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

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

(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.

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(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).

(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.

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(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.

(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.

(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.

(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.

(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.