1. Biochemistry and Chemical Biology
  2. Cell Biology

Release of cholesterol-rich particles from the macrophage plasma membrane during movement of filopodia and lamellipodia

  1. Xuchen Hu
  2. Thomas A Weston
  3. Cuiwen He
  4. Rachel S Jung
  5. Patrick J Heizer
  6. Brian D Young
  7. Yiping Tu
  8. Peter Tontonoz
  9. James A Wohlschlegel
  10. Haibo Jiang  Is a corresponding author
  11. Stephen G Young  Is a corresponding author
  12. Loren G Fong  Is a corresponding author
  1. University of California, Los Angeles, United States
https://doi.org/10.7554/eLife.50231
Share this article
Cite this article
  1. Xuchen Hu
  2. Thomas A Weston
  3. Cuiwen He
  4. Rachel S Jung
  5. Patrick J Heizer
  6. Brian D Young
  7. Yiping Tu
  8. Peter Tontonoz
  9. James A Wohlschlegel
  10. Haibo Jiang
  11. Stephen G Young
  12. Loren G Fong
(2019)
Release of cholesterol-rich particles from the macrophage plasma membrane during movement of filopodia and lamellipodia
eLife 8:e50231.
https://doi.org/10.7554/eLife.50231
  2. Download BibTeX
  3. Download .RIS

Abstract

Cultured mouse peritoneal macrophages release large numbers of ~30-nm cholesterol-rich particles. Here, we show that those particles represent fragments of the plasma membrane that are pulled away and left behind during the projection and retraction of filopodia and lamellipodia. Consistent with this finding, the particles are enriched in proteins found in focal adhesions, which attach macrophages to the substrate. The release of particles is abolished by blocking cell movement (either by depolymerizing actin with latrunculin A or by inhibiting myosin II with blebbistatin). Confocal microscopy and NanoSIMS imaging studies revealed that the plasma membrane–derived particles are enriched in 'accessible cholesterol' (a mobile pool of cholesterol detectable with the modified cytolysin ALO-D4) but not in sphingolipid-sequestered cholesterol (a pool detectable with ostreolysin A (OlyA)]. The discovery that macrophages release cholesterol-rich particles during cellular locomotion is likely relevant to cholesterol efflux and could contribute to extracellular cholesterol deposition in atherosclerotic plaques.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Xuchen Hu

    Department of Medicine, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0944-624X

  2. Thomas A Weston

    Department of Medicine, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    No competing interests declared.

  3. Cuiwen He

    Department of Medicine, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    No competing interests declared.

  4. Rachel S Jung

    Department of Medicine, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    No competing interests declared.

  5. Patrick J Heizer

    Department of Medicine, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    No competing interests declared.

  6. Brian D Young

    Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    No competing interests declared.

  7. Yiping Tu

    Department of Medicine, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    No competing interests declared.

  8. Peter Tontonoz

    Department of Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    Peter Tontonoz, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1259-0477

  9. James A Wohlschlegel

    Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    No competing interests declared.

  10. Haibo Jiang

    Department of Medicine, University of California, Los Angeles, Los Angeles, United States
    For correspondence
    haibo.jiang@uwa.edu.au
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2384-4826

  11. Stephen G Young

    Department of Medicine, University of California, Los Angeles, Los Angeles, United States
    For correspondence
    sgyoung@mednet.ucla.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7270-3176

  12. Loren G Fong

    Department of Medicine, University of California, Los Angeles, Los Angeles, United States
    For correspondence
    lfong@mednet.ucla.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4465-5290

Funding

National Heart, Lung, and Blood Institute (HL090553)

  • Stephen G Young

National Heart, Lung, and Blood Institute (HL087228)

  • Stephen G Young

National Heart, Lung, and Blood Institute (HL125335))

  • Stephen G Young

Fondation Leducq (12CVD04).)

  • Stephen G Young

Ruth L Kirschstein National Research Service Award (T32HL69766))

  • Xuchen Hu

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Fredric B Kraemer, Stanford University, United States

Ethics

Animal experimentation: Animal housing and experimental protocols were approved by UCLA's Animal Research Committee (ARC; 2004-125-51). The animals were housed in an AAALAC (Association for Assessment andAccreditation of Laboratory Animal Care International)-approved facility and cared for accordingto guidelines established by UCLA's Animal Research Committee.

Version history

  1. Received: July 16, 2019
  2. Accepted: September 4, 2019
  3. Accepted Manuscript published: September 5, 2019 (version 1)
  4. Version of Record published: September 18, 2019 (version 2)

Copyright

© 2019, Hu et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,118
    views
  • 318
    downloads
  • 27
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Xuchen Hu
  2. Thomas A Weston
  3. Cuiwen He
  4. Rachel S Jung
  5. Patrick J Heizer
  6. Brian D Young
  7. Yiping Tu
  8. Peter Tontonoz
  9. James A Wohlschlegel
  10. Haibo Jiang
  11. Stephen G Young
  12. Loren G Fong
(2019)
Release of cholesterol-rich particles from the macrophage plasma membrane during movement of filopodia and lamellipodia
eLife 8:e50231.
https://doi.org/10.7554/eLife.50231

Share this article

https://doi.org/10.7554/eLife.50231

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology

    Syntaxin-6 delays prion protein fibril formation and prolongs presence of toxic aggregation intermediates

    Daljit Sangar, Elizabeth Hill ... Jan Bieschke
    Research Article

    Prions replicate via the autocatalytic conversion of cellular prion protein (PrPC) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, non-physiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening suggests that variants that increase syntaxin-6 expression in the brain (gene: STX6) are risk factors for sporadic Creutzfeldt-Jakob disease (CJD). Analysis of the protein in NAA revealed, counterintuitively, that syntaxin-6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, syntaxin-6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that, instead of highly ordered fibrils, in the presence of syntaxin-6 PrP formed less-ordered aggregates containing syntaxin-6. These data strongly suggest that the protein can directly alter the initial phase of PrP self-assembly and, uniquely, can act as an 'anti-chaperone', which promotes toxic aggregation intermediates by inhibiting fibril formation.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    The ORP9-ORP11 dimer promotes sphingomyelin synthesis

    Birol Cabukusta, Shalom Borst Pauwels ... Jacques Neefjes
    Research Article

    Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Deciphering the actin structure-dependent preferential cooperative binding of cofilin

    Kien Xuan Ngo, Huong T Vu ... Taro Uyeda
    Research Article

    The mechanism underlying the preferential and cooperative binding of cofilin and the expansion of clusters toward the pointed-end side of actin filaments remains poorly understood. To address this, we conducted a principal component analysis based on available filamentous actin (F-actin) and C-actin (cofilins were excluded from cofilactin) structures and compared to monomeric G-actin. The results strongly suggest that C-actin, rather than F-ADP-actin, represented the favourable structure for binding preference of cofilin. High-speed atomic force microscopy explored that the shortened bare half helix adjacent to the cofilin clusters on the pointed end side included fewer actin protomers than normal helices. The mean axial distance (MAD) between two adjacent actin protomers along the same long-pitch strand within shortened bare half helices was longer (5.0–6.3 nm) than the MAD within typical helices (4.3–5.6 nm). The inhibition of torsional motion during helical twisting, achieved through stronger attachment to the lipid membrane, led to more pronounced inhibition of cofilin binding and cluster formation than the presence of inorganic phosphate (Pi) in solution. F-ADP-actin exhibited more naturally supertwisted half helices than F-ADP.Pi-actin, explaining how Pi inhibits cofilin binding to F-actin with variable helical twists. We propose that protomers within the shorter bare helical twists, either influenced by thermal fluctuation or induced allosterically by cofilin clusters, exhibit characteristics of C-actin-like structures with an elongated MAD, leading to preferential and cooperative binding of cofilin.