binds hypo-phosphorylated NS5A. Inhibition of DNA-PK with an inhibitor or via gene-specific knockdown significantly reduced S232 phosphorylation and NS5A hyper-phosphorylation. Because S232 phosphorylation initiates sequential S232/S235/S238 phosphorylation leading to NS5A hyper-phosphorylation, we identified a new protein kinase that regulates a delicate balance of NS5A between hypo- and hyper-phosphorylation states, respectively, involved in host antiviral responses and liver cancer progression.

A microfluidic device for in situ fixation and super-resolved mechanosensation studies of primary cilia.

The primary cilium plays an important role in mechanosensation in mammalian cells. To understand mechanosensation in the primary cilium, we combined a microfluidic device with super-resolution microscopy to study the primary cilium phenotypes. The microfluidic system enabled the precise control of the flow shear within a well-confined cell-culture environment. In addition, in situ cilia fixation was possible by switching from the culture medium to the fixation buffer instantaneously, which preserved the real-time cilium phenotype under the flow shear. After fixation, multiple cilium-specific proteins were immunostained to quantify the cilia bending behavior. We found that >50% of the primary cilia of mouse inner medullary collecting duct cells were highly aligned with the direction of flow under 11 Pa shear stress. Finally, we used super-resolution microscopy to observe the redistribution of two major cilium-specific proteins under flow shear, acetylated alpha-tubulin, and intraflagellar transport protein 88. To the best of our knowledge, this is the first platform to combine a microfluidic device with super-resolution microscopy to enable flow stimulation and in situ fixation for the observation of ciliary protein. This system can potentially be applied to the future development of a stimulation-enabled organ-on-a-chip to observe the intercellular signaling of primary cilia or for the analysis of disease mechanisms associated with ciliary mutations at the organ level.

Single-particle tracking localization microscopy reveals nonaxonemal dynamics of intraflagellar transport proteins at the base of mammalian primary cilia.

Primary cilia play a vital role in cellular sensing and signaling. An essential component of ciliogenesis is intraflagellar transport (IFT), which is involved in IFT protein recruitment, axonemal engagement of IFT protein complexes, and so on. The mechanistic understanding of these processes at the ciliary base was largely missing, because it is challenging to observe the motion of IFT proteins in this crowded region using conventional microscopy. Here, we report short-trajectory tracking of IFT proteins at the base of mammalian primary cilia by optimizing single-particle tracking photoactivated localization microscopy for IFT88-mEOS4b in live human retinal pigment epithelial cells. Intriguingly, we found that mobile IFT proteins "switched gears" multiple times from the distal appendages (DAPs) to the ciliary compartment (CC), moving slowly in the DAPs, relatively fast in the proximal transition zone (TZ), slowly again in the distal TZ, and then much faster in the CC. They could travel through the space between the DAPs and the axoneme without following DAP structures. We further revealed that BBS2 and IFT88 were highly populated at the distal TZ, a potential assembly site. Together, our live-cell single-particle tracking revealed region-dependent slowdown of IFT proteins at the ciliary base, shedding light on staged control of ciliary homeostasis.

Two separate functions of NME3 critical for cell survival underlie a neurodegenerative disorder.

We report a patient who presented with congenital hypotonia, hypoventilation, and cerebellar histopathological alterations. Exome analysis revealed a homozygous mutation in the initiation codon of the NME3 gene, which encodes an NDP kinase. The initiation-codon mutation leads to deficiency in NME3 protein expression. NME3 is a mitochondrial outer-membrane protein capable of interacting with MFN1/2, and its depletion causes dysfunction in mitochondrial dynamics. Consistently, the patient's fibroblasts were characterized by a slow rate of mitochondrial dynamics, which was reversed by expression of wild-type or catalytic-dead NME3. Moreover, glucose starvation caused mitochondrial fragmentation and cell death in the patient's cells. The expression of wild-type and catalytic-dead but not oligomerization-attenuated NME3 restored mitochondrial elongation. However, only wild-type NME3 sustained ATP production and viability. Thus, the separate functions of NME3 in mitochondrial fusion and NDP kinase cooperate in metabolic adaptation for cell survival in response to glucose starvation. Given the critical role of mitochondrial dynamics and energy requirements in neuronal development, the homozygous mutation in NME3 is linked to a fatal mitochondrial neurodegenerative disorder.

Adducin-1 is essential for spindle pole integrity through its interaction with TPX2.

Bipolar spindle assembly is necessary to ensure the proper progression of cell division. Loss of spindle pole integrity leads to multipolar spindles and aberrant chromosomal segregation. However, the mechanism underlying the maintenance of spindle pole integrity remains unclear. In this study, we show that the actin-binding protein adducin-1 (ADD1) is phosphorylated at S726 during mitosis. S726-phosphorylated ADD1 localizes to centrosomes, wherein it organizes into a rosette-like structure at the pericentriolar material. ADD1 depletion causes centriole splitting and therefore results in multipolar spindles during mitosis, which can be restored by re-expression of ADD1 and the phosphomimetic S726D mutant but not by the S726A mutant. Moreover, the phosphorylation of ADD1 at S726 is crucial for its interaction with TPX2, which is essential for spindle pole integrity. Together, our findings unveil a novel function of ADD1 in maintaining spindle pole integrity through its interaction with TPX2.

Super-Resolution Imaging Reveals TCTN2 Depletion-Induced IFT88 Lumen Leakage and Ciliary Weakening.

The primary cilium is an essential organelle mediating key signaling activities, such as sonic hedgehog signaling. The molecular composition of the ciliary compartment is distinct from that of the cytosol, with the transition zone (TZ) gated the ciliary base. The TZ is a packed and organized protein complex containing multiple ciliopathy-associated protein species. Tectonic 2 (TCTN2) is one of the TZ proteins in the vicinity of the ciliary membrane, and its mutation is associated with Meckel syndrome. Despite its importance in ciliopathies, the role of TCTN2 in ciliary structure and molecules remains unclear. Here, we created a CRISPR/Cas9 TCTN2 knockout human retinal pigment epithelial cell line and conducted quantitative analysis of geometric localization using both wide-field and super-resolution microscopy techniques. We found that TCTN2 depletion resulted in partial TZ damage, loss of ciliary membrane proteins, leakage of intraflagellar transport protein IFT88 toward the basal body lumen, and cilium shortening and curving. The basal body lumen occupancy of IFT88 was also observed in si-RPGRIP1L cells and cytochalasin-D-treated wild-type cells, suggesting varying lumen accessibility for intraflagellar transport proteins under different perturbed conditions. Our findings support two possible models for the lumen leakage of IFT88, i.e., a tip leakage model and a misregulation model. Together, our quantitative image analysis augmented by super-resolution microscopy facilitates the observation of structural destruction and molecular redistribution in TCTN2-/- cilia, shedding light on mechanistic understanding of TZ-protein-associated ciliopathies.

Super-resolution architecture of mammalian centriole distal appendages reveals distinct blade and matrix functional components.

Distal appendages (DAPs) are nanoscale, pinwheel-like structures protruding from the distal end of the centriole that mediate membrane docking during ciliogenesis, marking the cilia base around the ciliary gate. Here we determine a super-resolved multiplex of 16 centriole-distal-end components. Surprisingly, rather than pinwheels, intact DAPs exhibit a cone-shaped architecture with components filling the space between each pinwheel blade, a new structural element we term the distal appendage matrix (DAM). Specifically, CEP83, CEP89, SCLT1, and CEP164 form the backbone of pinwheel blades, with CEP83 confined at the root and CEP164 extending to the tip near the membrane-docking site. By contrast, FBF1 marks the distal end of the DAM near the ciliary membrane. Strikingly, unlike CEP164, which is essential for ciliogenesis, FBF1 is required for ciliary gating of transmembrane proteins, revealing DAPs as an essential component of the ciliary gate. Our findings redefine both the structure and function of DAPs.

STED and STORM Superresolution Imaging of Primary Cilia.

The characteristic lengths of molecular arrangement in primary cilia are below the diffraction limit of light, challenging structural and functional studies of ciliary proteins. Superresolution microscopy can reach up to a 20 nm resolution, significantly improving the ability to map molecules in primary cilia. Here we describe detailed experimental procedure of STED microscopy imaging and dSTORM imaging, two of the most powerful superresolution imaging techniques. Specifically, we emphasize the use of these two methods on imaging proteins in primary cilia.

Stage-Dependent Axon Transport of Proteasomes Contributes to Axon Development.

Axon extension at the growing tip requires elevated local protein supply, with a capability sustainable over long axons in varying environments. The exact mechanisms, however, remain elusive. Here we report that axon-promoting factors elicited a retrograde transport-dependent removal of proteasomes from nascent axon terminals, thereby increasing protein stability at axon tips. Such an effect occurred through phosphorylation of a dynein-interacting proteasome adaptor protein Ecm29. During the transition from immature neurites to nascent axons in cultured hippocampal neurons, live-cell imaging revealed a significant increase of the retrograde axonal transport of fluorescently labeled 20S proteasomes. This retrograde proteasome transport depended on neuron stage and increased with axon lengths. Blockade of retrograde transport caused accumulation of proteasomes, reduction of axon growth, and attenuation of growth-associated Par6 at the axon tip of newly polarized neurons. Our results delineate a regulatory mechanism that controls proteasome abundance via preferential transport required for axon development in newborn neurons.

Superresolution Pattern Recognition Reveals the Architectural Map of the Ciliary Transition Zone.

The transition zone (TZ) of primary cilia serves as a diffusion barrier to regulate ciliogenesis and receptor localization for key signaling events such as sonic hedgehog signaling. Its gating mechanism is poorly understood due to the tiny volume accommodating a large number of ciliopathy-associated molecules. Here we performed stimulated emission depletion (STED) imaging of collective samples and recreated superresolved relative localizations of eight representative species of ciliary proteins using position averages and overlapped with representative electron microscopy (EM) images, defining an architectural foundation at the ciliary base. Upon this framework, transmembrane proteins TMEM67 and TCTN2 were accumulated at the same axial level as MKS1 and RPGRIP1L, suggesting that their regulation roles for tissue-specific ciliogenesis occur at a specific level of the TZ. CEP290 is surprisingly localized at a different axial level bridging the basal body (BB) and other TZ proteins. Upon this molecular architecture, two reservoirs of intraflagellar transport (IFT) particles, correlating with phases of ciliary growth, are present: one colocalized with the transition fibers (TFs) while the other situated beyond the distal edge of the TZ. Together, our results reveal an unprecedented structural framework of the TZ, facilitating our understanding in molecular screening and assembly at the ciliary base.

TTBK2: a tau protein kinase beyond tau phosphorylation.

Tau tubulin kinase 2 (TTBK2) is a kinase known to phosphorylate tau and tubulin. It has recently drawn much attention due to its involvement in multiple important cellular processes. Here, we review the current understanding of TTBK2, including its sequence, structure, binding sites, phosphorylation substrates, and cellular processes involved. TTBK2 possesses a casein kinase 1 (CK1) kinase domain followed by a ~900 amino acid segment, potentially responsible for its localization and substrate recruitment. It is known to bind to CEP164, a centriolar protein, and EB1, a microtubule plus-end tracking protein. In addition to autophosphorylation, known phosphorylation substrates of TTBK2 include tau, tubulin, CEP164, CEP97, and TDP-43, a neurodegeneration-associated protein. Mutations of TTBK2 are associated with spinocerebellar ataxia type 11. In addition, TTBK2 is essential for regulating the growth of axonemal microtubules in ciliogenesis. It also plays roles in resistance of cancer target therapies and in regulating glucose and GABA transport. Reported sites of TTBK2 localization include the centriole/basal body, the midbody, and possibly the mitotic spindles. Together, TTBK2 is a multifunctional kinase involved in important cellular processes and demands augmented efforts in investigating its functions.

Effects of diallyl trisulfide on induction of apoptotic death in murine leukemia WEHI-3 cells in vitro and alterations of the immune responses in normal and leukemic mice in vivo.

Diallyl trisulfide (DATS), a chemopreventive dietary constituent and extracted from garlic, has been shown to against cultured many types of human cancer cell liens but the fate of apoptosis in murine leukemia cells in vitro and immune responses in leukemic mice remain elusive. Herein, we clarified the actions of DATS on growth inhibition of murine leukemia WEHI-3 cells in vitro and used WEHI-3 cells to generate leukemic mice in vivo, following to investigate the effects of DATS in animal model. In in vitro study, DATS induced apoptosis of WEHI-3 cells through the G0/G1 phase arrest and induction of caspase-3 activation. In in vivo study DATS decreased the weight of spleen of leukemia mice but did not affect the spleen weight of normal mice. DATS promoted the immune responses such as promotions of the macrophage phagocytosis and NK cell activities in WEHI-3 leukemic and normal mice. However, DATS only promotes NK cell activities in normal mice. DATS increases the surface markers of CD11b and Mac-3 in leukemia mice but only promoted CD3 in normal mice. In conclusion, the present study indicates that DATS induces cell death through induction of apoptosis in mice leukemia WHEI-3 cells. DATS also promotes immune responses in leukemia and normal mice in vivo.

SAS-6 assembly templated by the lumen of cartwheel-less centrioles precedes centriole duplication.

Centrioles are 9-fold symmetric structures duplicating once per cell cycle. Duplication involves self-oligomerization of the centriolar protein SAS-6, but how the 9-fold symmetry is invariantly established remains unclear. Here, we found that SAS-6 assembly can be shaped by preexisting (or mother) centrioles. During S phase, SAS-6 molecules are first recruited to the proximal lumen of the mother centriole, adopting a cartwheel-like organization through interactions with the luminal wall, rather than via their self-oligomerization activity. The removal or release of luminal SAS-6 requires Plk4 and the cartwheel protein STIL. Abolishing either the recruitment or the removal of luminal SAS-6 hinders SAS-6 (or centriole) assembly at the outside wall of mother centrioles. After duplication, the lumen of engaged mother centrioles becomes inaccessible to SAS-6, correlating with a block for reduplication. These results lead to a proposed model that centrioles may duplicate via a template-based process to preserve their geometry and copy number.

Morphological differences of primary cilia between human induced pluripotent stem cells and their parental somatic cells.

Human induced pluripotent stem cell (hiPSC) reprogramming possesses enormous potential in stem cell research and disease modeling. Chemical and mechanical signaling has been implicated in the maintenance of pluripotency of hiPSCs, as well as their differentiation pathways toward various lineages. Primary cilia have been shown to play a critical role in mechanochemical signaling across a wide spectrum of cell types. The functions of primary cilia in hiPSCs and their characteristic changes during the reprogramming process remain largely vague. This work focused on understanding how reprogramming affects the mechanical characteristics of primary cilia. Using immunofluorescence imaging assays, we validated the presence of primary cilia on reprogrammed cells. These reprogrammed cells had high expression levels of pluripotency markers, Nanog and Cripto, shown by quantitative polymerase chain reaction assays. We also found high expression of hedgehog signaling proteins Patched1 (Ptch1), Smoothened (Smo), Gli1, and Gli2 in reprogrammed cells. Stimulation of the hedgehog pathway resulted in the concerted movement of Ptch1 out of the cilia and Smo into the cilia, implying that the cilia on iPSCs contain functioning hedgehog machinery. The mean length of primary cilia in reprogrammed cells was shorter than those of parental human fibroblasts. Morphometric analyses revealed that reprogramming resulted in an increase in the curvature of primary cilia from ∼0.015 to 0.064 µm(-1), indicating an underlying approximately fourfold decrease in their rigidity, and a decrease in length of primary cilia from ∼2.38 to ∼1.45 µm. Furthermore, reprogramming resulted in fewer primary cilia displaying kinked geometries.