Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration

Abstract

Cells communicate with each other through secreting and releasing proteins and vesicles. Many cells can migrate. In this study, we report the discovery of migracytosis, a cell migration-dependent mechanism for releasing cellular contents, and migrasomes, the vesicular structures that mediate migracytosis. As migrating cells move, they leave long tubular strands, called retraction fibers, behind them. Large vesicles, which contain numerous smaller vesicles, grow on the tips and intersections of retraction fibers. These fibers, which connect the vesicles with the main cell body, eventually break, and the vesicles are released into the extracellular space or directly taken up by surrounding cells. Since the formation of these vesicles is migration-dependent, we named them "migrasomes". We also found that cytosolic contents can be transported into migrasomes and released from the cell through migrasomes. We named this migration-dependent release mechanism "migracytosis".

Figure 1

The structure of PLSs revealed by TEM and SEM. (A) TEM image of an ultra-thin section of a NRK cell cultured on a 35 mm dish for 12 h. PLSs are indicated by arrows. Scale bar, 5 μm. Enlarged PLSs are shown in the right panels. Scale bar, 200 nm. (B) TEM image of the lowest ultra-thin section (in contact with the culture dish) of a NRK cell. Scale bar, 5 μm. Enlarged PLSs are shown in the right panels. Scale bar, 200 nm. (C) TEM images of individual PLSs containing different numbers of vesicles. Scale bar, 200 nm. (D) PLSs from A were quantified for their size and the number of small internal vesicles. 120 PLSs were examined. (E) NRK cells were grown on cover slips and observed by field emission scanning electron microscopy. Boxed regions in the top left panel are enlarged in the small panels. Scale bar, 1 μm. (F) TEM images of PLSs in NRK cells and in the isolated PLS fraction. Scale bar, 500 nm.

Figure 2

Identification of PLS markers. (A) Outline of the screening strategy. MS, mass spectrometry. (B) Classification of the proteins identified by MS according to their function (left) and subcellular localization (right). (C) TSPAN4-mCherry-expressing NRK cells were cultured and then observed by confocal microscope. Scale bar, 5 μm. Enlarged PLSs are shown in the right panels. (D) Statistics of percentage of cells containing PLS and PLS number per cell in PLS-containing cells. TSPAN4-GFP-expressing NRK cells were cultured for 12 h and observed by confocal microscope. The upper panel show the percentage of cells containing PLS in total cells. The lower panel show the PLS number per cell. n = 150 from three independent experiments. (E) Correlative confocal and TEM images of the same NRK cell expressing TSPAN4-mCherry. TSPAN4-mCherry-expressing NRK cells were cultured for 12 h and observed by confocal microscope, then fixed in 2.5% glutaraldehyde for 10 min before preparing ultra-thin sections for observation by TEM. The TEM micrograph (right panel) shows the white boxed region in the confocal image (left panel). An enlarged PLS is shown in the top right of the TEM image. Scale bar, 5 μm.

Figure 3

Formation and release of PLSs. (A) 3D reconstruction of PLSs. TSPAN4-GFP-expressing NRK cells were cultured, then observed using a DeltaVision microscope. 3D reconstructions were generated by IMARIS software. Yellow, retraction fibers; red, PLSs. The right panels show enlarged regions of interest from the left panel. T, top view; L, lateral view; B, bottom view. Scale bar, 5 μm. (B) Formation of PLSs. TSPAN4-GFP-expressing NRK cells were cultured for 12 h, and time-lapse images were acquired with a NIKON A1 confocal microscope. The right panels show time-lapse images of three PLSs. The left panel shows a wide-field view at 120 min. Images were captured every 10 min for 12 h. Scale bar, 5 μm. (C) Individual PLSs from B were quantified for change of diameter over time. (D) The disintegration of retraction fibers and the release of PLSs. TSPAN4-GFP-expressing NRK cells was cultured for 12 h, and time-lapse images were acquired with a NIKON A1 confocal microscope. The main panel shows a PLS on a retraction fiber behind a migrating cell. Images were captured every 10 min for 12 h. Scale bar, 5 μm. Enlarged images of the same PLS at different times are shown in the right panels. (E) Duration of PLSs. Time-lapse images of TSPAN4-GFP-expressing NRK cells were acquired as described in D and the lifetime of individual PLSs was quantified. Error bar, SEM.

Figure 4

Formation of PLSs is dependent on cell migration. (A) TSPAN4-GFP-expressing NRK cells were cultured for 12 h and time-lapse images were acquired at the indicated times with a NIKON A1 confocal microscope. Images were captured every 10 min for 12 h. Scale bar, 10 μm. (B) Fibronectin (FN) increases the formation of PLSs. TSPAN4-GFP-expressing NRK cells were cultured for 12 h on 35 mm glass-bottom dishes pretreated with 10 μM FN or on untreated dishes (control). Cells were observed by confocal microscope. Scale bar, 10 μm. (C) Cells from B were assessed for the number of PLSs per cell. Y axis, number of PLSs in treated cell/number of PLSs in control cell. n = 100 cells from three independent experiments. Error bars indicate the SD. (D) TSPAN4-GFP-expressing NRK stable cell lines were transfected with NC (control) or SHARPIN siRNA. Cells were cultured for 12 h and observed by time-lapse confocal microscope. Images were captured every 3 min for 4 h. Scale bar, 10 μm. (E) Cells from D were assessed for the number of PLSs. Y axis, number of PLSs in SHARPIN-knockdown cell/number of PLSs in control cell. n = 100 cells from three independent experiments. Error bars indicate the SD. (F) TSPAN4-GFP-expressing NRK cell were treated with or without Blebbistatin and Dynasore and observed by time-lapse confocal microscope. Images were captured every 3 min for 4 h. The formation of individual PLSs within 5 regions of interest (indicated by colored boxes in the left panels) is shown in the small panels. Each region was monitored for 240 min. Scale bar, 10 μm. (G) Cells from F were assessed in a blind fashion for the total number of PLSs per cell (upper panel) and the number of newly-formed PLSs per cell (lower panel) during the observation period and quantified. Y axis, number of PLSs (or newly-formed PLS) in treated cell/number of PLSs (or newly-formed PLSs) in control cell. C, untreated (control) cells; BS, + Blebbistatin; DS, + Dynasore. n = 30 from three independent experiments. Error bars indicate SD.

Figure 5

Actin polymerization is required for migrasome formation. (A) TEM images of individual PLSs. Scale bar, 500 nm. (B) NRK cells stably expressing TSPAN4-mCherry were transfected with LifeAct-EGFP, then cultured for 12 h and observed by confocal microscope. (C) NRK cells stably expressing TSPAN4-mCherry were cultured for 12 h with or without Cytochalasin B, CK636 and Latruculin A, and observed by time-lapse confocal microscope. The formation of individual PLSs in 5 regions of interest (indicated by colored boxes in the left panels) is shown in the small panels. Each region was monitored for 550 min. Time interval, 6 min 20 s. Scale bar, 10 μm. (D) Cells from C were assessed in a blind fashion for the total number of migrasomes per cell (left panel) and the number of newly-formed migrasomes per cell (right panel) during the observation period and quantified. Y axis, number of migrasomes in treated cell/number of migrasomes in control cell. Cyto B, Cytochalasin B; Lat A, Latruculin A. n = 30 from three independent experiments. Error bars indicate SD.

Figure 6

Formation of migrasomes in various cell lines and under different growth conditions. (A) MEF (mouse embryonic fibroblast), NIH3T3 (mouse embryonic fibroblast), HaCaT (human keratinocyte), MDA-MB-231 (human breast cancer), HCT116 (human colon cancer), SW480 (human adenocarcinoma), MGC803 (human gastric carcinoma), SKOV-3 (human ovarian adenocarcinoma), and B16 (mouse melanoma) cells were transfected with mouse or human TSPAN4-GFP, cultured for 12 h and observed by confocal microscope. Insets show enlarged regions of interest. Scale bar, 2 μm. (B) Mouse embryonic stem cells, mouse hippocampal neurons and mouse bone marrow-derived macrophages were observed by TEM. Scale bar, 2 μm. (C) TSPAN4-GFP-expressing NRK cells were cultured in a 3D matrix of PEG (polyethylene glycol) mixed with collagen (1 mg/ml) and FN (10 μM/ml) for 20 h, then observed with a DeltaVision microscope. The inset shows an enlarged view of a migrasome. Scale bar, 5 μm. (D) Cells from C were observed by SEM. Scale bar, 5 μm. (E) The gastric carcinoma cell line MGC803 was cultured on glass-bottom dishes to 100% density and stained with CellTracker Green. NRK cells stably expressing TSPAN4-GFP were then added and co-cultured overnight. (F) Cells from E were observed by live-cell imaging (left) and SEM (right). Live-cell imaging was performed with a DeltaVision microscope and 3D reconstructions were generated by IMARIS software. Scale bar, 5 μm. (G) Ultra-thin sections of mouse or rat tissues were observed by TEM. Scale bar, 1 μm. LB, lamellar body; A, Alveolus; En, endothelial cell; C, collagen.

Figure 7

Release of cytosolic contents by migrasomes. (A) NRK cells stably expressing TSPAN4-mCherry were transfected with GFP and cultured for 20 h, then time-lapse images were acquired with a NIKON A1 confocal microscope. Images were captured every 6 min 20 s for 4 h. Scale bar, 20 μm. (B) Migrasomes from A were measured for GFP fluorescence intensity using NIKON A1 analysis 4.0 software. (C) Time-lapse images showing NRK-LAMP1-mCherry cells taking up migrasomes from NRK-TSPAN4-GFP cells. LAMP1-mCherry-expressing NRK cells were mixed with TSPAN4-GFP-expressing cells and co-cultured for 12 h, then time-lapse images were acquired with a NIKON A1 confocal microscope. Images were captured every 5 min for 10 h. Insets show enlarged regions of interest. Scale bar, 10 μm.

Figure 8

Migrasomes do not contain the MVB surface marker LAMP1. (A) NRK cells stably expressing TSPAN4-GFP were transfected with LAMP1-mCherry, then cultured for 12 h and observed by confocal microscope. Insets show migrasomes. (B) Illustration of the relationship between exosomes, MVBs and migrasomes.