scholarly journals Superresolution Microscopy Reveals Distinct Phosphoinositide Subdomains Within the Cilia Transition Zone

2021 ◽  
Vol 9 ◽  
Author(s):  
Sarah E. Conduit ◽  
Elizabeth M. Davies ◽  
Alex J. Fulcher ◽  
Viola Oorschot ◽  
Christina A. Mitchell
Keyword(s):  
Transition Zone ◽  
Mammalian Cells ◽  
Primary Cilia ◽  
Lipid Binding ◽  
Stimulated Emission ◽  
Phosphoinositide Metabolism ◽  
Inositol Polyphosphate ◽  
Superresolution Microscopy ◽  
Phosphoinositide Signaling ◽  
Ring Shape

Primary cilia are evolutionary conserved microtubule-based organelles that protrude from the surface of most mammalian cells. Phosphoinositides (PI) are membrane-associated signaling lipids that regulate numerous cellular events via the recruitment of lipid-binding effectors. The temporal and spatial membrane distribution of phosphoinositides is regulated by phosphoinositide kinases and phosphatases. Recently phosphoinositide signaling and turnover has been observed at primary cilia. However, the precise localization of the phosphoinositides to specific ciliary subdomains remains undefined. Here we use superresolution microscopy (2D stimulated emission depletion microscopy) to map phosphoinositide distribution at the cilia transition zone. PI(3,4,5)P3 and PI(4,5)P2 localized to distinct subregions of the transition zone in a ring-shape at the inner transition zone membrane. Interestingly, the PI(3,4,5)P3 subdomain was more distal within the transition zone relative to PtdIns(4,5)P2. The phosphoinositide effector kinase pAKT(S473) localized in close proximity to these phosphoinositides. The inositol polyphosphate 5-phosphatase, INPP5E, degrades transition zone phosphoinositides, however, studies of fixed cells have reported recombinant INPP5E localizes to the ciliary axoneme, distant from its substrates. Notably, here using live cell imaging and optimized fixation/permeabilization protocols INPP5E was found concentrated at the cilia base, in a distribution characteristic of the transition zone in a ring-shaped domain of similar dimensions to the phosphoinositides. Collectively, this superresolution map places the phosphoinositides in situ with the transition zone proteins and reveals that INPP5E also likely localizes to a subdomain of the transition zone membrane, where it is optimally situated to control local phosphoinositide metabolism.

scholarly journals Cholesterol Increases Lipid Binding Rate and Changes Binding Behavior of Bacillus thuringiensis Cytolytic Protein

2018 ◽  
Vol 19 (12) ◽  
pp. 3819 ◽  
Author(s):  
Sudarat Tharad ◽  
Öykü Üzülmez ◽  
Boonhiang Promdonkoy ◽  
José Toca-Herrera
Keyword(s):  
Bacillus Thuringiensis ◽  
Lipid Bilayers ◽  
Mammalian Cells ◽  
Layer Structure ◽  
Cholesterol Content ◽  
Lipid Binding ◽  
Lipid Layer ◽  
Binding Behavior ◽  
Binding Rate ◽  
Ring Shape

Cytolytic protein (Cyt) is a member of insecticidal proteins produced by Bacillus thuringiensis. Cyt protein has activity against insect cells and mammalian cells, which differ in lipid and cholesterol composition. This study presents the lipid binding behavior of Cyt2Aa2 protein on model membranes containing different levels of cholesterol content by combining Quartz Crystal Microbalance with Dissipation (QCM-D) and Atomic Force Microscopy (AFM). QCM-D results revealed that cholesterol enhances the binding rate of Cyt2Aa2 protein onto lipid bilayers. In addition, the thicker lipid bilayer was observed for the highest cholesterol content. These results were confirmed by AFM. The analysis of protein surface coverage as a function of time showed a slower process for 5:0 and 5:0.2 (POPC:Chol) ratios than for 5:1 and 5:2 (POPC:Chol) ratios. Significantly, the Cyt2Aa2-lipid binding behavior and the protein–lipid layer were different for the 5:3 (POPC:Chol) ratio. Furthermore, AFM images revealed a transformation of Cyt2Aa2/lipid layer structure from strip pattern to ring shape structures (which showed a strong repulsion with AFM tip). In summary, cholesterol increases the binding rate and alters the lipid binding behavior of Cyt2Aa2 protein, although it is not required for Cyt2Aa2 protein binding onto lipid bilayers.

scholarly journals INPP5E regulates phosphoinositide-dependent cilia transition zone function

2016 ◽  
Vol 216 (1) ◽  
pp. 247-263 ◽  
Author(s):  
Jennifer M. Dyson ◽  
Sarah E. Conduit ◽  
Sandra J. Feeney ◽  
Sandra Hakim ◽  
Tia DiTommaso ◽  
...  
Keyword(s):  
Transition Zone ◽  
Hedgehog Signaling ◽  
Joubert Syndrome ◽  
Mouse Genetics ◽  
Molecular Organization ◽  
Scaffolding Proteins ◽  
Inositol Polyphosphate ◽  
Wild Type ◽  
Essential Point ◽  
Phosphoinositide Signaling

Human ciliopathies, including Joubert syndrome (JBTS), arise from cilia dysfunction. The inositol polyphosphate 5-phosphatase INPP5E localizes to cilia and is mutated in JBTS. Murine Inpp5e ablation is embryonically lethal and recapitulates JBTS, including neural tube defects and polydactyly; however, the underlying defects in cilia signaling and the function of INPP5E at cilia are still emerging. We report Inpp5e−/− embryos exhibit aberrant Hedgehog-dependent patterning with reduced Hedgehog signaling. Using mouse genetics, we show increasing Hedgehog signaling via Smoothened M2 expression rescues some Inpp5e−/− ciliopathy phenotypes and “normalizes” Hedgehog signaling. INPP5E’s phosphoinositide substrates PI(4,5)P2 and PI(3,4,5)P3 accumulated at the transition zone (TZ) in Hedgehog-stimulated Inpp5e−/− cells, which was associated with reduced recruitment of TZ scaffolding proteins and reduced Smoothened levels at cilia. Expression of wild-type, but not 5-phosphatase-dead, INPP5E restored TZ molecular organization and Smoothened accumulation at cilia. Therefore, we identify INPP5E as an essential point of convergence between Hedgehog and phosphoinositide signaling at cilia that maintains TZ function and Hedgehog-dependent embryonic development.

scholarly journals Phosphatidylinositol-(4,5)-bisphosphate regulates clathrin-coated pit initiation, stabilization, and size

2011 ◽  
Vol 22 (14) ◽  
pp. 2588-2600 ◽  
Author(s):  
Costin N. Antonescu ◽  
François Aguet ◽  
Gaudenz Danuser ◽  
Sandra L. Schmid
Keyword(s):  
Mammalian Cells ◽  
Lipid Binding ◽  
Coated Pits ◽  
Inositol Phosphatase ◽  
Major Mechanism ◽  
Synaptojanin 1 ◽  
Phosphatidylinositol 4 ◽  
Sirna Knockdown ◽  
Interfering Rna ◽  
Pit Initiation

Clathrin-mediated endocytosis (CME) is the major mechanism for internalization in mammalian cells. CME initiates by recruitment of adaptors and clathrin to form clathrin-coated pits (CCPs). Nearly half of nascent CCPs abort, whereas others are stabilized by unknown mechanisms and undergo further maturation before pinching off to form clathrin-coated vesicles (CCVs). Phosphatidylinositol-(4,5)-bisphosphate (PIP2), the main lipid binding partner of endocytic proteins, is required for CCP assembly, but little is currently known about its contribution(s) to later events in CCV formation. Using small interfering RNA (siRNA) knockdown and overexpression, we have analyzed the effects of manipulating PIP2 synthesis and turnover on CME by quantitative total internal reflection fluorescence microscopy and computational analysis. Phosphatidylinositol-4-phosphate-5-kinase cannot be detected within CCPs but functions in initiation and controls the rate and extent of CCP growth. In contrast, the 5′-inositol phosphatase synaptojanin 1 localizes to CCPs and controls early stabilization and maturation efficiency. Together these results suggest that the balance of PIP2 synthesis in the bulk plasma membrane and its local turnover within CCPs control multiple stages of CCV formation.

scholarly journals Dynamic nanoscale morphology of the ER surveyed by STED microscopy

2018 ◽  
Vol 218 (1) ◽  
pp. 83-96 ◽  
Author(s):  
Lena K. Schroeder ◽  
Andrew E.S. Barentine ◽  
Holly Merta ◽  
Sarah Schweighofer ◽  
Yongdeng Zhang ◽  
...  
Keyword(s):  
Living Cells ◽  
Stimulated Emission ◽  
Membrane Structures ◽  
Spatiotemporal Resolution ◽  
Sted Microscopy ◽  
Superresolution Microscopy ◽  
Structure And Dynamics ◽  
First Time ◽  
Nanoscale Dynamics ◽  
Conventional Light Microscopy

The endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules. Superresolution microscopy recently revealed densely packed, rapidly moving ER tubules mistaken for sheets by conventional light microscopy, highlighting the importance of revisiting classical views of ER structure with high spatiotemporal resolution in living cells. In this study, we use live-cell stimulated emission depletion (STED) microscopy to survey the architecture of the ER at 50-nm resolution. We determine the nanoscale dimensions of ER tubules and sheets for the first time in living cells. We demonstrate that ER sheets contain highly dynamic, subdiffraction-sized holes, which we call nanoholes, that coexist with uniform sheet regions. Reticulon family members localize to curved edges of holes within sheets and are required for their formation. The luminal tether Climp63 and microtubule cytoskeleton modulate their nanoscale dynamics and organization. Thus, by providing the first quantitative analysis of ER membrane structure and dynamics at the nanoscale, our work reveals that the ER in living cells is not limited to uniform sheets and tubules; instead, we suggest the ER contains a continuum of membrane structures that includes dynamic nanoholes in sheets as well as clustered tubules.

scholarly journals The retromer complex regulates C. elegans development and mammalian ciliogenesis

2021 ◽  
Author(s):  
Shuwei Xie ◽  
Ellie Smith ◽  
Carter Dierlam ◽  
Danita Mathew ◽  
Angelina Davis ◽  
...  
Keyword(s):  
Potential Function ◽  
Primary Cilium ◽  
Mammalian Cells ◽  
Primary Cilia ◽  
Vulval Development ◽  
Sorting Nexin ◽  
Capping Protein ◽  
C Elegans ◽  
Retromer Complex ◽  
Mother Centriole

The mammalian retromer is comprised of subunits VPS26, VPS29 and VPS35, and a more loosely-associated sorting nexin (SNX) heterodimer. Despite known roles for the retromer in multiple trafficking events in yeast and mammalian cells, its role in development is poorly understood, and its potential function in primary ciliogenesis remains unknown. Using CRISPR-Cas9 editing, we demonstrated that vps-26 homozygous knockout C. elegans have reduced brood sizes and impaired vulval development, as well as decreased body length which has been linked to defects in primary ciliogenesis. Since many endocytic proteins are implicated in the generation of primary cilia, we addressed whether the retromer regulates ciliogenesis in mammalian cells. We observed VPS35 localized to the primary cilium, and depletion of VPS26, VPS35 or SNX1/SNX5 led to decreased ciliogenesis. Retromer also coimmunoprecipitated with the capping protein, CP110, and was required for its removal from the mother centriole. Herein, we characterize new roles for the retromer in C. elegans development and in the regulation of ciliogenesis in mammalian cells, and suggest a novel role for the retromer in CP110 removal from the mother centriole.

scholarly journals The Role of Sonic Hedgehog in Human Holoprosencephaly and Short-Rib Polydactyly Syndromes

2021 ◽  
Vol 22 (18) ◽  
pp. 9854
Author(s):  
Christine Loo ◽  
Michael Pearen ◽  
Grant Ramm
Keyword(s):  
Human Development ◽  
Sonic Hedgehog ◽  
Mammalian Cells ◽  
Animal Studies ◽  
Primary Cilia ◽  
Genetic Mutation ◽  
Wide Variability ◽  
Using Data ◽  
Differentiation And Proliferation

The Hedgehog (HH) signalling pathway is one of the major pathways controlling cell differentiation and proliferation during human development. This pathway is complex, with HH function influenced by inhibitors, promotors, interactions with other signalling pathways, and non-genetic and cellular factors. Many aspects of this pathway are not yet clarified. The main features of Sonic Hedgehog (SHH) signalling are discussed in relation to its function in human development. The possible role of SHH will be considered using examples of holoprosencephaly and short-rib polydactyly (SRP) syndromes. In these syndromes, there is wide variability in phenotype even with the same genetic mutation, so that other factors must influence the outcome. SHH mutations were the first identified genetic causes of holoprosencephaly, but many other genes and environmental factors can cause malformations in the holoprosencephaly spectrum. Many patients with SRP have genetic defects affecting primary cilia, structures found on most mammalian cells which are thought to be necessary for canonical HH signal transduction. Although SHH signalling is affected in both these genetic conditions, there is little overlap in phenotype. Possible explanations will be canvassed, using data from published human and animal studies. Implications for the understanding of SHH signalling in humans will be discussed.

scholarly journals Down-Regulation of Inpp5e Associated With Abnormal Ciliogenesis During Embryonic Neurodevelopment Under Inositol Deficiency

2021 ◽  
Vol 12 ◽  
Author(s):  
Huixuan Yue ◽  
Shen Li ◽  
Jiaxing Qin ◽  
Tingting Gao ◽  
Jianjun Lyu ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Cell Model ◽  
Gas Chromatography Mass Spectrometry ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inositol Polyphosphate ◽  
Cell Models ◽  
Down Regulation ◽  
Nih3t3 Cells ◽  
Expression Levels ◽  
Brain Tissues

The inositol polyphosphate-5-phosphatase E (Inpp5e) gene is located on chromosome 9q34.3. The enzyme it encodes mainly hydrolyzes the 5-phosphate groups of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5) P3) and phosphatidylinositol (4,5)-bisphosphate (PtdIns (4,5)P2), which are closely related to ciliogenesis and embryonic neurodevelopment, through mechanisms that are largely unknown. Here we studied the role of Inpp5e gene in ciliogenesis during embryonic neurodevelopment using inositol-deficiency neural tube defects (NTDs) mouse and cell models. Confocal microscopy and scanning electron microscope were used to examine the number and the length of primary cilia. The dynamic changes of Inpp5e expression in embryonic murine brain tissues were observed during Embryonic Day 10.5–13.5 (E 10.5–13.5). Immunohistochemistry, western blot, polymerase chain reaction (PCR) arrays were applied to detect the expression of Inpp5e and cilia-related genes of the embryonic brain tissues in inositol deficiency NTDs mouse. Real-time quantitative PCR (RT-qPCR) was used to validate the candidate genes in cell models. The levels of inositol and PtdIns(3,4) P2 were measured using gas chromatography-mass spectrometry (GC-MS) and enzyme linked immunosorbent assay (ELISA), respectively. Our results showed that the expression levels of Inpp5e gradually decreased in the forebrain tissues of the control embryos, but no stable trend was observed in the inositol deficiency NTDs embryos. Inpp5e expression in inositol deficiency NTDs embryos was significantly decreased compared with the control tissues. The expression levels of Inpp5e gene and the PtdIns (3,4) P2 levels were also significantly decreased in the inositol deficient cell model. A reduced number and length of primary cilia were observed in NIH3T3 cells when inositol deficient. Three important cilia-related genes (Ift80, Mkks, Smo) were down-regulated significantly in the inositol-deficient NTDs mouse and cell models, and Smo was highly involved in NTDs. In summary, these findings suggested that down-regulation of Inpp5e might be associated with abnormal ciliogenesis during embryonic neurodevelopment, under conditions of inositol deficiency.

scholarly journals Multiple Parameters Beyond Lipid Binding Affinity Drive Cytotoxicity of Cholesterol-Dependent Cytolysins

Toxins ◽  
2018 ◽  
Vol 11 (1) ◽  
pp. 1 ◽  
Author(s):  
Sucharit Ray ◽  
Roshan Thapa ◽  
Peter Keyel
Keyword(s):  
Cell Death ◽  
Binding Affinity ◽  
Hemolytic Activity ◽  
Mammalian Cells ◽  
Pathogenic Bacteria ◽  
Calcium Influx ◽  
Membrane Binding ◽  
Lipid Binding ◽  
Membrane Repair ◽  
Death Kinetics

The largest superfamily of bacterial virulence factors is pore-forming toxins (PFTs). PFTs are secreted by both pathogenic and non-pathogenic bacteria. PFTs sometimes kill or induce pro-pathogen signaling in mammalian cells, all primarily through plasma membrane perforation, though the parameters that determine these outcomes are unclear. Membrane binding, calcium influx, pore size, and membrane repair are factors that influence PFT cytotoxicity. To test the contribution of membrane binding to cytotoxicity and repair, we compared the closely related, similarly-sized PFTs Perfringolysin O (PFO) from Clostridium perfringens and Streptolysin O (SLO) from Streptococcus pyogenes. Cell death kinetics for PFO and SLO were different because PFO increased in cytotoxicity over time. We introduced known L3 loop mutations that swap binding affinity between toxins and measured hemolytic activity, nucleated cell death kinetics and membrane repair using viability assays, and live cell imaging. Altered hemolytic activity was directly proportional to toxin binding affinity. In contrast, L3 loop alterations reduced nucleated cell death, and they had limited effects on cytotoxicity kinetics and membrane repair. This suggests other toxin structural features, like oligomerization, drives these parameters. Overall, these findings suggest that repair mechanisms and toxin oligomerization add constraints beyond membrane binding on toxin evolution and activity against nucleated cells.

scholarly journals Up-Regulation of Phosphoinositide Metabolism in Tobacco Cells Constitutively Expressing the Human Type I Inositol Polyphosphate 5-Phosphatase

2002 ◽  
Vol 129 (4) ◽  
pp. 1795-1806 ◽  
Author(s):  
Imara Y. Perera ◽  
John Love ◽  
Ingo Heilmann ◽  
William F. Thompson ◽  
Wendy F. Boss
Keyword(s):  
Type I ◽  
Phosphoinositide Metabolism ◽  
Inositol Polyphosphate ◽  
Tobacco Cells ◽  
Human Type

scholarly journals Current understandings of the relationship between extracellular vesicles and cilia

2020 ◽  
Author(s):  
Koji Ikegami ◽  
Faryal Ijaz
Keyword(s):  
Extracellular Vesicles ◽  
Primary Cilium ◽  
Mammalian Cells ◽  
Primary Cilia ◽  
Research Field ◽  
Historical Background ◽  
Unicellular Organism ◽  
The Relationship

Abstract Mammalian cells have a tiny hair-like protrusion on their surface called a primary cilium. Primary cilia are thought to be the antennae for the cells, receiving signals from the environment. In some studies, extracellular vesicles (EVs) were found attached to the surface of the primary cilium. An idea for the phenomenon is that the primary cilium is the receptor for receiving the EVs. Meanwhile, a unicellular organism, Chlamydomonas, which has two long cilia, usually called flagella, release EVs termed ectosomes from the surface of the flagella. Accumulating evidence suggests that the primary cilium also functions as the ‘emitter’ of EVs. Physiological and pathological impacts are also elucidated for the release of EVs from primary cilia. However, the roles of released cilia-derived EVs remain to be clarified. This review introduces the historical background of the relationship between EVs and cilia, and recent progresses in the research field.

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