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Intercellular transfer to signalling endosomes regulates an ex vivo bone marrow niche.

Gillette JM, Larochelle A, Dunbar CE, Lippincott-Schwartz J - Nat. Cell Biol. (2009)

Bottom Line: Here, we use live-cell imaging to characterize both the site of contact between osteoblasts and haematopoietic progenitor cells (HPCs) and events at this site that result in downstream signalling responses important for niche maintenance.This caused osteoblasts to downregulate Smad signalling and increase production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HSPC homing to bone marrow.These findings identify a mechanism involving intercellular transfer to signalling endosomes for targeted regulation of signalling and remodelling events within an ex vivo osteoblastic niche.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Program, National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892, USA.

ABSTRACT
Haematopoietic stem-progenitor cells (HSPCs) reside in the bone marrow niche, where interactions with osteoblasts provide essential cues for their proliferation and survival. Here, we use live-cell imaging to characterize both the site of contact between osteoblasts and haematopoietic progenitor cells (HPCs) and events at this site that result in downstream signalling responses important for niche maintenance. HPCs made prolonged contact with the osteoblast surface through a specialized membrane domain enriched in prominin 1, CD63 and rhodamine PE. At the contact site, portions of the specialized domain containing these molecules were taken up by the osteoblast and internalized into SARA-positive signalling endosomes. This caused osteoblasts to downregulate Smad signalling and increase production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HSPC homing to bone marrow. These findings identify a mechanism involving intercellular transfer to signalling endosomes for targeted regulation of signalling and remodelling events within an ex vivo osteoblastic niche.

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Intercellular transfer occurs between HPC and osteoblastic cells in a contact dependent manner. (a) KG1a cell labeled with QDs (red) and co-cultured with osteoblastic cells stably transfected with tubulin-YFP (green) for 2 h before live-cell imaging. The white box indicates the zoomed region and arrows indicate transferred QDs internalized within the osteoblasts. KG1a cells transiently transfected with (b) prominin1-GFP (green) or (c) CD63-cherry (red) and co-cultured with osteoblasts for one hour before live cell confocal imaging. Arrows indicate transferred protein. CD63-cherry transfected cells were co-cultured with osteoblasts stably transfected with tubulin-YFP (green). (d) Live-cell confocal microscopy of N-Rh-PE (red) transfer from KG1a cells to osteoblastic cells. Arrows indicate transferred lipid. (e) The percentage of osteoblastic cells (black, n=120) or HeLa cells (grey, n=75) that acquired N-Rh-PE transfer following contact with an N-Rh-PE labeled HPC after either 1 or 3 h of co-culture. (n, number of HPC/osteoblast or HPC/Hela contacted cells scored over three independent experiments). (f) Live cell imaging of N-Rh-PE (red) labeled CD34+ cell contacting an osteoblastic cell stably transfected with GFP (green). The asterisk indicates the site of CD34+ cell contact and the white lines outline the osteoblasts in the field of view. (g) The percentage of osteoblastic cells that acquired N-Rh-PE transfer following: direct contact with an N-Rh-PE labeled HPC, no contact with a labeled HPC (osteoblasts neighboring HPC contacted cells), or labeled HPC co-culture with osteoblasts through a 0.4μm membrane filter. (n > 100 osteoblasts for each condition). (h) The percentage of osteoblastic cells that acquired N-Rh-PE transfer following contact with a labeled HPC treated with control, 10 mM methyl β cyclodextrin (MβCD), 2 μm cytocholasin D (Cyto D), or 80 μm Dynasore (n > 100 for each condition). (n, number of HPC/osteoblast contacted cells counted over three independent experiments). (i) Live cell confocal microscopy of CD34+ cells labeled with N-Rh-PE and co-cultured with osteoblastic cells. Cells were co-cultured for 1 h before imaging began and intercellular transfer was observed. (j) Live cell confocal microscopy of CD34+CD38- cells labeled with N-Rh-PE and co-cultured with primary human osteoblasts. Scale bars – 5μm.
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Figure 3: Intercellular transfer occurs between HPC and osteoblastic cells in a contact dependent manner. (a) KG1a cell labeled with QDs (red) and co-cultured with osteoblastic cells stably transfected with tubulin-YFP (green) for 2 h before live-cell imaging. The white box indicates the zoomed region and arrows indicate transferred QDs internalized within the osteoblasts. KG1a cells transiently transfected with (b) prominin1-GFP (green) or (c) CD63-cherry (red) and co-cultured with osteoblasts for one hour before live cell confocal imaging. Arrows indicate transferred protein. CD63-cherry transfected cells were co-cultured with osteoblasts stably transfected with tubulin-YFP (green). (d) Live-cell confocal microscopy of N-Rh-PE (red) transfer from KG1a cells to osteoblastic cells. Arrows indicate transferred lipid. (e) The percentage of osteoblastic cells (black, n=120) or HeLa cells (grey, n=75) that acquired N-Rh-PE transfer following contact with an N-Rh-PE labeled HPC after either 1 or 3 h of co-culture. (n, number of HPC/osteoblast or HPC/Hela contacted cells scored over three independent experiments). (f) Live cell imaging of N-Rh-PE (red) labeled CD34+ cell contacting an osteoblastic cell stably transfected with GFP (green). The asterisk indicates the site of CD34+ cell contact and the white lines outline the osteoblasts in the field of view. (g) The percentage of osteoblastic cells that acquired N-Rh-PE transfer following: direct contact with an N-Rh-PE labeled HPC, no contact with a labeled HPC (osteoblasts neighboring HPC contacted cells), or labeled HPC co-culture with osteoblasts through a 0.4μm membrane filter. (n > 100 osteoblasts for each condition). (h) The percentage of osteoblastic cells that acquired N-Rh-PE transfer following contact with a labeled HPC treated with control, 10 mM methyl β cyclodextrin (MβCD), 2 μm cytocholasin D (Cyto D), or 80 μm Dynasore (n > 100 for each condition). (n, number of HPC/osteoblast contacted cells counted over three independent experiments). (i) Live cell confocal microscopy of CD34+ cells labeled with N-Rh-PE and co-cultured with osteoblastic cells. Cells were co-cultured for 1 h before imaging began and intercellular transfer was observed. (j) Live cell confocal microscopy of CD34+CD38- cells labeled with N-Rh-PE and co-cultured with primary human osteoblasts. Scale bars – 5μm.

Mentions: To study the contact site membrane dynamics, we imaged QD-labeled HPCs in contact with osteoblastic cells. Strikingly, QDs transferred from the HPC uropod into the cytoplasm of tubulin-YFP expressing osteoblasts (Fig. 3a) (Supp. Mov. 4). The cytoplasmic localization of the transferred QDs was confirmed by Z series confocal imaging through the osteoblast, which showed that the QDs were in the same plane as the intercellular marker, tubulin-YFP, and not detected on the osteoblast surface (Fig. 3a, zoom). This suggested that intercellular transfer was occurring at the contact site. Imaging of prominin 1-GFP-labeled HPCs in contact with osteoblasts revealed punctate structures enriched in the marker being transferred to the osteoblast over a 20 min contact period (Fig. 3b). Unlike QDs, which reside peripherally on the surface of HPCs and so might be transferred to osteoblasts by a sticking and release process, prominin-1-GFP is embedded in the HPC bilayer and so can only be transferred along with HPC membrane. Co-culturing of CD63-cherry-labeled HPCs with tubulin-YFP osteoblasts also revealed uptake of CD63-cherry into the osteoblast (Fig. 3c) (Supp. Mov. 5). Because both prominin 1-GFP and CD63-cherry proteins were engineered with the fluorescent proteins fused to the carboxy-terminus, which is located in the cytosol, their detection in the osteoblast cells after transfer from HPCs suggested that the entire protein was transferred, not a cleaved fragment. Intercellular transfer of N-Rh-PE into osteoblasts also occurred when N-Rh-PE-labeled HPCs contacted osteoblasts (Fig. 3d). This indicated that both lipid and protein components from the HPC uropod were transferred to osteoblasts.


Intercellular transfer to signalling endosomes regulates an ex vivo bone marrow niche.

Gillette JM, Larochelle A, Dunbar CE, Lippincott-Schwartz J - Nat. Cell Biol. (2009)

Intercellular transfer occurs between HPC and osteoblastic cells in a contact dependent manner. (a) KG1a cell labeled with QDs (red) and co-cultured with osteoblastic cells stably transfected with tubulin-YFP (green) for 2 h before live-cell imaging. The white box indicates the zoomed region and arrows indicate transferred QDs internalized within the osteoblasts. KG1a cells transiently transfected with (b) prominin1-GFP (green) or (c) CD63-cherry (red) and co-cultured with osteoblasts for one hour before live cell confocal imaging. Arrows indicate transferred protein. CD63-cherry transfected cells were co-cultured with osteoblasts stably transfected with tubulin-YFP (green). (d) Live-cell confocal microscopy of N-Rh-PE (red) transfer from KG1a cells to osteoblastic cells. Arrows indicate transferred lipid. (e) The percentage of osteoblastic cells (black, n=120) or HeLa cells (grey, n=75) that acquired N-Rh-PE transfer following contact with an N-Rh-PE labeled HPC after either 1 or 3 h of co-culture. (n, number of HPC/osteoblast or HPC/Hela contacted cells scored over three independent experiments). (f) Live cell imaging of N-Rh-PE (red) labeled CD34+ cell contacting an osteoblastic cell stably transfected with GFP (green). The asterisk indicates the site of CD34+ cell contact and the white lines outline the osteoblasts in the field of view. (g) The percentage of osteoblastic cells that acquired N-Rh-PE transfer following: direct contact with an N-Rh-PE labeled HPC, no contact with a labeled HPC (osteoblasts neighboring HPC contacted cells), or labeled HPC co-culture with osteoblasts through a 0.4μm membrane filter. (n > 100 osteoblasts for each condition). (h) The percentage of osteoblastic cells that acquired N-Rh-PE transfer following contact with a labeled HPC treated with control, 10 mM methyl β cyclodextrin (MβCD), 2 μm cytocholasin D (Cyto D), or 80 μm Dynasore (n > 100 for each condition). (n, number of HPC/osteoblast contacted cells counted over three independent experiments). (i) Live cell confocal microscopy of CD34+ cells labeled with N-Rh-PE and co-cultured with osteoblastic cells. Cells were co-cultured for 1 h before imaging began and intercellular transfer was observed. (j) Live cell confocal microscopy of CD34+CD38- cells labeled with N-Rh-PE and co-cultured with primary human osteoblasts. Scale bars – 5μm.
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Figure 3: Intercellular transfer occurs between HPC and osteoblastic cells in a contact dependent manner. (a) KG1a cell labeled with QDs (red) and co-cultured with osteoblastic cells stably transfected with tubulin-YFP (green) for 2 h before live-cell imaging. The white box indicates the zoomed region and arrows indicate transferred QDs internalized within the osteoblasts. KG1a cells transiently transfected with (b) prominin1-GFP (green) or (c) CD63-cherry (red) and co-cultured with osteoblasts for one hour before live cell confocal imaging. Arrows indicate transferred protein. CD63-cherry transfected cells were co-cultured with osteoblasts stably transfected with tubulin-YFP (green). (d) Live-cell confocal microscopy of N-Rh-PE (red) transfer from KG1a cells to osteoblastic cells. Arrows indicate transferred lipid. (e) The percentage of osteoblastic cells (black, n=120) or HeLa cells (grey, n=75) that acquired N-Rh-PE transfer following contact with an N-Rh-PE labeled HPC after either 1 or 3 h of co-culture. (n, number of HPC/osteoblast or HPC/Hela contacted cells scored over three independent experiments). (f) Live cell imaging of N-Rh-PE (red) labeled CD34+ cell contacting an osteoblastic cell stably transfected with GFP (green). The asterisk indicates the site of CD34+ cell contact and the white lines outline the osteoblasts in the field of view. (g) The percentage of osteoblastic cells that acquired N-Rh-PE transfer following: direct contact with an N-Rh-PE labeled HPC, no contact with a labeled HPC (osteoblasts neighboring HPC contacted cells), or labeled HPC co-culture with osteoblasts through a 0.4μm membrane filter. (n > 100 osteoblasts for each condition). (h) The percentage of osteoblastic cells that acquired N-Rh-PE transfer following contact with a labeled HPC treated with control, 10 mM methyl β cyclodextrin (MβCD), 2 μm cytocholasin D (Cyto D), or 80 μm Dynasore (n > 100 for each condition). (n, number of HPC/osteoblast contacted cells counted over three independent experiments). (i) Live cell confocal microscopy of CD34+ cells labeled with N-Rh-PE and co-cultured with osteoblastic cells. Cells were co-cultured for 1 h before imaging began and intercellular transfer was observed. (j) Live cell confocal microscopy of CD34+CD38- cells labeled with N-Rh-PE and co-cultured with primary human osteoblasts. Scale bars – 5μm.
Mentions: To study the contact site membrane dynamics, we imaged QD-labeled HPCs in contact with osteoblastic cells. Strikingly, QDs transferred from the HPC uropod into the cytoplasm of tubulin-YFP expressing osteoblasts (Fig. 3a) (Supp. Mov. 4). The cytoplasmic localization of the transferred QDs was confirmed by Z series confocal imaging through the osteoblast, which showed that the QDs were in the same plane as the intercellular marker, tubulin-YFP, and not detected on the osteoblast surface (Fig. 3a, zoom). This suggested that intercellular transfer was occurring at the contact site. Imaging of prominin 1-GFP-labeled HPCs in contact with osteoblasts revealed punctate structures enriched in the marker being transferred to the osteoblast over a 20 min contact period (Fig. 3b). Unlike QDs, which reside peripherally on the surface of HPCs and so might be transferred to osteoblasts by a sticking and release process, prominin-1-GFP is embedded in the HPC bilayer and so can only be transferred along with HPC membrane. Co-culturing of CD63-cherry-labeled HPCs with tubulin-YFP osteoblasts also revealed uptake of CD63-cherry into the osteoblast (Fig. 3c) (Supp. Mov. 5). Because both prominin 1-GFP and CD63-cherry proteins were engineered with the fluorescent proteins fused to the carboxy-terminus, which is located in the cytosol, their detection in the osteoblast cells after transfer from HPCs suggested that the entire protein was transferred, not a cleaved fragment. Intercellular transfer of N-Rh-PE into osteoblasts also occurred when N-Rh-PE-labeled HPCs contacted osteoblasts (Fig. 3d). This indicated that both lipid and protein components from the HPC uropod were transferred to osteoblasts.

Bottom Line: Here, we use live-cell imaging to characterize both the site of contact between osteoblasts and haematopoietic progenitor cells (HPCs) and events at this site that result in downstream signalling responses important for niche maintenance.This caused osteoblasts to downregulate Smad signalling and increase production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HSPC homing to bone marrow.These findings identify a mechanism involving intercellular transfer to signalling endosomes for targeted regulation of signalling and remodelling events within an ex vivo osteoblastic niche.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Program, National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892, USA.

ABSTRACT
Haematopoietic stem-progenitor cells (HSPCs) reside in the bone marrow niche, where interactions with osteoblasts provide essential cues for their proliferation and survival. Here, we use live-cell imaging to characterize both the site of contact between osteoblasts and haematopoietic progenitor cells (HPCs) and events at this site that result in downstream signalling responses important for niche maintenance. HPCs made prolonged contact with the osteoblast surface through a specialized membrane domain enriched in prominin 1, CD63 and rhodamine PE. At the contact site, portions of the specialized domain containing these molecules were taken up by the osteoblast and internalized into SARA-positive signalling endosomes. This caused osteoblasts to downregulate Smad signalling and increase production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HSPC homing to bone marrow. These findings identify a mechanism involving intercellular transfer to signalling endosomes for targeted regulation of signalling and remodelling events within an ex vivo osteoblastic niche.

Show MeSH