Menu

Close

Long-term live imaging of the Drosophila adult midgut reveals real-time dynamics of cell division, differentiation, and loss

Judy Martin, Erin Nicole Sanders, Paola Moreno-Roman, Shruthi Balachandra, XinXin Du, Leslie Ann Jaramillo Koyama, Lucy Erin O'Brien

Preprint posted on February 26, 2018 https://www.biorxiv.org/content/early/2018/02/26/271742

A new method captures the dynamics of tissue homeostasis in the stem-cell based organ of the adult fly midgut

Selected by Natalie Dye

Background:

To maintain tissue homeostasis, many adult organs harbour pools of stem cells that divide and differentiate to replenish cells that are lost through ageing and/or injury. How these division and differentiation events are regulated is an important open question in biology.

Due to its genetic tractability, the adult Drosophila midgut (analogous to the mammalian small intestine) has been a powerful model system for identifying the conserved signalling pathways underlying both normal tissue homeostasis and its dysfunction during disease, infection, and ageing. Nonetheless, without methods to support extended live imaging of this tissue (longer than 90min), it has been difficult to use this powerful genetic model to study the cell biology and signalling dynamics underlying stem-cell-based homeostasis.

Summary of the preprint:

The authors of this preprint create a new and vastly improved protocol for imaging the midgut. They first stabilize the abdomen of the animal while providing a feeding tube; then they remove part of the dorsal cuticle, immerse this exposed part in media, and image through this media (Fig 1). The authors develop an image processing pipeline to compensate for global movements of the tissue and use a combination of fluorescent genetic markers and quantification of nuclear size to simultaneously identify and quantify the dynamics of different midgut cell types on a tissue scale for 12-16 hrs.

Fig 1: Method schematic (Fig 1A-C of the preprint): (A) Illustration of the fly digestive track; midgut in white. (B) Mounting the animal for imaging (with an upright microscope). The midgut is accessed through a hole in the cuticle, and the animal is fed through a tube. (C) Steps in sample preparation.

Movie S5 from the preprint showing the a ‘fate sensor’ midgut (esgGal4, UAS-his2b::CFP, GBE-Su(H)-GFP:nls; ubi-his2av::mRFP). With the combination of nuclear size and genetic labels, four midgut cell types can be identified and tracked: stem cells (red), enteroblasts (yellow-green), enterocytes (gray, polyploid – and thus larger), and enteroendocrine cells (gray, diploid – and thus smaller).

 

To demonstrate the power of their technique, the authors then provide new observations related to three processes occurring during normal tissue homeostasis:

  • Loss. Novel observations regarding the extrusion of enterocytes (terminally differentiated, nutrient-absorbing cells) include:
    1. Basal cross sectional cell area shrinks in a rachet-like pulsatile fashion
    2. The nucleus rapidly shoots out of the tissue toward the lumen before recoiling back toward the epithelial surface.
  • Division. Stem cell divisions occur less frequently during live imaging than would be expected based on staining of fixed tissues. Nonetheless, the authors report observations from 39 mitosis events—far more than ever before. Results not only confirm previous work using fixed tissues but also include the following new observations:
    1. Dividing stem cells can rapidly reorient before division.
    2. Neighbouring enteroblasts influence stem cell division orientation.
  • Differentiation. While genetic experiments have clearly implicated Notch signalling in stem cell differentiation, without live imaging data, the dynamics have been elusive, limiting our understanding of the regulatory mechanisms. Here, the authors calibrate a live Notch activity reporter and make three new discoveries:
    1. Notch activation and stem cell differentiation can be directly observed live (Fig 2) and occurs over a time-scale of hours (based on observation of 5 events).
    2. Proximity between stem cell siblings after division does not correlate with Notch activity.
    3. The kinetics of Notch activation and cell differentiation imply a latency period after a cell is born, whereby its differentiation is delayed for several hours.

Fig 2: Direct observation of Notch activation during cell fate transitions (Fig 5D from the preprint). GBE-Su(H)-GFP:nls reports Notch transcriptional activity, while ubi-his2av::mRFP provides a reference signal. Cells additionally express esg>his2ab::CFP to mark progenitor cells. The authors use the GFP:RFP ratio to quantify Notch activation and directly observe transitions between stem and enteroblast states, finding that these transitions require several hours (2.4-6.9hrs).

 

Why I chose this preprint:

In my humble opinion, methods papers are where the preprint servers are especially valuable. Getting a new method out in the open as soon as possible is a great thing. Included in this paper are videos and detailed diagrams to help the reader use the method. Slight modifications may enable imaging of different parts of the adult fly, making the approach potentially even more widely applicable. Drosophila genetics has proven to be extremely powerful over the decades, but I’m excited to see more and more recent efforts to add live imaging to the toolbox – from the embryo, to the brain, wing disc, and now gut.

While this paper is first and foremost a method paper, there’s also a lot of tantalizing new biology presented. Their new data demonstrate how certain kinds of information can really only be obtained by direct observation over time, something I myself have been aiming for in my own work. The authors provide solid, valuable characterization of the dynamics—but nevertheless their data seem to generate more questions than answers. Thus, I think the best is yet to come. There is great potential for the live imaging approach to synergize with existing genetic tools in Drosophila to make really fundamental mechanistic discoveries. In principle, their techniques may also be combined with laser ablation, to probe the influence of mechanical forces, and pharmacological perturbations to rapidly, locally, and reversibly inhibit various proteins.

 

Future Directions:

Here are some of the questions I’m looking forward to hearing about in the future:

  • What in the world is going on with the nucleus in those extruding cells?
  • Since the duration of stem cell sibling contact (at least at the level of the nucleus) does not seem to be important, what regulates the when and whether Notch is activated?
    • Related question: are there filopodia-like protrusions in these cells that may signal long distances through Notch, as in the Drosophila notum?
  • What is the molecular mechanism underlying the apparent latency phase in stem cell differentiation?
  • What happens to these dynamics upon injury, infection, ageing, and with varying diets?
    • Related comment: another recent preprint demonstrates that dietary lipids affect cell differentiation and Notch signalling in the midgut (Obniski et al 2018: https://www.biorxiv.org/content/early/2018/02/28/273813). This is just one example of the interesting perturbations that can be now studied live using the techniques from O’Brien and colleagues.

 

Further reading:

  • Cohen M, Georgiou M, Stevenson NL, Miodownik M, Baum B. “Dynamic filopodia transmit intermittent Delta-Notch signaling to drive pattern refinement during lateral inhibition.” Dev Cell. 2010. doi: 10.1016/j.devcel.2010.06.006.
  • Gervais L, Bardin AJ. “Tissue homeostasis and aging: new insight from the fly intestine.” Curr Opin Cell Biol. 2017. doi: 10.1016/j.ceb.2017.06.005.
  • Guo Z, Lucchetta E, Rafel N, Ohlstein B.“Maintenance of the adult Drosophila intestine: all roads lead to homeostasis.” Curr Opin Genet Dev. 2016. doi: 10.1016/j.gde.2016.06.009.
  • Obniski R, Sieber M, and Spradling A. “Dietary lipids modulate Notch signaling and influence adult intestinal development and metabolism in Drosophila.” BioRxiv 2018. https://www.biorxiv.org/content/early/2018/02/28/273813

 

Tags: cell extrusion, differentiation, homeostasis, intestine, microscopy, notch, organ renewal, stem cells

Posted on: 20th March 2018

Read preprint (No Ratings Yet)




  • Have your say

    Your email address will not be published. Required fields are marked *

    This site uses Akismet to reduce spam. Learn how your comment data is processed.

    Sign up to customise the site to your preferences and to receive alerts

    Register here

    Also in the cancer biology category:

    Oncogenic hijacking of a developmental transcription factor evokes therapeutic vulnerability for ROS-induction in Ewing sarcoma

    Aruna Marchetto, Shunya Ohmura, Martin F. Orth, et al.



    Selected by Hannah Brunsdon

    Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

    Adam K. Glaser, Nicholas P. Reder, Ye Chen, et al.



    Selected by Tim Fessenden

    1

    Myc instructs and maintains pancreatic adenocarcinoma phenotype

    Nicole M Sodir, Roderik M Kortlever, Valentin JA Barthet, et al.



    Selected by William Hill

    Quantification of microenvironmental metabolites in murine cancer models reveals determinants of tumor nutrient availability

    Mark R Sullivan, Laura V Danai, Caroline A Lewis, et al.



    Selected by Maria Rafaeva

    1

    Mechanical Stretch Kills Transformed Cancer Cells

    Ajay Tijore, Mingxi Yao, Yu-Hsiu Wang, et al.



    Selected by Joseph Jose Thottacherry

    Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

    Jayashree Chadchankar, Victoria Korboukh, Peter Doig, et al.



    Selected by Mila Basic

    A conserved MFS orchestrates a subset of O-glycosylation to facilitate macrophage dissemination and tissue invasion

    Katarina Valoskova, Julia Biebl, Marko Roblek, et al.



    Selected by Giuliana Clemente

    1

    MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

    Emily Yun-Chia Chang, James P Wells, Shu-Huei Tsai, et al.



    Selected by Katie Weiner

    1

    Applications, Promises, and Pitfalls of Deep Learning for Fluorescence Image Reconstruction

    Chinmay Belthangady , Loic A. Royer



    Selected by Romain F. Laine

    Basal extrusion drives cell invasion and mechanical stripping of E-cadherin

    John Fadul, Gloria M Slattum, Nadja M Redd, et al.



    Selected by William Hill

    Molecularly distinct models of zebrafish Myc-induced B cell leukemia

    Chiara Borga, Clay Foster, Sowmya Iyer, et al.



    Selected by Hannah Brunsdon

    1

    The landscape of antigen-specific T cells in human cancers

    Bo Li, Longchao Liu, Jian Zhang, et al.



    Selected by Rob Hynds

    1

    Unjamming overcomes kinetic and proliferation arrest in terminally differentiated cells and promotes collective motility of carcinoma.

    Andrea Palamidessi, Chiara Malinverno, Emanuela FRITTOLI, et al.



    Selected by Tim Fessenden

    1

    STAT3 promotes melanoma metastasis by CEBP-induced repression of the MITF pigmentation pathway

    Alexander Swoboda, Robert Soukup, Katharina Kinslechner, et al.



    Selected by Hannah Brunsdon

    Memory sequencing reveals heritable single cell gene expression programs associated with distinct cellular behaviors

    Sydney M Shaffer, Benjamin L Emert, Ann E. Sizemore, et al.



    Selected by Leighton Daigh

    2

    Anti-angiogenic effects of VEGF stimulation on endothelium deficient in phosphoinositide recycling

    Amber N Stratman, Olivia M Farrelly, Constantinos M Mikelis, et al.



    Selected by Coert Margadant

    Also in the cell biology category:

    The autophagic membrane tether ATG2A transfers lipids between membranes

    Shintaro Maeda, Chinatsu Otomo, Takanori Otomo



    Selected by Sandra Malmgren Hill

    LTK is an ER-resident receptor tyrosine kinase that regulates secretion

    Federica G. Centonze, Veronika Reiterer, Karsten Nalbach, et al.



    Selected by Nicola Stevenson

    1

    Distinct RhoGEFs activate apical and junctional actomyosin contractility under control of G proteins during epithelial morphogenesis

    Alain Garcia De Las Bayonas, Jean-Marc Philippe, Annemarie C. Lellouch, et al.



    Selected by Ivana Viktorinová

    1

    In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor

    Jacob P. Keller, Jonathan S. Marvin, Haluk Lacin, et al.



    Selected by Stephan Daetwyler

    1

    The spindle assembly checkpoint functions during early development in non-chordate embryos

    Janet Chenevert, Marianne Roca, Lydia Besnardeau, et al.



    Selected by Maiko Kitaoka

    Blue light induces neuronal-activity-regulated gene expression in the absence of optogenetic proteins

    Kelsey M. Tyssowski, Jesse M. Gray



    Selected by Zheng-Shan Chong

    Mutations in the Insulator Protein Suppressor of Hairy Wing Induce Genome Instability

    Shih-Jui Hsu, Emily C. Stow, James R. Simmons, et al.



    Selected by Maiko Kitaoka

    1

    Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

    Adam K. Glaser, Nicholas P. Reder, Ye Chen, et al.



    Selected by Tim Fessenden

    1

    ATAT1-enriched vesicles promote microtubule acetylation via axonal transport

    Aviel Even, Giovanni Morelli, Chiara Scaramuzzino, et al.



    Selected by Stephen Royle

    1

    HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

    C. Favard, J. Chojnacki, P. Merida, et al.



    Selected by Amberley Stephens

    Hepatocyte-specific deletion of Pparα promotes NASH in the context of obesity

    Marion Regnier, Arnaud Polizzi, Sarra Smati, et al.



    Selected by Pablo Ranea Robles

    Mitochondrial biogenesis is transcriptionally repressed in lysosomal lipid storage diseases

    King Faisal Yambire, Lorena Fernandez-Mosquera, Robert Steinfeld, et al.



    Selected by Sandra Franco Iborra

    1

    Thyroid hormone regulates distinct paths to maturation in pigment cell lineages

    Lauren Saunders, Abhishek Mishra, Andrew J Aman, et al.



    Selected by Hannah Brunsdon

    1

    Kinesin-6 Klp9 plays motor-dependent and -independent roles in collaboration with Kinesin-5 Cut7 and the microtubule crosslinker Ase1 in fission yeast

    Masashi Yukawa, Masaki Okazaki, Yasuhiro Teratani, et al.



    Selected by I. Bouhlel

    A pair of E3 ubiquitin ligases compete to regulate filopodial dynamics and axon guidance

    Nicholas P Boyer, Laura E McCormick, Fabio L Urbina, et al.



    Selected by Angika Basant

    1

    SorCS1-mediated Sorting of Neurexin in Dendrites Maintains Presynaptic Function

    Luis Filipe Ribeiro, Ben Verpoort, Julie Nys, et al.



    Selected by Carmen Adriaens

    1

    Also in the developmental biology category:

    The visual system of the genetically tractable crustacean Parhyale hawaiensis: diversification of eyes and visual circuits associated with low-resolution vision

    Ana Patricia Ramos, Ola Gustafsson, Nicolas Labert, et al.



    Selected by Alexa Sadier

    Distinct RhoGEFs activate apical and junctional actomyosin contractility under control of G proteins during epithelial morphogenesis

    Alain Garcia De Las Bayonas, Jean-Marc Philippe, Annemarie C. Lellouch, et al.



    Selected by Ivana Viktorinová

    1

    The Hunchback temporal transcription factor determines motor neuron axon and dendrite targeting in Drosophila

    Austin Q Seroka, Chris Q Doe



    Selected by Abagael Lasseigne

    1

    Preformed Chromatin Topology Assists Transcriptional Robustness of Shh during Limb Development

    Christina Paliou, Philine Guckelberger, Robert Schöpflin, et al.



    Selected by Rafael Galupa

    1

    Molecular Logic of Spinocerebellar Tract Neuron Diversity and Connectivity

    Myungin Baek, Vilas Menon, Thomas Jessell, et al.



    Selected by Yen-Chung Chen

    In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor

    Jacob P. Keller, Jonathan S. Marvin, Haluk Lacin, et al.



    Selected by Stephan Daetwyler

    1

    Active behaviour during early development shapes glucocorticoid reactivity

    Luis A. Castillo-Ramírez, Soojin Ryu, Rodrigo J. De Marco



    Selected by Kathleen Gilmour

    A unicellular relative of animals generates an epithelium-like cell layer by actomyosin-dependent cellularization

    Omaya Dudin, Andrej Ondracka, Xavier Grau-Bové, et al.



    Selected by Paul Gerald L. Sanchez and Stefano Vianello

    1

    Members of the Arabidopsis auxin receptor gene family are essential early in embryogenesis and have broadly overlapping functions

    Michael J Prigge, Nikita Kadakia, Kathleen Greenham, et al.



    Selected by Chandra Shekhar Misra

    1

    The spindle assembly checkpoint functions during early development in non-chordate embryos

    Janet Chenevert, Marianne Roca, Lydia Besnardeau, et al.



    Selected by Maiko Kitaoka

    Over-activation of BMP signaling in neural crest cells precipitates heart outflow tract septation

    Jean Francois Darrigrand, Mariana Valente, Pauline Martinez, et al.



    Selected by Giulia Boezio

    Mutations in the Insulator Protein Suppressor of Hairy Wing Induce Genome Instability

    Shih-Jui Hsu, Emily C. Stow, James R. Simmons, et al.



    Selected by Maiko Kitaoka

    1

    Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

    Adam K. Glaser, Nicholas P. Reder, Ye Chen, et al.



    Selected by Tim Fessenden

    1

    Dynamic Erasure of Random X-Chromosome Inactivation during iPSC Reprogramming

    Adrian Janiszewski, Irene Talon, Juan Song, et al.



    Selected by Sergio Menchero

    MicroRNA-mediated control of developmental lymphangiogenesis

    Hyun Min Jung, Ciara Hu, Alexandra M Fister, et al.



    Selected by Rudra Nayan Das

    Endogenous CRISPR arrays for scalable whole organism lineage tracing

    James Cotterell, James Sharpe



    Selected by Irepan Salvador-Martinez

    Also in the genetics category:

    The Hunchback temporal transcription factor determines motor neuron axon and dendrite targeting in Drosophila

    Austin Q Seroka, Chris Q Doe



    Selected by Abagael Lasseigne

    1

    Preformed Chromatin Topology Assists Transcriptional Robustness of Shh during Limb Development

    Christina Paliou, Philine Guckelberger, Robert Schöpflin, et al.



    Selected by Rafael Galupa

    1

    Molecular Logic of Spinocerebellar Tract Neuron Diversity and Connectivity

    Myungin Baek, Vilas Menon, Thomas Jessell, et al.



    Selected by Yen-Chung Chen

    Over-activation of BMP signaling in neural crest cells precipitates heart outflow tract septation

    Jean Francois Darrigrand, Mariana Valente, Pauline Martinez, et al.



    Selected by Giulia Boezio

    Functional dissection of TADs reveals non-essential and instructive roles in regulating gene expression

    Alexandra Despang, Robert Schöpflin, Martin Franke, et al.



    Selected by Clarice Hong

    Crowdfunded whole-genome sequencing of the celebrity cat Lil BUB identifies causal mutations for her osteopetrosis and polydactyly

    Mike Bridavsky, Heiner Kuhl, Arthur Woodruf, et al.



    Selected by Jesus Victorino, Gabriel Aughey

    1

    Dynamic Erasure of Random X-Chromosome Inactivation during iPSC Reprogramming

    Adrian Janiszewski, Irene Talon, Juan Song, et al.



    Selected by Sergio Menchero

    Kinesin-6 Klp9 plays motor-dependent and -independent roles in collaboration with Kinesin-5 Cut7 and the microtubule crosslinker Ase1 in fission yeast

    Masashi Yukawa, Masaki Okazaki, Yasuhiro Teratani, et al.



    Selected by I. Bouhlel

    Human Handedness: Genetics, Microtubules, Neuropsychiatric Diseases and Brain Language Areas

    Akira Wiberg, Gwenaelle Douaud, Michael Ng, et al.



    Selected by Jose Guerrero

    The Spatio-Temporal Control of Zygotic Genome Activation

    George Gentsch, Nick D. L. Owens, James C. Smith



    Selected by Meng Zhu

    Multilevel regulation of the glass locus during Drosophila eye development

    Cornelia Fritsch, F. Javier Bernardo-Garcia, Tim Humberg, et al.



    Selected by Gabriel Aughey

    1

    Distinct ROPGEFs successively drive polarization and outgrowth of root hairs

    Philipp Denninger, Anna Reichelt, Vanessa Aphaia Fiona Schmidt, et al.



    Selected by Marc Somssich

    A direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila

    Christopher M Uyehara, Daniel J McKay



    Selected by Natalie Dye

    MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

    Emily Yun-Chia Chang, James P Wells, Shu-Huei Tsai, et al.



    Selected by Katie Weiner

    1

    Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

    Quynh Nguyen, Zhen Liu, Rashmi Nanjundappa, et al.



    Selected by Robert Mahen

    Single cell RNA-Seq reveals distinct stem cell populations that drive sensory hair cell regeneration in response to loss of Fgf and Notch signaling

    Mark E. Lush, Daniel C. Diaz, Nina Koenecke, et al.

    AND

    Distinct progenitor populations mediate regeneration in the zebrafish lateral line.

    Eric D Thomas, David Raible



    Selected by Rudra Nayan Das

    2

    Also in the physiology category:

    In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor

    Jacob P. Keller, Jonathan S. Marvin, Haluk Lacin, et al.



    Selected by Stephan Daetwyler

    1

    Active behaviour during early development shapes glucocorticoid reactivity

    Luis A. Castillo-Ramírez, Soojin Ryu, Rodrigo J. De Marco



    Selected by Kathleen Gilmour

    Optical determination of absolute membrane potential

    Julia R. Lazzari-Dean, Anneliese M.M. Gest, Evan Miller



    Selected by James Marchant

    Hepatocyte-specific deletion of Pparα promotes NASH in the context of obesity

    Marion Regnier, Arnaud Polizzi, Sarra Smati, et al.



    Selected by Pablo Ranea Robles

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    1

    Quantification of microenvironmental metabolites in murine cancer models reveals determinants of tumor nutrient availability

    Mark R Sullivan, Laura V Danai, Caroline A Lewis, et al.



    Selected by Maria Rafaeva

    1

    Regulation of modulatory cell activity across olfactory structures in Drosophila melanogaster

    Xiaonan Zhang, Kaylynn Coates, Andrew Dacks, et al.



    Selected by Rudra Nayan Das

    1

    EHD2-mediated restriction of caveolar dynamics regulates cellular lipid uptake

    Claudia Matthaeus, Ines Lahmann, Severine Kunz, et al.



    Selected by Andreas Müller

    1

    Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

    Quynh Nguyen, Zhen Liu, Rashmi Nanjundappa, et al.



    Selected by Robert Mahen

    Defining the design requirements for an assistive powered hand exoskeleton

    Quinn A Boser, Michael R Dawson, Jonathon S Schofield, et al.



    Selected by Joanna Cross

    Reduced mitochondrial lipid oxidation leads to fat accumulation in myosteatosis

    Jonathan P Gumucio, Austin H Qasawa, Patrick J Ferrara, et al.



    Selected by Pablo Ranea Robles

    The Toll pathway inhibits tissue growth and regulates cell fitness in an infection-dependent manner

    Federico Germani, Daniel Hain, Denise Sternlicht, et al.



    Selected by Rohan Khadilkar

    Optogenetic manipulation of medullary neurons in the locust optic lobe

    Hongxia Wang, Richard B. Dewell, Markus U. Ehrengruber, et al.



    Selected by Ana Patricia Ramos

    Targeting light-gated chloride channels to neuronal somatodendritic domain reduces their excitatory effect in the axon

    Jessica Messier, Hongmei Chen, Zhao-Lin Cai, et al.

    AND

    High-efficiency optogenetic silencing with soma-targeted anion-conducting channelrhodopsins

    Mathias Mahn, Lihi Gibor, Katayun Cohen-Kashi Malina, et al.



    Selected by Mahesh Karnani

    2

    Rearing temperature and fatty acid supplementation jointly affect membrane fluidity and heat tolerance in Daphnia

    Dominik Martin-Creuzburg, Bret L. Coggins, Dieter Ebert, et al.



    Selected by Alexander Little

    Long-term live imaging of the Drosophila adult midgut reveals real-time dynamics of cell division, differentiation, and loss

    Judy Martin, Erin Nicole Sanders, Paola Moreno-Roman, et al.



    Selected by Natalie Dye
    Close