Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Newest Articles
    • Current Issue
    • Methods & Resources
    • Author Interviews
    • Archive
    • Subjects
  • Collections
  • Submit
    • Submit a Manuscript
    • Author Guidelines
    • License, Copyright, Fee
    • FAQ
    • Why submit
  • About
    • About Us
    • Editors & Staff
    • Board Members
    • Licensing and Reuse
    • Reviewer Guidelines
    • Privacy Policy
    • Advertise
    • Contact Us
    • LSA LLC
  • Alerts
  • Other Publications
    • EMBO Press
    • The EMBO Journal
    • EMBO reports
    • EMBO Molecular Medicine
    • Molecular Systems Biology
    • Rockefeller University Press
    • Journal of Cell Biology
    • Journal of Experimental Medicine
    • Journal of General Physiology
    • Journal of Human Immunity
    • Cold Spring Harbor Laboratory Press
    • Genes & Development
    • Genome Research

User menu

  • My alerts

Search

  • Advanced search
Life Science Alliance
  • Other Publications
    • EMBO Press
    • The EMBO Journal
    • EMBO reports
    • EMBO Molecular Medicine
    • Molecular Systems Biology
    • Rockefeller University Press
    • Journal of Cell Biology
    • Journal of Experimental Medicine
    • Journal of General Physiology
    • Journal of Human Immunity
    • Cold Spring Harbor Laboratory Press
    • Genes & Development
    • Genome Research
  • My alerts
Life Science Alliance

Advanced Search

  • Home
  • Articles
    • Newest Articles
    • Current Issue
    • Methods & Resources
    • Author Interviews
    • Archive
    • Subjects
  • Collections
  • Submit
    • Submit a Manuscript
    • Author Guidelines
    • License, Copyright, Fee
    • FAQ
    • Why submit
  • About
    • About Us
    • Editors & Staff
    • Board Members
    • Licensing and Reuse
    • Reviewer Guidelines
    • Privacy Policy
    • Advertise
    • Contact Us
    • LSA LLC
  • Alerts
  • Follow LSA on Bluesky
  • Follow lsa Template on Twitter
Research Article
Source Data
Transparent Process
Open Access

Multiple membrane extrusion sites drive megakaryocyte migration into bone marrow blood vessels

View ORCID ProfileEdward Brown, Leo M Carlin, Claus Nerlov, Cristina Lo Celso, View ORCID ProfileAlastair W Poole  Correspondence email
Edward Brown
1 School of Physiology and Pharmacology, Faculty of Medical and Veterinary Sciences, University of Bristol, Bristol, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Edward Brown
Leo M Carlin
2 Cancer Research UK Beatson Institute, Garscube Campus, Glasgow, UK
3 Inflammation, Repair, and Development, National Heart and Lung Institute, London, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Claus Nerlov
4 MRC Molecular Hematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Cristina Lo Celso
5 Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, UK
6 The Francis Crick Institute, London, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Alastair W Poole
1 School of Physiology and Pharmacology, Faculty of Medical and Veterinary Sciences, University of Bristol, Bristol, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Alastair W Poole
  • For correspondence: a.poole@bristol.ac.uk
Published 21 May 2018. DOI: 10.26508/lsa.201800061
  • Article
  • Figures & Data
  • Info
  • Metrics
  • Reviewer Comments
  • PDF
Loading

Article Figures & Data

Figures

  • Supplementary Materials
  • Figure 1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 1. Megakaryocytes predominantly enter the circulation as a large protrusion.

    (A) Megakaryocyte (M)-intravasating large protrusion (arrowhead) extending into sinusoid lumen (SL). (B) Megakaryocyte (M)-intravasating proplatelet (arrowhead) extending into SL. (C) Free large protrusion (Pr) within SL. (D) Free proplatelet within SL. Scale bars represent 10 μm. (E) Observations of intravasating large protrusions, intravasating proplatelets, free large protrusions, and free proplatelets per 1 mm3 of bone marrow. Bone marrow of the diaphysis region obtained from flushed femurs. Data presented are mean ± SEM. **P < 0.005. *P < 0.01 (unpaired two-tailed t test, three independent experiments).

  • Figure S1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure S1. Single 2-μm histological section from 140 serial sections used to quantify relative abundance of intravasating large protrusions, intravasating proplatelets, free large protrusions, and free proplatelets.

    Bone marrow of the diaphysis region obtained from flushed femurs. Scale bar represents 100 μm. Image representative of three independent experiments.

  • Figure 2.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 2. Proplatelets and large protrusions are morphologically distinct and possess different MT arrangements.

    (A) Selected images from time-lapse DIC microscopy of proplatelet formation in vitro. One proplatelet tip has been followed (arrowhead). (B) Model constructed from TEM section of the same megakaryocyte (green). (C) MT (red) arrangement within the proplatelet tip. (D) MTs at the leading edge of the proplatelet. MTs within 50 nm of the plasma membrane are colored blue. (E) Model of large protrusion constructed from serial TEM sections showing the megakaryocyte (green) protrusion entering the sinusoid (pink). Free proplatelet-like structures (blue) are also present. (F) MT (red) arrangement within the protrusion tip. (G) MTs at the leading edge of the protrusion. MTs within 50 nm of the plasma membrane are colored blue. Scale bars represent 50 μm in (A), 10 μm in (B, E), and 1 μm in (C, D, F, G). Bone marrow of the diaphysis region obtained from flushed femurs. Images representative of three independent experiments.

    Source data are available for this figure.

    Source Data for Figure 2[LSA-2018-00061_SdataF1.pdf]

  • Figure S2.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure S2. Procedure for intravital CLEM.

    (A) Intravital microscopy of bone marrow within murine calvarium. (B) Single frame from time-lapse imaging of mTmG-Tomato–expressing mouse showing a megakaryocyte positive for glycoprotein 1b (green) entering a sinusoid. (C) Extended focus-stitched tile scan of the calvarium showing the marrow (red) and vasculature (white), which will be used to locate the area observed during intravital imaging (box) and the location of the protrusion (*). (D) Calvarium is removed and immersion-fixed. (E) Calvarium is decalcified, stained, and embedded in resin. (F) Resin-embedded calvarium showing the same area viewed for live imaging (box) and the location of the protrusion (*). (G) The desired area trimmed down for sectioning. (H) Thick section showing sinusoids (SL), bone marrow (BM), and bone (B), which when viewed in combination with z-stacks acquired during live imaging allows determination of the z-height within the tissue and hence is when serial 300-nm sections should be taken. (I) Serial 300-nm sections taken just before the region of interest is reached. (J) Low-magnification TEM image confirming the location viewed with intravital imaging has been found. (K) Selected TEM images from the serial sections at the protrusion location. (L) Alignment of the images acquired from the serial sections. (M) Segmentation of structures of interest within the aligned stack of images. (N) 3D rendering to generate a model of the structures. (O, P) Models generated from the segmented image stack. (Q) Tomography can be performed on sections corresponding to regions of interest within the model. (R) This allows high-resolution models to be generated revealing cytoskeletal structure. (S) Model generated from large-volume tomography showing MT (red) arrangement. Scale bars represent 20 μm in (B, K, O, P); 500 μm in (C, F); 100 μm in (H); 50 μm in (J); and 2 μm in (S).

  • Figure 3.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 3. Megakaryocyte large protrusions can be observed by intravital correlative EM.

    (A) Selected images from time-lapse intravital microscopy showing three megakaryocytes expressing vWF-tdTomato (red) (M1–M3) adjacent to a sinusoid and two proplatelet-like structures (yellow and blue arrowheads) positive for glycoprotein 1b (green) within the lumen. (B) 3D model generated from a stack of TEM images of the area observed in (A). The three megakaryocytes (green with black nuclei) adjacent to the sinusoid (pink) are all extending protrusions into the sinusoid lumen (SL). (C) Single TEM image and 3D model of M3. (D) Single tomographic slices showing the IMS, MTs (red), and a centriole (yellow). (E) 3D model of the nucleus (black), MTs, and centriole. (F) Single TEM image of M1 showing the detached large protrusion. (G) Single tomographic slice from boxed region in (F) of the interface between M1 and the detached large protrusion. Bone marrow from intravital CLEM of calvarium. Scale bars represent 25 μm in (A, B); 5 μm in (C, F); and 2 μm in (D, E, G). Images representative of a total of six megakaryocytes from three independent experiments.

    Source data are available for this figure.

    Source Data for Figure 3[LSA-2018-00061_SdataF2.tif]

  • Figure 4.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 4. Megakaryocytes can produce multiple large protrusions simultaneously that anchor into the endothelium.

    (A) Histological sections of a single megakaryocyte (M) extending three separate large protrusions into the sinusoid lumen (SL). (B, D) TEM images of protruding megakaryocytes, highlighting an area selected for tomography (red box). (C, E) Single tomographic slices and respective models of the protrusion membrane (green) overlapping or inserting into the luminal surface of the endothelium (pink). Bone marrow of the diaphysis region obtained from flushed femurs. Scale bars represent 10 μm in (A); 5 μm in (B, D); 500 nm in (C); and 1 μm in (D). Images representative of five independent experiments.

    Source data are available for this figure.

    Source Data for Figure 4[LSA-2018-00061_SdataF3.tif][LSA-2018-00061_SdataF4.tif]

  • Figure 5.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 5. The peripheral zone (PZ) is lost in protruding megakaryocytes.

    (A) Two regions (red boxes) selected for tomography from a non-protruding megakaryocyte (middle panel) with normal PZ (blue) and IMS at both the rear (left panel) and front (right panel) of the cell. (B) Two regions (red boxes) selected for tomography from a protruding megakaryocyte (middle panel) lacking a PZ and allowing the IMS closer to the plasma membrane at both the rear (left panel) and front (right panel) of the cell. Bone marrow of the diaphysis region obtained from flushed femurs. Scale bars represent 2 μm. Images representative of three independent experiments.

    Source data are available for this figure.

    Source Data for Figure 5[LSA-2018-00061_SdataF5.zip][LSA-2018-00061_SdataF6.tif][LSA-2018-00061_SdataF7.tif]

  • Figure 6.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 6. The IMS fuses to the plasma membrane at the protrusion tip.

    (A) Area of protrusion (box) selected for large-volume tomography. (B) Large-volume tomographic reconstruction of the protrusion tip showing the IMS throughout. (C) Tomographic section showing different membrane types. (D) Model showing multiple sites of fusion (arrows) between the IMS (blue) and the plasma membrane. (E) Tomographic slices of selected fusion sites. (F) Model showing the relationship between a partial reconstruction of the IMS (blue) and MTs (red) at a fusion site. Bone marrow of the diaphysis region obtained from flushed femurs. Scale bars represent 5 μm in (A); 1 μm in (B, C); and 500 nm in (D–F). Images representative of three independent experiments.

  • Figure 7.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 7. Megakaryocyte total surface area increases with protrusion extension.

    (A) Selected images of model generated from time-lapse intravital z-stacks of a megakaryocyte protrusion extending to the left into a sinusoid (not shown). (B) A graph of megakaryocyte surface area and protrusion length over time. (C) A dot plot of protrusion length against surface area. Images obtained from calvarium bone marrow. Scale bar represents 20 μm. Data representative of four independent experiments.

  • Figure S3.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure S3. Megakaryocyte surface area increases with protrusion length.

    (A–C) Three additional graphs showing protrusion length and surface area over time, and protrusion length against surface area. Data obtained from three independent experiments.

  • Figure 8.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 8. Diagrammatic representation of thrombopoiesis in vivo.

    (A) Non-protruding megakaryocyte with intact peripheral zone and densely packed IMS. (B) Loss of peripheral zone allows IMS to fuse with plasma membrane and a protrusion to form, which anchors to the luminal surface of the endothelium. (C) IMS trafficked along MTs continues to extrude into the protrusion plasma membrane resulting in extension into the sinusoid lumen in the direction of flow. After release of the large protrusion, MT and membrane reorganization must occur to form proplatelets and ultimately platelets.

Supplementary Materials

  • Figures
  • Video 1

    Time-lapse DIC imaging of proplatelet formation in vitro. This video shows a megakaryocyte extending multiple proplatelets over a 17-h period. CLEM has been performed on one of the proplatelet tips.Download video

  • Video 2

    Reconstruction of a megakaryocyte large protrusion and the MTs at the tip. This video shows the TEM stack of images and the model of the megakaryocyte/protrusion (green), sinusoid (pink), and free proplatelets (blue) constructed from them.Download video

  • Video 3

    TEM reconstruction of megakaryocyte from intravital CLEM. This video shows the TEM stack of images and the model of the megakaryocyte constructed from them.Download video

  • Video 4

    Time-lapse intravital microscopy from an intravital CLEM experiment. This video shows two visible megakaryocyte extensions (red) positive for glycoprotein 1b (green) within a bone marrow sinusoid.Download video

  • Video 5

    TEM reconstruction of megakaryocytes from an intravital CLEM experiment. This video shows the TEM stack of images and the model of the megakaryocytes (green) and sinusoid (pink) constructed from them. These are the same cells observed with intravital microscopy in Video 4.Download video

  • Video 6

    Large-volume electron tomography of a protrusion tip. This video shows the model of the MTs (red) and a portion of IMS (blue) constructed from large-volume tomography at the tip of a large protrusion. Fusions between the IMS and the plasma membrane are shown in red.Download video

  • Video 7

    Time-lapse intravital microscopy of megakaryocyte protrusion. This video shows a model generated from intravital microscopy used to calculate protrusion surface area and length.Download video

PreviousNext
Back to top
Download PDF
Email Article

Thank you for your interest in spreading the word on Life Science Alliance.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Multiple membrane extrusion sites drive megakaryocyte migration into bone marrow blood vessels
(Your Name) has sent you a message from Life Science Alliance
(Your Name) thought you would like to see the Life Science Alliance web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Megakaryocytes migrate by membrane extrusion
Edward Brown, Leo M Carlin, Claus Nerlov, Cristina Lo Celso, Alastair W Poole
Life Science Alliance May 2018, 1 (2) e201800061; DOI: 10.26508/lsa.201800061

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Share
Megakaryocytes migrate by membrane extrusion
Edward Brown, Leo M Carlin, Claus Nerlov, Cristina Lo Celso, Alastair W Poole
Life Science Alliance May 2018, 1 (2) e201800061; DOI: 10.26508/lsa.201800061
Twitter logo Facebook logo Mendeley logo
  • Tweet Widget
Issue Cover

In this Issue

Volume 1, No. 2
May 2018
  • Table of Contents
  • Cover (PDF)
  • About the Cover
  • Masthead (PDF)
Advertisement

Jump to section

  • Article
    • Abstract
    • Introduction
    • Results
    • Discussion
    • Materials and Methods
    • Supplementary Information
    • Acknowledgements
    • References
  • Figures & Data
  • Info
  • Metrics
  • Reviewer Comments
  • PDF

Subjects

  • Cell Biology
  • Methods & Resources
  • Physiology

Related Articles

  • No related articles found.

Cited By...

  • Studies of infused megakaryocytes into mice support a "catch-and-release" model of pulmonary-centric thrombopoiesis
  • Blood platelet formation at a glance
  • Flow-accelerated platelet biogenesis is due to an elasto-hydrodynamic instability
  • Using genome editing to engineer universal platelets
  • Google Scholar

More in this TOC Section

  • HU modulates thiol–disulfide homeostasis
  • GSK3A, a proviral host factor for HAdV-B7 replication
  • PSME3 regulates myogenesis
Show more Research Article

Similar Articles

EMBO Press LogoRockefeller University Press LogoCold Spring Harbor Logo

Content

  • Home
  • Newest Articles
  • Current Issue
  • Archive
  • Subject Collections

For Authors

  • Submit a Manuscript
  • Author Guidelines
  • License, copyright, Fee

Other Services

  • Alerts
  • Bluesky
  • X/Twitter
  • RSS Feeds

More Information

  • Editors & Staff
  • Reviewer Guidelines
  • Feedback
  • Licensing and Reuse
  • Privacy Policy

ISSN: 2575-1077
© 2025 Life Science Alliance LLC

Life Science Alliance is registered as a trademark in the U.S. Patent and Trade Mark Office and in the European Union Intellectual Property Office.